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Different roles of p160 coregulators in myogenesis

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Different roles of p160 coregulators in myogenesis

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Content DIFFERENT ROLES OF P160 COREGULATORS IN
MYOGENESIS
by
Hung-Yi Wu
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements of the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY & MOLECULAR BIOLOGY)
December 2002
Copyright 2002 Hung-Yi Wu
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UMI Number: 3093938
UMI
UMI Microform 3093938
Copyright 2003 by ProQuest Information and Learning Company.
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UNIVERSITY OF SOUTHERN CALIFORNIA
The Graduate School
University Park
LOS ANGELES, CALIFORNIA 90089-1695
This dissertation, w ritten b y
Hung-Yi W u _____ ______________ _____
U nder th e direction o f h. D issertation
Com m ittee, an d approved b y a ll its m em bers,
has been p resen ted to an d accepted b y The
G raduate School, in p a rtia l fu lfillm en t o f
requirem ents fo r th e degree o f
DOCTOR OF PHILOSOPHY
D ate Decem ber 1 8 , 2002
Dean o f Graduate Studies
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DEDICATION
I would like to dedicate this thesis to my parents for their efforts to
raise me up and continuous support that I can complete my graduate
studies.
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ACKNOWLEDGEMENTS
I would like to express my greatest gratitude toward my mentor. Dr.
Larry Kedes, for all his inspiration, guidance, criticism,
encouragement, and support that I can complete the Ph.D. training in
his laboratory.
In addition, I would like to thank Yasuo Hamamori for his guidance
at the initial stage of my graduate studies. Also, I would thank all the
members of Kedes’s laboratory, especially Vittorio Sartorelli, Coralie
Poizat and Tatsuya Iso, for their comments on this project. I would
also like to thank Shin Chang and Ming-Fu Chang for their generous
support each time when I stayed in Taiwan.
At last, I would like to thank the coordinator of the Master program,
Dr. Zoltan Tokes for his help when I was in the program. I would also
give my greatest gratitude to the members of my thesis committee, Dr.
Michael Stallcup, Dr, Robert Maxson, and Dr. Michael Lai for their
suggestions and comments. Hung-Yi would like to thank Dr. Michael
Stallcup for his pioneering work on the p i60 coregulators mostly on
my behalf.
iii
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TABLE OF CONTENTS
PAGE
Dedication ................................................. ii
Acknowledgements ........................................................... iii
List of Figures ................................................. v
Abstract............................................................................... vii
Chapter 1: Introduction......................................... 1
Chapter 2: Functions Of P160 Coregulators On
MyoD Transactivation...................... 7
Chapter 3: The Interactions Between PI60
Coregulators And MyoD....................30
Bibliography...................... ..................................................118
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LIST OF FIGURES
Figure Page
1. SRC1A And CIP Coactivate, Whereas GRIP1
Represses MyoD On Muscle Specific Promoters... 12
2. The Transactivation Of MyoD Significantly
Diminished in SRC1A-/- Mouse Embryonic
Fibroblasts (MEFs)................................................. 18
3. Adenovirus E1A Interacts With P160 Coregulators
Both In Vitro And In Vivo......................................21
4. Myogenic Factors Directly Interact With P160
Coregulators In V itro............................................ 37
5. P160 Coregulators Associate With MyoD In Vivo..41
6. Domains Of PI 60s That Interact With MyoD In
Vitro........................................................................ 48
7. N-termini of SRC1A And p/CIP Are Not Involved
In The Interactions With MyoD In Vivo..................59
8. A(MID 1/2+C) Mutants Of PI60 Coregulators
Significantly Lose Ability To Interact With MyoD
Both In Vitro And In Vivo....................................... 64
9. GRIP 1 A(MID 1 /2+C) Mutant Loses The Repressor
Activity Toward MyoD, While Both SRC1AA
(MID 1/2+C) And p/CIPA(MID 1/2+C) Mutants Fail
To Coactivate MyoD On Muscle Specific
Promoters..................................................................68
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10. Both N Terminus And BHLH Domain Of MyoD
Interact With P160 Coregulators In Vitro................78
11. PI60 Coregulators And P300 Differentially
Interact With Various Regions Of The N-terminal
Activation Domain Of MyoD.................................. 84
12. PCAF Acetylation Sites Of MyoD Are Not
Responsible To Interact With P160 Coregulators... 91
13. Two Subregions Of The Basic Domain Of MyoD
Are Required To Interact With SRC1A...................95
14. Cyclic AMP-Dependent Protein Kinase Potentiates
The Transactivations Of LexA-P160 Coregulators
And Synergizes The Interactions Between
p/CIP And MyoD In Vivo....................................... 102
v i
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ABSTRACT
P I60 coregulators were initially identified as nuclear hormone
receptor coactivators. In this study, functional data support the notion
that the three p i60 family members have different roles in the
transactivation of MyoD. Both SRC1A and p/CIP function as
coactivators on MyoD-mediated transcription, while GRIP1 acts
negatively as a (co)repressor. In addition, biochemical and mutational
analysis demonstrate that myogenic factors and El A are both able to
directly associate with pi 60s. The regions responsible for mutual
protein-protein interactions between MyoD and individual pi 60s were
characterized. Novel myogenic factors interacting domains (MID)
together with the C terminus of pi 60s have been identified as
essential for binding to MyoD. The N-terminal activation domain and
the basic region of MyoD are critical for interacting with the p i60
coregulators. In addition, cAMP-dependent protein kinase A
specifically enhances the protein-protein interaction between MyoD
and p/CIP in vivo.
vii
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CHAPTER 1
INTRODUCTION
1.1 Myogenic Factors:
The myogenic bHLH proteins initiate and direct the differentiation
of muscle progenitor cells by regulating muscle specific gene
expression. Targeted-inactivation in mice of the four mammalian
myogenic bHLH proteins, MyoD, Myogenin, Myf5, and MRF4 (for
review see (Olson, 1990)), revealed their functional redundancy as
well as their differences (for reviews see (Olson and Klein, 1994; Yun
and Wold, 1996)). The mechanisms by which these proteins activate
muscle specific gene expression involve several concerted processes.
The characteristic helix-loop-helix region of MyoD preferentially
forms a heterodimer with a ubiquitously expressed basic helix-loop-
helix E protein, E12 or E47 (Lassar et al., 1991). The basic regions of
the MyoD-E 12/47 heterodimer forms a DNA-protein complex on E
boxes (CANNTG) (Murre et al., 1989; Murre et al., 1989)— a cis
element in the regulatory regions of many muscle-specific genes, such
as creatine kinase (MCK) (Lassar et al., 1989), human cardiac a-actin
(HCA) (Sartorelli et al., 1990)—to activate transcription. It has been
proposed that the docking of the DNA binding domain of MyoD
involves recognition factors that are able to decode a specific protein-
1
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DNA conformation comprising the basic domain of MyoD and the E
box because the binding itself seems to precede unmasking of the N-
terminal activation domain of MyoD (Weintraub et al., 1991). Two
amino acids, alanine 114 and threonine 115 of MyoD (alanine 86 and
threonine 87 of myogenin), located in the basic region of myogenic
bHLH proteins are intrinsic to this myogenic code (Brennan et al.,
1991; Davis and Weintraub, 1992). This model was further
strengthened by studies involving MyoDAl 14N (a substitution of
alanine 114 to asparagine 114). A conformational change in the basic
region of MyoDAl 14N indeed affects its transcriptional activity since
it can no longer be potentiated by the coactivators p300 or TAFII40
(Huang et al., 1998) that are known to target MyoD’s N-terminal
activation domain. Several candidates for the role of recognition
factor include the MADS box proteins, MEF2C (Black et al., 1998;
Molkentin et al., 1995), and the coactivator p300/CBP (Sartorelli et
al., 1997). Importantly, the acidic activation domain of the Herpes
Simplex Virus protein VP16 can substitute for the N-terminal
activation domain of MyoD to restore its transactivation activity on
either the synthetic MyoD-responsive 4R-TK reporter (Weintraub et
al., 1991) or the natural human cardiac a-actin promoter (Huang et al.,
1998). These results suggest a model in which specific cofactors are
common to the activation domains of both MyoD and VP 16. Indeed,
p300 was shown able to potentiate MyoDANVP16 but not MyoDAN
2
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on an artificial E box reporter (4RE) (Sartorelli et al., 1997).
Interestingly, at least one other class of coactivators can potentiate
Gal-VP16: the pl60 proteins. One of the pl60 proteins, SRCA1, acts
as a coactivator by associating with p300/CBP (Onate et al., 1995)
and we have tested its role and that of other p i60 proteins in
regulating the transcriptional activity of MyoD.
1.2 P160 Coregulator Family:
1.2.1 The Structures And Functions Of PI 60 Coregulators
Three stractually related classes of pl60 coregulators have been
identified to date and are designated as SRC-l/NCoA-1 (Kalkhoven et
al., 1998; Onate et al., 1995; Torchia et al., 1997; Yao et al., 1996),
TIF2/GRIPl/NCoA-2 (Hong et al., 1996; Torchia et al., 1997; Voegel
et al., 1996), and p/CIP/ACTR/AIB 1/RAC-3/TRAM- 1/SRC3 (Anzick
et al., 1997; Chen et al., 1997; Li et al., 1997; Takeshita et al., 1997;
Torchia et al., 1997). The pl60 family members share several similar
structural and functional features. A basic helix-loop-helix (bHLH)
domain followed by a PAS (Pert/Amt/Sim) domain is conserved at the
amino-terminus of all three members. Three nuclear receptor (NR)
boxes, I, II, III, located in the middle region are crucial for
potentiating transactivation by nuclear receptors and are denoted as
LXXLL motifs (Heery et al., 1997). These NR boxes affect the
3
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affinity and specificity of interaction with various nuclear receptors
(Ding et al., 1998; Heery et al., 2001; Leers et al., 1998; Voegel et al.,
1998). A fourth NR box (IV) was found only at the very C terminus of
SRC-1A but not in the SRC1-E isoform (Kalkhoven et al., 1998) or
the other p i60 family members. Another feature that SRC1 (Spencer
et al., 1997) and ACTR (Chen et al., 1997) shares in common with the
myogenic coactivators, p300 and pCAF, is the presence of a histone
acetyltransferase (HAT) activity, located at their respective C Termini.
The HAT activity of the coactivator pCAF is essential for MyoD to
activate transcription of the cyclin-dependent kinase inhibitor, p21
(Puri et al., 1997), an early marker for myogenic differentiation as
well as a late one for terminal cell cycle arrest (Parker et al., 1995).
The importance of the HAT activity of p300/CBP was also shown to
be required for myotube formation as well as the expression of late
markers of muscle differentiation, e.g. muscle creatine kinase (MCK)
and myosin heavy chain (MHC) (Lau et al., 2000; Polesskaya et al.,
2001). Moreover, MyoD has also been demonstrated to be a substrate
of the HAT activities of both p300/CBP (Polesskaya et al., 2000) and
pCAF (Sartorelli et al., 1999) although only the latter appears to be
required for MyoD activation. PCAF and p300 cooperate to mediate
acetylation of MyoD, thus increasing the ability of MyoD to bind
DNA and transactivate muscle specific promoters. Given that
p300/CBP and pCAF are both required for MyoD-mediated muscle
4
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specific gene expression and that p i60 coregulators can interact with
both p300/CBP and pCAF, it seems likely that p i60 proteins may also
be involved in the MyoD-dependent components of myogenic
differentiation.
1.2.2 The Phenotypes O f PI 60 Inactivated Mice
SRC-1 null mice (Qi et al., 1999; Xu et al., 1998) and p/CIP/SRC-3
null mice (Wang et al., 2000; Xu et al., 2000) have only subtle
phenotypes suggesting functional redundancy or compensation among
the p i60 family members. Indeed there is increased TIF2 expression
in SRC-1 -/- mice (Xu et al., 1998) as well as increased expression of
both p300 and TIF2/GRIPl/NCoA2 in p/CIP deficient mice (Wang et
al., 2000). Interestingly, studies in p/CIP knockout mice consistently
demonstrated significant decreases of insulin-like growth factor 1
(IGF-1) in serum (Wang et al., 2000; Xu et al., 2000), which could
partially account for the growth defect (dwarfism) phenotype. IGF-1
is required for both muscle proliferation and differentiation because of
its dual roles as both a mitogen and in differentiation (for review, see
(Florini et al., 1996)). IGF-1 -/- dwarf mice die immediately after
birth, and the muscle hypolasia of these null mice likely contributes to
their failure to breathe (Liu et al., 1993; Powell-Braxton et al., 1993).
Also IGF-1 postmitotically induces expressions of the myogenic
activators myogenin (Florini et al., 1991) and MEF2C (Musaro and
5
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Rosenthal, 1999) and in addition potentiates the expressions of MRF4
(Musaro and Rosenthal, 1999). Since p i60 knock-out mice have
reduced levels of IGF-1,1 speculate that signaling pathways involving
both IGF-1 and the p i60 coregulatoirs promote myogenic
differentiation and maturation.
1.3 Adenovirus E1A Inhibits Myogenesis:
Myogenic differentiation and muscle specific transcription is
suppressed by Adenovirus E1A (Webster et al., 1988). Several
molecular mechanisms have been proposed, including direct physical
competition between El A and p300/CBP or between El A and pCAF.
Given that El A also perturbs the binary assembly between p/CIP and
p300/CBP (Kurokawa et al., 1998) as well as the association between
pCAF and p300/CBP (Puri et al., 1997; Yang et al., 1996), I
speculated that if p300/CBP-pCAF formed a functional ternary
coactivator complex with pi 60s, it would likely be disrupted by E1A.
This would provide an additional mechanism by which El A targets
myogenic transcription and would provide additional evidence for the
importance of pi 60s in myogenesis as well as the modulation of
muscle specific transcriptional regulation.
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CHAPTER 2
FUNCTIONS OF P160 COREGULATORS ON
MYOD TRANSACTIVATION
2.1 Introduction
When they were initially discovered, the p i60 family of coactivators
were thought to interact only as coactivators of nuclear hormone
receptor-mediated transcription in a ligand-dependent manner (for
review, see (Lee et al., 2001; McKenna et al., 1999; Rosenfeld and
Glass, 2001; Stallcup et al., 2000)). However, evidence rapidly
accumulated to suggest that their functions are not limited to
potentiation of nuclear receptor-activated transcription but extended to
interactions with other transcriptional activators, e.g. Spl (Onate et
al., 1995), NFkB (Na et al., 1998; Sheppard et al., 1999; Werbajh et
al., 2000), serum response factor (SRF) (Kim et al., 1998), API (Lee
et al., 1998), CREB (Korzus et al., 1998), and STAT-1 (Korzus et al.,
1998). Moreover, pl60 family members associate with p300 or CBP:
SRC-l/p300 (Yao et al., 1996); SRC-1/CBP (Kamei et al., 1996);
TIF2/CBP (Voegel et al., 1998); GRIPl/p300 (Hong et al., 1999);
p/CIP/CBP (Torchia et al., 1997); and with the pCAF coactivator
SRC-l/pCAF (Korzus et al., 1998; Spencer et al., 1997);
ACTR/pCAF (Chen et al., 1997); p/CIP/pCAF (Korzus et al., 1998).
In addition, immunostaining demonstrates that both SRCA1 and
7
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GRIP1 can colocalize with p300/CBP in nuclei (Baumann et al., 2001;
Yao et al., 1996). Given that both p300/CBP (Eckner et al., 1996; Puri
et al., 1997; Sartorelli et al., 1997; Yuan et al., 1996) and pCAF (Puri
et al., 1997) are required as coactivators for MyoD-mediated
transcription, I speculated that the p i60 coregulators might also be
involved in the regulation of muscle specific transcription since they
interact with both p300/CBP and pCAF. The aim of the experiments
reported in this chapter therefore is to study the potential roles of each
of the p i60 coregulator families on MyoD-activated muscle specific
transcription. In addition, because of its previously demonstrated
ability to disrupt myogenic coactivator complexes. I studied the ability
of adenovirus El A protein to associate with pi 60s.
2.2 Materials and Methods
2.2.1 Constructions O f Plasmids
GST-E1A, mammalian expression vectors of El A, VP16E1A,
VP16E1AA2,3 were generously provided by H-Y Kao (Case Western
University). The cDNAs for GRIP1, SRC1A and p/CIP were provided
by M. Stallcup, B. O'Malley, M-J Tsai, and M. Rosenfeld were
subcloned into pSG5 (Stratagene). pSG5GRIPl was kindly provided
by M. Stallcup. 4RELUC, MCKLUC, pCDNA3MyoD, have been
described previously (Hamamori et al., 1997; Sartorelli et al., 1997).
HCALUC was made by substituting the chloramphenicol
8
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acetyltransferase (CAT) cassette of HCACAT with luciferase (LUC)
cassette derived through pGL2-Basic (Promega). The proteins
expressed through eukaryotic expression vectors were confirmed by
western blotting of in vitro translation products and showed proteins
of the expected molecular sizes.
2.2.2 Cell Culture, Transient Transfections, And Luciferase
Assays
Mouse C3H10T1/2 embryo fibroblasts were obtained from the
American Type Culture Collection. Cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
fetal bovine serum (FBS, 10%, heat-inactivated). Mouse C2C12
myoblasts are a subclone obtained from a myoblast cell line derived
from regenerating adult mouse skeletal muscle (Yaffe and Saxel,
1977). Mononucleated myoblasts were grown in growth medium
(DMEM supplemented with 20% FBS, 0.5% chicken embryo
extracts) and maintained at low density by frequent passaging.
Transient transfections were carried out by the BES-buffered saline
(BBS) method as described (Hamamori et al., 1997). When cells
reached approximately 40-60% confluence, plasmid (total of 9 fig per
60 mm-diameter dish) was added as a calcium phosphate precipitate.
Medium was replenished 16-20 hrs later. Luciferase assays were
carried out after an additional 48 hrs of incubation for the transfected
9
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10T1/2 cells following the manufacturer's protocol. Transfected
C2C12 cells were maintained in growth medium for an additional 16-
24 hrs and then replenished with differentiation medium (DMEM
supplemented with 2% horse serum) for 16-24 hrs before luciferase
assays. All luciferase assays were done either in duplicate or triplicate
with each of two to three independent plasmid preps. All results were
normalized to total protein in cell lysates.
To test the functions of pl60 coregulators on MyoD transactivation,
1 fig of 4RELUC, MCKLUC, or HCALUC reporter, plus
pCDNA3MyoD expression vector (0.1 jig), were cotransfected into
C3H10T1/2 or C2C12 cells in the presence or absence of
pSG5GRIPl, pSG5mSRClA, or pSG5mCIP expression vectors
(amounts as indicated in each figure) as described above.
Both SRC1A +/+ and -/- mouse embryonic fibroblasts ( MEFs) were
maintained in DMEM supplemented with FBS (10%) and 6-
mercaptoethanol (10'4M). MEFs were refed with fresh medium
without 2-mercaptoethanol when transient transfections were carried
out. To measure the transcription activity of MyoD in SRC1A -/-
MEFs, 4RELUC, MCKLUC, or HCALUC (1 jig each) reporter, plus
pCDNA3MyoD expression vectors (amount as shown in the figure),
were cotransfected into the MEFs.
10
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2.3 Results
2.3.1 Functions O f P160 Coregulators Towards MyoD On
Muscle Specific Promoters
To test whether or not p i60 coregulators play roles in myogenesis, I
investigated their effects on the ability of MyoD to transactivate three
different muscle specific promoters. These promoters were fused to a
luciferase reporter. 4RELUC contains 4 reiterated copies of a MyoD
binding site, the right E box of the enhancer of the muscle creatine
kinase gene. It’s known to be activated only by MyoD family
members but not other myogenic activators. MCKLUC carries a
muscle specific regulatory enhancer of the muscle creatine kinase
gene. HCALUC contains the muscle specific regulatory enhancer of
the human cardiac a-actin gene. Mouse CH310T1/2 fibroblasts were
transiently transfected with one of the reporters plus various
combinations of a MyoD expression vector and pi 60s expression
vectors. MyoD alone activates 4RELUC (Fig. 1A, lane 2, 6, 10).
Importantly, both SRC1A (Fig. 1A, lane 7, 8) and p/CIP (Fig. 1A,
lane 12) were capable of potentiating MyoD transactivation on
4RELUC in C3H10T1/2 fibroblasts. Surprisingly, however, GRIP1
did not do the same. It not only failed to increase MyoD-mediated
induction of 4RELUC reporter activity but repressed it significantly
(Fig. 1A, lane3, 4). Similar results were obtained when the artificial
reporter, 4RELUC, was replaced with physiological reporters,
11
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Fig. 1. SRC1A And CIP Coactivate, Whereas GRIP1 Represses
MyoD On Muscle Specific Promoters.
(A) Mouse C3H10T1/2 cells were transiently transfected with
4RELUC reporter (1 jig), expression vectors of MyoD (0.1 jig),
and indicated coregulators GRIP1/SRC1A/CIP (the amounts as
shown in the panel). The up or down regulation of MyoD
transactivation was measured by luciferase activity 48 hrs after
transfection.
(B) Transient transfection experiments were carried out as shown in
(A) with MCKLUC reporter instead.
(C) Transient transfection experiments were carried out as shown in
(A) with HCALUC reporter instead.
(D) Transient transfection experiments were carried out as shown in
(A) 4RELUC (B) MCKLUC (C) HCALUC with C2C12
myoblasts instead. C2C12 myoblasts were normally cultured in
the growth medium until the differentiation medium was
replenished. Luciferase assays were performed after myoblasts
were switched to the differentiation medium for an additional 24
hrs.
12
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10T1/2
(A)
RLU
3000
1 2 3 4
4RELUC (1) + + + +
MyoD (0.1)
-
+ + +
GRIP1 - - 1 2
RLU
10T1/2
22000
20000
18000
16000
14000
12000
10000
5 6 7 6
4RELUC (1) + + + +
MyoD (0.1)
-
+ + +
SRC1A
-
2 4
10T1/2
60000
50000
40000
RLU 30000
20000
10000
9 10 11 12
4RELUC (1) + + + +
MyoD (0.1) + + +
CIP 2 4
Fig. 1. SRC1A And CIP Coactivate, Whereas GRIP1 Represses MyoD On
Muscle Specific Promoters.
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(Continued)
10T1/2
100000
90000
80000
70000
60000
RLU 50000
40000
30000
20000
10000
1 2 3 4
MCKLUC (1) + + + +
MyoD (0.1) . + + +
GR9P1 - 2 4
10T1/2
300000
250000
200000
RLU 150000
100000
50000
5 6 7 8
MCKLUC (1) + + + +
MyoD (0.1) - + + +
SRC1A - 2 4
120000
100000
80000
RLU 60000
40000
20000
10T1/2
I
l
■
1
9 10 11 12
MCKLUC (1) + + + +
MyoD (0.1)
-
+ + +
CIP - -
2 4
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(Continued)
(C)
60000001 10X1/2
5000000-
4000000
RLU 3000000-
2000000-
1000000-
HCALUC (1)
MyoD (0.1)
GRIP1
10T1/2
8000000
7000000
6000000
5000000
RLU 4000000
3000000
2000000
1000000
0
HCALUC (1) +
MyoD (0.1)
SRC1A
10T1/2
9 10 11 12
HCALUC (1) + + + +
MyoD (0.1) - + + +
CIP 2 4
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(Continued)
C2C12
(D)
RLU
1 2 3 4 5 6 7 8
4R E L uc (1) + + + + + + + +
M yoD (0.1)
-
+ + + + + + + •
GRIP1
-
1 2
- -
SR C 1A - - - 1 2
CIP 2 4
140000
120000
100000
80000
60000
40000
20000
C 2C 12
3 5 0 0000
3 0 0 0000
2 5 0 0000
2000000
1 5 0 0 0 0 0
1000000
50 0 0 0 0
13 14 15
MCKLuc (1) + + + + + + + +
M yoD (0.1) - + + + + + +
GRIP1
- .
1 2 - - -
SR C 1A - _ - 1 2 - -
CIP 1 2
40000000
35000000
30000000
25000000
RLU 20000000
15000000
10000000
5000000
0
17 18 19 2 0 21 2 2 2 3 24
HCALuc (1) + + ■ f + + + +
MyoD (0.1) + + + + + + +
GRIP1
- 1 2
- - - -
SRC1A . - -
1 2
- -
CIP 1 2
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MCKLUC (Fig. IB) and HCALUC (Fig. 1C). These results strongly
imply that SRC1A and CIP function as positive regulators for MyoD,
while GRIP1 acts negatively at least in myogenically undifferentiated
cells. The full maturation of myoblasts to myotubes requires mitogen
withdrawal. To further investigate whether the effects on MyoD
dependent transcription by pi 60s also occurs in differentiated muscle
cells, I carried out similar experiments in differentiated C2C12
myotubes. C2C12 cells were continuously maintained under growth
conditions, transfected and switched to differentiation medium 16-24
hrs later. The results of these transfection experiments are shown in
Fig. ID. Again, both SRC1A and CIP potentiate MyoD-activated
reporter activity in differentiated muscle cells but GRIP1 represses.
These data strongly suggest a functional association between MyoD
and p i60 coregulators, with both SRC1A and CIP acting as
coactivators, and GRIP1 as a (co)repressor in myogenesis.
2.3.2 The Transactivation Activity O f MyoD Is Diminished
In SRC1A - / - Mouse Embryonic Fibroblasts
The positive effect of SRC1A on MyoD-dependent transcription led
me to test whether SRC1A is essential for MyoD transactivation of
muscle specific promoters. SRC1A -/- mouse embryonic fibroblasts
(MEFs) derived from the SRC1A -/- mice were employed to test this
hypothesis. I compared the ability of MyoD to transactivate the target
17
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Fig. 2. The Transaetivation Of MyoD Is Significantly Diminished
in SRC1A-/- Mouse Embryonic Fibroblasts (MEFs)
(A) SRC1A +/+ and -/- MEFs were respectively transiently
transfected with 4RELUC reporter (1 fig), and a MyoD expression
vector of MyoD (amount as indicated in the figure). The
transaetivation of MyoD was measured by luciferase reporter assay
48hrs after transfection. SW3T3 stands for SRC1A +/+ MEFs shown
as black bar, SM3T3 for SRC1A -/- MEFs shown as gray bar.
(B) Transient transfection experiments were carried out as shown in
(A) with MCKLUC reporter instead.
(C) Transient transfection experiments were carried out as shown in
(A) with HCALUC reporter instead.
18
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(A)
FOLD
14
12
1 0
8
6
4
2
0
4RELUC (1)
ISW3T3 I ISM3T3
:
MyoD
(B)
o .i 0.5 1.0
J S W 3 T 3 Q SM 3T 3
MCKLUC (1)
0.1 1.0 MyoD 0.5
(C)
ISW3T3 SM3T3
FOLD 4
HCALUC (1)
MyoD 0.1 0.5 1.0
Fig. 2. The Transaetivation Of MyoD Is Significantly Diminished in
SRC1A -/- Mouse Embryonic Fibroblasts (MEFs)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
reporters in wild type versus SRC1A-/- MEFs. As shown in fig. 2, the
MyoD-dependent reporter activity was 5 fold less on the 4RE
promoter (fig. 2A, lane 3), 20 fold less on the MCK promoter (fig. 2B,
lane 4), and 3-4 fold less on the HCA promoter (fig. 2C, lane 4).
Indeed, the activation of the muscle creatine kinase promoter
mediated by MyoD was completely silenced in the SRC1A -/- MEFs
(fig. 2B). These data demonstrated that the ability of MyoD to
transactivate is significantly diminished in the absence of SRC1A, and
that SRC1A is essential for certain types of muscle specific gene
expression.
2.3.3 E l A Directly Interacts With PI 60 Coregulators Both
In Vitro And In Vivo
The adenovirus El A protein inhibits the activity of MyoD to
transactivate muscle specific promoters by mechanisms that include
disruption of myogenic transcription complexes. Such events preclude
directly testing the effects of E1A on the ability of pi 60s to effect
MyoD dependent transcription that I have observed. Instead I
investigated whether El A physically interacts with any of the pi 60
proteins. Protein-protein interactions were evaluated using in vitro
GST affinity binding assays (Fig. 3A). 3 5 S-methionine-labeled GRIP1,
SRC1A, or p/CIP was incubated with glutathione-S-transferase (GST)
agarose beads coupled to 12S E1A. As shown in Figure 3A, there was
20
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Fig. 3. Adenovirus E1A Interacts With P160 Coregulators
Both In Vitro And In Vivo.
(A) GST-E1A immobilized on glutathione agarose beads were
incubated with in vitro-translated [3 5 S] GRIP1, SRC1A, and CIP. The
bound proteins were resolved by autoradiography after SDS-PAGE.
Glutathione agarose-immobilized GST protein serves as the control.
10% INPUT stands for 10% amount of in vitro-translated pi 60s used
in the binding reactions was loaded.
(B) Mammalian two-hybrid assay was carried out in C3H10T1/2 cells,
The interaction between GALSRC1A and VP16E1A/VP16E1A A2,3
was measured using G4TATALUC reporter for luciferase activity.
The amount (/xg) of each individual expression vector transfected has
been indicated in the figure.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
GST-E1A GST 10% INPUT
3 5 S Met
GRIPl
SRC1A
p/CIP
GRIPl
SRC1A
p/CIP
GRIPl
SRC1A
p/CIP
184 -----
86 ------
69 -----
IW * * * ^
123 456 789
Fig. 3. Adenovirus E1A Interacts With P160 Coregulators Both In Vitro And
In Vivo.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(B)
10T1/2
5000001
450000
400000
350000
300000
RLU 250000
200000
150000
100000
50000
1 2 6 3 4 5 7 8
G4TATALUC(1) + + + + + -t- + +
GALSRC1 A(1) + + + + + + + +
E1A
-
2 4
- - - -
VP16E1A
- - -
2 4
- -
VP16E1A A2,3 -
- - - - -
2 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
substantial binding of all three pi 60s to GST-E1A (lanes 1, 2, 3),
compared to background binding to GST alone (lanes, 4, 5, 6). Thus,
E1A is capable of directly interacting with pi 60s in vitro and this
interaction does not require mediation by any other p i60 associated
coactivators, like p300/CBP or pCAF, since they were not included in
the binding reaction.
To determine whether this protein-protein interaction can also occur
in a cellular context, I used a mammalian two-hybrid assay. Full
length SRC1A was fused to the GAL4 DNA binding domain (GAL4-
DBD) to generate GALSRC1A. Wild type 12S E1A or E1A A2,3, a
deletion mutant of El A at the 2nd and 3rd amino acids, were fused to
the VP16 activation domain (VP 16-AD) to construct VP16E1A. As
expected GALSRC1A itself generated significant reporter activity
(Fig. 3B, lane 2) as previously reported (Onate et al., 1998). This
background reporter activity was dramatically increased upon
cotransfection with VP16E1A (lane 5, 6) in 10T1/2 cells, but there
was little or no significant increase in reporter activity when
cotransfected with E1A alone (lane 3, 4). The binding of p300 by E1A
is specifically dependent on the positively charged 2nd and 3rd amino
acids of the N terminus of E1A proteins (Wang et al., 1993).
However, it remains unknown which regions of E1A are critical for
binding to p i60 coregulators. I also tested the effects in this p i60
assay of a VP16E1AA2,3 mutant fusion construct. Surprisingly,
24
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reporter activities were dramatically decreased upon cotransfecting
with VP16E1AA2,3 (lane 7, 8). These results not only demonstrate
that E1A is indeed capable of associating with SRC1A in mammalian
cells but suggest that the 2nd and 3rd amino acids of E1A are also
critically involved in its interaction with SRC1A.
2.4 Discussion
The functions of pl60 coregulators on MyoD-mediated transcription
have been demonstrated as shown in Fig.l. Both SRC1A and p/CIP
are capable of potentiating MyoD transaetivation in both C3H10T1/2
and C2C12 cells. I observed increases by SRC1A of up to 9 fold in
10T1/2 cells (fig. 1C), and 5-6 fold by p/CIP in C2C12 cells (fig. ID)
using the HCA promoter. Thus, I concluded that both SRC1A and
p/CIP function as coactivators for MyoD-dependent transcription. On
some occasions, there was a slight down regulation of MyoD
transaetivation by p/CIP at low dosage and the reason for that is
unclear. In addition, ACTR, the human homologue of CIP, was unable
to potentiate MyoD-mediated activation in differentiated C2C12 cells
but to activate transcription like p/CIP in myoblasts (data not shown).
In marked contrast, GRIPl which coactivates ligand-mediated
nuclear hormone receptor-dependent transcription, does not act as a
coactivator to potentiate MyoD-mediated transcription. Indeed,
25
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GRIPl significantly down regulates MyoD-dependent reporter
activity on both 4RE and MCK promoters in both fibroblasts and
differentiating myoblasts by at least 80-100% when compared to
MyoD alone. This negative regulation is less on the HCA promoter.
Nevertheless, it still shows 40-50% reduction of MyoD transaetivation
in both cell lines. Considering the compositions of cis-elements
identified on both MCK and HCA promoters, this milder repressor
effect caused by GRIPl on HCA promoter could possibly be
attributed to the facts that the complex MCK promoter contains both
AP2 and MEF2 binding sites, whereas the HCA promoter does not.
Thus, GRIPl might also modify MEF2 or AP2 dependent
transaetivation. My preliminary results testing artificial MEF2Luc
reporter suggest that GRIPl (co)represses MEF2-mediated
transcription on MEF2Luc reporter (data not shown). However, this
negative regulation of MEF2 transaetivation caused by GRIPl is in
direct contrast to the results published by Muscat’s group on the
positive effects of GRIPl on MEF2 mediated myogenic gene
expression (Chen et al., 2000). The reasons for this discrepancy are
not known. XMyoD, the Xenopus homologue of MyoD is a lateral
and ventral mesoderm marker but is ectopic ally expressed in the
ectoderm or the animal caps of Xenopus laevis embryos when
dominant negative mutants of XTIF2 were injected (de la Calle-
Mustienes and Gomez-Skarmeta, 2000). This suggests that wild type
26
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XTIF2 acts as a negative regulator on the endogenous expression of
XMyoD in Xenopus. Therefore, the molecular mechanisms by which
GRIPl behaves differently from SRC1A and p/CIP on MyoD
transaetivation are still unclear and remain to be investigated further.
The opposing effects on MyoD-dependent transcription caused by
p i60 coregulators not only demonstrates their functions in modulating
muscle specific gene expressions but reveals their independent and
different roles on MyoD activity. In addition, these characteristics
don’t seem to be affected by the profound changes of cellular context
that occur when myoblasts exit from the cell cycle and start
differentiating to become myotubes.
MyoD transaetivation is significantly decreased on both 4RE and
HCA promoters in SRC1A -/- MEFs, suggesting SRC1A is required
for full MyoD transaetivation (fig. 2A and C). The transaetivation
activity of both GALMyoD (full length of MyoD fused to Gal-DBD;
4 fold decreased) and GALMyoD-N (N-terminal activation domain of
MyoD fused to Gal-DBD; 2 fold decreased) was also decreased using
a G4TATALUC reporter in SRC1A -/- MEFs when compared to wild
type MEFs (data not shown). In addition, SRC1A appears to be
essential for MyoD transaetivation activity on the MCK promoter in
SRC1A -/- MEFs (fig. 2B). The mechanisms by which MyoD acts
differently on various muscle specific promoters in SRC1A -/- MEFs
27
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without SRC1A backgrounds still remains to be investigated. The
transcriptional activity of the PPARy coactivator-1 (PGC-1) in the
SRC1A -/- MEFs can be further potentiated by overexpressing
SRC1A in the SRC1A -/- MEFs (Puigserver et al., 1999). Therefore, I
made several attempts to introduce the SRC1A expression vector back
into the SRC1A -/- MEFs to see whether MyoD-mediated
transcription can be increased. Surprisingly, there was no significant
increase of transaetivation by MyoD in SRC1A -/- MEFs
overexpressing SRC1A (data not shown). This suggests (1) the
expressions of essential factors for MyoD-mediated transcription lost
in SRC1A -/- MEFs may not be immediately or completely
complemented in the transient transfection conditions; (2) other
coactivators like p300/CBP, pCAF, or p/CIP might have
complemented the coactivator activity of SRC1A when it is absent.
Therefore, it still remains to be investigated whether other
downstream targets of pl60 coregulators are required to constitute the
full activity of muscle specific gene expression.
A model in which E1A targets a p300/CBP-pCAF-pl60s ternary
complex is strengthened by the demonstration of direct physical
contacts between E1A and pl60 coregulators using GST pull down
assays in vitro (Fig. 3A). Moreover, this interaction between E1A and
SRC1A was recapitulated in vivo (Fig. 3B). Both the 2nd and the 3rd
28
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amino acids of E1A that are responsible for binding to p300, are also
involved in this in vivo interaction. However, whether or not both
amino acids are directly responsible for binding to pi 60s still remains
unproven until protein-protein interaction experiments are carried out
in vitro, because p300 or other proteins could possibly function as a
bridging adaptor between GALSRC1A and VP16E1A in the cellular
context. Furthermore, it’s been shown that E1A can inhibit both
skeletal and cardiac a-actin promoter activities (Webster et al., 1988).
Therefore, I also tested whether overexpressions of SRC1A can rescue
the inhibition of MyoD transaetivation caused by E1A on a HCALUC
reporter. The results showed only 9-16% of the reporter activity can
be rescued by overexpressing SRC1A in the presence of El A (data
not shown), indicating that the full recovery of MyoD transaetivation
activity may require additional supplements of other coactivators like
p300/CBP or pCAF because they are also targeted by El A.
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CHAPTER 3
THE PHYSICAL INTERACTIONS
BETWEEN P160 COREGULATORS AND
MYOD
3.1 Introduction
Previous studies demonstrated that both SRC1A and p/CIP are able
to potentiate MyoD transaetivation, while GRIPl downregulates. To
further investigate whether these functions could be exerted directly
through physical interaction of MyoD with the p i60 coregulators,
several protein-protein interaction assays were carried out. Thus, the
goal of the experiments reported in this chapter is to determine the
peptide binding affinities between pi 60s and MyoD. In addition, the
peptide domains of either pi 60s or MyoD involved in the protein-
protein interactions are documented. I have identified novel myogenic
factor interacting domains (MID) of pi 60s essential for binding to
MyoD. Further studies of the MIDI domain implicate c-AMP
dependent transducing signals as playing an important in vivo role in
mediating protein-protein interaction between MyoD and p/CIP.
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3.2 Materials and Methods
3.2.1 Constructions Of Plasmids
pCDNA3MyoDflag has been described previously (Hamamori et al.,
1997). The cDNAs for GRIPl, SRC1A and p/CIP were subcloned into
pCMX-Gal4N (a gift from R. Evans, The Salk Institute), or pCMX-
LexA respectively. pCMX-LexA was made by substituting the Gal4
DNA binding domain of pCMX-Gal4N with the LexA DNA binding
domain of pEG202 (a gift from E. Golemis). L8G5LUC was
generously provided by S. Hollenberg (The Volume Institute).
G4TATALUC was made by digesting out 17mer-TATA fragment
through 17-TATA polyA vector (a gift from M-J Tsai, B. O'Malley,
Baylor College of Medicine) and subcloning it into pGL2-Basic.
GST-MyoD was originally provided by H. Weintraub (Hutchinson
Cancer Institute). pCMXVP 16MyoD was made by subcloning PCR
amplified MyoD fragment into pCMXVP16 (a gift from R. Evans,
The Salk Institute). The cDNAs for myogenin, myf5, mrf4 generously
provided by E. Olson (University of Texas, Southwestern), A.
Buonanno (NIH), S. Konieczny (Indiana) were subcloned into
pGEX2TK respectively (Amersham Pharmacia). Various deletion as
well as site specific mutants of GST-MyoD were created by
subcloning different PCR amplified DNA fragments into pGEX2TK.
Each set of deletion mutants of GRIPl, SRC1A, p/CIP, as well as
Gal4DBD fusion constructs were made by subcloning various PCR
31
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amplified DNA fragments into either pSG5, pCDNA3 or pCMX-
Gal4N expression vectors. The nucleotide sequences of all the
mutants have been determined at least for the critical regions
subjected to changes. All the expression vectors, either prokaryotic or
eukaryotic, were tested for production of a protein of the expected
molecular size determined either by commassie blue staining for
bacterial produced proteins or in vitro translation for mammalian
expressed proteins.
3.2.2 Cell Culture, Transient Transfections, And Luciferase
Assays
The maintenance of mouse C3H10T1/2 embryo fibroblasts, C2C12
myoblasts, the transfection assay, and luciferase assay has been
described in 2.2.2
In mammalian two-hybrid assays, G4TATALUC (1 /xg)/L8G5LUC
(ljiig) reporter, GAL/LexA chimeras (1 pg), VP 16 chimeras (amounts
as indicated in each figure) were cotransfected into C3H10T1/2 cells
as described above.
To test the effects of functional mutants of pl60 coregulators on
MyoD transaetivation of 4RELUC, MCKLUC, or HCALUC (1 jiig
each) reporters, pCDNA3MyoD expression vectors (0.1 /rg), were
cotransfected into C3H10T1/2 or C2C12 cells in the absence or
32
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presence of pSG5GRIPlA(MIDl/2+C), pSG5mSRClAA(MIDl/2+C),
or pSG5mCIPA(MID 1 /2+C) expression vectors respectively.
3.2.3 Immunoprecipitation and Western Blotting
Both immunoprecipitation and Western Blot experiments were
carried out as described previously (Hamamori et al., 1999;
Hamamori et al., 1997) with slight modifications. 293 cells were
grown to 40-60% confluency in 10-cm dishes for transfection using
the calcium-phosphate protocol described above. Fresh medium was
replenished 16-24hrs after transfection and the cells were incubated
for another 36 hrs before metabolic labeling or direct affinity
precipitations. For labeling, cells were metabolically starved in
methionine-free DMEM medium for 2 hrs. After decanting serum-free
medium, cells were then labeled with methionine-free DMEM
supplemented with 10% dialyzed fetal bovine serum and 0.37mCi of
L-[3 5 S] Methionine (>1000Ci/mmole; Amersham) for 3-4 hrs. After
being washed with PBS several times, cells were lysed in RIP A buffer
(Tris-HCl, pH7.4, lOmM; NaCl, 150 mM; EDTA, 2mM;
deoxycholate, 0.5%; Nonidet P-40, 0.5%; protease inhibitors,
aprotinin. leupeptin, pepstatin) with gently rocking (4°C, 1 hr). The
lysate was then centrifuged to pellet debris (14,000g, lOmin) and the
supernatants were collected and counted for radioactivity. An aliquot
of supernatant, approximately 106-107 cpm of radioactivity, was
3 3
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incubated with a 1st antibody with gentle rocking (4°C, overnight) and
followed by addition of protein G-agarose beads (Sigma) with
continuous rocking (4°C, 4 hrs). The matrix was then washed with
RIP A buffer three times and the bound proteins were released by
boiling (95°C, 10 min) in the same buffer for second antibody
incubation and agarose-bead precipitation. The final bound proteins
were fractionated by SDS-PAGE and the dried gels were subjected to
autoradiography. Antibodies used in immunoprecipitation assays are
goat polyclonal anti-SRC 1A antibody (Santa Cruz, SC-6096), mouse
monoclonal anti-FlagM2 antibody (Sigma F-3165). For GST fusion
protein precipitation, the nuclei were extracted by Dounce
homogenization in hypotonic buffer (HEPES, lOmM; MgCl2, 1.5mM;
KC1, lOmM; PMSF, 0.2mM; DTT, 0.5mM). The nuclei were
collected (4,000 rpm, 15 min) and lysed in RIP A buffer as described.
The nuclear extracts (supernatant obtained after centrifugation of
lysed nuclear pellet at 14,500 rpm for 30 min), were incubated with
GST-MyoD. Bound proteins were seperated by SDS-PAGE followed
by Western Blot analysis using goat polyclonal anti-GRIPl antibody
(Santa Cruz, SC-6264) or mouse monoclonal anti-p/CIP antibody
(Santa Cruz, SC-5305). Detection of immunocomplexes was carried
out using ECL chemiluminescence (Amersham) according to the
manufacturer’s protocol.
34
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3.2.4 In Vitro Protein-Protein Interaction Assays
In vitro transcription and translation were carried out using ljag of
various supercoiled DNA plasmids, TNT® Coupled
Transcription/Translation System (Promega), and L-[3 5 S] Methionine
(>1000Ci/mmole; Amersham) according to the manufacturer's
protocol. The expression and purification of various glutathione-S-
transferase (GST) fusion proteins as well as protein-protein interaction
assays were performed as described previously (Hamamori et al.,
1997; Sartorelli et al., 1997) with slight modifications. The GST and
GST fusion proteins were expressed in E sch erich ia coli (BL21) and
purified using glutathione-agarose affinity matrix (Sigma®). The
molecular weight of each individual purified protein was analyzed,
and confirmed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Normalized amounts of agarose-bound
GST fusion proteins were incubated with 10/xL of [35S] methionine-
labeled translation products in NETN binding buffer (Nonidet P-40,
0.5%; EDTA, ImM; Tris-HCl, 20mM, pH8.0; NaCl, lOOmM;
ethidium bromide, 12.5/rg) at 4°C for 1 hr. The beads were washed
three times with iced NETN buffer and the bound proteins were
released by incubating at 95°C for 3 min. The released proteins were
then analyzed on 10% SDS-PAGE. The gels were then fixed (H2 0,
50%; methanol, 40%; acetic acid, 10%; 20min), treated with
35
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Enlightning™ (DuPont, 20 min), dried, and subjected to
autoradiography.
3.3 Results
3.3.1 Myogenic Factors Directly Associate With PI 60
Coregulators In Vitro
The p i60 coregulators appear to modulate MyoD-dependent
transcription but the data accumulated to this point leaves uncertain
whether these effects are indirect, e.g. through their interactions with
the p300/CBP-pCAF complex, or are directly related to the physical
contacts between pi 60s and MyoD. I used GST-affinity binding
assays to test whether MyoD and pi 60s can directly interact in vitro.
In vitro- translated [35S] GRIPl (fig. 4A, lane 1), SRC1A (fig. 4A,
lane 4), and p/CIP proteins (fig. 4A, lane 7) all bind to GST-MyoD
significantly when compared to GST alone (fig. 4A, lanes 2, 5, and 8).
I also examined the ability of the three p i60 molecules to interact with
other myogenic factors since they each function differently in
activating as well as stabilizing the myogenic program. The three
myogenic factors, Myf5 (fig. 4B, lane 1, 4, and 7), Myogenin (fig. 4C,
lane 1, 4, and 7), and MRF4 (fig. 4D, lane 1, 4, and 7) also interact
directly with the p i60 proteins. These data establish that the p i60
36
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Fig. 4. Myogenic Factors Directly Interact With P160
Coregulators In Vitro.
(A) GST-MyoD immobilized on glutathione agarose beads were
incubated with in vitro-translated [3 5 S] GRIPl, SRC1A, and CIP. The
bound proteins were resolved by autoradiography after SDS-PAGE.
Glutathione agarose-immobilized GST protein serves as control. 10%
INPUT stands for 10% amount of in vitro-translated pi 60s used in the
binding reactions were loaded.
(B) GST affinity binding assay was carried out as shown in (A) with
GST-Myf5 instead.
(C) GST affinity binding assay was carried out as shown in (A) with
GST-Myogenin instead.
(D) GST affinity binding assay was carried out as shown in (A) with
GST-MRF4 instead.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
35,
S Met
(B)
35
SMet
GRIPl
GST-
MyoD
GST
10%
INPUT
W M fff
SRC1A
GST-
MyoD
GST
10%
INPUT
CIP
GST-
MyoD
GST
10%
INPUT
m
8
GRIPl
GST- 10% GST-
Myf5 GST INPUT Myf5
SRC1A
10%
GST INPUT
CIP
GST-
Myf5
GST
10%
INPUT
•200
-200
1 2 3 4 5 6 7 8 9
Fig. 4. Myogenic Factors Directly Interact With P160 Coregulators In Vitro.
38
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(Continued)
(C)
35,
S Met
(D)
GRIPl
GST-
Myogenin
GST
GST-
Hyogenin
10%
INPUT
SRC1A
GST
10%
INPUT
CIP
GST-
Myogenin
GST
10%
INPUT
■200
35,
S Met GRIPl
GST-
MRF4
GST
10%
INPUT
lit
SRC1A
GST
MRF4
GST
10%
INPUT
CIP
GST-
MRF4
GST
10%
INPUT
-200
39
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coregulators and myogenic bHLH proteins can bind directly to each
other in vitro. However, the central test is whether these interactions
can also occur in a cellular context.
3.3.2 PI 60 Coregulators Associate With MyoD In Vivo
The effects of the p i60 coregulators on MyoD-dependent gene
activation (fig. 1) implicate their involvement with MyoD containing
transcription complexes. To test the notion that p i60 coregulators are
able to associate with MyoD in a cellular context, I used the
immunoprecipitation assays. To study the potential association in vivo
between one of the pi 60s, SRC1A and MyoD, I cotransfected
MyoDflag and SRC1A expression vectors into 293 cells which were
then metabolically labeled with 3 5 S-methionine. The cell lysate
derived from them were subjected first to immunoprecipitation using
beads coupled to flag antibody. Bound proteins (fig. 5A, lane 1) were
then immediately transferred for subsequent immunoprecipitation
using SRC1A antibody-beads. The data shown in fig. 5A reveals that
SRC1A associates with exogenous MyoD in vivo (lane 2). When I
repeated the immunoprecipitation but first used SRC1A antibody
beads followed by a second precipitation using flag antibody beads, I
was able to demonstrate that SRC1A is found in complexes (fig. 5,
lane 3) that also contain MyoD (fig. 5, lane 4). To determine whether
40
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Fig. 5. P160 Coregulators Associate With MyoD In Vivo.
(A) Nuclear extracts were derived from [3 5 S] methionine-labeled 293
cells transiently transfected with the expression vectors of MyoDFlag
and SRC1A. Radiolabeled extracts were subjected to double
immunoprecipitation, first with Flag Ab-protein G-agarose beads and
then SRC1A Ab-protein G-agarose beads (or vice versa). The bound
proteins were analyzed by autoradiography after SDS-PAGE.
(B) 293 cells were transiently transfected with p/CIP and MyoDflag
expression vectors. Nuclear extracts derived from these cells were
incubated with Flag Ab-protein G-agarose beads. Bound proteins
were analyzed by Western Blot analysis using anti-CIP antibodies.
10% INPUT stands for 10% of nuclear extracts loaded. In vitro
translated CIP (nonradiolabeled) prepared from TNT couple
transcription-translated system serves as the size marker as well as the
positive control for the antibody detection (indicated as IVT CIP).
(To be continued)
4 1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(C) Mammalian two-hybrid assay was carried out in C3H10T1/2 cells.
The interaction between GALGRIP1 and VP16MyoD was measured
using G4TATALUC reporter for luciferase activity 48 hrs after
transfection. The amount (/xg) of each individual expression vector
being transiently transfected has been indicated in the figure.
(D) Transient transfection experiments were carried out as shown in
(C) with L8G5LUC reporter and GALSRC1A instead.
(E) Transient transfection experiments were carried out as shown in
(C) with GALCIP instead.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
Ip: 1st Ab
Flag SRC1A -
Ip: 2nd Ab
SRC1A
Flag
SRC1A —
M
* 1
MyoDFlag —
■
J M H k
su s? ?
n
------------------------
-------------------------
•200
■97
•68
-43
(B)
Western:
a-CIP
Ip: Flag Ab 10%Input
1 2 3 4
Fig. 5. P160 Coregulators Associate With MyoD In Vivo.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(C)
10T1/2
300000 -n
250000
200000 -
RLU150000^
50000 -j
G4TATALUC (1) + + + + + + + +
GALGRIP1 (1)
VP16MyoD
MyoD
+ + + + +
1 2
(D)
(E)
10T1/2
120000n
100000 -
80000
RLU 60000
40000-
20000 -
2 3 4 5 6 7 8 1
L8G5LUC (1)
LexASRCIA (1) - + + + + +
VP16MyoD
MyoD 1 2 4
10T1/2
2500000n
2000000
1500000-
RLU
1000000
500000-
1 2 3 4 5 6 7 8
G4TATALUC (1) + + + + + + + +
GALCIP (1) + + + + + + +
VP16MyoD 1 2 4
. . -
MyoD 1 2 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
p/CIP also can interact with MyoD in cells, the p/CIP and MyoDflag
expression vectors were cotransfected into 293 cells. Nuclear extracts
were incubated with anti-flag agarose beads followed by isolation on
an affinity matrix. Bound proteins were then subjected to Western
Blot analysis using an antibody against the p/CIP peptide (aa 455-
851). As shown in fig. 5, MyoDflag is able to associate with cellular
p/CIP (fig. 5B, lane 3). In vitro translated CIP derived from rabbit
lysates was also loaded as a positive control for immunodetection as
well as acting as a size maker. These results demonstrate that the p i60
coregulators, SRC1A and p/CIP are capable of associating with MyoD
in the cellular environment. The in vivo interaction between MyoD
and all three pi 60s was further confirmed by mammalian two-hybrid
assays. Full length GRIP1 and p/CIP were fused to GAL4-DBD to
generate GALGRIP1 and GALCIP, and SRC1A was fused to LexA-
DBD to generate LexASRClA. These were individually tested for
their ability to effect the transcriptional activity of VP16MyoD. As
reported previously (Font de Mora and Brown, 2000; Lopez et al.,
2001; Onate et al., 1998), GALGRIP1, LexASRClA and GALCIP
can themselves contribute significantly to the reporter activity (lane 2
of fig. 5 C, D, E). Strikingly, the activities of all the reporters were
increased in a dose dependent manner when cotransfected with
VP16MyoD (lanes 3, 4, 5 of fig. 5 C, D, E). No substantial increases
in reporter activities were seen following cotransfection with a MyoD
45
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
expression vector alone (lanes 6, 7, 8 of fig. 5 C, D, E). The results of
these mammalian two-hybrid experiments strongly support the
conclusion that p i60 coregulators and MyoD associate with each
other in vivo.
3.3.3 SRC1A Interacts With MyoD In Vitro Through Its
MIDI/2 Domains And C-Terminus
I next mapped a number of distinct interaction domains on the p i60
family members by creating a variety of deletions or segments (fig.
6A, C, E). Deletions of large segments of the SRC1A protein at either
the amino (AN) or carboxyl termini (AC) diminishes but does not fully
eliminate the ability of SRC1A to bind MyoD (figure 6B, lane 2, 5).
Neither of these segments alone (N, C) binds MyoD (figure 6B, lane
6, 12). In contrast, deletion of a large middle segment (AM) all but
eliminates the SRClA:MyoD interaction (figure 6B, lane 3).
Conversely, the middle segment alone (M) retains the ability to bind
MyoD (figure 6B, lane 9). Taken together, these data suggest that the
central portion of SRC1A contains one or more regions required for
MyoD interaction. This conclusion is supported by the observation
that a slightly smaller central deletion of the central portion of
SRClA(AAval) restores strong MyoD binding ability (figure 6B, lane
4). This focused my attention on the relatively short regions that
differed between AM and AAval. The information obtained from these
46
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
deletion mutations also suggests that the NR boxes essential for
interaction between pi 60s and nuclear receptors are not required for
SRC1A interaction with MyoD. Thus, we turned our attention to
junction regions delineated by amino acids 325-385 and 1028-1104
either or both of which appear to play important roles in the
interaction between SRC1A and MyoD. I named these regions as
MyoD Interacting Domains 1 and 2, respectively.
Accordingly, I evaluated several SRC1A fragments with or without
these junctional domains. As shown in fig. 6B, adding MIDI to the N
domain (N+MID1) strongly restores the binding capability (lane 7).
Similarly, adding MID2 to the C terminus also restores binding
capability (figure 6B, lane 10). Thus, both MIDI and MID2 appear to
independently restore MyoD binding ability. The critical roles of these
two junctional domains in binding to MyoD were confirmed when
their deletion from the middle segment (MAMID 1/2) led to loss of
MyoD binding (fig.6B, lane 8) compared with an intact middle
segment (M; fig. 6B, lane 9).
The contributions of MIDI and MID2 appear to be independent
since a polypeptide consisting of just the two MID segments plus the
C terminus was found to bind MyoD as well as wild type protein (fig.
6B, lane 11). Thus, I defined the domain spanning amino acids 325-
385 as the first Myogenic factor Interaction Domain, (MIDI), and
amino acids 1028-1104 as MID2. Since simple deletion of the N or C
47
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fig. 6. Domains Of P160s That Interact With MyoD In
Vitro
(A) Schematic representations of SRC1A deletion mutants employed
in these experiments. The coordinates (amino acids) of the deletion
mutants of SRC1A are indicated in the figure. I simply divide it as
three components designated as N-terminal (N), middle (M), and C-
terminal (C) portions. N-terminal region starts with 1st methionine
followed by the bHLH domain and ends with the PAS-B domain (aa
1-324). The middle segment encompasses the nuclear receptor (NR
boxes I, II, III) and p300/CBP interaction domains and consists of
amino acids 325-1104. The C-terminal fragment, which contains the
HAT domain and the pCAF and the other binding domains, includes
amino acids 1105-1441.
(To be continued)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(B) GST-MyoD immobilized on glutathione agarose beads were
incubated with in vitro-translated [3 5 S] SRC1A deletion mutants. The
bound proteins were resolved by autoradiography after SDS-PAGE
(upper panel). GST protein serves as the control (middle panel). 10%
INPUT stands for 10% amount of in vitro-translated SRC1A used in
the binding reactions was loaded (lower panel).
(C) Schematic representations of CIP deletion mutants employed in
these experiments. The coordinates (amino acids) of the deletion
mutants of CIP are indicated in the figure.
(D) GST affinity binding assays were carried out as shown in (B) with
CIP deletion mutants instead.
(E) Schematic representations of GRIP1 deletion mutants employed in
these experiments. The coordinates (amino acids) of the deletion
mutants of GRIP1 are indicated in the figure.
(F) GST affinity binding assays were carried out as shown in (B) with
GRIP1 deletion mutants instead.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
SRC1A
F u ll bHLH PASA/B MIDI 3 LxxLLs MID2 HAT LxxLL
1 --------------------------------------------------------------------------------------------------------1441
A N 325 1441
AM l 324 1105 1441
AAval 1 385 1028____________________ 1441
AC 1 ---------------------------------------------------------------------------------1104
N 1 324
N+MID1 1 _____________________385
MAMID1/2
(Aval frag)
MID1/2+C
386 1027
M-----------------------------325-------------------------------------------------- 1104
MID2+C
1028------------------------------- 1441
325 385 1028_____________________ 1441
C 1105_________________1441
Fig. 6. Domains Of P160s That Interact With MyoD In Vitro
50
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(B)
SRC1A SRC1A
w +
+ C J
GST-
1 2 3 4 5 6 7 8 9 10 11 12
184
GST
184
10%
Input
isa
5 1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(C)
CIP
_ j bHLH PASA/B MIDI 3 LxxLLs HAT MID2
A N 3 2 0 _________________________________I3 9 8
AC2 i ________________________________________ 1 0 93
N l ___________ 3 1 9
N+MID1 l _______________ 3 84
MAMID1______________ 385____________________ 9 97
M 320__________________________________ 1093
C l 998_____________________ 1398
C 2 1094__________________ 1398
(To be continued)
52
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(D)
CIP
s T. U . +
to < < i £ £
GST-
MyoD
184-
GST
10% 8 4 ‘
Input
1 2 3 4 5 6 7 8 9
*5?
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(E)
GRIPl
H A T
F u ll j bHLH PASA/B MIDI 3 LxxLLs p300ID MID2
AbHLH 1 0 1---------------------------------------------------------------------
APASA/B,
’1-----------105 221
-1462
AM 1---------------------------------- 481--------------766-------------------------------------------- 1462
Ap300ID 1 ______________________________________767 1122____________ 1462
AC
1118
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(F) GRIPl
ffl
-O
m
<
Ph
o
o
A O
< < 1 < 1 < < 3
10%
Input
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
terminus (AN, AC) decreased the MyoD interaction, somewhat the
roles of these segments in either binding MyoD or in stablizing
required conformations of the MIDI or MID2 domains remains
unresolved.
Using the information derived from SRC1A as a template, I used the
same approach to determine whether the two other p i60 proteins had
analogous MyoD binding domains. The scheme of p/CIP deletion
mutants is shown in fig. 6C. As shown in fig. 6D, deletion of either
the N-terminus (AN; aa 320-1398) or the C-terminus (AC2) had no
serious effect on binding (lane 2). Consistent with this, neither the N-
terminus (N; aa 1-319) nor the C-terminus (Cl and C2) was capable
of binding MyoD (fig. 6D, lane 4, 8, 9). The central portion of the
molecule (M) retains strong binding which is greatly diminished when
it is shortened at each end (MAMID) (figure 6D, lane 6). Amino acids
998-1093 in M do not appear to be involved since segment Cl does
not appear to bind MyoD (fig. 6D, lane 8). However amino acids 320-
384 do appear to effect MyoD binding capability since their addition
to the N domain (N+MID1) restores binding capability (fig. 6D, lane
5). I then concluded that there is a MID domain in p/CIP (located in
aa 319-384) important in participating in protein-protein interaction
between p/CIP and MyoD.
I have also carried out a limited analysis on the in vitro interactions
between GRIP1 and MyoD. I tested several deletion mutants of
56
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GRIP1 either devoid of its bHLH domain (designated as AbHLH), or
its p300 interacting domain (designated as Ap300ID). A middle
portion deletion and a C-terminal deletion were also studied (AM and
AC). The scheme of these GRIP1 deletion mutants is shown in fig. 6E.
As demonstrated in fig. 6F, only the AC mutant unequivically lost its
ability to interact with MyoD (lane 6), suggesting an essential role of
the C terminus of GRIP1. This deletion removed the HAT domain.
Further studies are needed to delineate the involved region.
Taken together these data demonstrate that the MyoD interaction
domains of the three p i60 molecules studied are not located at
precisely analogous positions on the molecules. Whether the precise
binding regions have sequences of conformational similarities remains
to be determined. However the apparent variations of the interactions
may ultimately explain my demonstration of differential effects of
different p i60 family members on the function of myogenic factors.
3.3.4 N-Termini Of SRC 1A And p/CIP Do Not Interact
With MyoD In Vivo
To further evaluate the role of the C-terminus of SRC1A and p/CIP
in mediating interactions between p i60 coactivators and MyoD. I
carried out mammalian two-hybrid assays in C3H10T1/2 fibroblasts.
A series of deletions of SRC1A and p/CIP fused with the GAL-DNA
binding domain were created and tested for their abilities to interact
5 7
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
with VP16MyoD based on the readouts of the G4TATALUC reporter.
The fold activation for each set of GALSRC1A or GALCIP deletions
versus VP16MyoD or MyoD alone was normalized to the expression
level observed with GALASRC1A or GALCIP deletions alone (1
fold). Unlike the in vitro results, the C-terminus of SRC1A bound
strongly to VP16MyoD (fig. 7A, 28.1 fold, lane 17) and to MyoD (5.8
fold, lane 18). Surprisingly, neither the MIDI nor the MID2 domain
that is critical for the SRC1A interaction with MyoD in vitro, effect
the ability of the C-terminus of SRC1A to interact with VP16MyoD in
vivo when GALSRC1A-MID1/2+C was cotransfected with
VP16MyoD (5.8 fold, fig. 7A, lane 20) or MyoD (1.1 fold, fig. 7A,
lane 21). Moreover, the MIDI domain does not effect interaction of
the N-terminus of SRC1A with MyoD as shown in the GST binding
assay (fig. 6B, lane 6, 7) when both GALSRC 1A-N (fig. 7A, lane 2)
and GALSRC 1A-N+MID1 (fig. 7A, lane 5) were tested. However, the
in vivo role of these segments may still be important since the middle
segment of SRC1A is both sufficient and necessary for interaction
with MyoD.
I next examined p/CIP domains for their ability to interact with
MyoD using a series of GALCIP deletion mutants. The C-terminus of
p/CIP also has significant binding activities with VP16MyoD (13 fold,
fig. 7B, lane 17) or MyoD (3.7 fold, fig. 7B, lane 20). As expected
from the in vitro data (fig. 6D, lane 4), the N-terminus of p/CIP alone
58
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fig. 7. N-termini of SRC1A And p/CIP Are Not Involved
In The Interactions With MyoD In Vivo
(A) Mammalian two-hybrid assays were carried out in C3H10T1/2
cells. The binding activities between deletion mutants of GALSRC 1A
and VP16MyoD or MyoD were measured using G4TATALUC
reporter 48 hrs after transfection. Results were normalized to the
activity of each individual GALSRC 1A mutant (defined as lx fold).
The amount (/xg) of each individual expression vector transfected is
indicated in the figure.
(B) Mammalian two-hybrid experiments were carried out as shown in
(A) with deletion mutants of GALCIP instead.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
10T1/2
30-,
25-
20 -
F O L D 1 5 -
10 -
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
G4TATALUC (1) + + + ++ + + + + + + + + + + ++ + ++ +
MyoD (4) + - - + + -
GALSRC1A-N (1) + + +
GALSRC1A
1-385 aa (1) + + +
GALSRC1A-AN (1) + + +
GALSRC1A-M (1) + + +
GALSRC1A-AM (1) + + +
GALSRC1A-C (1) + + +
GALSRC1A
MID1/2+C (1) + + +
Fig. 7. N-termini of SRC1A And p/CIP Are Not Involved In The Interactions
With MyoD In Vivo
60
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(B)
10T1/2
14-,
1 2 -
1 0 -
8 -
6 -
4 -
2 -
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
G4TATALUC(1) + + + + + + + + + + + + + + + + + + + + +
VP16MyoD (4) - +
GALCIP-N (1) + + +
GALCIP-AN (1) - - - + + +
GALCIP-
N+MID1 (1) + + +
GALCIP-M (1) + + + ........................................................
GALCIP-
MAMID1 (1) + + + .....................................
GALCIP-C2 ( 1 ) .............................................................................................. + + + - - -
GALCIP-AC2 ( 1 ) ................................................................................................................. + + +
6 1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
is not able to interact with MyoD in vivo (fig. 7B, lane 2, 3).
Noticeably, the MIDI domain of p/CIP, unlike that of SRC1A, does
enable the N-terminus of p/CIP to interact with VP16MyoD (4.1 fold,
fig. 7B, lane 8) and MyoD (2.7 fold, fig. 7B, lane 9). Most
importantly, the essential role of MIDI in directing the middle portion
of p/CIP to contact MyoD was revealed when both GALCIP-M and
GALCIP-MAMID1 were tested. GALCIP-M showed a 6.2 fold
increase of binding activity with VP16MyoD (fig. 7B, lane 11).
GALCIP-MAMID 1 had a minimal effect on interaction with either
VP16MyoD or MyoD (1.9 fold, fig. 7B, lane 14, 15). The N-terminus
of p/CIP alone is not involved in mediating the p/CIP and MyoD
interaction in vitro (fig. 6D). Therefore, the involvement of the middle
portion of p/CIP in interactions with MyoD is further confirmed when
the deletion mutant GALCIP-AC2 was examined (fig. 7B, lane 20,
21).
Taken together, the N-termini of SRC1A and p/CIP are not directly
involved with interactions with MyoD, whereas the C-termini of both
SRC1A and p/CIP demonstrate significant binding activities with
MyoD in vivo. In addition, the MIDI domain of p/CIP is essential in
mediating its interaction with MyoD in vivo.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.3.5 PI 60A(MID1/2+C) Mutants Fail To Interact With
MyoD Both In Vitro and In Vivo
The MIDI and MID2 domains as well as the C terminus of SRC1A
were identified as essential for SRC1A interactions with MyoD in
vitro. I continued to test whether the SRC1A mutants devoid of these
essential segments lose their ability to interact with MyoD in vitro. I
also applied the same approach to test whether GRIP1 and p/CIP
deleted of these SRC1A analogous regions would also render them
unable to contact MyoD in vitro. Therefore, three mutants,
GRIP 1 A(MID+C), SRC 1 AA(MID 1 /2+C), and p/CIP A(MID+C), were
created to test this using the GST binding assay. As shown in fig. 8A,
B & C (left panels), all full length pi 60s were able to bind GST-
MyoD. All three deletion mutants had either greatly diminished or
total loss of ability to interact with MyoD (fig. 8A, B & C, right side
panels). I next used the mammalian two-hybrid assay to see whether
A(MIDl/2+C) mutations also eliminate MyoD binding in vivo.
Expression vectors of GALGRIP1 A (M ID l/2+C) and
GALCIPA(MID 1/2+C) were generated by fusing the A(MIDl/2+C)
mutants of GRIP1 and p/CIP to GAL4-DBD. As shown in fig. 8, the
wild type GALGRIP1 and GALCIP increased expression on
G4TATALUC reporter 6-8 fold and 20 fold respectively upon
cotransfection with VP16MyoD. Deletion of MID 1/2+C domains in
either molecule abrogated its ability to elicit a transcriptional
63
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fig. 8. A(MIDl/2+C) Mutants Of PI60 Coregulators
Significantly Lose Ability To Interact With MyoD Both
In Vitro And In Vivo
(A) GST-MyoD immobilized on glutathione agarose beads were
incubated with in vitro-translated [3 5 S] GRIP1 or GRIP1
A(MID 1/2+C). The bound proteins were resolved by autoradiography
after SDS-PAGE. Glutathione agarose-immobilized GST protein
serves as control. 10% INPUT stands for 10% amount of in vitro-
translated pi 60s used in the binding reactions were loaded.
(B) GST affinity binding assays were carried out as shown in (A) with
SRC1A and SRC 1AA(MID 1/2+C).
(C) GST affinity binding assays were carried out as shown in (A) with
CIP and CIPA(MID 1/2+C).
(D) Mammalian two-hybrid assays were carried out in C3H10T1/2
cells. The interactions between GALGRIP1/ GALGRIP1A
(MID 1/2+C) and VP16MyoD were measured using G4TATALUC
reporter for luciferase activity 48 hrs after transfection. The amount
(fig) of each individual expression vector transfected has been
indicated in the figure.
(E) Transient transfection experiments were carried out as shown in
(D) with GALSRC 1 A/ GALSRC 1 AA(MID 1/2+C).
64
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A) 3 5 S Met G RIPl
G RIPl A(M IDl/2+C)
200 —
GST-
MyoD
GST
10%
Input
GST-
MyoD
GST
10%
Input
igi
( B ) 3 5 S Met SRC1A SRClAA(M IDl/2+C)
200----
GST-
MyoD
GST
10%
Input
GST-
MyoD
GST
10%
Input
(C)
35,
S Met
2 0 0 -
Fig. 8. A(MID 1/2+C) Mutants
Of P160 Coregulators
Significantly Lose Ability To
Interact With MyoD Both
In Vitro And In Vivo
CIP
GST-
MyoD
GST
10%
Input
CIPA(MID 1/2+C)
GST-
MvoD
GST
10%
Input
65
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(D)
10T1/2
300000
250000
200000 -
RLU150000
100000
50000 -
1 2 3 4 5 6 7
G4TATALUC (1) + + + + + + +
GALGRIP1 (1) + +
- - - -
GALGRIP1
A(MID1/2+C) (1) -
m
+ + + +
VP16MyoD
-
4
- 1 2 4
(E)
RLU
2500000
2000000
1500000
1000000
500000
10T1/2
u — —
1 2 3
—
4 5
-------
6
i
7
G4TATALUC (1) + + + + + + +
GALCIP (1) + +
- - - -
GALCIP
A(MID1/2+C) (1) - + + + +
VP16MyoD - 4 - 1 2 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
response. These results from both in vitro binding reactions and in
vivo mammalian two-hybrid assays suggest p i60 mutants devoid of
MIDI and MID2 domains as well as the C terminus are unable to
interact with MyoD. Since the C terminus alone is able to interact
with MyoD (fig. 7), its role cannot be to merely stablize the
conformation of segments bearing the MID domains.
3.3.6 SRC1AA(MID1/2+C) And CIPA(MIDl/2+C) Fail To
Coactivate MyoD, While GRIP1 A(MIDl/2+C) Upregulates
MyoD On Muscle Specific Promoters
MIDI and MID2 domains as well as the C terminus of pl60
coregulators appear to be crucial MyoD interaction domains. All three
P160A(MIDl/2+C) mutants significantly lose the ability to interact
with MyoD both in vitro and vivo. To further characterize whether
these essential interaction domains also play functional roles in
mediating MyoD-activated transcription, transient transfection assays
were carried out to compare the A(MIDl/2+C) mutants with the wild
type pl60s. As shown in fig. 9A, GRIPlA(MIDl/2+C) loses
(co)repressor activity on MyoD-directed activation on 4RE (lane 5),
MCK (lane 9, 10), and HCA (lane 14, 15) promoters in C3H10T1/2
fibroblasts when compared to wild type GRIP1 (lane 3, 8). The same
approach was applied to differentiating myoblasts and the results
showed that mutant GRIP 1 A(MID 1 /2+C) also loses its (co)repressor
67
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Fig. 9. GRIPlA(MIDl/2+C) Mutant Loses The Repressor
Activity Toward MyoD, While Both SRClAA(MIDl/2+C)
And p/CIPA(MID 1/2+C) Mutants Fail To Coactivate
MyoD On Muscle Specific Promoters.
(A) C3H10T1/2 cells were transfected with 4RELUC, MCKLUC, or
HCALUC reporter (1 fig), MyoD expression vectors (0.1 ju,g), and
either the wild type GRIP1 or mutant GRIP 1 A(MID 1 /2+C) (amounts
as indicated in the figure). The regulation of MyoD transactivation
was measured by luciferase activity 48 hrs after transfection.
(B) Transient transfection experiments were carried out either using
4RELUC or MCKLUC reporter as shown in (A) with C2C12
myoblasts. C2C12 myoblasts were cultured in growth medium
transfected and switched to differentiation medium. Luciferase assays
were performed 24 hrs later.
(To be continued)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(C) Transient transfection experiments were carried out as shown in
(A) with the wild type SRC 1 A/mutant SRC 1AA(MID 1/2+C).
(D) Transient transfection experiments were carried out as shown in
(B) with the wild type SRC 1 A/mutant SRC 1AA(MID 1/2+C).
(E) Transient transfection experiments were carried out as shown in
(A) with the wild type p/CIP/mutant p/CIPA(MID 1/2+C).
(F) Transient transfection experiments were carried out as shown in
(B) with the wild type p/CIP/mutant p/CIPA(MID 1/2+C).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1 o r i /2
(A)
40000
35000
30000
25000-
RLU 20000
15000
10000 -
5 0 0 0 -
1 2 3 4 5
4RELUC (1) + + + + +
MyoD (0.1)
-
+ + + +
GR1P1
. 4 -
GR1P1
A(MID!/2+C) 2 4
RLU
RLU
Fig. 9. GRIP1A(MID 1/2+C)
Mutant Loses The Repressor
Activity Toward MyoD, While
Both SRClAA(MIDl/2+C)
And p/CIPA(MIDl/2+C)
Mutants FailTo Coactivate
MyoD On Muscle Specific Promoters.
10T1/2
180000
160000-
140000
120000
100000
80 000-
60000
40000
20000
6 7 8 9 10
MCKLUC (1) + • + + + +
MyoD (0.1)
_ + + + +
GRIP1
- 4 -
GR1P1
A(MlD1/2+C) 1 2
10T1/2
18000000-
16000000
14000000
12000000
10000000
8000000
6000000
4000000
2000000
11 12 13 14 15
HCALUC (1) + + + + +
M yoD (0.1)
_
+ + + +
GRIP1 4
_
GR1P1
A(MlD1/2+C)
. .
1 2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(B)
„„„„„ C2C12
40000-3
35000-
30000-i
25000-
RLU 20000-
15000-
10000-
5000-
1 2 3 4 5
4RELUC (1) + + + + +
MyoD (0.1) - + + + +
GRIP1 - - 2 - -
GRIP1
A(MID1/2+C)
„ _
1 2
4500000-;
4000000-i
3500000-;
3000000-
RLU 2500000-i
2 0 0 0 0 0 0 - :
1500000-i
1000000-
500000-
0-
MCKLUC (1) + + + + +
MyoD (0.1) - + + + +
GRIP1 - - 2 -
GRIP1
A(MID1/2+C) 1 2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(C)
10T1/2
22000-
20000 -
18000-
160004
14000
120004
100004
80004
60004
4000
2000
0
1 2 3 4 5
4RELUC (1) + + + + +
MyoD (0.1) - + • + + +
SRC1A - 4 -
SRC1A
A(MlD1/2+C)
_
2 4
300000-
250000-
200000- ^
RLU150000-;
1 0 0 0 0 0 - J
50000-
8000000
7000000-
6000000-
5000000-
RLU 4000000-
3000000-
2000000-
1 0 0 0 0 0 0 4
0 -
10T1/2
8 9 10
MCKLUC (1) + + + + +
MyoD (0.1) - + + + +
SRC1A - - 4 - -
SRC1A
A(MID1/2+C)
. .
2 4
10T1/2
11 12 13 14 15
HCALUC (1) + + + + +
MyoD (0.1) - + +
SRC1A - - 4 -
SRC1A
A(MID1/2+C)
_
2 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(D)
35000
30000
25000
20000
RLU
15000
10000
5000
0
1 2 3 4 5
4RELUC (1) + + + + +
MyoD (0.1) - + + + +
SRC1A - - 2 - -
SRC1A
A(MID1/2+C)
_ _ _
1 2
C2C12
300000
250000 -
200000
RLU 150000
100000
50000
MCKLUC (1) + + + + +
MyoD (0.1) - + + + +
SRC1A - 2 -
SRC1A
A(MID1/2+C)
_
1 2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(E)
10T1/2
1 2 3 4 5
4RELUC (1) + + + + +
MyoD (0.1)
.
+ + + +
CIP - 4
CIP
A(MID1/2+C) 2 4
10T1/2
6 7 8 9 10
MCKLUC (1) + + + + +
MyoD (0.1)
-
+ + + +
CIP - 4 -
CIP
A(MIDt/2+C) 2 4
10T1V2
11 12 13 14 15
HCALUC (1) + + + + +
MyoD (0.1) - + + + +
CIP - - 4 - -
CIP
A(MID1/2+C) 2 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(F)
700000- 3
600000-
500000-
400000-
RLU :
300000-
C2C12
1 2 3 4 5
4RELUC (1) + + + + +
MyoD (0.1) - + + + +
CIP - 4 - -
CIP
A(MID1/2+C)
_
2 4
200000
180000
160000
140000
120000
RLU 100000
80000
60000
40000
20000
0-
C2C12
MCKLUC (1) + + + + +
MyoD (0.1) - + + + +
CIP - - 4 - -
CIP
A(MID1/2+C)
. _
2 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
activity towards MyoD on the 4RE (fig. 9B, lane 1, 2) and MCK (fig.
9B, lane 9, 10) promoters. As shown earlier SRC1A and p/CIP
upregulate MyoD-dependent transcription (fig. 9C, D, E, F).
However, mutant SRC 1AA(MID 1 /2+C) no longer potentiates MyoD
transactivation on the 4RE (fig. 9C, lane 4, 5), MCK (fig. 9C, lane 9,
10), or HCA (fig. 9C, lane 14, 15) promoters in 10T1/2 cells. They
also fail to activate 4RE (fig. 9D, lane 4, 5) and MCK (fig. 9D, lane 9,
10) promoters in differentiating myoblasts when compared to wild
type SRC1A (fig. 9C, lane 3, 8, 13; fig. 9D, lane 3, 8).
Likewise, CIPA(MID 1/2+C) was unable to coactivate MyoD-
dependent transcription on 4RE (fig. 9E, lane 4, 5), and MCK (fig. 9E,
lane 9, 10) promoters in 10T1/2 cells as well as 4RE (fig. 9F, lane 4,
5) and MCK (fig. 9F, lane 10) promoters in differentiating myoblasts
when compared to wild type p/CIP (fig. 9E, lane 3, 8; fig. 9F, lane 3,
8). Taken together, these transient transfection assays established that
the functional effects of the p i60 coregulators on MyoD mediated
transcription requires the presence of the MIDI, MID2 and the C-
terminus. These observations are consistent with the notion that the
functional effects of pl60s require direct pl60:MyoD physical
interaction.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.3.7 Both N-Terminus And bHLH Domains Of MyoD Are
Targeted by p i 60 Coregulators In Vitro
The N-terminus of MyoD is a transcriptional activation domain
(Weintraub et al., 1991) that can be coactivated by p300/CBP
(Sartorelli et al., 1997). This coactivation requires binding to the N-
terminus of MyoD (aa 1-100) by either of two p300 segments (aa 1-
596 or aa 1572-2370). When both an FYD motif and phenylalanine at
position 42 of the MyoD-N terminus (3-53) are mutated, designated
GAL-N MD(PPA+P), the N-terminus loses its ability to transactivate
or to be coactivated by p300 on a G5E1B reporter (Sartorelli et al.,
1997). I have demonstrated that the functions of p i60 coregulators
towards MyoD are mediated by MIDI and MID2 domains as well as
by the C terminus. However, the regions of MyoD that interact with
pi 60s are unknown. Therefore, I tested three segments of MyoD for
their ability to bind pl60s: N-terminus (1-36 aa), bHLH domain (37-
72 aa), and C-terminus (73-109 aa). I created a set of deletion mutants
of these three domains as well as testing the N and bHLH domains
alone. All were cloned into GST-fusion vectors. The schematic
representation of GST-MyoD domain mutants is shown in fig. 10A.
As shown in fig. 10B-F, the N-terminal domain of MyoD is both
required and sufficient to interact with either of the three p i60
coregulators. There is no evidence of significant interaction with
either the bHLH or C-terminal domains. However, I cannot exclude
77
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fig. 10. Both N Terminus And BHLH Domain Of MyoD
Interact With P160 Coregulators In Vitro.
(A) Schematic representations of GST-MyoD mutants.
(B) GST-MyoD-AN (indicated as AN in the figure) immobilized on
glutathione agarose beads were incubated with in vitro-translated [3 5 S]
GRIP1, SRC1A, and CIP. The bound proteins were resolved by
autoradiography after SDS-PAGE. Glutathione agarose-immobilized
GST protein serves as control. 10% INPUT stands for 10% amount of
in vitro-translated pi 60s used in the binding reactions was loaded.
(C) GST affinity binding assays were carried out as shown in (B) with
GST -My oD-AbHLH (indicated as AbHLH in the figure).
(D) GST affinity binding assays were carried out as shown in (B) with
GST-MyoD-bHLH (indicated as bHLH in the figure).
(E) GST affinity binding assays were carried out as shown in (B) with
GST-MyoD-AC (indicated as AC in the figure).
(F) GST affinity binding assays were carried out as shown in (B) with
GST-MyoD-N (indicated as N in the figure).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
GST MYOD
N bHLH C
Full Length ©©©©[
A N
AbHLH
AC
bHLH
* aa
B
N
(B)
35,
SMet P300
AN
GRIP1
AN
GST
10%
input
SRC1A
AN
GST
10%
input
CIP
AN
_ 10%
GS r input
<*»
1 2 3 4 5 6 7 8 9 10
■200
■97
■68
•43
Fig. 10. Both N Terminus And BHLH Domain Of MyoD Interact With P160
Coregulators In Vitro.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(C)
3 5 SMet P300 GRIP1 SRC1A CIP
AbHLH
3 GST io %
§ input
<
j GST io %
® input
< 1
I GST 1 0 %
5 input
<
■ -
1 2 3 4 5 6 7 8 9 10
(D)
35SMet P300 GRIP1 SRC1A CIP
bHLH
3 GST 10%
5 input
3 GST 10%
* input
3 GST . 10%
X input
a
f
*
* * I f f l
1 2 3 4 5 67 8 9 10
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(E)
CIP SRC1A P300 GRIP1 ’ SMet
GST AC AC AC AC
10%
input
10%
in p u t
10%
input
GST GST
m mm
1 2 3 4 5 6 7 8 9 10
(F)
3 5 SMet P300 GRIP1 SRC1A CIP
N N GST
10%
input N GST
10%
input
N GST
10%
input
m m
m
1 2 3 4 5 6 7 8 9 10
200
97
200
97
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
the possibility that either or both the bHLH and C-terminus can effect
the strength of N-terminal interaction with pi 60s.
3.3.8 P300 And The PI 60 Coregulators Bind To Different
Subdomains O f The MyoD Amino Terminus.
The primary target of both the p i60 coregulators and p300 is the N-
terminus of MyoD. This raises the issue whether SRC1A and p/CIP
compete or collaborate with p300/CBP when exerting their functions
as coactivators of MyoD. In addition, if the pi 60s and p300 share the
same target on MyoD, it would raise the possibility that GRIP1
competes with the coactivators for binding to the N-terminal
activation domain of MyoD, thus repressing MyoD transactivation. To
test these hypotheses, I carried out GST affinity binding assays using
GST-MyoD fusion proteins with various segments of the N-terminal
region 109 aa of MyoD. I divided the N-terminus into three
contiguous subregions, N1 (aa 1-36), N2 (aa 37-72), and N3 (aa 73-
109). In addition, a site-specific mutant with a mutation at the 42
position (aa) of MyoD, phenylalanine to proline, was also tested. The
schematic representation of these MyoD N-terminal variants is shown
in fig. 11A. As shown in fig. 11B, p300 does not bind to the N1
segments (lane 2) when compared to GST alone (lane 8). This result
was confirmed when the mutant AN1 (lane 4) showed that no
significant p300 binding activity was lost, comparing to full length
82
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(lane 1). P300 appears to bind to GST-MyoD-N3 (fig. 11B, lane 3)
efficiently and this result is confirmed by substantial loss of p300
binding activity when N3 is deleted (fig. 11B, lane 6). The N2 region
of MyoD may also interact with p300 because some binding activity
is lost in the mutant AN2 (fig. 11B, lane 5). This result is consistent
with the previous results (Sartorelli et al., 1997). Our data reconfirm
the earlier observation that p300 interacts at residues 42 since its
binding is diminished using GST-MyoD-N(F42P) (fig. 11B, lane 7).
Thus, I conclude that both the N3 and N2 regions are both required for
full binding to P300.
The same experimental approach was applied to the p i60
coregulators. As shown in fig. 11, the N1 region of MyoD is neither
required nor capable of interacting with GRIP1, SRC1A, or p/CIP.
GST-MyoD-Nl alone was not sufficient to bind and no substantial
binding activities were lost using the mutant GST-MyoD-AN 1 (fig. 11
C, D, E, lane 4).
The N3 region of MyoD appears to interact with GRIP1 (fig. 11C,
lane 3) and p/CIP (fig. 1 IE, lane 3). Reciprocally, substantial binding
activities are lost by deletion of N3 (fig. 11C, E, lane 6) when
compared to the wild type binding (fig. 11C, E, lane 1). In contrast,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fig. 11. P160 Coregulators And P300 Differentially
Interact With Various Regions Of The N-terminal
Activation Domain Of MyoD.
(A) Schematic representation of GST-MyoD-N terminus internal
deletion mutants employed in these experiments. The coordinates
(amino acids) of the regions deleted at the N-terminus of MyoD are
indicated in the figure. F42P represents the mutant GST-MyoD-N (aa
1-109) with phenylalanine substituted to proline at the position 42
(amino acid).
(B) In vitro-translated [3 5 S] P300 was incubated with various GST-
MyoD-N terminal mutants immobilized on glutathione agarose beads.
The bound proteins were resolved by autoradiography after SDS-
PAGE. Glutathione agarose-immobilized GST protein serves as
control. 10% INPUT stands for 10% amount of in vitro-translated
pi 60s used in the binding reactions was loaded.
(C) GST affinity binding assays were carried out as shown in (B) with
GRIP1.
(D) GST affinity binding assays were carried out as shown in (B) with
SRC1A.
(E) GST affinity binding assays were carried out as shown in (B) with
CIP.
84
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
N bHLH
GSTMvoD
OOOO
109
1-109
N1
1-36
N3
73-109
AN1
37-109
AN2
1-36/73-109
AN3
1-72
oooo
OOQO
Fig. 11. PI60 Coregulators And P300 Differentially Interact With Various
Regions Of The N-terminal Activation Domain Of MyoD
85
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(B)
35
SMet
AN2
N
1-109
N1
1-36
N3
73-109
AN1
37-109
1-36/
73-109
AN3
1-72
F42P GST
Input
10%
200
97
P300
m m
■1SI
(C)
1 2 3 4 6 7 8 9
35,
SMet
GRIP1
N
1-109
N1
1-36
N3
73-109
AN1
37-109
AN2
1-36/
73-109
AN3
1-72
F42P GST
Input
10%
200
97
■
1 2 3 4 5 6 7 8 9
86
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(D)
35,
SMet SRC1A
N
1-109
M N3 AN1
AN2
1-36/
AN3
F42P
1-36 73-109 37-109
73-109
1-72
GST
Input
10%
200
97
«r i|^|§ I P
■ P
(E)
1 2 3 4 5 6 7 8 9
35,
SMet CIP
200
97
N
1-109
.
N1
1-36
N3
73-109
ANl
37-109
AN2
1-36/
73-109
AN3
1-72
F42P GST
• * »
SfiMii#
'SiSSSt
♦V .JuJA - n a? J
Input
10%
8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
there is only weak binding activity of SRC1A to the N3 region of
MyoD (fig. 11D, lane 3) and no substantial binding is lost when N3 is
added (fig. 11D, lane 6). Remarkably, N3 interacts strongly with
p/CIP and GRIP1, but has little or no interaction with SRC1A.
However, it seems hard to completely exclude the possibility of the
N3 region of MyoD to be involved.
I next tested the ability of the N2 region of MyoD to interact with the
pi 60s. GST-MyoD AN2 loses significant binding activity to SRC1A
(fig. 11D, lane 5) when compared to GST-MyoD-N (fig. 11D, lane 1).
The N2 region of MyoD also appears involved in the GRIP 1-MyoD
interaction (fig. 11C, lane 5) when compared to wild type GST-
MyoD-N (fig 11C, lane 1). However, p/CIP does not bind strongly to
the N2 region because no substantial binding activity of p/CIP is lost
using GST-MyoD-AN2 (fig. 11E, lane 5) when compared to the wild
type GST-MyoD-N (fig. 11E, lane 1). In addition, the phenylalanine
at MyoD residue 42 appeared to be involved in the GRIP 1-MyoD
interaction but does not appear to play a major role in either the
SRClA-MyoD, or p/CIP-MyoD interaction.
In summary, the N1 (1-36 aa) region of MyoD is not a target of any
of the pi 60s nor p300. SRC1A interacts predominantly with the N2
(aa 37-72) region of MyoD and p/CIP interacts predominantly with
MyoD residues in the N3 (aa 73-109) region. GRIP1 appears to
interact with both the N2 and N3 regions (aa 37-109). P300 binds
88
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
predominantly to the N3 domain but also interacts with N2. These
results suggest that the pi 60s each interact with different domains
within the amino terminal activation domain of MyoD. SRC1A and
p/CIP binding targets do not appear to overlap. These observations
leave open the possibility that the SRC1A, p/CIP, and p300 could
directly interact with MyoD in combination.
The binding targets of GRIP1 are less easily defined but could well
overlap those of both SRC1A and p/CIP as well as p300. In addition,
whereas binding of SRC1A and p/CIP are unaffected by the mutation
of MyoD at residue 42 F to P, both p300 and GRIP! are. The
functional repression of MyoD activated transcription by GRIP1
stands in stark contrast to the coactivation properties of p300, p/CIP,
and SRC1A. That GRIP1, the repressor, binds to MyoD’s activation
domain at regions that fully overlap those required for binding by the
coactivators p300, SRC1A, and p/CIP has not escaped our attention.
3.3.9 Acetylation Sites O f MyoD Are Not Required To
Interact With PI 60 Coregulators
The activity of MyoD as a transcription coactivator requires its
acetylation by pCAF (Sartorelli et al., 1999). While p300 is required
to tether pCAF to MyoD, it cannot substitute for the acetylaion
function of pCAF. It appears that only three lysines are acetylated and
are located at the positions 99, 102, 104 aa in the N-terminus of
89
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
MyoD as shown in fig. 12A. A nonacetylable MyoD mutant,
designated as MyoD (RRR) cannot transactivate and cannot induce
gene expression of the late muscle maker, myosin heavy chain. In
addition, acetylated MyoD, also binds DNA more tightly either as
MyoD-MyoD homodimers or MyoD-E12 heterodimers (Sartorelli et
al., 1999). Thus, acetylation of MyoD plays a critical role in muscle
specific gene expression. SRC1A and ACTR (human homologue of
p/CIP) have been demonstrated to possess endogenous histone
acetyltransferase activities acting on histone 2B (core histones only),
and both histones 3 and 4 (nucleosomes). Moreover, the N3 region (aa
73-109), encompassing the acetylatable lysines (shown in fig 11) also
happens to be the docking sites for at least 2 pl60 coregulators as well
as p300 as shown in section 3.3.8. Nonhistone proteins have not yet
been reported to be the substrates of the acetyltransferase abilities of
p i60 coregulators. Nevertheless, the ability of SRC1A and p/CIP to
bind MyoD’s N-terminal activation domain to coactivate MyoD-
mediated transcription might be involved in MyoD acetylation. Thus,
I created the mutant GST-MyoD-N(3K/3A) mutant, lysine to alaline
substitution at the 99, 102, 104 aa positions, and tested its binding
activity with pl60 coregulators and p300. As shown in fig. 12B,
neither the pl60 coregulators (lane 2, 3, 4) nor p300 (lane 1) lost their
binding activities to the mutant GST-MyoD-N(3K/3A) when
90
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fig. 12. PCAF Acetylation Sites Of MyoD Are Not
Responsible To Interact With P160 Coregulators.
(A) Schematic representation of MyoD major acetylation sites and the
3K/3A mutant employed in the experiment. The coordinates (amino
acids) of 3 acetylation sites of MyoD are indicated in the figure.
3K/3A stands for MyoD with lysine substituted as alanine at position
99, 102, 104.
(B) GST-MyoD-N (wt) and GST-MyoD-N (3K/3A) immobilized on
glutathione agarose beads were incubated with in vitro-translated [3 5 S]
P300, GRIP1, SRC1A, and CIP respectively. The bound proteins were
resolved by autoradiography after SDS-PAGE. GST serves as control.
10% INPUT stands for 10% amount of in vitro-translated p300 and
pi 60s used in the binding reactions was loaded.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
N bHLH
MyoD
WT
3K/3A
-WACKACKRKTTNAD-
■WACAACARATTNAD-
(B)
GST-M D-N(3K/3A) GST-M D-N
Input 10%
iWWI
lilM p H
43
m
m m M B m
mpHMi
Jlill
HI
1 2 3 4 5 6 8 10 11 12
Fig. 12. PCAF Acetylation Sites Of MyoD Are Not Responsible To Interact
With P160 Coregulators.
92
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
compared to wild type GST-MyoD-N (lane 5, 6, 7, 8) in vitro. Thus,
three lysines at the positions 99, 102, 104 aa acetylated by HATs are
not required for interaction with either the p i60 coregulators or p300.
The results of my previous studies showed that the functions of pl60
as coregulators of MyoD are coupled to their physical interactions
with MyoD (fig. 8, 9). Thus, SRC1A and p/CIP can still interact with
MyoD regardless of the acetylation status of MyoD. This data
demonstrated that binding of MyoD by pl60’s and acetylation of
MyoD are two independent functions. Thus, pi 60s may engage MyoD
but MyoD must be acetylated before the pi 60s can coactivate.
3.3.10 Basic Domains O f MyoD Are Involved In Interacting
With PI 60 Coregulators
The basic domain of MyoD binds the DNA E box target (Lassar et
al., 1989) and the AT (alanine 114, threonine 115) motif of the basic
domain is critical for myogenic conversion (Davis and Weintraub,
1992) through a conformational mechanism that makes the N-
terminus available as a transcriptioanl activator (Huang et al., 1998).
The helix-loop-helix region is used to heterodimerize with E proteins,
and the heterodimer, thus formed, can participate in forming a
stablized protein-DNA complex upon docking to the cis elements of
muscle specific genes.
93
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The essential regions of p i60 coregulators required to interact with
MyoD have been delimited to the MIDI and 2 domains as well as the
C terminus. Data presented earlier (fig. 10D) leaves open the
possibility that pi 60s, especially p/CIP, may bind to the bHLH
domain of MyoD. Thus, I wished to further examined whether the N-
terminus of MyoD is the only region responsible to contact with p i60
coregulators. Accordingly, I created a mutant GST-SRC1A
(MID1/2+C) with minimal essential MyoD anchoring regions and
tested its ability to bind various deletion mutants of MyoD. The VP 16
activation domain was introduced at the N-terminus of each MyoD
mutant, N, bHLH, C, AN, AbHLH, and AC to increase [3 5 S1
methionine signal. As expected, the mutant VP16MyoDAN
substantially lost binding activity to GST-SRC 1 A(MID 1/2+C) (fig.
13B, lane 1) compared to the AC segments. Surprisingly, the mutant
VP 16MyoDAbHLH also significantly lost its binding ability to GST-
SRC1A(MID 1/2+C) (fig. 13B, lane 2), suggesting the low level
binding activity of SRC1A to GST-MyoD-bHLH shown previously
(fig. 10D, lane 5) cannot be dismissed. I next verified this interaction
by showing that both VP 16MyoD-bHLH as well as VP16MyoD-N
bound to GST-SRC1 A(MID 1 /2+C) (fig. 13C, lane 1, 2) but
VP16MyoD-C does not (fig. 13C, lane 3). These results demonstrate
that the bHLH domain of MyoD as well as its N-terminus is involved
in the interaction between SRC1A and MyoD.
94
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Fig. 13. Two Subregions Of The Basic Domain Of MyoD
Are Required To Interact With SRC1A.
(A) Schematic representation of MyoD basic domain internal deletion
mutants employed in the experiment.
(B) GST-SRC 1AA(MID 1/2+C) immobilized on glutathione agarose
beads were incubated with in vitro-translated [3 5 S] VP16MyoDAN,
VP16MyoD AbHLH, and VP16MyoDAC respectively. The
VP16MyoD deletion mutants employed here are the same as shown in
the (A) of figure 10 except VP 16 activation domain replacing GST in
front of MyoD gene. The bound proteins were resolved by
autoradiography after SDS-PAGE. 10% INPUT stands for 10%
amount of in vitro-translated VP16MyoD deletion mutants used in the
binding reactions was loaded.
(To be continued)
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(Continued)
(C) GST affinity binding assays were carried out as shown in (B) with
deletion mutants VP16MyoD-N, VP 16MyoD-bHLH, and
VP16MyoD-C instead.
(D) GST affinity binding assays were carried out as shown in (B) with
MyoD wild type, deletion mutants MyoDAbHLH, and MyoDAbasic
instead.
(E) GST affinity binding assays were carried out as shown in (B) with
MyoD wild type, basic-domain-deletion mutants of MyoD shown in
(A) instead.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
MyoD
N
WT -K R K TTN A D R R K A A TM R ER R R LSK -
Abasie -K RK TTN A D LSK -
A103-112 -K A A TM R ER R R LSK -
A112 116 -K R K TTN A D R R R E R R R L SK -
A117-121 -K RK TTN A D R RK A A TM LSK-
A122-124 -K R K TTN A D R R K A A TM R ER R R
Fig. 13. Two Subregions Of The Basic Domain Of MyoD Are Required To
Interact With SRC1A.
bHLH
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(B) GST-SRClA(MIDl/2+C) 10% Input
3 5 S VP16MyoD
AN AbHLH AC AN AbHLH AC
68
43 ----- 4
* •
I
29
1 2 3 4 5 6
( C )
GST-SRClA(MIDl/2+C) 10% Input
3 5 S VP16MyoD
N bHLH C N bHLH C
43 —
29 -----
1
M i;: w m m
A l e 1 . - ■ ■ ;
1 2 3 4 5 6
98
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(Continued)
(D)
GST-SRClA(MIDl/2+C)
10% Input
35.
SMyoD
W t AbHLH Abasic W t AbHLH Abasic
43
29
(E)
GST-SRClA(MIDl/2+C)
10% Input
3 5 S MyoD Wt
A103
-112
A112
-116
A117
-121
A122
-124
Wt
A103
-112
A112
-116
A117
-121
A122
-124
43
29
■ m «#* m . IMf
1 2 3 4 6 7 8 9 10
99
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To determine whether it is the basic domain, the helix-loop-helix
region or both that are involved in binding to SRC1A, the same
mutant GST-SRC1 A(MID 1/2+C) was tested for its ability to interact
with bHLH and basic region deletion mutants of MyoD. As shown in
fig. 13D, both mutants MyoDAbHLH and MyoDAbasic no longer
bind GST-SRClA(MIDl/2+C) (lane 2, 3), which confirmed the data
obtained using VP 16MyoDAbHLH (fig. 13R, lane 2). I next used a
series of small internal deletion mutants, spanning residues 103-123 of
the basic region of MyoD to narrow down which residues actually
make contact with SRC1A. The schematic representation of these
MyoD basic region mutants is shown in fig. 13A. As shown in fig.
13E, two of these MyoD mutants, with deletion of aa 103-112 (lane 2)
or aa 117-121 (lane 4), are both unable to interact with SRC1A,
suggesting that the residues within these two regions play critical roles
in mediating MyoD-SRCIA interaction. Deletions of residues 112-
116 or 122-124 of MyoD does not affect binding to SRC1A (fig. 13E,
lane 3, 5). Taken together, the basic domain, specifically at the
regions at aa 103-112 and aa 117-121 of MyoD seem to interact with
SRC1A.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.3.11 c-AMP-Dependent Protein Kinase Potentiates Gal-
p i 60s and Enhances The Interaction Between MyoD and
p/CIP
The function of pl60 coregulators is related to their phosphorylation
by several signaling pathways. SRC-1 is the target of the mitogen-
activated protein kinase (MAPK) pathway (Rowan et al., 2000). SRC1
can also be phosphorylated by the cAMP signalling pathway
following treatment of cells with 8-bromo-cAMP (Rowan et al., 2000)
or overexpression of PKA. When the MAP kinase pathway is
activated, it can be crossly provoked by the cAMP signaling pathway
upon treatment with 8-bromo-cAMP (Rowan et al., 2000). In addition,
GRIP1 is also regulated by MAPK following stimulation of the
extracellular signal-regulated kinase (ERK) by epidermal growth
factor (Lopez et al., 2001). AIB1 also has been identified as a
phosphoprotein. It can be phosphorylated by Erk2 of the MAP kinase
family (Font de Mora and Brown, 2000).
These observation raised the possibility that phosphorylation may
effect the ability of pi 60s to interact with MyoD. I used mammalian
two-hybrid experiments to address this hypothesis. Full length GRIP1,
SRC1A, and p/CIP were fused to the LexA DNA binding domain
(LexA-DBD) to generate LexAGRIPl, LexASRCIA, and LexACIP.
These were cotransfected with a reporter gene carrying a LexA
binding domain. As shown in Fig. 14B, C, and D, the transcriptional
101
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Fig. 14. Cyclic AMP-Dependent Protein Kinase
Potentiates The Transactivations Of LexA-P160
Coregulators And Synergizes The Interactions Between
p/CIP And MyoD In Vivo.
(A) Alignments of MIDI domain of P160 coregulators and the
consensus sequences of the kinase induced domain (KID) of CREB.
(B) C3H10T1/2 cells transiently transfected with L8G5LUC reporter
and the expression vectors of LexAGRIPl (1/rg), LexA, and the
catalytic subunit of cyclic AMP-dependent protein kinase (PKA,
amount as indicated in the figure). The potentiation of LexAGRIPl
transactivation by PKA was measured by luciferase activity 48 hrs
after transfection.
(To be continued)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Continued)
(C) Transient transfection experiments were carried out as shown in
(B) with LexASRCIA instead.
(D) Transient transfection experiments were carried out as shown in
(B) with LexACIP instead.
(E) C3H10T1/2 cells transiently transfected with G4TATALUC
reporter and the expression vectors of GALCIP (1/xg), VP16MyoD
(ljLtg), and PKA (1 fig). The synergistic effect between CIP and MyoD
interaction was measured by luciferase activity 48 hrs after
transfection.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(A)
[GENETYX-MAC: Multiple Alignment]
D ate : 2001.10.21
hSRCIA
mSRClA
hTIF2
mGRIPl
hACTR
mCIP
KID
hSRCIA
mSRClA
hTIF2
mGRIPl
hACTR
mCIP
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tH e p q l v i
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NPV B> DRHGFV
I I I RE TNSGMSI
H REQr V1
HREQIJV
QREQN'
RECW
61 PR
61 PR
59 —
59 —
59 —
59 —
16 —
60
60
58
58
58
58
15
62
62
58
58
58
58
15
(B)
10T1/2
RLU
180000
160000
140000
120000
100000
80000
60000
40000
20000 -
i —— i r i
1 2 3 4 5 6 7
L8G5LUC (1)
LexAGRIPl (1)
LexA (1)_______
PKA
Fig.14. Cyclic AMP-Dependent Protein Kinase Potentiates The
Transactivations Of LexA-P160 Coregulators And Synergizes The
Interactions Between p/CIP And MyoD In Vivo
10 4
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(Continued)
(C)
m
10T1/2
80000-.
70000-
60000-
50000
RLU 40000
30000
20000
10000
L8G5LUC (1)
LexASRCIA (1)
LexA (1)
PKA
500000
450000-^
400000
350000•:
300000- j
RLU 250000
200000
150000
100000
50000
0
10T1/2
1 2 3 4 5 6 7
L8G5LUC (1) + + + + + + +
LexACIP (1) + + +
„ „
LexA (1)
. .
+ + +
PKA
-
2 4
.
2 4
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(Continued)
(E)
Annnnnn 10T1/2
4000000-
3500000-
3000000-
2500000-
RLU 2000000-;
1500000-i
1000000 -
500000-
1 2 3 4 5
G4TATALUC (1) + + + + +
GALCIP (1) + + + +
VP16MyoD (1)
- -
+ +
PKA (1)
- + - +
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
activities of all three pi 60s were highly potentiated 10 fold, 17 fold,
and 43 fold respectively when an expression vector carrying the
catalytic subunit of PKA was cotransfected. I next addressed whether
the protein-protein interactions between a p i60 corgulator and MyoD
is affected by their phosphorylation status. As shown in fig. 14E, the
transcriptional activity engendered by the interaction of LexACIP and
VP16MyoD (lane 4) was greatly enhanced at least 4 fold (lane 5).
These data suggest that the interaction between MyoD and p/CIP may
be subjected to regulation by PKA in vivo. In addition, this PKA-
regulated protein-protein interaction is more specific between MyoD
and p/CIP than the other two p i60 coregulators (data not shown).
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3.4 Discussion
The HAT activity of pCAF but not that of p300/CBP is required to
coactivate MyoD-dependent transcription (Puri et al., 1997). Given
that (1) the in vitro interactions between MyoD and pCAF are much
weaker than between MyoD and p300 and (2) there is no detectable
interaction between pCAF and MyoD in mammalian two-hybrid
assays, it seems likely that the role of pCAF in MyoD-activated
transcription must be mediated by other associated factors. To date it
has been concluded that p300/CBP plays that role (Puri et al., 1997). I
have shown here that in addition to p300/CBP, all p i60 coregulators
are able to associate with MyoD in vivo (fig. 4). Thus, it is possible
that the pi 60s, SRC1A and p/CIP, and p300/CBP, either together or
individually, may stablize the interactions required to complex pCAF
and its acetylation activity with MyoD. Further support for this notion
is provided by my observation that El A, a direct inhibitor of
myogenesis and MyoD-dependent gene transcription targets pi 60s
(data not shown). The important role of pl60 in the function of such
myogenic coactivator complexes is underscored by my discovery of
the direct physical interactions between p i60 coregulators and
myogenic bHLH proteins in vitro (fig. 5). Importantly, these physical
interactions between pi 60s and MyoD can occur independently of
both E2A gene products, the heterodimer partners of myogenic bHLH
108
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proteins, and MEF2 since they are not necessarily included in the in
vitro GST binding reactions. In addition, MyoD and Myf5 are
important for cell fate determination of myogenic progenitors, while
myogenin is essential for skeletal muscle differentiation (Relaix and
Buckingham, 1999). Thus, the ability of SRC1A and p/CIP to interact
with all four myogenic factors implicates them in both the
proliferation and maturation of myoblasts through the completion of
terminal skeletal muscle differentiation.
Several transcription factors interact with different domains of pl60
coregulators. ERR3 was identified by its ability to bind to the N-
terminus of GRIP1 in yeast two-hybrid screenings and bound to the
NR boxes (Hong et al., 1999). The C-terminus has the most reported
interactions and has been shown to bind with C-Jun/Fos (Lee et al.,
1998), Zac-1 (Huang and Stallcup, 2000), CARM-1 (Chen et al.,
1999), PRMT-1 (Koh et al., 2001), pCAF (Chen et al., 1997; Korzus
et al., 1998). The region located between the NR boxes and the HAT
domain is bound by the p50 subunit of NFkB (Na et al., 1998) and by
p300/CBP (O’Malley, ref; Yao et al., 1996; Torchia et al., 1997; Hong
et al., 1999; Voegel et al., 1998). The NR boxes mediate the
interactions with various nuclear hormone receptors (Ding et al.,
1998; Heery et al., 2001; Heery et al., 1997; Leers et al., 1998; Voegel
et al., 1998) in a ligand-dependent manner. Finally, SRF uses multiple
109
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interfaces to contact SRC1A (Kim et al., 1998). Here I have
demonstrated that the C termini of all three p i60 coregulators are
required for interaction with MyoD (fig. 6B, D, F). In addition, other
regions, MIDI and MID2 domains for SRC1A, and MIDI for p/CIP,
play critical roles in the interaction with MyoD (fig. 6B, D).
P300/CBP has been proposed as an adaptor or mediator between the
enhancesome and the basal transcriptional machinery because it
transactivates and integrates upstream signals and relays them to
multiple downstream transcriptional activators (Vo and Goodman,
2001). Similarly, the complexity and number of transcriptional
activators that interact with p i60 proteins raises the possibility that
they too might be similar signal integrators (Rosenfeld and Glass,
2001), although the association between basal transcriptional factors
(TFIIB and TBP), and p i60 coregulators is still in debate (Kalkhoven
et al., 1998; Takeshita et al., 1996). Indeed, the limited binding
activity between p300/CBP and nuclear hormone receptors suggests
that either p i60 coregulators or pCAF (Blanco et al., 1998) are more
likely to be recruited when the nuclear hormone receptors dock on
their cis elements following ligand binding. In the case of MyoD
docking on E boxes, p i60 coregulators and p300/CBP rather than
pCAF are likely to be preferentially recruited with because of stronger
binding affinities. In this way differential usage of various
110
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coregulators could contribute to the fine modulation of expression of
individual genes.
The results of my experiments using the mammalian two-hybrid
assay, confirmed that the C-terminus of SRC1A and p/CIP are
important to mediate protein-protein interaction with MyoD in vivo,
where the N-terminus seems dispensable (fig. 7 A, B). The middle
segment (aa 325-1104) of SRC1A also showed some ability to bind
VP16MyoD (3 fold, fig. 7A, lane 11) when compared to MyoD (1.4
fold, fig. 7A, lane 12). This suggests that the three NR boxes (LXXLL
motifs) in this middle segment that p i60 coactivators use to interact
with nuclear hormone receptors also might contribute to a weak
interaction with MyoD in vivo. On the contrary, the MID 1 domain of
p/CIP plays an essential role in directing the interaction between
MyoD and GalCIP-M in vivo, which excludes the possibility that the
three LXXLL motifs in the middle segment are involved in the p/CIP-
MyoD interaction in vivo. Thus, the roles of LXXLL motifs in
mediating the interaction between MyoD and the p i60 coactivators
awaits finescale careful mutational studies. The mutant fusion protein
GAL-SRC1 A-AM includes the C-terminus of SRC1A but is unable to
interact with VP16MyoD in vivo (fig. 7A, lane 14). The reasons for
this apparent paradox are unclear. However, the N-terminus of
SRC1A might inhibit C-terminus interaction since both GALSRC1A-
111
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N and GALSRC1A-N+MID1 mutants were unable to interact with
VPlbMyoD (fig. 7A, lane 2, 5). Alternatively, the middle segment
might simply be required to hold SRC1A in a conformation necessary
for binding.
The essential roles of the MIDI and MID2 domains as well as the C
terminus of SRC1A in mediating its interaction with MyoD led me to
create the mutant p 160A(MID 1/2+C) with which MyoD is unable to
contact. Similarly configured A(MID 1/2+C) mutations in all three
pi 60s are unable to bind to MyoD as strongly as wild type both in
vitro and vivo (fig. 8). Importantly the functions of the pl60
coregulators on MyoD-dependent transcription require these essential
domains (fig. 9). The mutant GRIP 1 A(MID 1/2+C) not only loses its
(co)repressor activity but now coactivates MyoD on muscle specific
promoters in both 10T1/2 cells and differentiating myoblasts. This
acquired up-regulation activity of this GRIP1 mutant likely involves
its residual binding activity to MyoD and p300/CBP. Similarly, the
residual potentiation of MyoD transactivation by the mutant
CIPA(MID 1/2+C) is consistent with its residual binding to MyoD (fig.
9E, lane 4, 5).
The activation domains of transcription factors are the binding
targets of co-activating adaptor proteins such as p300/CBP. SRC1A
112
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and p/CIP coactivators follow this rule since they bind to the N-
terminal activation domain of MyoD (fig. 10B, F). P300, SRC1A and
p/CIP all coactivate MyoD-dependent transcription through
interactions at the same N-terminal activation domain of MyoD. This
raises the question of whether their binding is coordinated and
cooperative or whether their binding activities and docking sites are
mutually interchangeable. My experiments suggest that each has its
individual binding positions on the N-terminus of MyoD (fig. 11).
Thus, it is likely that these molecules interact and regulate MyoD
transactivation in a coordinated manner rather than competing for
binding. GRIP1 appears to be an exception since it binds throughout
the N-terminus (fig. 11C). This suggests it could compete for binding
by the coactivators thus providing a possible explanation for the
(co)repressor activity of GRIP1 on MyoD-mediated transcription.
The predicted bHLH domain located at the N-terminus of all three
p i60 coregulators, was initially thought to interact with the bHLH
domain of MyoD. However, the in vitro binding data didn’t support
this hypothesis because N termini of the pi 60s are not required to
interact with MyoD (fig.6 B D, F).
Differential usage of acetylated lysines of MyoD by different types
of HATs has implicated that two concerted events, coactivation and
acetylation of MyoD, are tightly associated to regulate MyoD-
113
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dependent transcription. PCAF which is unable to directly associate
with MyoD in vitro is still able to coactivate MyoD in vivo and
acetylate MyoD in vitro. The roles of pCAF as a coactivator can be
excerted through its associating partners like p300/CBP, SRC1A, or
p/CIP. However, it is still an enigma how pCAF selects and acetylates
the substrates without binding them as a prerequisite condition. Thus,
I think both coactivation and acetylation can be mutually and
independently functioning because the acetylated sites of MyoD are
also not required to interact with all pl60 coregulators (fig. 12). In
addition, it is still unclear whether MyoD can behave as the substrate
for the pl60HATs, SRC1A or ACTR.
It has been shown that MIDI, MID2 domains and the C-terminus of
SRC1A are the essential regions responsible to interact with MyoD.
However, the C-terminus alone does not interact with MyoD in vitro
(data not shown), suggesting that the MIDI and MID2 domains are
the actual binding components. Wild type MyoD and the mutant
VP16MyoD-N, but not the mutant MyoDAN, are capable of
interacting with GST-SRC 1 A(MID 1/2+C) (fig. 13), reinforcing the
conclusion that the N-terminus of MyoD interacts with SRC1A (fig.
10B, F). By using the GST-SRC 1 A(MID 1/2+C) mutant I was able to
show that the basic domain of MyoD, specifically aa 103-112 and aa
117-121, can also interact with SRC1A (fig. 13D, E). Muscle
1 1 4
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differentiation could be inhibited when the bHLH domain of
myogenic factors are targeted by inhibitors (Braun et al., 1992; Taylor
et al., 1993). Thus, description of the involvement of the basic domain
of MyoD with MyoD-SRCIA becomes another possible way in which
El A inhibits MyoD transactivation .
A plausible explanation for the discrepancy derived from two
independent GST pull down experiments using GST-MyoDbHLH
(fig. 10 D), or GST-SRC1A(MID 1/2+C) (fig. 13B, D) respectively
could be that the N-terminus but not the basic region of MyoD itself is
competent to bind SRC1A. However, a certain degree of cooperation
between basic region and the N-terminus of MyoD to maintain a fully
functional scaffold to interact with SRC1A seems necessary. It has
been known that the myogenic codes (alanine 114, threonine 115) in
the basic and junction regions of MyoD direct the programming of
myogenic conversion (Davis and Weintraub, 1992), and yet the
mechansims by which recognition of these amino acids by as-yet-
undefined molecules can ignite myogenic programs are still unknown.
Some models have been proposed that the recognition of myogenic
code might unmask the N-terminal activation domain of MyoD
(Huang et al., 1998), which could only partially support the targeting
model built through SRClA-MyoD interaction because all these three
codes are mutually excluded from the SRC1A docking sites at the
115
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basic domain of MyoD. Nevertheless, interesting findings came out
when I examined both subregions of the basic domain, aa 103-112 and
aa 117-121 and they turned out to be the same residues that MyoD
uses to interact with Twist (Hamamori et al., 1997). This coincidence
indicates that similar residues might reside in the p i60 coregulators
and Twist. In addition, the conservation of aa 103-112 of MyoD in
myogenic factors but not in E12, Myc, or T4 bHLH proteins also
suggests that the targeting of MyoD by SRC1A is specific to
myogenic rather than general bHLH proteins. Moreover, several
methyltransferases have been demonstrated to modify histones
(Bannister et al., 2001; Lachner et al., 2001; Nishioka et al., 2002; Rea
et al., 2000; Strahl et al., 2001), as well as regulate nuclear hormone
receptor-mediated transcription (Chen et al., 1999; Ma et al., 2001;
Wang et al., 2001). Arginines were also found in the regions of aa
103-112 and aa 117-121, making me wonder about the roles of
arginine-methyltransferases like CARM1, PRMT1, or others in
regulating MyoD-mediated transcription because p i60 coregulators
are able to associate with these methyltransferases (Chen et al., 1999).
CARM1 has been shown able to associate with MEF2C (Chen et al.,
2002).
Whether p i60 coregulators really contains the kinase induced
domain still remains investigated, and yet the transactivations of
116
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GRIP1, p/CIP and SRC1A are indeed regulated by PKA (fig. 14B, C,
D). In addition, phosphorylation by PKA can enhance the protein-
protein interaction between MyoD and p/CIP (fig. 14E) but not in
MyoD-GRIPl or MyoD-SRCIA interaction (data not shown). This
synergism can be attributed to a more stablized coactivator complex
thus formed upon phosphorylation by PKA as hypothesized (Rowan
et al., 2000). It has been shown that MyoD can be phosphorylated by
PKA in vitro and it is the c-AMP signaling activity rather than the
phosphorylation status of MyoD that was important for the nuclear
entry of MyoD (Vandromme et al., 1994). Some proteins responsible
for nuclear import mechinery were proposed to be involved but not
proved yet. Besides, the polyglutamine stretch of p300/CBP was
shown to be important to suck in the protein causing Hungtinton
disease (Steffan et al., 2001). Thus, the unique characteristics of
polyQ in p/CIP might possibly explain why this synergism was only
specific towards p/CIP-MyoD rather than GRIP 1-MyoD or SRC1A-
MyoD complex.
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Asset Metadata

Creator Wu, Hung-Yi (author)

Core Title Different roles of p160 coregulators in myogenesis

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Kedes, Laurence H. (committee chair), Lai, Michael M.C. (committee member), Maxson, Robert E. (committee member), Stallcup, Michael R. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-266237

Unique identifier UC11339415

Identifier 3093938.pdf (filename),usctheses-c16-266237 (legacy record id)

Legacy Identifier 3093938.pdf

Dmrecord 266237

Document Type Dissertation

Rights Wu, Hung-Yi

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA