Mixed lineage leukemia proteins (MLLs), their effect as coregulators on target gene expression and global histone methylation

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Content

Mixed
Lineage
Leukemia
Proteins
(MLLs),

Their
Effect
as
Coregulators
on
Target
Gene

Expression
and
Global
Histone
Methylation

By

Farah
Alammari

A
Thesis
Presented
to
the

FACULTY
OF
THE
USC
GRADUATE
SCHOOL

UNIVERSITY
OF
SOUTHERN
CALIFORNIA

In
Partial
Fulfillment
of
the

Requirements
for
the
Degree

MASTER
OF
SCIENCE

(BIOCHEMISTRY
AND
MOLECULAR
BIOLOGY)

December
2013

Copyright
2013

Farah
Alammari

i

ACKNOWLEDGMENTS

First and foremost, I would like to thank Dr. Michael Stallcup for the
great opportunity he gave me by working in his laboratory. He was a
wonderful professor, mentor, and friend. I learned a lot from him and
appreciate his patience and support. I also would like to thank my committee
members Dr. Zoltan Tokes for his advice, help, and encouragement during
my master’s study and Dr.Wei lei for his discussions and comments.

I would like to express my sincere appreciation to kwang Jeong, I
came to the lab as a fresh graduate and he introduced me to basic research,
I’m very thankful for his assistance and encouragement. Special thanks to the
sweetest person Chen-yin Ou who was a teacher and a friend to me. She
helped me a lot with my work and always been there for me. Also my deepest
appreciation to my friend Rajas Chodankar, I learned a lot from her and she
supported me in all aspects. I would like to thank my colleagues Daniele
Gerke, Dai-ying Wu, Danielle Bittencourt for their assistance and friendship.

ii

Finally, I would like to thank my Mother (Rugayah Alrumaih), my father
(Mohammed Alammari), my brothers Hisham and Essam, and my sister
Muneerah for their support and love. Thus, I dedicate this thesis to them.

iii

TABLE OF CONTENTS

Acknowledgments……………………………………….………....i
List of Figures…………………………………………..………......iv
List of Tables………………………………………………………..vi
Abstract…………………………………………..………………...vii
Chapter 1: Introduction……………………………...…………….1
Chapter 2: Results ……………………………………………….15
Chapter 3: Discussion ……………………………………………45
Chapter 4: Materials and Methods……………………………...50
Bibliography……………………………………………………….56

iv

LIST OF FIGURES

Figure Page

Fig.1: Structure of nuclear receptors. 3

Fig.2: Structure of MLL1, MLL2, MLL3, MLL4, and MLL5. 7

Fig.3: Multiprotein complex of MLLs. 8

Fig.4: Mechanism of multiple lysine methylations by MLL family. 11

Fig.5: Effect of MLL1 depletion on PgR gene expression. 12

Fig.6: Effect of MLL1 depletion on ER alpha and RNA 14
polymerase II recruitment to PgR gene.

Fig.7: Figure 7: Effect of MLL1 and SETD1A depletion
on global mono-, di-, and tri-H3K4 methylation. 14

Fig.8: Effect of MLL1 depletion on protein and mRNA levels. 19-20

Fig.9: Effect of MLL2 depletion on protein and mRNA levels. 21-22

v

Fig.10: Effect of SETD1A depletion on protein and mRNA levels. 23-24

Fig.11: Effect of SETD1B depletion on protein and mRNA levels. 25-26

Fig.12: Effect of SETD7/9 depletion on protein and mRNA levels. 27-28

Fig.13: Effect of coregulator depletion by two different
siRNAs on the E2-regulated expression TFF1 gene. 33-34

Fig.14: Effect of coregulator depletion by two different
siRNAs on the E2-regulated expression GREB1 gene. 35-36

Fig.15: Effect of coregulator depletion by two different
siRNAs on the E2-regulated expression PgR gene. 37-38

Fig.16: Effect of coregulator depletion on Histone 3. 41

Fig.17: Effect of coregulator depletion on global
H3K4 mono-methylation. 42

Fig.18: Effect of coregulator depletion on global
H3K4 di-methylation. 43

Fig.19: Effect of coregulator depletion on global
H3K4 tri-methylation 44

vi

LIST OF TABLES

Table

Page

Table 1: summary of the effect of coregulators on
TFF1, GREB1, and PgR genes 49
Table 2: Summary of the effect of coregulators on
H3K4 methylation mark 50

vii

ABSTRACT

Mixed lineage leukemia proteins (MLLs) belong to the evolutionarily
conserved trithorax family of human genes that play important roles in
development and HOX gene regulation. MLL genes are usually
rearranged in myeloid and lymphoid leukemias implicating them in these
diseases. There are several MLL family proteins, including MLL1, MLL2,
MLL3, MLL4, MLL5, Set1A, Set1B, and Set7/9. Each possesses histone
H3 lysine 4 (H3K4)-specific methyltransferase activity, which is an
epigenetic mark that presents in open and transcriptionally active
chromatin. Some MLL proteins interact with nuclear receptors such as
estrogen receptor (ER alpha) and play an important role in steroid
hormone-mediated gene activation. In this study, we deplete five different
members of the MLL family in MCF-7 breast cancer cells and compare
the effect of each on the total cellular levels of mono-, di-, and
trimethylation of H3K4, and on expression of the Ps2, GREB1, and PgR
genes, which are ER alpha target genes. We found that even though
they are all members of the same family, each one of them has its own
unique pattern of regulation.

1

CHAPTER 1

INTRODUCTION

Steroid hormones are important substances for the proper
function of the body. They are synthesized and secreted into the blood
by endocrine glands such as the gonads and the adrenal cortex. They
consist of five classes: Androgens, Estrogens, Progestin,
Mineralocorticoids, and Glucocorticoids. Androgens and Estrogens
regulate sexual development and functions, Progestin helps in
controlling the menstrual cycle and pregnancy, Mineralocorticoid
regulates water and salt excretion from the kidney, and glucocorticoids
regulate immune function and metabolism of glucose, lipids, and
proteins (Cinsidine et al., 1984). Steroid hormones function as genetic
regulators and control the rate of synthesis of particular mRNAs and
the proteins they encode. Steroids penetrate the cell membrane to
couple with Steroid Receptors (SR) forming hormone-SR complex.
Steroid Receptors belong to the nuclear receptor (NR) family of
proteins.

2

Nuclear receptors, NRs, are transcription factors that are crucial for
development, metabolism, cell death and many other functions.
Dysfunction of NRs may lead to dysregulated proliferation and
metabolic diseases like diabetes. NRs bind to their specific DNA
sequences and regulate the expression of target genes. They are
divided into 3 major classes (Chen et al., 2006). Class I receptors are
steroid hormone receptors that are being activated by their ligands and
then bind as homodimer to inverted repeats of DNA called hormone
response elements (HRE). Examples of this class include estrogen
receptor (ER), glucocorticoid receptor (GR), and progesterone receptor
(PR). Class II receptors bind to their DNA sequence without ligand but
are activated by ligands; examples include the retinoic acid receptors.
Class III nuclear receptors are orphan receptors because their ligands
are unknown. Examples of this class are the testis receptors.

Nuclear receptors contain N-terminal domain, DNA binding
domain, hinge region, ligand binding domain, and C-terminal domain
(Chen et al., 2006). The amino-terminal regulatory domain contains
activation function 1 (AF1) that synergizes with AF-2 in the ligand
binding domain (LBD) to regulate

3

target gene expression. The central DNA binding domain (DBD) is
highly conserved among different NRs and contains two zinc-
fingers that binds to HRE. Hinge region is a flexible domain that
connects DBD to LBD.
The LBD contributes to the dimerization of the receptor, and along with
AF-1 and other domains binds to coactivator and corepressor proteins.
The carboxyl-terminal domain is highly variable in sequence between
different NRs (Figure 1 a & b).

Figure 1: Structure of Nuclear Receptors
A) Domain structure of a nuclear receptor.
(B) Nuclear receptors can regulate gene expression by binding to the hormone
response element (HRE) as homodimers, RXR heterodimers, or monomers.
(Wada et al., 2008)

4

Cis-regulatory elements include the enhancer elements which are
200-500 bp in length and contain recognition sites for multiple
transcription factors to bind. They have important roles in controlling
gene expression and activate or repress target genes from a great
distance. Two mechanisms were proposed to explain the long-range
communication between the enhancer and the promoters of target
genes, which are looping and tracking (Calo et al., 2013). The looping
mechanism postulates that the enhancer-associated factors are delivered
to the promoter by direct interaction between the enhancer and the
promoter with looping out of the intervening DNA sequence. In contrast
the tracking mechanism postulates that the enhancer activates
transcription by tracking of RNA polymerase II down the intervening DNA
to connect with the promoter.

The chromatin at the enhancer must become accessible for
different factors to bind. Different models are proposed to cause this
opening. One model proposes that the cooperative binding of
transcription factors (TF) can overcome the nucleosomal barriers and
allow binding. The second model proposes that Pioneers factors
e.g.FOXA1 can directly associate with nucleosomal DNA, decompact the

5

chromatin, reposition nucleosomes and recruit other TFs. Alternative
models postulate that the incorporation of H2A.Z histone variant,
mediated by TIP60/p400 complex, facilitates transcription factor binding.
(Calo et al., 2013). No matter which model is correct, transcription factors
do not act alone to accomplish these tasks. They recruit numerous
coregulators, which provide these functions.

Transcription coregulators are proteins that interact with TFs to
either activate or repress the transcription of specific genes. Transcription
coregulators that activate target gene expression are coactivators while
coregulators that repress gene expression are referred to as
corepressors (Glass et al., 2000). Some Transcription coregulators
modify chromatin structure to either open up the chromatin and make the
DNA more accessible for transcription or compact the chromatin to make
it less accessible. Many transcription coregulators are known to play
important roles. One class of coregulators modify the structure of
chromatin by covalent modification of histones, while another class
modifies the chromatin conformation in an ATP dependent manner.

6

In 1991, scientists identified a gene that spans the breakpoint in the
11q23 translocation found in human leukemia, and they called it MLL
(Poel et al., 1991). Mixed lineage leukemia (MLL) is a family of proteins
that are H3K4-specific methyltransferases. They play a critical role in
gene regulation and epigenetics, and are usually rearranged in myeloid
and lymphoid leukemia. There are several family members of MLLs
including MLL1, MLL2, MLL3, MLL4, Set1A, Set1B, and Set7/9
(Figure 2) (Ansari et al., 2010). Some of the MLL family members interact
with nuclear receptors and play an important role in steroid hormone-
mediated gene regulation.
Figure 2: Structure of MLL1, MLL2, MLL3, MLL4, and MLL5
(Ansari et al., 2010)

7

Studies showed that MLL members exist as a multiprotein complex
and some of them share common protein subunits like Ash2, Wdr5,
Rbbp5, and Dpy30 (Figure 3). Each member of the MLL family contains a
catalytic SET domain that is responsible for the histone
methyltrasnferase activity of the member (Ansari et al., 2010).

Figure 3: MLLs excist as a multiprotein complex (Ansari et al, 2010).

8

MLL members excist as a multiprotein complex with common protein
subunits. They bind to the nuclear receptors either through their LXXLL
motif or through other proteins like Menin and ASC2/INII.

SET domain enzymes differ in their ability to add single, double, or
multiple methyl groups on the lysine side chain, and this phenomenon is
known as “product specifity”. The methyl group status is determined by
the “Phe/Tyr switch position” which is the presence of either tyrosine or
phenylalanine in the SET domain active site. Enzymes with a
phenylalanine at the switch position has larger substrate binding pocket
and can accommodate the addition of more than one methyl group; on
the other hand the SET domain enzyme with a tyrosine at the switch
position has smaller substrate binding pocket and so will only add one
methyl group on the lysine side chain. However, the MLL family
contradicts this hypothesis. Since MLL family members have a
conserved tyrosine residue at the switch position, they were expected to
be mono-methyltransferases but mono, di, and tri methyltransferase
activity has been observed in vitro and in vivo. In order to understand

9

this, scientists hypothesized that the proteins that bind to the MLL
members to make the multiprotein complex are what control the product
specifity of MLL family members.

In vitro experiments were done to test this hypothesis and to determine
the product specificity of the SET domain of MLL family members and
MLL interacting proteins like WDRb, RbBP5, Ash2L, and DPY-30.They
found that the SET domain is a mono-methyltransferase as the switch
hypothesis indicates, but when they added other subunits of the MLL
complexes, the enzymatic activity increased to the dimethylated histone
products were formed. Scientists also speculate that there are additional
unidentified proteins that may be required for tri methylation of histones
(Cosgrove et al., 2010). In conclusion scientists propose that the
mechanism that controls the formation of the MLL multiprotein complex
regulates H3K4 methylation status in the cell (figure 4).

10

Figure 4: Mechanism of multiple lysine methylations by MLL family
(Cosgrove et al., 2010).

11

In previous studies, Jeong et al. (Jeong et al., 2012) demonstrated that
MLL1 coregulator is required for the expression of estrogen-induced
genes like TFF1, PgR, and GREB1 in the MCF-7 breast cancer cell line
(Figure 5).

Figure 5: Effect of MLL1 depletion on PgR gene expression
(Kwang et al, 2012).

12

Moreover, previous studies reported that H3K4 methylation is
required for ER alpha dependent gene transcriptional activation (Lee et
al., 2007). Jeong et al. showed that after depletion of MLL1 in MCF-7
breast cancer cells there was a reduction in global mono-, di- and tri-
H3K4 methylation, (Jeong et al., 2011) (Figure 6), and after depletion of
MLL1 there was a reduction of ER alpha recruitment to TFF1 and PgR
genes (Jeong et al., 2012) (Figure 7). From these data, Jeong et al.
studied the mechanism of MLL1 requirement in more details and found
that the protein acetyltransferase TIP60 recruitment requires H3K4
methylation by MLL1; and this was required to remodel the chromatin
and led to transcription complex assembly (Jeong et al., 2011).

13

Figure 6: Effect of MLL1 and SETD1A depletion on global mono-, di-,
and tri-H3K4 methylation (Jeong et al., 2011).

Figure 7: Effect of MLL1 depletion on ER alpha and RNA polymerase II
recruitment to PgR gene (Jeong et al., 2012).

14

This led us to investigate the effect of four other members of the MLL
family of H3K4-specific histone methyltransferases on target gene
expression and global H3K4 methylation. Here we provide data that
should stimulate further study in more detail on the requirement of each
coregulator, its effect on the promoter architecture and its regulation of
target genes.

15

CHAPTER 2
RESULTS

I) The endogenous mRNA and protein levels of MLL1,
MLL2, SETD1A, SETD1B, and SETD7/9 were reduced by
siRNA against each coactivator

First, in order to study the role of each member of the MLL family in
the regulation of target gene expression by ER and methylation status of
H3K4, we reduced the level of endogenous MLL1, MLL2, SETD1A,
SETD1B, and SETD7/9 by two different siRNAs. Previous studies by
Jeong et al. characterized the results of depleting MLL1 (Jeong et al.,
2012), so one siRNA against MLL1 was used in this study as a control.
The results were compared with non-specific siRNA (siNS) in the MCF-7
breast cancer cell line.

To detect endogenous mRNA levels, the cells were treated with
10 nM of estradiol (E2) for 16 hours, and mRNA level was determined by
qRT-PCR and expressed relative to β-actin and GAPDH mRNA.

16

And in order to detect the levels of protein, whole cell lysates were
extracted and assessed by SDS-PAGE immunoblotting, and histone H3
or actin was used as a loading control.

MLL1 gene encodes a large protein composed of ~ 4000 amino
acids and when MLL1 gene is expressed, the full length protein is
cleaved by endogenous protease 1 enzyme into to MLL-N and MLL-C
fragments that associate in vivo. The molecular weight that is predicted is
~ 180 kDa and ~ 320 kDa. (Cosgrove et al., 2010)

The full length MLL2 is an uncleaved protein with a predicted molecular
weight of ~ 290 kDa. MLL2 protein also undergoes proteolytic cleavage
by taspase 1 enzyme, and its consensus cleavage site (D/GVDD) is at
a.a. 2063. This cleavage generates a large N-terminal fragment with a
predicted molecular weight of 215 kDa, and a smaller C-terminal
fragment which separates at ~75 kDa on denaturing gels (Natarajan et
al., 2010 ).

17

SETD1A, SETD1B and SET7/9 express as full length proteins and are
not cleaved, and the predicted molecular weights are
~ 250 kDa, 450 KDa and 43.4 kDa respectively.

From the real-time PCR results and immunoblot analysis, the
endogenous levels of each member were shown to be specifically
reduced by the siRNA against each coactivator, compared with the non-
specific siRNA. Moreover, the fact that these two siRNAs did not affect
the expression of endogenous actin, GAPDH or H3 suggests that the
inhibition of each coactivator was highly specific (Figures 8-12).
Thus, the siRNAs shown were judged to be suitable for the proposed
studies.

18

Anti-β-Actin

Anti-MLL1

siNS siMLL1

Figure 8: Effect of MLL1 depletion on protein and mRNA levels.

A) MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of 20 µM of siRNA against MLL1. Cells were
harvested followed by loading the samples on the 4% SDS-PAGE and
immunoblotting with anti-MLL1, anti-β-actin, or anti-H3 antibodies.

19

E2 - + - +
siNS siMLL1

B) MCF-7 cells were transfected using oligofectamine with 3 µL of 20 uM
of either siRNA against MLL1 or siNS. 72 hours after transfection, cells
were either treated with E2 (+) or Ethanol (-) and harvested after
additional 16 hours. Total RNA was prepared and used for reverse
transcription to synthesize first strand cDNA. 2 µL of the reverse
transcription reaction were utilized as template in the quantitative real-
time PCR to measure the level of β-actin and MLL1 mRNA. Results
shown are mean and standard deviation of CT values from three
technical replicates for one experiment, which is a representative of five
independent experiments
p-value ≤ 0.001 (***).
-second siRNA results are not shown.

***

Relative
mRNA
normalized
by
actin

Relative
mRNA
normalized
by
actin

0

0.2

0.4

0.6

0.8

1

1.2

20

Anti-H3

Anti-MLL2

siNS siMLL2-A siMLL2-B

Figure 9: Effect of MLL2 depletion on protein and mRNA levels.

A) MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of two different siRNA (20 µM) against MLL2.
Cells were harvested followed by loading the samples on the 8% SDS-
PAGE and immunoblotting with anti-MLL2 and anti-H3 antibody.

21

E2 - + - +
siNS siMLL2

B) MCF-7 cells were transfected using oligofectamine with 3 µL of 20 µM
of two siRNA against MLL2 or siRNA against siNS. 72 hours after
transfection, cells were treated with E2 or Ethanol and harvested after
additional 16 hours. Total RNA was prepared and used for reverse
transcription to synthesize first strand cDNA. 2 ul of the reverse
transcription reaction were utilized as template in the quantitative real-
time PCR to measure the level of β-actin and MLL2 mRNA. Results
shown are mean and standard deviation of CT values from three
technical replicates for one experiment, which is a representative of ten
independent experiments p-value ≤ 0.001(***).

***

Relative
mRNA
normalized
by
actin

0

0.5

1

1.5

2

2.5

3

22

Anti-H3

Anti-SET1A

siNS siSETD1A siSETD1A
(A) (B)

Figure10: Effect of SETD1A depletion on protein and mRNA levels.

A) MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of two different siRNAs (20 µM) against
SETD1A. Cell were harvested followed by loading the samples on the
4% SDS-PAGE and immunoblotting with anti-SETD1A and anti-H3
antibodies

23

E2 - + - + - +
siNS SETD1A SETD1A
(A) (B)

B) MCF-7 cells were transfected using oligofectamine with 3 µL of 20 µM
of two siRNA against SETD1A or siRNA against siNS. 72 hours after
transfection, cells were treated with E2 or Ethanol and harvested after an
additional 16 hours. Total RNA was prepared and used for reverse
transcription to synthesize first strand cDNA. 2 µL of the reverse
transcription reaction were utilized as template in the quantitative real-
time PCR to measure the level of β-actin and SETD1A mRNA. Results
shown are mean and standard deviation of CT values from three
technical replicates for one experiment, which is a representative of three
independent experiments, p-value ≤ 0.01 (**).

**

Relative
mRNA
normalized
by
actin

0

0.2

0.4

0.6

0.8

1

1.2

**

24

Anti-H3

Anti-SETD1B

siNS siSETD1B siSETD1B
(A) (B)

Figure 11: Effect of SETD1B depletion on protein and mRNA levels.

A) MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of two different siRNAs (20 µM) against
SETD1B. Cells were harvested followed by loading the samples on the
4% SDS-PAGE and immunoblotting with anti-SETD1B and anti-H3
antibodies.

25

E2 - + - + - +
siNS SETD1B SETD1B
(A) (B)

B) MCF-7 cells were transfected using oligofectamine with 3 µL of 20 µM
of two siRNA against SETD1B or siRNA against siNS. 72 hours after
transfection, cells were treated with E2 or Ethanol and harvested after an
additional 16 hours. Total RNA was prepared and used for reverse
transcription to synthesize first strand cDNA. 2 µL of the reverse
transcription reaction were utilized as template in the quantitative real-
time PCR to measure the level of β-actin and SETD1B mRNA. Results
shown are mean and standard deviation of CT values from three
technical replicates for one experiment, which is a representative of three
independent experiments p-value ≤ 0.001 (***), p-value ≤ 0.01 (**).

**

0

0.2

0.4

0.6

0.8

1

1.2

***

Relative
mRNA
normalized
by
actin

26

Anti-H3

Anti-SETD7/9

siNS siSETD7/9 siSETD7/9
(A) (B)

Figure12: Effect of SETD7/9 depletion on protein and mRNA levels.

A) MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of two different siRNAs (20 µM) against
SETD7/9. Cells were harvested followed by loading the samples on the
10% SDS-PAGE and immunoblotting with anti-SETD7/9 and anti-H3
antibodies.

27

E2 - + - + - +
siNS SETD7/9 SETD7/9
(A) (B)

B) MCF-7 cells were transfected using oligofectamine with 3 µL of 20 µM
of two siRNA against SETD7/9 or siRNA against siNS. 72 hours after
transfection, cells were treated with E2 or Ethanol and harvested after an
additional 16 hours. Total RNA was prepared and used for reverse
transcription to synthesize first strand cDNA. 2 µL of the reverse
transcription reaction were utilized as template in the quantitative real-
time PCR to measure the level of β-actin and SETD7/9 mRNA. Results
shown are mean and standard deviation of CT values from three
technical replicates for one experiment, which is representative of three
independent experiments p-value ≤ 0.001 (***).

***
***

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

Relative
mRNA
normalized
by
actin

28

II) Differential requirement of nuclear receptor coactivators
for E2-regulated expression of three different genes

The product of trefoil factor 1 gene, TFF1, (also called Ps2) functions
to stabilize the mucous overlying the gastrointestinal mucosa and provides a
physical barrier against foreign agents. GREB1 protein (Growth Regulation
By Estrogen In Breast Cancer 1) may play a role in estrogen-stimulated cell
proliferation in breast cancer. PgR (progesterone receptor) controls the
menstrual cycle and pregnancy and has an important role in breast cancer
development and progression. These genes are all induced by estrogen in
MCF-7 cancer cells, however, the requirements of coregulators for
estrogen-induced expression of these three genes have not been fully
explored.

In this study, we explore the gene specific requirement for each
coregulator and we selected five coregulators of the MLL family with
previously established roles in ER alpha-mediated transcription and

29

examined their requirement for E2-induced expression of three different
target genes of ER alpha: GREB1, PgR, and TFF1 .

MCF-7 cells were transfected with nonspecific siRNA (siNS) or siRNA
specific for each coregulator, and depletion of each coregulator was
monitored by immunobloting and qRT-PCR as shown in results section 1.
Similar levels of depletion were obtained in multiple experiments.

After transfection with siNS, expression of the three target genes of ER
alpha was determined by quantitative RT-PCR before and after 16 hours of
E2 treatment, and the level of mRNA observed after E2 treatment was used
as a baseline for comparison with the level of mRNA observed after E2
treatment of cells transfected with the coregulator-directed siRNA.

Among the three ER alpha target genes tested, we found that each gene
has a unique pattern of coregulator requirements. Each coregulator was
depleted by using two siRNAs (except MLL1) in MCF-7 cells and then
treated with E2.

30

First by looking at the mRNA level of TFF1 gene, we found that E2
treatment induced the expression of TFF1 gene but this induction was then
significantly reduced by the siMLL1, siMLL2, siSETD1B, and siSETD7/9
treatment (Figure 13). However, after treatment with two different
siSETD1A, we found that each siRNA has an opposite effect on the mRNA
level. The first siSETD1A significantly reduced mRNA level, while the
second siRNA significantly increased mRNA level, and so more
experiments should be done in order to accurately understand the effect
and make a conclusion.

Then we examined GREB1 gene expression in breast cancer cells after
depleting the coregulators and found that there was significant reduction in
mRNA level after depleting MLL2, SETD1A, SETD1B, and to a greater
extent MLL1. However, depleting SETD7/9 has a small but not significant
reduction of GREB1 mRNA level (Figure 14).

Finally, we examined PgR gene expression in the same cell line after
depleting the coregulators (Figure 15). There was a significant reduction of
PgR mRNA after siMLL1 and siMLL2 and SETD1A treatment, although one
siRNA against SETD1A caused a reduction that was not significant.

31

After siSETD1B treatment we found that the first siRNA has a small and not
significant increase of mRNA level while the second siRNA significantly
increased mRNA level. More experiments should be done to make an
accurate conclusion. Moreover, when the cells were treated with siSEDT7/9
we had a significant increase of mRNA level.

In conclusion, MLL1 and MLL2 seem to have similar effects on target gene
expression, while SETD1A, SETD1B, and SETD7/9 each has its own
pattern of regulating target genes either by reducing or inducing them.

32

- + - + - + - +
siNS siMLL1 siNS siMLL2

- + - + - + - + - + - +
siNS siSETD1A siSETD1A siNS siSETD1B siSETD1B
(A) (B) (A) (B)

Figure 13:
Effect of coregulators depletion by two different siRNAs on the E2-
regulated expression of TFF1 gene.

0

0.5

1

1.5

2

2.5

3

0

0.5

1

1.5

2

2.5

3

3.5

0

0.5

1

1.5

2

2.5

3

3.5

0

0.5

1

1.5

2

2.5

3

***

**

*

*

*

**

Relative
mRNA
normalized
by
actin
Relative
mRNA
normalized
by
actin

33

- + - + - +
NS SETD7/9 SETD7/9
(A) (B)

Continue figure 13:
Effect of coregulators depletion by two different siRNAs on the E2-
regulated expression of TFF1 gene.

siRNA transfection and mRNA measurement were carried out as
described in Figures 8-12. Data shown are mean and standard deviation
of three technical replicates from a single experiment, which is
representative of three independent experiments. Depletion efficiency for
this experiment is shown in Figures 8-12.
*, p-value ≤ 0.05. **, p-value ≤0.01. ***, p-value ≤ 0.001.

***

***

**

Relative
mRNA
normalized
by
actin

0

50

100

150

200

34

- + - + - + - +
siNS siMLL1 siNS siMLL2

- + - + - + - + - + - +
siNS siSETD1A siSETD1A siNS siSETD1B siSETD1B
(A) (B) (A) (B)

Figure 14:

Effect of coregulators depletion by two different siRNAs on the E2-
regulated expression of GREB1 gene.

0

1

2

3

4

5

0

2

4

6

8

10

12

14

0

2

4

6

8

10

12

14

*
*

*
*

0

0.5

1

1.5

2

2.5

3

3.5

4

***

**

*
**

**

***

Relative
mRNA
normalized
by
actin

Relative
mRNA
normalized
by
actin

35

- + - + - +
NS SETD7/9 SETD7/9
(A) (B)

Continue figure 14:

Effect of coregulators depletion by two different siRNAs on the E2-
regulated expression of GREB1 gene.

siRNA transfection and mRNA measurement were carried out as
described in Figures 8-12. Data shown are mean and standard deviation
of three technical replicates from a single experiment, which is
representative of three independent experiments. Depletion efficiency for
this experiment is shown in Figures 8-12.
*, p-value ≤ 0.05. **, p-value ≤0.01. ***, p-value ≤ 0.001.
NS, not significant.

NS

NS

Relative
mRNA
normalized
by
actin

0

0.5

1

1.5

2

36

- + - + - + - +
siNS siMLL1 siNS siMLL2

- + - + - + - + - + - +
siNS siSETD1A siSETD1A siNS siSETD1B siSETD1B
(A) (B) (A) (B)

Figure 15: Effect of coregulators depletion by two different siRNAs on
the E2-regulated expression of PgR gene.

0

0.5

1

1.5

2

2.5

3

3.5

4

0

0.5

1

1.5

2

2.5

0

1

2

3

4

5

6

7

8

0

2

4

6

8

10

12

***

***

***

**

NS

NS

Relative
mRNA
normalized
by
actin
Relative
mRNA
normalized
by
actin

37

- + - + - +
siNS siSETD1A siSETD1A
(A) (B)

Continue figure 15: Effect of coregulators depletion by two different
siRNAs on the E2-regulated expression of PgR gene.

siRNA transfection and mRNA measurement were carried out as
described in Figures 8-12. Data shown are mean and standard deviation
of three technical replicates from a single experiment, which is
representative of three independent experiments. Depletion efficiency for
this experiment is shown in Figures 8-12.
*, p-value ≤ 0.05. **, p-value ≤0.01. ***, p-value ≤ 0.001.
NS, not significant.

**

**

0

0.5

1

1.5

2

Relative
mRNA
normalized
by
actin

38

III) Each coregulator supports a different global
pattern of H3K4 methylation in MCF-7 cells

In previous studies, methylation of H3K4 has been linked
to transcriptional activation in different eukaryotic species.
Lysine residues of histones can be mono-, di-, or tri-
methylated. Recent genomic analysis of histone modifications
show that there is a correlation between different methylation
states of H3K4, their genomic loci, and gene expression level.
(Ruthenburg et al., 2007). MLL protein family has been
implicated in gene activation, and the members have H3K4
methyltransferase activity. However, the enzymes responsible
for maintaining global H3K4 methylation in cells have not been
determined.

In this study, we investigated the effect of depletion of
each coregulator of the MLL family on H3K4 methylation by
immunoblotting analysis using antibody against each

39

methylation mark. Total H3 levels were used as an internal
control for loading of the samples (Figure 16).

H3K4 mono-methylation was greatly reduced after depleting
MLL1 and MLL2 only. However, after depleting SETD1B no
effect was shown on global H3K4 mono-methylation in MCF-7
cells. Moreover, SETD1A and SETD7/9 seem to have
insignificant effect on global mono methylation after treating
the cells with siRNAs (Figure 17).

H3K4 di-methylation mark was reduced after MLL1, MLL2, and
SETD1A depletion, while SETD1B has the greatest effect in
which the mark was completely abolished after depleting it.
SETD7/9 has no effect at all on H3K4 di-methylation (Figure
18).

H3K4 tri-methylation was almost completely abolished after
MLL2, SETD1A, and SETD1B depletion and was reduced after
MLL1

40

depletion, while SETD7/9 had a slight effect on H3K4 tri-
methylation (Figure 19).

In conclusion, our data suggest that each coregulator modifies
the chromatin by adding different number of methyl groups on
H3K4.

41

Anti-β-Actin

siNS siMLL1

Anti-H3

siNS siMLL2A siMLL2-B siNS SETD1A SETD1
(A) (B)

siNS siSETD1B siSETD1B siNS siSETD7/9 siSETD7/9
(A) (B) (A) (B)

Figure 16:Effect of coregulator depletion on Histone 3.

MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of 20 µM of siRNA against MLL1, MLL2, SETD1A,
SETD1B, and SETD7/9. Cells were harvested followed by loading the
samples on the 12% SDS-PAGE and immunoblotting with anti-H3 or anti-β-
Actin antibody as a control. Results shown are representative of three
independent experiments.

42

siNS siMLL1

siNS siMLL2-A siMLL2-B siNS siSETD1A siSETD1A
(A) (B)

siNS siSETD1B siSETD1B siNS siSETD7/9 siSETD7/9
(A) (B) (A) (B)

Figure 17:Effect of coregulator depletion on global H3K4 mono-methylation

MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of 20 µM of siRNA against MLL1, MLL2, SETD1A,
SETD1B, and SETD7/9. Cells were harvested followed by loading the
samples on the 12% SDS-PAGE and immunoblotting with anti-H3K4 mono-
methylation antibody. Results shown are representative of three independent
experiments. H3 levels on this blot were shown to be equal across all lanes.

43

siNS siMLL1

siNS siMLL2 siMLL2 siNS siSETD1A siSETD1A
(A) (B) (A) (B)

siNS siSETD1B siSETD1B siNS siSETD7/9 siSETD7/9
(A) (B) (A) (B)

Figure 18: Effect of coregulator depletion on global H3K4 di-methylation

MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of 20 µM of siRNA against MLL1, MLL2,
SETD1A, SETD1B and SETD7/9. Cells were harvested followed by
loading the samples on the 12% SDS-PAGE and immunoblotting with
anti-H3K4 di-methylation antibody. Results shown are representative of
three independent experiments. H3 levels on this blot were shown to be
equal across all lanes.

44

siNS siMLL1

siNS siMLL2 siMLL2 siSETD1A siSETD2A siSETD1B siSETD1B
A B A B A B

siNS siSETD7/9 siSETD7/9
(A) (B)

Figure 19: Effect of coregulator depletion on global H3K4 tri-methylation

MCF-7 cells were grown in 6-well plates and then transfected using
oligofectamine with 3 µL of 20 µM of siRNA against MLL1, MLL2, SETD1A,
SETD1B, and SETD7/9. Cells were harvested followed by loading the
samples on the 12% SDS-PAGE and immunoblotting with anti-H3K4 tri-
methylation antibody. Results shown are representative of three independent
experiments. H3 levels on this blot were shown to be equal across all lanes.

45

CHAPTER 3

DISCUSSION

Mammalian cells contain many enzymes that modify histones and one class
of them is the H3K4 methyltransferase enzymes like MLL family. It has been
reported previously that there is an association between histone H3K4
methylation, and euchromatin of active genes (Lee et al., 2007). However,
the molecular mechanisms that control H3K4 methyltransferases and their
effect on target genes are still unclear. In addition, the importance of
multiple histone H3K4 mehtyltransferases is still not well understood.

In this study, we try to answer the question if MLLs are primarily histone
methylases or do they play an important role in regulation of genes by
comparing the effect of depleting each member of the MLL histone H3K4
methyltransferases in order to provide data to facilitate study of the
mechanism of each coregulator and its contribution to gene regulation in
more detail in the future.

46

The work led to several major conclusions:
First, among the three ER alpha target genes tested (TFF1, GREB1, and
PgR), we found that each gene has a unique pattern of coregulator
requirements. Full estrogen-induced expression of TFF1 required four
coregulators MLL1, MLL2, SETD1B, and setd7/9 while SETD1A is still
unresolved. GREB1 expression required only four of these coregulators,
MLL1, MLL2, SETD1A and SETD1B. While PgR expression required only
three coregulators, MLL1, MLL2 and SETD1A (Table 1).

Second, H3K4 mono-methylation mark was reduced after depletion of MLL1
and MLL2 only and was slightly affected by SETD1A and SETD7/9 and not
effected by SETD1B. H3K4 di- methylation mark was reduced after
depletion of MLL1, MLL2, and SETD1A, while it was completely abolished
after SETD1B depletion. However, it was not affected by SETD7/9
depletion. Moreover, H3K4 tri-methylation mark was reduced by MLL1
depletion and dramatically affected by MLL2, SETD1A, SETD1B and slightly
affected by SETD7/9 depletion (Table 2).

47

In conclusion, our results indicate that the reduction of endogenous
coregulator levels and the inhibition of some of the endogenous ER-
regulated gene expression (TFF1, GREB1, and PgR) prove that MLL
coregulators are required for the efficient hormonal induction of these genes
controlled by ER in MCF-7 breast cancer cells, and that each of the three
different E2-induced genes has a different pattern of coregulator
requirement and so has a different effect on the expression of target genes
after adding estrogen hormone. In addition, several of the H3K4
methyltransferases are involved in maintaining global cellular methylation,
each member of the MLL family of histone methyltransferses adds a
different number of methyl groups to H3K4 and so may affect chromatin
architecture differently.

Further studies are needed to establish the mechanism of each member of
the MLL family regarding what protein-protein interactions are involved in
their recruitment mechanism, what they recruit to the enhancers of target
genes, and how they modify the chromatin to make it more or less
accessible for transcription. Moreover, studying the effect of these
coregulators in different cell lines might be important in providing more detail
about their roles in the cell

48

Table 1: summary of the effect of coregulators on TFF1, GREB1, and PgR
genes

MLL1

MLL2

SETD1A

SETD1B

SETD7/9

TFF1

GREB1

PgR

== Reduced mRNA level of target gene

== Increased mRNA level of target gene

==No effect

== Conflicting results

49

Table 2: Summary of the effect of coregulators on H3K4 methylation mark

MLL1

MLL2

SETD1A

SETD1B

SETD7/9

H3K4 Mono
methylation

H3K4 Di
methylation

H3K4 Tri
methylation

***: Almost completely abolished

**: Reduced

*: Slightly affected

-: No effect

50

CHAPTER 4
METHODS AND MATERIALS

Cell Culture
MCF-7 cells were cultured and maintained in Dulbecco’s modified
Eagle’s medium (DMEM) from Invitrogen. Medium was supplemented
with penicillin and streptomycin and 10% fetal bovine serum.

RNA interference
Small interfering RNA specific for each coactivator was designed by
Sigma- Aldrich.The siRNA sequences used were as follows:

MLL1, 5’-GAUUCGAACACCCAGUUAUdTdT-3’ (sense)
5’-AUAACUGGGUGUUCGAAUCdTdT-3’ (antisense)
MLL2-A, 5’-CCAAGAUGGUGGCUUUGAAdTdT-3’ (sense)
5’-UUCAAAGCCACCAUCUUGG dTdT-3’ (antisense)
MLL2 B, 5’-GUCAACUGCUUCUUCCAUUdTdT-3’ (sense)
5’-AAUGGAAGAAGCAGUUGAC dTdT-3’ (antisense)

51

SETD1A-A, 5’-CUCAGAAGGUGUACCGCUAdTdT-3’ (sense)
5’-UAGCGGUACACCUUCUGAGdTdT-3’ (antisense)
SETD1A -B, 5’-CGGAAGAAGAAGCUCCGAUdTdT-3’ (sense)
5’-AUCGGAGCUUCUUCUUCCGdTdT-3’ (anti-sense)
SETD1B-A, 5’CACAUUUGCCCACACUCCAdTdT-3’ (sense)
5’-UGGAGUGUGGGCAAAUGUGdTdT-3’ (antisense)
SETD1B -B, 5’-CAGAAUAUCCGUCAGGUGA dTdT-3’(sense)
5’-UCACCUGACGGAUAUUCUGdTdT-3’ (antisense)
SET7/9-A, 5’-GAGUUUACACUUACGAAGAdTdT-3’ (sense)
5’-UCUUCGUAAGUGUAAACUCdTdT-3’ (antisense)
SETD7/9-B, 5’-CCCUUAUGUCCACUGAAGAdTdT-3’ (sense)
5’-CUUCAGUGGACAUAAGGGdTdT-3’ (antisense)
Negative control siNS, 5’-UUCUCCGAACGUGUCACGUdTdT-3’ (sense)
5’-ACGUGACACGUUCGGAGAAdTdT-3’ (antisense)

Transient transfection
MCF-7 cells were plated (1.5 x 10^5 cell/ well) in 6-well plates in
hormone free media containing 5% charcoal – dextran- stripped FBS and
grown until reaching 20-30 % confluence at the day of siRNA

52

transfection. The transfection mixture of siRNA-against each coactivator
or non-specific control siRNA - and equal amount of transfection reagent
(oligofectamin , Invitrogen , cat # : 12252-011) was added into each well
directly without removal of the medium. Three days later, some cells
were lysed and protein was isolated, and other cells were treated with 10
nM of estradiol for 16 hours and then total RNA was isolated.

Reverse transcriptase PCR and quantitative real-time PCR

After hormone treatment, total RNA were extracted using Trizol reagent
(Invitrogen), and cDNA was synthesized by reverse transcribing 0.8 µg of
total RNA using iScript cDNA synthesis kit (Bio-Rad). 2 µL of the reverse
transcription reaction were used as template in the quantitative real-time
PCR with 5 µL Sybr Green qPCR Master Mix (Roche) and 5 µM of
forward and reverse primers. qPCR condition is the following:
Pre-incubation: 1 cycle, 95 C for 5 mins.
Amplification: 45 cycles, 95 C for 10 seconds, 60 C for 10 seconds, and 72 C
for 10 seconds.

53

Melting curve: 1 cycle, 95 C for 5 seconds, and 65 C for 1 minute.
Cooling: 1 cycle, 40 C for 30 seconds.
The primers used were as follows:
β-Actin, 5’-ACCCCATCGAGCACGGCATCG-3’ (forward)
5’-GTCACCGGAGTCCATCACGATG-3’ (reverse)
GAPDH, 5’-TCTGGTAAAGTGGATATTGTTG-3’ (forward)
5’-GATGGTGATGGGATTTCC-3’ (reverse)
PS2 (TFF1), 5’-GAACAAGGTGATCTGCG-3’ (forward)
5’-TGGTATTAGGATAGAAGCACCA-3’ (reverse)
GREB1, 5’-CAAAGAATAACCTGTTGGCCCTGC-3’(forward)
5’-GACATGCCTGCGCTCTCATACTTA-3’ (reverse)
PgR, 5’-GTGCCTATCCTGCCTCTCAATC-3’ (forward)
5’-CCCGCCGTCGTAACTTTCG-3’ (reverse)
MLL1, 5’-GAGGACCCCGGATTAAACAT-3’ (forward)
5’-GGAGCAAGAGGTTCAGCATC-3’ (reverse)
MLL2, 5’-GTGCAGCAGAAGATGGTGAA-3’(forward)
5’-GCACAATGCTGTCAGGAGAA-3’(reverse)
SETD1A, 5’-CAGTGGCGGAACTACAAGCTC-3’ (forward)
5’-CATAGCGGTACACCTTCTGAGA-3’ (reverse)

54

SETD1B, 5’-AGGGGCATCATAAACTGTACCG-3’ (forward)
5’-GGGGATCTTCGACAATTTCCAC-3’ (reverse)
SETD7/9, 5’-ATGGATAGCGACGACGAGATG-3’ (forward)
5’-GCAGAACCCGTGCGGTAAT-3’ (reverse)
After the end of the amplification, a standard curve analysis was done to
confirm the accuracy of the results from each reaction. Each sample was run
in triplicate to obtain average Ct values and standard deviation. All samples Ct
values were normalized to that of β-Actin. Results shown are from a single
experiment that is representative of multiple experiments.

Protein Immunoblot analysis (western blot)

Cells were harvested in 100 µL/well of RIPA buffer (50 mM Tris-Cl),
pH 8.0, 120 mM NaCl, 0.1% NP-40, 1 % sodium dodecyl sulfate) and
protease inhibitor cocktail tablets (Roche), followed by 20 min
centrifugation at maximum speed. The supernatant was removed for
protein assay and immunoblot analysis. Protein samples were resolved
by SDS-polyacrylamide gel electrophoresis and then immunoblotting was
performed using primary antibody against the indicated protein and
horseradish peroxidase-conjugated antibody as the secondary antibody,
and ECL reagent was used for detection.

55

Antibody conditions are the following:

Antibody Company Catalogue
number
Dilution Gel % Loading
volume
β-Actin Sigma A5441 1:10 000 10% 10 µg

ER
alpha
Santa cruz Sc-543 1:1000 10% 10 µg
MLL1

Bethyl A300-086A 1:500 4% 25 µg
MLL2

Bethyl A300-113A 1:500 8 % 25 µg
SET1A

Bethyl A300-289A 1:2000 4% 25 µg
SET1B

Protein
technology
55005-1-AP 1:200 4 % 25 µg
SET7/9

Bethyl A301-747A 1:5000 10% 25 µg
H3

Santa
Cruz
Sc-10809 1:5000 12 % 10 µg
MONO
M
Active
motif
39297 1:10 000 12 % 10 µg
DI M

Abcam 1347-1 1: 10
000
12 % 10 µg
TRI M

Active
motif
39159 1:5000 12 % 10 µg

56

BIBLIOGRAPHY

Ansari KI & Mandal SS (2010) Mixed lineage leukemia: roles in gene
expression, hormone signaling and mRNA processing. FEBS J
277(8):1790-1804.

Calo E & Wysocka J (2013) Modification of enhancer chromatin: what,
how, and why? Mol Cell 49(5):825-837.

Chen J, Kinyamu HK, & Archer TK (2006) Changes in attitude, changes
in latitude: nuclear receptors remodeling chromatin to regulate
transcription. Mol Endocrinol 20(1):1-13.

Cosgrove MS & Patel A (2010) Mixed lineage leukemia: a structure-
function perspective of the MLL1 protein. FEBS J 277(8):1832-
1842.

Gronemeyer H, Gustafsson JA, & Laudet V (2004) Principles for
modulation of the nuclear receptor superfamily. Nat Rev Drug
Discov 3(11):950-964.

Hsieh JJ, Cheng EH, & Korsmeyer SJ (2003) Taspase1: a threonine
aspartase required for cleavage of MLL and proper HOX gene
expression. Cell 115(3):293-303.

Hsieh JJ, Ernst P, Erdjument-Bromage H, Tempst P, & Korsmeyer SJ
(2003) Proteolytic cleavage of MLL generates a complex of N- and
C-terminal fragments that confers protein stability and subnuclear
localization. Mol Cell Biol 23(1):186-194.

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Jeong KW, et al. (2011) Recognition of enhancer element-specific
histone methylation by TIP60 in transcriptional activation. Nat
Struct Mol Biol 18(12):1358-1365.

Lee JH, Tate CM, You JS, & Skalnik DG (2007) Identification and
characterization of the human Set1B histone H3-Lys4
methyltransferase complex. J Biol Chem 282(18):13419-13428.

Natarajan TG, et al. (2010) Epigenetic regulator MLL2 shows altered
expression in cancer cell lines and tumors from human breast and
colon. Cancer Cell Int 10:13.

Patel A, Dharmarajan V, Vought VE, & Cosgrove MS (2009) On the
mechanism of multiple lysine methylation by the human mixed
lineage leukemia protein-1 (MLL1) core complex. J Biol Chem
284(36):24242-24256.

Ruthenburg AJ, Allis CD, & Wysocka J (2007) Methylation of lysine 4 on
histone H3: intricacy of writing and reading a single epigenetic
mark. Mol Cell 25(1):15-30.

Wada T, Kang HS, Jetten AM, & Xie W (2008) The emerging role of
nuclear receptor RORalpha and its crosstalk with LXR in xeno- and
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233(10):1191-1201.

Won Jeong K, Chodankar R, Purcell DJ, Bittencourt D, & Stallcup MR
(2012) Gene-specific patterns of coregulator requirements by
estrogen receptor-α in breast cancer cells. Mol Endocrinol
26(6):955-966.

Asset Metadata

Creator Alammari, Farah (author)

Core Title Mixed lineage leukemia proteins (MLLs), their effect as coregulators on target gene expression and global histone methylation

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 11/21/2013

Defense Date 10/21/2013

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

Tag coregulators,epigenetics,histone modification,MLLs,oai:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Advisor Stallcup, Michael R. (committee chair), Iei, Wei (committee member), Tokes, Zoltan A. (committee member)

Creator Email alammari@usc.edu,f.alammari@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-348907

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Abstract (if available)

Abstract Mixed lineage leukemia proteins (MLLs) belong to the evolutionarily conserved trithorax family of human genes that play important roles in development and HOX gene regulation. MLL genes are usually rearranged in myeloid and lymphoid leukemias implicating them in these diseases. There are several MLL family proteins, including MLL1, MLL2, MLL3, MLL4, MLL5, Set1A, Set1B, and Set7/9. Each possesses histone H3 lysine 4 (H3K4)-specific methyltransferase activity, which is an epigenetic mark that presents in open and transcriptionally active chromatin. Some MLL proteins interact with nuclear receptors such as estrogen receptor (ER alpha) and play an important role in steroid hormone-mediated gene activation. In this study, we deplete five different members of the MLL family in MCF-7 breast cancer cells and compare the effect of each on the total cellular levels of mono-, di-, and trimethylation of H3K4, and on expression of the Ps2, GREB1, and PgR genes, which are ER alpha target genes. We found that even though they are all members of the same family, each one of them has its own unique pattern of regulation.