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Characterizing the physiological roles and regulatory mechanisms of Maf1

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Characterizing the physiological roles and regulatory mechanisms of Maf1

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Content Characterizing the physiological roles and
regulatory mechanisms of Maf1

by

Akshat Khanna

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)

May 2015

Akshat Khanna

ii

TABLE OF CONTENTS Page No.

Approval of dissertation.........................................................................................iv
Dedication……………………………………………………………….……………......v
Acknowledgements...…………………………………………………………………..vi
List of Tables and Figures……………………………………......……………..….…viii
Abstract...………………………………………………………………………………...x

CHAPTER I: Introduction……………………………………………………...1
Cellular homeostasis……………………………………………………………………2
TOR signaling pathway…………………………………………………………………4
Insulin/TOR signaling pathway………………………………………………………...6
RNA Pol III transcription & Stress……………………………….…………………….7
Canonical role of Maf1………………………………………………………………….8
Maf1 regulation of localization and function………………………………………...10
Non-canonical role of Maf1………………………………………………………...…12
References……………………………………………………………………………...14

CHAPTER II: Identification of C. elegans Maf1…………...……………...21
Abstract……………………………………………………………………………...….22
Introduction……..………………………………………………………………………23
Material and Methods………………………………………………………………….25
Results………………………………………………………………………………..…28
Discussion………………………………………………………………………………35
References……………………………………………………………….……………..36

CHAPTER III: New physiological roles of MAFR-1…..…………………..38
Abstract………………………………………………………………………………….39
Introduction……………………………………………………………………………..40
Material and Methods………………………………………………………………….42
Results…………………………………………………………………………………..47
Discussion………………………………………………………………………………74
References……………………………………………………………….……………..84

iii

CHAPTER IV: Molecular regulation on MAFR1.....................................93
Abstract………………………..………………………………………….……………94
Introduction……………………..………………………………………….…………..95
Material and Methods…………..………………………………………….………….99
Results……………………………..………………………………………….……....102
Discussion………………………...………………………………………….……….111
References………………………..……………………………………….…….…...115

iv

The dissertation of Akshat Khanna is approved.

Sean P. Curran (Committee
chair)

lte-Laird
Offringa

v

DEDICATION

To my parents, my brother, my extended family and all my loved ones
for believing in me and for their unconditional love and support.

vi

ACKNOWLEDGEMENTS
I would like to thank my mentor, Sean Curran, for giving me the
wonderful opportunity to be his student. As an excellent scientist and advisor
Sean has always been exceptionally patient with me and has kept me motivated
throughout and provided me with much needed encouragement. I appreciate
very much how he has always been available for any kind of help, no matter how
busy he might be. No matter how tough this whole journey has been, I have
always felt that Sean believes in me. I have learned a lot from him and I hope
one day I can be the kind of person he is. I am very happy and proud to be a
member of the Curran lab.
I am thankful to Deborah Johnson for setting the bar so high. I never
would have worked so hard, if it weren’t for you. My meetings with the Johnson
lab have always helped look at the bigger picture of my project. Thank you for all
your valuable inputs and all your support in helping me plan and prioritize my
project.
I am very lucky that I had Ite-Laird Offringa since the time I started at
USC. Not only has she always been really supportive, but helped take the right
steps towards my Ph.D. She has always been there to discuss my science and
give me her unique perspective that I have always appreciated. I thank her very
much for having my best interests in her mind.
A big thank you to John Tower and Ian Ehrenreich for all their critical
suggestions and encouragement through all stages of my Ph.D. They are the
kind of committee members every graduate student needs.

vii

I have to pay a big thank you to Lori Thomas, for being a second mother
to me all these years. You have always pushed me to do things right and
definitely made me a better person than before. Thank for always being there for
me. I thank Lori Thomas, Meagan He and Andres Ixtlahuac for all their technical
help. I would like to acknowledge Shanshan Pang, Dana Lynn, Jackie Lo, Hans
Dalton for all the invaluable discussions and all the fun times outside lab. Your
support has been crucial in helping me get through the last 5 years.
Thank you to Hitesh Arora for the countless hours he spent on the phone
talking to me to drive me to spend those long hours in the lab and thanks to Hank
Cheng for giving me company in those late hours and always lending me an ear
to hear me out. Thank you for also being so willing to help me troubleshoot
experiments that have gone wrong. Thank you Priyamvada Jayaprakash for your
unconditional help with my experiments and all the discussions on cloning. Thank
you Rohit, Richa, Sapna, Ayesha, Keerthi and Aditi for all the encouragement.
I’d like to also acknowledge help from the Molecular Biology and the Gerontology
staff who have helped me stay on track over the years.
Chapter II and III are versions of the journal article “Physiological roles for
mafr-1 in reproduction and lipid homeostasis” Cell Reports (2014)
Vol.9, p2180–
2191 by Akshat Khanna, Deborah L. Johnson and Sean Curran. We thank Emily
Griffin, Meng Li for technical assistance; members of the Curran laboratory for all
the useful inputs and discussions.

viii

LIST OF TABLES AND FIGURES Page No.

CHAPTER II: Identification of C. elegans Maf1…………...……………...21
Fig. 1: Identification of C. elegans Maf1 homolog using BLASTP ………………..29
Fig. 2: MAFR-1 is a conserved modulator of RNA Polymerase-III and -II……….31
Fig. 3: Deregulated mafr-1 levels alters the expression of RNA Polymerase III
genes……………………………………………………………………………………32

CHAPTER III: New physiological roles of MAFR-1…..………………….38
Fig. 1: Temporal and Spatial expression of MAFR-1. ……………………………..48
Table 1/S1: GO-term analysis of altered genes in mafr-1 O/E …………………....50
Fig. 2: Impact of mafr-1 expression on lipid transport and oocyte
development. …………………………………………………………………………..52
Fig. 3: Changes to vitellogenin gene expression is specific to mafr-1 levels but
not are result of global deregulation of tRNA biosynthesis.…………………….……53
Fig. 4: Cell non-autonomous impact of altered mafr-1 expression in the intestine
on reproductive output.………………………………………..………………………56
Fig. 5: Decreased expression of mafr-1 does not increase maximal progeny
production. ……………………………………………………………………………..57
Fig. 6: mafr-1 expression levels alter organismal lipid homeostasis. ……………59
Fig. 7: Changes in stored lipids resulting from deregulated mafr-1 levels are not a
product of changed expression of altered lipid utilization genes. ………………..61

ix

Fig. 8: Glucose and insulin signaling pathways regulate the expression
of mafr-1.………………………………………………………………………………..66
Fig. 9: Nutrient availability and insulin signaling regulate mafr-1.…….……….......67
Fig. 10: Model of the central role of MAFR-1 in organismal
physiology. …………………………………………………………………………….73

CHAPTER IV: Molecular regulation on MAFR-1.....................................93
Fig. 1: Maf1 protein contains regions of high similarity.……………….…………..96
Fig. 2: Gene expression profile of downregulated genes in
mafr-1 ΔC / mafr-1 O/E..……………….………….……………….………………..103
Fig. 3: MAFR-1 CTD regulates it’s physiological functions……………………….105
Fig. 4: MAFR-1 CTD is a regulator of subcellular localization………………..….106
Fig. 5: MAFR-1 CTD has high similarity to the human Maf1 Protein…………....108
Fig. 6: Serine
182
, Serine
185
regulate subcellular localization……………………....109
Fig. 7: Serine
182
, Serine
185
regulate MAFR-1 function……………………………110

x

ABSTRACT

Obesity has become markedly more prevalent over the past two decades among
developed countries however, the molecular events that connect obesity, lipid
deregulation and human diseases are still unclear. Mammalian Maf1 was initially
identified as a repressor of RNA Polymerase III-transcribed genes and studies
have focused on examining it’s role in repressing RNA pol III-dependent targets.
Our work surprisingly revealed that Maf1 is also able to directly repress RNA pol
II genes, although little is known regarding these specific gene targets.
Furthermore, we have identified a novel and conserved function for Maf1 in the
maintenance of intracellular lipid pools in C. elegans and human cell culture.
These results are the first to define a specific physiologic role for Maf1 in a
multicellular organism. Maf1 negatively regulates lipid accumulation, in part, by
repressing the expression of lipid biosynthesis genes. We hypothesize that Maf1
is a central node in the maintenance of organismal lipid homeostasis. These
studies define Maf1 as an important new player in lipid metabolism and will be
critical towards our future understanding of the roles that Maf1 plays in human
diseases such as diabetes, obesity, and cancer, which display prominent lipid
dysregulation phenotypes. Understanding the regulation of Maf1 will help us
understand how these phenotypes are being impacted by Maf1. The Maf1
protein from yeast to mammals has been shown to have three regions of high
similarity, but it’s still unclear how these three regions might regulate the signals
that Maf1 responds to or the way Maf1 might regulate reproduction and lipid

xi

metabolism. We have found that removing the C-box domain from both Maf1 and
MAFR-1, results in a constitutively nuclear protein. Moreover, this MAFR-1
mutant seems to act as a gain of function mutant as it represses the RNA Pol III
targets in C. elegans just as the mafr-1 O/E strain. This mutant also negatively
impacts reproduction and the lipid phenotypes. This domain is highly conserved
with the human Maf1 and we believe it contains important residues that have not
been implicated in regulating either Maf1 function or localization or both.

1

CHAPTER I: Introduction

2

Cellular Homeostasis

Despite having the same number of genes in each cell, multicellular organisms
are able to develop several hundred different types of cells, each one programed
to perform a specialized function. This level of complexity is achieved via
intricately coordinated gene expression changes in the cell [1], which results in
the expression of a specific subset of genes that also helps it to gauge and
respond to physiological and environmental cues. This signature gene
expression pattern is regulated at multiple levels--including transcriptional, post-
transcriptional, epigenetic, translational, and post-translational. What makes this
model even more complicated is that some of these processes can talk to each
other [2].

Cells are highly sensitive and respond promptly to environmental changes, such
as presence of stress, growth factors, nutrients, temperature, chemical agents,
cytokines, hormones and many more. These signals are communicated between
tissues and cell types, at times via certain secreted ligands. As gene regulation
can happen at multiple levels, the cells must chose a response that is most
efficient for them but also control this response spatially and temporally. A
translational response to control the expression of other mRNA’s allows the cells
to initiate a more rapid regulatory response on gene expression [3].

3

This involves activation of a wide signaling network that integrates this sensing
with the energy status of a cell and then relays it to various metabolites to
regulate anabolic and/or catabolic processes. This is critical for a cell to survive
and respond to those changes and to maintain homeostasis, the failure of which
is a cause for a wide array of metabolic, inflammatory, autoimmune diseases [4].
Cellular homeostasis is highly dependent on the energy status of a cell, which is
a balance of the cell’s energy production, storage, and utilization.

Much research has shown a link between the nutritional status of the cell
and it’s energy status [5],[6] and so the focus has been to identify pathways that
mediate nutrient signaling and characterize downstream effectors of such
pathways. In the presence of nutrients, these pathways promote energy storage
in the form of glycogen and triglycerides, while allowing cells to undergo anabolic
processes. Whereas in the absence of nutrients, major anabolic processes are
inhibited, and the stored energy is mobilized. For example, protein synthesis is
one of the most energy intensive process, requiring up to ~30% of total energy
available [5][6]. At the same time the cell maintains a balance the rate of protein
synthesis and protein degradation, and it’s mis-regulation can cause various
diseases including cancer [7].

4

Nutrient and stress sensing
TOR Signaling pathway
The mTOR (mammalian target of rapamycin) pathway is a well studied nutrient
sensing pathway and is activated in the presence of ample nutrients particularly
amino acids and growth factors. Upon unfavorable conditions, TOR is inactive,
leading to a reduction in protein synthesis and upregulation of protein
degradation.

TOR proteins are serine/threonine kinases of the PIKK (phosphoinositide-3-
kinase-related protein kinase) family, with orthologs found in all eukaryotes.
mTOR exists as two distinct multi-protein complexes that exist in all cells from
yeast to mammals [8]- raptor-TOR (mTORC1) and rictor-TOR (mTORC2) [9].
mTORC1 is rapamycin sensitive and has been shown to link nutrient sensing to
protein synthesis [10]. mTORC2 is rapamycin insensitive, is regulated by growth
factors, and is implicated in cytoskeletal organization. While the mTORC1
phosphorylation targets are widely characterized, few studies have identified very
few mTORC2 substrates.

The TOR pathway connects nutrient sensing to cellular pathways that control
growth, proliferation, differentiation, transcription, autophagy, and mRNA
translation [11]. One key regulatory pathway is by regulation of mRNA
translation, and in the context of the TOR pathway it is done via phosphorylation

5

of it’s two major downstream targets: the eIF4E-binding proteins (4E-BPs) and
the ribosomal protein S6 kinases (S6K1 and S6K2).

The 4E-BPs are a family of inhibitory proteins that negatively control the
assembly of the eIF4F complex by competing with eIF4G for binding to eIF4E
[12]. The binding of the 4E-BPs to eIF4E is regulated by mTORC1-mediated
phosphorylation. Upon stimulation by growth factors, nutrients, or hormones,
mTORC1 phosphorylates 4E-BP1, leading to it’s release from eIF4E, and a
subsequent increase in cap-dependent translation. mTORC1 directly
phosphorylates 4E-BP1 on Thr37 and Thr46. Subsequent phosphorylation of
residues Thr70 and Ser65 leads to the ultimate release of 4E-BP1 from eIF4E
[13].

The ribosomal S6 kinases are direct targets of mTORC1 and have
elaborate roles in the regulation of mRNA translation. Two S6 kinase proteins,
S6K1 and S6K2 (in mammalian cells), are phosphorylated and activated by
mTORC1 [14]. mTORC1 phosphorylates S6K1 at Thr389 but complete
activation of S6K is achieved after subsequent phosphorylation of Thr229 by
PDK1 [15]. S6K1 promotes cell growth by increasing mRNA translation via
phosphorylation of it’s downstream targets rpS6, eIF4B, and eEF2 kinase [15].

6

Rapamycin treatment or TOR depletion also inhibit’s cell cycle progression
by arresting cells at the G1/S boundary [16]. Studies in yeast and mammals have
demonstrated that TOR signaling regulates ribosome synthesis in response to
nutrient availability [17]. Ribosome synthesis requires the coordinated activities
of all three nuclear RNA Polymerases, that is, Pol I for the synthesis of rRNA, Pol
II for transcription of ribosomal protein genes and Pol III for the synthesis of 5S
RNA [18]. Whether or not the cell coordinates transcription by all three nuclear
RNA Polymerases and the post-transcriptional machinery, is unknown but an
area of immense interest.

Insulin/TOR Signaling pathway
Insulin is a peptide hormone that is made in the pancreas and secreted in to the
blood stream in response to glucose to promote nutrient uptake and growth.
Some other effects of insulin include glucose utilization, protein synthesis [19],
and triglyceride metabolism [20]. When dietary nutrients are abundant, levels of
circulating insulins are high and the insulin/TOR pathway promotes cell and
tissue growth and upon starvation the insulin secretion is reduced [21].

Insulin/IGF pathway activates a cascade of phosphorylation events that
lead to phosphorylation of PI3K and Akt [22], that in turn phosphorylates and
activates the mTORC1 pathway and at the same time represses a second
branch containing members of the forkhead-like transcription factor family [23].
Signaling through PI3K and Akt leads to the inactivation of GSK3, which in turn

7

inhibit’s eIF2B. eIF2B regulates levels of eIF2, a translation initiation factor. This
results in increased cellular protein synthesis [24]. Other studies have shown that
in drosophila, FOXO positively regulates the expression of 4E-BP [25] via Lk6
[26]. Whether FOXO might have other roles in the control of protein biosynthesis
has not been investigated. Defects in insulin/TOR signaling may be causative for
many cancers [26].

RNA Pol III transcription and stress
The major biosynthetic function of RNA Polymerase III (pol III) is the production
of small, untranslated structural RNAs for protein synthesis. It’s products 5S
rRNA and tRNA contribute to~ 15% of total RNA by weight. Other pol III
transcripts like RNAseP, have roles in pre-rRNA or pre-tRNA processing
complexes. 7SL RNA is required for co-translational insertion of nascent
polypeptides across the membrane of the endoplasmic reticulum [27].

The Pol III transcriptional machinery is well conserved from yeast to
humans [28]. The transcripts synthesized by pol III are around 100 nucleotides,
unlike the transcripts from other nuclear Polymerases [29]. More studies have
focused on how the TOR pathway regulates Pol I and Pol II mediated
transcription, but relatively less work has characterized how the TOR pathway
regulates Pol III transcription.

8

Introduction to Maf1
Under conditions of stress, namely DNA damage, nutrient limitation or defects in
secretory pathways, the cellular response is to activate pathways that tackle the
stress, and inhibiting growth during this unfavorable time, by repressing rRNA’s
and tRNA’s by RNA Polymerases I and III. Different types of stress signals relay
onto and activate different pathways, but ultimately seem to converge to inhibit
growth. Maf1 has been reported to integrate stress signaling pathways to growth
and has been characterized in yeast [30] and subsequently in mammalian cell
culture [31], drosphila [32] and elegans [33].

Canonical role of Maf1
In yeast, tRNA levels were elevated 2 fold in Maf1 mutant cells [34]. The invitro
rate of Pol III RNA was significantly increased in Maf1 mutant cell extracts.
Upadhay et al. [30] found the signaling pathways activated by nutrient limitation,
DNA damage, secretory defects, and yeast growth cycle, required Maf1 to
repress RNA Pol III repression. Upadhay et al. [30] reported Maf1 to be either at
or below the point of convergence of these stress pathways. Even outside the
context of stress, they identified Maf1 as the factor that is necessary to
coordinate RNA Pol III transcription with cell growth i.e. high Pol III activity during
active cell division and low activity thereafter.

9

A small portion of Maf1 was found associated with Pol III under
favourable conditions, but how this interaction changes under stress was not
looked at [34]. Upadhay et al. [30] identified TFIIIB as a target of Maf1-dependent
repression. They found casein kinase 2 (CK2) activity to be correlated with
elevated levels of Pol III and higher levels of TFIIIB-DNA assembly. The first
evidence that human Maf1 can also bind to Pol III came from Reina et al. [35]
Using whole cell extracts from HeLa cells, the authors showed that Maf1
interacts strongly with RPAC2, the alpha like subunit of Pol III and weakly with
RPC1 (the largest subunit of Pol III) and Brf1. The authors mention no reduction
in Pol III transcription in HEK293 cells after adding MMS, but interestingly they
used the nuclear extracts of the same cells to look for Maf1 dephosphorylation
upon treatment with MMS. They propose a second step of Maf1 activation after it
is localized to the nucleus, required to inhibit Pol III.

They also go on to show that it is the dephosphorylated form of Maf1 that
interacts with RPC1, which was an unusual given the fact that the authors report
this interaction to be weak, when done with whole cell extracts from HeLa cells.
Suprisingly, there was no mention of the interaction of dephosphorylated Maf1 or
after treating the cells with stress, with RPAC2 or Brf1.

10

Maf1 regulation of localization and function
Roberts et al. [36] and Oficjalska-Pham et al. [37] showed biochemically that
yeast Maf1 was phosphorylated under favorable conditions, whereas the
unfavorable conditions used by Upadhay et al. [30] rapidly dephosphorylated
Maf1. At the time, they reported Maf1 to be a phosphoprotein that integrated
stress mediated signals through it’s phosphorylation status. Moir et al. [38]
reported cAMP-dependent protein kinase A (PKA) sites on Maf1 that are
phosphorylated under normal conditions, keeping it in the cytoplasm; mutants
with high PKA activity blocked Maf1-dependent Pol III repression. The PKA sites
that were identified were linked to the NLS (near the N-terminal), and PKA
activity negatively regulated it’s action. No phosphorylation sites were found near
the NLS (near the C-terminal) and so the authors couldn’t really explore the
regulation on this NLS. Interestingly, when a PKA site on Maf1 was mutated to
an alanine (S65A), the mutant was seen to be nuclear, but it’s levels of RNA Pol
III transcripts were comparable to wild type, indicating the possibility of an
activation step on Maf1, after it is dephosphorylated.

This observation was intriguing because it highlighted the complex
regulation that is potentially relayed onto Maf1, under conditions unfavorable for
growth. What was unknown here was if there are any other PTM on Maf1, what
other pathways are involved in regulating Maf1, whether modifications happen in
the cytoplasm or the nucleus and what other proteins Maf1 might bind to.

11

Going by stress signals that dephosphorylate Maf1, Roberts et al. [36]
addressed some of these aspects. They began to explore components of
previously identified stress response pathways [34] that are required for Maf1-
dependent Pol III repression. Protein Kinase C1 (Pkc1) was found to be
necessary, as cells lacking Pkc1 failed to cause Maf1 dephosphorylation in
response to nutrient deprivation. Unexpectedly, Pkc1 was not required for
rapamycin mediated Pol III repression indicating that nutrient deprivation and
rapamycin converge on Maf1 through separate pathways [39]. Although Upadhay
et al. [30] had identified TFIIIB as a target of Maf1-dependent repression of Pol
III, there was no link between TFIIIB and the phosphorylation status of Maf1.

To explore this, Desai et al. [40] used Chlorpromazine (CPZ), that induces
membrane stretching, to show that it rapidly dephosphorylates Maf1, but showed
no increase in the affinity of dephosphorylated Maf1 to bind to TFIIIB, specifically
to Brf1 and Pol III (Rpc82). According to their model, Maf1 prevents the
assembly of TFIIIB at Pol III promoters and also interferes with the recruitment of
Pol III to it’s gene promoters.

Around the same time, Roberts et al. [36] contradicted these results. They
reported that under stress induced by CPZ, nutrient limitation and methyl
methanesulfonate (MMS), Maf1 gets dephosphorylated, greatly increasing it’s
affinity for Pol III. The authors do not discuss the reasons for the discrepancy
between the two results but looking at the methods used, there are some

12

differences that could be the reason for this difference. Desai et al. [40] did not
use cross-linking of cells before the immunoprecipitation. Moreso, Desai et al.
only used CPZ induced stress for Maf1 interactions with Pol III, despite the fact
that different stress signals use different pathways to relay their signals to Maf1.
Therefore, the Model that Roberts et al. [36] proposed was that components of
TFIIIB remain fully associated with Pol III promoters during acute repression.
Maf1-Pol III interactions largely increase, compensating for Pol III-DNA
interactions, which is the reason for the observed increase in Maf1 occupancy at
Pol III promoters during repression. They say Maf1 only prevents the assembly of
new TFIIIB complexes under prolonged repression, but is not a feature of acute
repression, where TFIIIB remains bound to Pol III promoters. The
characterization of several kinases and the other observations strongly
suggested a regulation of Maf1 activity by a phosphorylation-dephosphorylation
mechanism. Oficjalska-Pham et al. [37] reported that the protein phosphatase 2A
(PP2A) opposed the activity of PKA on Maf1 and the hyper-repression of Pol III
genes in the PP2A-defective cells under rapamycin.

Non-canonical Maf1 function
Johnson et al. [41] showed that human MAF1 negatively regulated transcription
by all 3 nuclear RNA Polymerases. Changes in MAF1 expression affected not
just pol III-dependent but pol I-dependent transcription in human glioblastoma
cell lines. These effects were mediated partially through the ability of MAF1 to
repress transcription of TATA-binding protein. MAF1 occupancy of Pol III genes

13

was inversely correlated with that of the initiation factor TFIIIB and Pol III.
Reducing MAF1 expression induced changes in cell morphology and
accumulation of actin stress fibers, whereas MAF1 overexpression suppressed
anchorage-independent growth. Most studies on Maf1 have been in single cell
model systems that have their own limitations. For example, any cell-non
autonomous effects that Maf1 might have, or when and how do different tissues
express Maf1 and many more. It still needs to be explored whether altering levels
of Maf1, might alter the physiology of an organism in any other way, given the
fact that Maf1 levels have shown to impact select Pol II genes.

14

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Cancer Cell. (2005); pp. 179–183.

[23] E.L. Greer, A. Brunet, “FOXO transcription factors at the interface between
longevity and tumor suppression.” Oncogene. (2005); 24,pp.7410–7425.

[24] Proud et al., “Regulation of protein synthesis by insulin” Biochem.” Soc.
Trans. (2006); 34,pp. 213–216.

18

[25] M.A. Junger, F. Rintelen, H. Stocker, J.D. Wasserman, M. Vegh, T.
Radimerski, M.E. Greenberg, E. Hafen., “The Drosophila forkhead transcription
factor FOXO mediates the reduction in cell number associated with reduced
insulin signaling.” J. Biol. (2003); 2,p. 20.

[26] R.J. Shaw, L.C. Cantley., “Ras PI(3)K and mTOR signalling controls tumour
cell growth.” Nature. (2006); 441,pp. 424–430.

[27] G. Dieci, G. Fiorino, M. Castelnuovo, M. Teichmann, A. Pagano, ”The
expanding RNA Polymerase III transcriptome.” Trends Genet. (2007); 23,pp.
614-622.

[28] D.A. Guertin, K.V. Guntur, G.W. Bell, C.C. Thoreen, D.M. Sabatini.,
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[29] G. Dieci, S. Giuliodori, M. Catellani, R. Percudani, S. Ottonello., “Intragenic
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19

[30] Upadhya R, Lee J, Willis IM., “Maf1 is an essential mediator of diverse
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1489–1494.

[31] Reina JH, Azzouz TN, Hernandez N., ”Maf1, a new player in the regulation
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[32] Rideout EJ, Marshall L, Grewal SS., ”Drosophila RNA polymerase III
repressor Maf1 controls body size and developmental timing by modulating
tRNAiMet synthesis and systemic insulin signaling.” Proc Natl Acad Sci. (2012);
109: 1139–1144.

[33] Khanna A, Curran SP, Johnson D., “Physiological roles for mafr-1 in
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[34] Pluta K, Lefebvre O, Martin NC, Smagowicz WJ, Stanford DR., “Maf1p, a
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[35] Michels AA, Robitaille AM, Buczynski-Ruchonnet D, Hodroj W, Reina JH, et
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20

[36] Roberts, D. N., Wilson, B., Huff, J. T., Stewart, A. J., Cairns, B. R.,
“Dephosphorylation and genome-wide association of Maf1 with Pol III-transcribed
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[37] Oficjalska-Pham, D., Harismendy, O., Smagowicz, W. J., Gonzalez de
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[38] Moir RD, Willis IM., “Regulation of pol III transcription by nutrient and stress
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[39] Willis IM, Desai N, Upadhya R., “Signaling repression of transcription by
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[40] Desai N, Lee J, Upadhya R, Chu Y, Moir RD, Willis IM., “Two steps in Maf1-
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[41] Johnson, S. S., C. Zhang, J. Fromm, I. M. Willis, and D. L. Johnson.,
“Mammalian Maf1 is a negative regulator of transcription by all three nuclear
RNA polymerases.” Mol. Cell. (2007); 26: 367-379.

21

CHAPTER II: Identification of C. elegans Maf1

22

Abstract

Maf1 is an evolutionary conserved repressor of RNA Polymerase-III. The current
knowledge on Maf1 comes from work mostly done in yeast and mammalian cell
culture. More recently drosophila Maf1 had been characterized and shown to be
a conserved repressor of RNA Polymerase III. The majority of the work on Maf1
has given new insights into it’s canonical role as a Pol III repressor, in part
because of the limitations of a single cell model system. To advance our
knowledge on Maf1, we used C. elegans, which as a model system is ideal for
studying genetic interactions and whole organism phenotypes. To begin a more
detailed study on Maf1, we identify the C. elegans homolog of Maf1, C43H8.2.
This locus is now annotated as mafr-1 and we show that mafr-1 is functionally
orthologous to human Maf1, can repress the expression levels of both RNA
Polymerase-III and Polymerase-II transcripts.

23

Introduction

C. elegans has been extensively used as a genetically tractable organism for the
discovery of some highly conserved signaling pathways and fundamental
processes underlying metazoan reproduction, development, metabolism,
neurobiology and behavior. The worm genome is fully sequenced and is thirty
times smaller than the human genome. Moreover, 40% of this genome is
predicted to be homologous to that of humans. The short life cycle of 3 days
makes it ideal for a wide array of studies.

C. elegans has more recently been exploited to study the core metabolic
pathways that are highly conserved. Any effects of the fat regulatory pathway on
other phenotypes like feeding, excretion, reproduction or lifespan can be looked
at in parallel. The transparent body also makes it easy to track changes in tissue
morphology and image after staining the intact animal using dyes, as well as
enabling visualization of transgenic genes. C. elegans is amenable to an
extensive array of forward and reverse genetic screens using genome wide RNAi
and mutagenesis screens, making it a useful model to study complex pathways.

Initially characterized in S. cerevisiae, Maf1 is an evolutionarily conserved
transcriptional co-repressor of RNA Polymerase (pol) III-dependent genes, such
as tRNA and 5S rRNA, which impact the biosynthetic capacity of the cell
(Upadhya et al., 2002; Vannini et al., 2010). Previous studies on Maf1 regulation
and function have primarily utilized in vitro and cell culture models. Recent

24

characterization of Drosophila Maf1 has revealed common regulatory
mechanisms in invertebrates. However, mammalian Maf1 has been shown to
regulate certain RNA pol II-dependent promoters, such as a subset of Elk-1-
regulated promoters, while fly and yeast Maf1 have only been shown to
represses RNA pol III genes (Rideout et al.)(Johnson et al., 2007). We have
identified MAFR-1, the C. elegans homolog of Maf1. Here we show that MAFR-1
can repress RNA Pol III targets and select Pol II targets both in the worm and
mammalian cell culture, indicating MAFR-1 to be functionally orthologous to
human Maf1.

25

Material and Methods

Standard Growth Conditions
C. elegans were raised on standard 6 cm nematode growth media plates
supplemented with streptomycin and seeded with Escherichia coli OP50. For
RNAi experiments NGM plates containing 5 mM IPTG and 100ug
ml
−1
carbencillin were seeded with overnight cultures of double-stranded RNAi-
expressing HT115 bacteria. Plates were allowed to induce overnight followed by
transfer of age-synchronous populations of C. elegans. All behavioral and
metabolism analyses of mutant animals are fed E. coli OP50, while all RNAi
based assays are done in animals fed HT115. Animals were fed either food
source for at least two generations before analysis to avoid diet dependent
effects.

Strains use in this study
N2 Bristol (wild type), SPC328[mafr-1p::mafr-1-GFP::mafr-13’UTR(laxIs004)].

Cell lines and culture conditions
293T cells were cultured in DMEM supplemented with 10% fetal bovine serum.
At 50-70% confluence, cells were transfected with human/C. elegans Maf1 genes
by using Lipofectamine 2000 (Life Technologies). After twenty-four hours, cells
were washed and collected in Trizol reagent (Invitrogen) for RNA extraction.

26

Gene Expression
Developmentally synchronous worms of indicated genotype and developmental
stages were collected, washed in M9 buffer and then homogenized in Trizol
reagent (Invitrogen). RNA was extracted according to manufacturer’s protocol.
DNA contamination was digested with DNase I (New England Biolabs) and
subsequently RNA was reverse-transcribed to cDNA by using the SuperScript®
III First-Strand Synthesis System (Life Technologies). Prepared RNA was further
purified using the RNAse easy kit. Samples were hybridized to
Affymetrix C. elegans Gene 1.0 ST Arrays. Data and statistics were analyzed
with Partek Genomics Suite Software version 6.6.

Quantitative PCR was performed by using SYBR Green (BioRad). The
expression of snb-1 was used to normalize C. elegans RNA samples and
GAPDH from human cell culture samples. The efficiencies of all primers used
were within 5% of each other. These genes showed minimal variation after
changes in mafr-1/Maf1 expression. All samples were run in triplicate for
quantifying the steady state mRNA levels. Human Pol III primer sequences were
taken from Johnson et al (Johnson et al.).

Primers for C. elegans genes were designed using the Primer3plus
algorithm(Untergasser et al., 2012). C.elegans pol III primers were designed
using the Genomic tRNA database(Chan and Lowe, 2009). A list of all qPCR
primers can be accessed in Supplementary Table S2.

27

Statistics
Data presented reflect biological replicates as indicated in each sample’s n.
Sample sizes were determined to reliably reveal the statistic significance given
the magnitude of the changes expected in each experiment. No randomization
was used. Data were presumed to be normally distributed. Data were presented
as mean ± SEM. Data were analyzed by using unpaired student t test. P < 0.05
was considered as significant.

28

Results

C. elegans MAFR-1 is a conserved modulator of RNA Pol-III and Pol-II transcript
levels.

We used BLAST to look for the Maf1 homolog in C. elegans. The hit with
the most similarity was C43H8.2, annotated on wormbase as mafr-1. We began
our studies with this gene (Fig. 1)
Given the conserved role of Maf1 as a negative regulator of RNA
Polymerase III in yeast, flies, and mammals, we investigated whether C. elegans
MAFR-1 functions in an orthologous manner. We reduced mafr-1 expression by
RNAi and measured the transcript levels of established RNA Pol-III transcripts,
such as tRNAs. As predicted, when mafr-1 expression was reduced by
approximately 50% (Fig. 3), the expression of most tRNAs was significantly
increased as compared to the internal normalization control, snb-1 whose
expression was stable (Fig. 2A and Fig. 3A). We further examined animals
harboring an additional chromosomally integrated copy of mafr-1, which results in
~80% increase in mafr-1 overexpression (mafr-1 O/E) (Fig. 3B) and observed a
striking reduction in all tRNAs tested (Fig. 2B and Fig. 3B). Furthermore, the
reduction of tRNA levels observed in mafr-1 O/E animals were restored when
animals were fed dsRNA targeting mafr-1, which lead us to conclude that the
effects on RNA Pol-III transcripts were specific to mafr-1 levels (Fig. 3C).

29

Fig. 1 Identification of C. elegans Maf1 homolog using BLASTP. C43H8.2 in
C. elegans has highest similarity to human Maf1 protein; 33% identical residues.
* represents identical residues, : represents residues of similar chemical nature.

30

Inhibition of RNA Pol-III through dMaf1 in Drosophila has been shown to control
body size and developmental timing by specifically regulating tRNAiMet
synthesis (Rideout et al.). In C. elegans, mafr-1 expression is inversely
correlated with the synthesis of multiple tRNAs in addition to the initiator
methionine tRNA (Fig. 2A). Similar to flies, C. elegans body size is also sensitive
to mafr-1 expression; mafr-1 RNAi increases animal body area by ~4% while
mafr-1 O/E leads to a ~7% decrease in body area (Fig. 3D). Intriguingly and
unlike flies, modulation of mafr-1 levels does not alter developmental timing in
the worm (Fig. 3E). The time required to progress through embryonic and larval
development, adult sexual maturation, and the laying of the first egg is
statistically indistinguishable between wild type animals and animals either
overexpressing mafr-1 or with reduced mafr-1 expression by RNAi (Fig. 3E).

To identify functional conservation of C. elegans MAFR-1, we tested the ability of
MAFR-1 to regulate the expression of mammalian RNA-Pol III targets.
Overexpression of either MAFR-1 or human Maf1 in human 293T cells was
sufficient to significantly reduce the expression of multiple human RNA Pol-III
targets (Fig. 2C). This finding indicates that C. elegans MAFR-1 function is
conserved across metazoans. Human Maf1 is recruited to the promoters of
select RNA Understanding the regulation on Maf1 Understanding the regulation
on Maf1 II genes such as TBP1, to regulate expression (Johnson et al., 2007). In
light of the conserved function of worm MAFR-1 in the regulation of human RNA
pol III targets in 293T cells, we examined the ability of MAFR-1 to regulate tbp-1.

31

Fig. 2. MAFR-1 is a conserved modulator of RNA Polymerase-III and -II.
Expression of RNA Polymerase III targets in mafr-1 RNAi treated animals (A) or
animals overexpressing (O/E) mafr-1 (B). Expression of human RNA Polymerase
III targets in 293T cells transfected with mafr-1 or Maf1 (C). Expression of the
RNA Polymerase II target tbp-1/TBP (D).
Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001 versus
respective controls.

32

Fig. 3. Deregulated mafr-1 levels alters the expression of RNA Polymerase
III genes. Expression of mafr-1 and non-protein coding RNAs in mafr-1 RNAi
treated animals (A) and mafr-1 O/E animals (B). (C) RNAi of mafr-1 reverses the
transcriptional repression of tRNAs in mafr-1 O/E animals. Measurements of
body size (D) and developmental timing (E) in animals with altered mafr-1
expression.
Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001 versus
respective controls.

33

Fig.3 (continued). Deregulated mafr-1 levels alters the expression of RNA
Polymerase III genes. (F) Expression of TBP-like factor, tlf-1.Expression of
tRNAs in in brf-1 RNAi or tbp-1 RNAi treated animals in wild type (G) or mafr-1
O/E (H) animals. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01,
***P<0.001 versus respective controls.

34

Similar to mammalian Maf1, MAFR-1 is also capable of negatively regulating the
expression of the RNA pol II target tbp-1 in worms as well as human TBP1 in
293T cells (Fig. 2D), further supporting conservation of MAFR-1 activity. One
model for Maf1 function is as a transcriptional repressor by interacting with
components of the Transcription Factor for Polymerase III B (TFIIIB) complex,
which contains RNA Polymerase III, TBP1 and Brf1(Boguta; Boguta and
Graczyk; Marshall et al.). Consistent with previous reports in other organisms,
we find that in C. elegans, decreased expression of tbp-1 or brf-1 effectively
reduces the expression of RNA Polymerase III transcripts, similar to mafr-1 O/E
(Fig. 3F). Importantly, RNAi of tbp-1 or brf-1 in the mafr-1 O/E strain could not
further reduce the expression of tRNAs (Fig. 3G), indicative of MAFR-1, TBP-1,
and BRF-1 functioning in the same pathway to regulate RNA Pol-III. Taken
together, these results support a role for MAFR-1 as a negative regulator of RNA
Pol-III, which impacts biosynthetic capacity, RNA Pol-II transcript levels, and
identify MAFR-1 as a functional ortholog of mammalian Maf1.

35

Discussion

We have identified C. elegans MAFR-1 as the functional ortholog of mammalian
Maf1. Maf1 proteins are evolutionarily conserved in humans, animals, plants, and
single cell eukaryotes (Boguta, 2013). We find that MAFR-1 can function in an
orthologous manner to human Maf1 (hMaf1) in cell culture. Specifically, we find
that MAFR-1 is capable of repressing the expression of human RNA Pol-III
transcribed targets, which indicates functional conservation between
invertebrates and mammals. Moreover, MAFR-1 can alter the transcript levels of
RNA pol II targets such as tbp-1, similar to mammalian Maf1 (Johnson et al.,
2007). tbp-1 RNAi reduces tRNA transcript levels in a manner similar to the
effect observed in mafr-1 O/E. The C. elegans genome encodes one TBP-related
factor (TRF), tlf-1 that could possibly compensate for the absence of tbp-1. We
find that although tbp-1 levels are responsive to mafr-1, tlf-1 levels are not altered
when mafr-1 is reduced or overexpressed (Figure S1). Intriguingly tlf-1 levels are
also lower in tbp-1 RNAi treated animals and therefore not likely compensating
for tbp-1. (Figure 3). This is consistent with previous studies that suggest TLF-1
performs a unique function in activating bulk Pol II transcription during
embryogenesis that is distinct from TBP-1.(Kaltenbach et al.)

36

References

Upadhya, R., Lee, J., and Willis, I.M. (2002). Maf1 is an essential mediator of
diverse signals that repress RNA Polymerase III transcription. Mol Cell 10, 1489-
1494.
Vannini, A., Ringel, R., Kusser, A.G., Berninghausen, O., Kassavetis, G.A., and
Cramer, P. (2010). Molecular basis of RNA Polymerase III transcription
repression by Maf1. Cell 143, 59-70.
Boguta, M. (2013). Maf1, a general negative regulator of RNA Polymerase III in
yeast. Biochim Biophys Acta 1829, 376-384.
Boguta, M., and Graczyk, D. (2011). RNA Polymerase III under control:
repression and de-repression. Trends Biochem Sci 36, 451-456.
Marshall, L., Rideout, E.J., and Grewal, S.S. (2012). Nutrient/TOR-dependent
regulation of RNA Polymerase III controls tissue and organismal growth in
Drosophila. Embo J 31, 1916-1930.
Rideout, E.J., Marshall, L., and Grewal, S.S. (2012). Drosophila RNA
Polymerase III repressor Maf1 controls body size and developmental timing by
modulating tRNAiMet synthesis and systemic insulin signaling. Proceedings of

37

the National Academy of Sciences of the United States of America 109, 1139-
1144.
Johnson, S.S., Zhang, C., Fromm, J., Willis, I.M., and Johnson, D.L. (2007).
Mammalian Maf1 is a negative regulator of transcription by all three nuclear RNA
Polymerases. Mol Cell 26, 367-379.
Kaltenbach, L., Horner, M.A., Rothman, J.H., and Mango, S.E. (2000). The TBP-
like factor CeTLF is required to activate RNA Polymerase II transcription during
C. elegans embryogenesis. Mol Cell 6, 705-713.

38

CHAPTER III: New physiological roles of MAFR-1

39

Abstract

mafr-1 is a conserved repressor of RNA Polymerase-III, however it’s
physiological role in the context of a multicellular organism is not well
understood. Here we show that MAFR-1 mediates organismal reproductive
capacity and lipid homeostasis. MAFR-1 impacts lipid transport by modulating
the intestinal expression of the vitellogenin family of proteins; resulting in cell
non-autonomous defects in the developing reproductive system. MAFR-1 levels
inversely correlate with stored intestinal lipids, in part by influencing the
expression of de novo lipogenesis enzymes fasn-1/FASN and pod-2/ACC1.
Animals fed a high carbohydrate diet transcriptionally repress mafr-1 and
mutations in the insulin signaling pathway genes daf-18/PTEN and daf-16/FoxO
abrogate the lipid storage defects associated with deregulated mafr-1
expression. Our results reveal novel physiological roles for mafr-1 in the
regulation of organismal lipid homeostasis, which ensure reproductive success.

40

Introduction

The ability of Maf1 to impact the protein synthesis capacity of a cell integrates it’s
established cellular function as a repressor of RNA Pol-III with the regulation of
oncogenic transformation. A hallmark of cancer cells is the exceptionally high
rate of catabolism of glucose (Vander Heiden et al., 2009). Similarly, cancer
cells display an increase in de novo fatty acid synthesis; however, a role for Maf1
in lipid metabolism has not been previously established. As such, we were
curious whether Maf1 could function as a central node in modulating lipid
metabolism in a multicellular system at the level of de novo lipogenesis,
transport, and/or utilization.

We were keen to investigate the physiological role of Maf1 in a genetically
tractable system such as C. elegans. As such, to better define the biological
function of Maf1 in the context of a multicellular organism, we investigated the
role of the related C. elegans Maf1 (MAFR-1) protein and examined the
functional consequences of altered mafr-1 expression on development,
reproduction, and lipid homeostasis.

In C. elegans, lipid homeostasis is maintained by multiple evolutionarily
conserved mechanisms (Barros et al., 2012; Brey et al., 2009; Brock et al., 2006,
2007; O'Rourke et al., 2009; Soukas et al., 2009; Walker et al., 2011; Watts,
2009; Zheng and Greenway, 2012) and C. elegans has become exceptionally
useful for high-throughput screening studies of complex cellular processes

41

relevant to human diseases (Anastassopoulou et al., 2011; Squiban et al., 2012;
Wahlby et al., 2012). We have discovered that MAFR-1 negatively regulates
intracellular lipid accumulation and influences reproductive capacity. Taken
together, these studies define these novel biological roles for Maf1 and indicate
the potential targeting of Maf1 for new therapeutic strategies for the prevention
and treatment of metabolic diseases with deregulated lipid phenotypes.

42

Material and Methods

Standard Growth Conditions
C. elegans were raised on standard 6 cm nematode growth media plates
supplemented with streptomycin and seeded with Escherichia coli OP50. For
RNAi experiments NGM plates containing 5 mM IPTG and 100 ug
ml
−1
carbencillin were seeded with overnight cultures of double-stranded RNAi-
expressing HT115 bacteria. Plates were allowed to induce overnight followed by
transfer of age-synchronous populations of C. elegans. All behavioral and
metabolism analyses of mutant animals are fed E. coli OP50, while all RNAi
based assays are done in animals fed HT115. Animals were fed either food
source for at least two generations before analysis to avoid diet dependent
effects.

Strains use in this study
N2 Bristol (wild type), SPC328[mafr-1p::mafr-1-GFP::mafr-13’UTR(laxIs004)],
CF1038[daf-16(mu86)], RB712[daf-18(ok480)], DH1033[vit-2::gfp], MGH168[sid-
1(qt9); vha-6p::sid-1]. Double mutants were generated by standard genetic
techniques.

43

Cell lines and culture conditions
293T cells were cultured in DMEM supplemented with 10% fetal bovine serum.
At 50-70% confluence, cells were transfected with vectors harboring human Maf1
or C. elegans mafr-1 genes by using Lipofectamine 2000 (Life Technologies).
After twenty-four hours, cells were washed and collected in Trizol reagent
(Invitrogen) for RNA extraction.

Gene Expression
Developmentally synchronous worms of indicated genotype and developmental
stages were collected, washed in M9 buffer and then homogenized in Trizol
reagent (Invitrogen). RNA was extracted according to manufacturer’s protocol.
DNA contamination was digested with DNase I (New England Biolabs) and
subsequently RNA was reverse-transcribed to cDNA by using the SuperScript®
III First-Strand Synthesis System (Life Technologies). Prepared RNA was further
purified using the RNAse easy kit. Samples were hybridized to
Affymetrix C. elegans Gene 1.0 ST Arrays. Data and statistics were analyzed
with Partek Genomics Suite Software version 6.6. Quantitative PCR was
performed by using SYBR Green (BioRad). The expression of snb-1 was used to
normalize C. elegans RNA samples and GAPDH from human cell culture
samples. The efficiencies of all primers used were within 5% of each other.
These genes showed minimal variation after changes in mafr-1/Maf1 expression.
For quantifying the stage specific transcript levels of mafr-1, RNA was extracted

44

from eggs and from larvae, 13hrs/22hrs/31hrs/43hrs and 49hrs post hatching.
We took worms at each time point, mounted them on a slide, and imaged the
gonad to confirm each stage. All samples were run in triplicate for quantifying the
steady state mRNA levels. Human Pol III primer sequences were taken from
Johnson et al (Johnson et al.). Primers for C. elegans genes were designed
using the Primer3plus algorithm(Untergasser et al., 2012). C.elegans pol III
primers were designed using the Genomic tRNA database(Chan and Lowe,
2009). A list of all qPCR primers can be accessed in Supplementary Table S2.

Protein extraction
Using the same time points as mentioned for the qPCR, worms were washed
twice with PBS to get rid of the bacteria and once with PBS supplemented with
protease inhibitors. LDS buffer was added to a final dilution of 1x and worms
were frozen using dry ice and then thawed at 90 degrees. This was done 3 times
and finally they were passed through a 26G needle before spinning them down at
max speed for 20mins. The supernatant was used for the protein analysis.

GFP expression and immunoanalysis
A GFP construct with a 6X-Histidine tag, DNA fragments of mafr-1 5' and 3’
regulatory sequence, and coding sequence introns and exons was PCR
amplified, gateway cloned into pDONR plasmids and lastly recombined into
vector pCFJ150. Animals harboring a single copy integration by MoSCI were

45

selected for further study (Frokjaer-Jensen et al., 2008). Immunochemistry was
performed with monoclonal antibodies specific for either GFP (Life Technologies)
or the 6XHistidine tag (Pierce) in the fusion construct.

Staining of fat
Nile red staining was performed as previously described (Pino et al., 2013).
Briefly, animals of indicated genotypes were collected, fixed in 40% isopropanol,
and stained in Nile red working solution in dark for two hours. Then worms were
washed once with PBS/0.01% triton-X and kept in dark for 30 min, mounted on
slides and imaged with the green fluorescent protein (GFP) channel of
microscope Zeiss Axio Imager with Zen software package. Fluorescent density
was measured by using NIH ImageJ software. In brief, images were converted
to a 32-bit format and then the background/GFP intensity was measured using
the Integrated Density (ID)method. The Region of interest tool was used to
measure body size, for normalization with intensity. For Oil Red O staining
animals of indicated genotypes were collected and fixed in 1% formaldehyde in
PBS. Then samples were frozen and thawed three times in a dry ice/ethanol
bath. Worms were washed with PBS three times before staining with freshly
prepared Oil Red O working solution. After staining for half an hour, worms were
washed again for 15 minutes, mounted on slides, and imaged under a bright field
illumination.

46

Microscopy
Images were captured on a Zeiss Axio Imager with Zen software package.
Fluorescent density was measured by using NIH ImageJ software.

Statistics
Data presented reflect biological replicates as indicated in each sample’s n.
Sample sizes were determined to reliably reveal the statistic significance given
the magnitude of the changes expected in each experiment. No randomization
was used. For Nile Red staining, strain genotype was blinded prior to staining
and only decoded after quantification of all samples was complete. Data were
presumed to be normally distributed. Data were presented as mean ± SEM. Data
were analyzed by using unpaired student t test. P < 0.05 was considered as
significant.

47

Results

MAFR-1 expression in C. elegans fat storage tissues alters the expression of
metabolism and reproduction genes.

To begin identifying the physiological roles of MAFR-1 in a multicellular
organism, we first examined the temporal expression of mafr-1 transcripts and a
MAFR-1::GFP protein throughout development, and documented the tissue
expression patterns of MAFR-1::GFP. mafr-1 expression is highest in embryos
but post-hatching is reduced and remains relatively constant throughout larval
development and into adulthood (Fig. 1E). After embryogenesis, the levels of
MAFR-1 protein are reduced and stabilize (31 hours post hatching) in the L3/L4
larval stage (Fig. 1F). We next examined the tissue expression pattern of
MAFR-1 using transgenic animals expressing a MAFR-1::GFP fusion construct.
We observed the strongest expression of MAFR-1::GFP in the intestine and
hypodermis, which are the two major sites of fat storage in C. elegans (Mak,
2012; O'Rourke et al., 2009) (Fig. 1G-I).

48

Fig. 1. Temporal and Spatial expression of MAFR-1.
Developmental expression pattern of mafr-1 mRNA (E) and MAFR-1::GFP
protein (F). MAFR-1 tissue expression revealed by imaging animals expressing
MAFR-1::GFP DIC (G) (A, anterior and P, posterior), intestinal cells (H) (arrows),
and hypodermal cells (I) (arrow heads).
Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001 versus
respective controls.

49

Our discovery that MAFR-1 can modulate the expression of RNA Pol-II genes,
such as tbp-1, fits previous reports reporting the localization of human Maf1 at
select RNA Pol-II promoters (Johnson et al., 2007). However, it remains unclear
if worm MAFR-1 directly regulates these genes and the extent to which Maf1 can
impact the expression of RNA Pol-II transcriptional targets is not fully understood.
To further define the physiological impact of mafr-1, we compared the steady
state gene expression profiles of a single copy mafr-1 overexpression (mafr-1
O/E) model as compared to control animals. The evolutionarily conserved
function of MAFR-1 to negatively regulate transcript levels drove our initial
analysis of identifying genes whose expression level was reduced when mafr-1
was overexpressed (Table 1). A bioinformatic examination of these genes
identified a significant enrichment for gene ontology (GO) terms related to
reproduction enrichment score = 24.51, p-value 10E
-11
and metabolism
enrichment score = 5.46, p-value 0.004 (Table 1). Enrichment for factors
associated with the negative regulation of translation and lipid transport were
also identified. GO-terms linked to the regulation of RNA Pol-III target expression
(4.17% of genes of this GO-term were represented) and RNA Polymerase
activity (5.26% of genes of this GO-term were represented) were also identified
(data not shown), which are consistent with the canonical role of Maf1. We also
identified transcripts that were increased in the mafr-1 O/E animals (Table S1),
which may represent homeostatic responses to deregulated mafr-1 expression.
These findings support a role for MAFR-1 in modulating RNA Pol-III and protein
synthesis capacity, but also reveal the ability to influence RNA Pol-II targets with

50

51

new physiological functions. We investigated a selection of transcripts whose
expression was deregulated when mafr-1 levels are altered. These transcripts
were selected based on the degree and significance of the change, the
identification of a representative portion of the genes within the gene family or
GO-term group, and in the case of the enrichment for metabolism genes, we
selected genes that were also found to be regulated by Maf1 in mammals
(Johnson et al. 2007)(Palian et al, 2014)

mafr-1 negatively regulates lipid transport and fecundity.

Our analysis of the transcripts sensitive to mafr-1 expression identified
components of the C. elegans vitellogenin (vit) lipid transport system, which
provides nutrients to the developing germline during reproduction (Fig. 2A).
Vitellogenins are lipid-binding proteins that are produced in the intestine,
secreted as yolk into the body cavity, and diffused into the gonad where they are
endocytosed by developing oocytes via the low density lipoprotein receptor,
RME-2 (Grant and Hirsh, 1999; Kimble and Sharrock, 1983). We measured the
expression of vit genes in mafr-1 RNAi treated (Fig. 2B) and mafr-1 O/E animals
(Fig. 2C) and discovered MAFR-1 could negatively regulate the expression vit-2,
vit-4, vit-5, and vit-6. The reduction in vit expression in mafr-1 O/E animals was
specific to mafr-1 expression levels as RNAi targeting mafr-1 could reverse this
reduction (Fig. 3A). The abundance of RNA Pol-III transcripts is an established
measure of biosynthetic capacity.

52

Fig. 2. Impact of mafr-1 expression on lipid transport and oocyte
development. The vitellogenin lipid transport system was significantly repressed
in microarrays of mafr-1 O/E animals as compared to non-transgenic siblings (A).
qPCR analysis reveals the lipid transport genes vit-2, -4, -5, and -6 are
differentially regulated by mafr-1 levels in RNAi (B) or overexpression (C)
animals. (D) Altered vitellogenin gene expression correlates with localization and
abundance of VIT-2::GFP in developing oocytes. Data are presented as mean ±
SEM. *P < 0.05, **P < 0.01, ***P<0.001 versus respective controls.

53

Fig. 3. Changes to vitellogenin gene expression is specific to mafr-1 levels
but not are result of global deregulation of tRNA biosynthesis. (A) RNAi of
mafr-1 reverses the transcriptional repression of most vit genes in mafr-1 O/E
animals. (B) vit gene expression levels in brf-1 RNAi or tbp-1 RNAi treated
animals. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001
versus respective controls.

54

We next tested if vit gene expression was tied to the change in biosynthetic
capacity of the organism, as measured by tRNA abundance. As reported above,
RNAi of tbp-1 or brf-1 reduced the expression of tRNAs, similar to mafr-1 O/E.
However, unlike mafr-1 O/E, which dramatically reduced vit gene expression, brf-
1 RNAi treated animals in general have normal levels of vit expression (Fig. 3B).
Surprisingly, animals with reduced expression of tbp-1 actually increase vit gene
expression (Fig. 3B). The mechanism of this regulation is unknown but of
particular interest. Taken together these findings implicate a role for MAFR-1 in
the expression of vit genes that is not simply a result of altered tRNA expression.

To further examine the effects of mafr-1 expression on VIT-2 function we
utilized a transgenic strain expressing a VIT-2 fusion to GFP to allow
visualization of lipid transport to the reproductive system (Grant and Hirsh, 1999).
Consistent with our gene expression analysis, oocytes in mafr-1 O/E animals
contained less intracellular VIT-2::GFP when compared to controls (Fig. 2D).
Similarly, RNAi of mafr-1 led to a modest increase in VIT-2::GFP in oocytes.
These data suggest mafr-1 expression levels regulate the abundance of the
vitellogenin yolk precursor proteins in the intestine, which function in a cell non-
autonomous manner in the developing germline of the reproductive system. We
hypothesized that the diminished abundance of VIT-2 lipid particles in the
developing oocytes of mafr-1 O/E animals would result in a strong reproduction
phenotype. As predicted, mafr-1 O/E animals display a >50% reduction in
fecundity as compared to wild type controls (Fig. 4A). In addition, mafr-1 O/E

55

had no measurable effect on embryo viability as all eggs that were laid properly
develop through embryogenesis and hatch (Fig. 4B). The diminished
reproduction phenotype is tied to MAFR-1 overexpression as RNAi of mafr-1 in
the mafr-1 O/E strain could partially restore progeny production (Fig. 4C).
Intriguingly, in wild type animals, RNAi of mafr-1 was not capable of increasing
total brood size (Fig. 5A), which is determined by the number of sperm generated
in a hermaphrodite (Hughes et al., 2007; Ward and Carrel, 1979). brf-1 RNAi
animals have a small reduction in fecundity that is synergistically reduced when
combined with mafr-1 O/E, which suggests two parallel pathways converging
upon reproductive output (Fig. 5B). This finding is consistent with our observation
that the altered expression of vit genes in the mafr-1 O/E strain could not be
phenocopied by simply reducing the expression of tRNAs following tbp-1 or brf-1
RNAi. We could not test the relationship with tbp-1 in this assay since tbp-1 RNAi
treatment in worms leads to embryonic lethality, consistent with previous reports
(Gonczy et al.; Kamath et al.; Piano et al., 2000).

Since vitellogenin biosynthesis occurs in the intestine we next tested
whether mafr-1 expression specifically in the intestine was causal for the
observed decline in reproductive output. The intestinal specific RNAi strain has a
reduced brood as compared to wild type animals. This is perhaps due to the sid-
1(qt9) genetic background compounded with the intestinal sid-1(+) rescue array.
However, mafr-1 O/E in this genetic background is still capable of reducing
fecundity, demonstrating that reproductive output in this strain remains sensitive

56

Fig. 4. Cell non-autonomous impact of altered mafr-1 expression in the
intestine on reproductive output.Total brood size (A) and embryo viability (B)
of animals with mafr-1 O/E. Restoration of fecundity by tissue general (C) or
intestine specific (D) RNAi of mafr-1. Model for cell non-autonomous role of
mafr-1 expression on fecundity (E). Data are presented as mean ± SEM.
*P < 0.05, **P < 0.01, ***P<0.001 versus respective controls.

57

Fig. 5. Decreased expression of mafr-1 does not increase maximal progeny
production.
Total progeny production in (A) animals with tissue general mafr-1 RNAi (B) brf-1
RNAi in wild type and mafr-1 O/E animals and (C) intestinal specific mafr-1 RNAi
treated animals. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01,
***P<0.001 versus respective controls.

58

to mafr-1 levels. Importantly, when we lower mafr-1 expression by RNAi
specifically in the intestine of animals with mafr-1 O/E, we observed an increase
in fecundity (Fig. 4D and 5C). Together, these results suggest that mafr-1
expression exerts a cell non-autonomous influence on reproductive success by
altering lipid transport and not through pleiotropic effects on zygote formation or
embryonic development (Fig. 4E).

mafr-1 negatively regulates lipid homeostasis.
Our microarray analysis of mafr-1 overexpression animals identified a
significant enrichment for lipid, amino acid, and carbohydrate metabolism genes
(Fig. 6A). As this finding implies a novel function for Maf1 outside the canonical
role in the regulation of RNA Pol-III transcripts, we examined the expression of
established lipid metabolism genes in mafr-1 RNAi treated animals and mafr-1
single copy overexpression strains (Fig. 6B, 6C, and 6D). We increased the
specificity of our gene targets whose expression is sensitive to MAFR-1 by
imposing a requirement for reciprocal effects on transcript levels: increased
expression in mafr-1 RNAi animals and decreased expression in mafr-1 O/E
animals. The enrichment for lipid metabolism genes was intriguing, but lipid
homeostasis is exceptionally complex and requires the coordination of hundreds
of lipid pathway genes. As such, we first focused on lipid biosynthesis, which is
initiated from the irreversible enzymatic carboxylation of acetyl-CoA by acetyl-
CoA carboxylase (ACC1) followed by the synthesis of palmitate by fatty acid
synthase (FASN) (Girard et al., 1994).

59

60

Fig. 6. mafr-1 expression levels alter organismal lipid homeostasis.
(A), Multiple GO-terms tied to cellular metabolism were significantly repressed in
microarrays of mafr-1O/E animals as compared to non-transgenic siblings.
Expression of the lipid biosynthesis genes pod-2/ACC and fasn-1 in animals
overexpressing mafr-1 (B) or with RNAi-reduction of mafr-1 (C). Expression of
human ACC and FASN in human 293T cells transfected with either MAFR-1 or
hMaf1 constructs (D). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01,
***P<0.001 versus respective controls, (n) number of animals used for statistical
analysis in each condition.

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Fig. 7. Changes in stored lipids resulting from deregulated mafr-1 levels are
not a product of changed expression of altered lipid utilization genes. (A)
RNAi of mafr-1 reverses the transcriptional repression of pod-2 and fasn-1 genes
in mafr-1 O/E animals. (B) pod-2 and fasn-1 gene expression levels in brf-1 RNAi
or tbp-1 RNAi treated animals. Expression of the indicated mitochondrial and
peroxisomal fatty acid oxidation genes and the lipid degradation gene atgl-1 are
not differentially regulated by mafr-1 levels in overexpression (C) or RNAi treated
(D) animals.

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Fig. 7. Changes in stored lipids resulting from deregulated mafr-1 levels are
not a product of changed expression of altered lipid utilization genes.
(E) Oil red O staining of wild type animals fed control or mafr-1 RNAi bacteria. (F)
Oil red O staining of OP50 fed wild type and mafr-1 O/E animals. Comparison of
Nile red stained lipids in worms fed OP50 and HT115 diets. The representative
images are shown in (G) and quantitative data are shown in (H). (I) Food
consumption rates as measured by pharyngeal pumping in animals with altered
mafr-1 expression levels. Data are presented as mean ± SEM. *P < 0.05, **P <
0.01, ***P<0.001 versus respective controls.

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The expression of the C. elegans lipid biosynthesis genes pod-2/ACC1 and fasn-
1/FASN, were repressed in animals with increased mafr-1 expression (Fig. 6B)
and were higher when mafr-1 expression was reduced by RNAi (Fig. 6C). The
repression in mafr-1 O/E could be reversed by mafr-1 RNAi (Fig. 7A). It is
notable that an identical relationship exists between mammalian Maf1 levels and
the expression of mammalian ACC1 and FASN in mouse and human cells
(Palian et al, 2014). To determine the possible role of RNA pol III-dependent
transcription on mafr-1-mediated regulation of the lipogenic enzyme genes, brf-1
reduction by RNAi revealed that similar to our findings for the expression of the
vit genes, pod-2 and fasn-1 transcript levels were not altered in brf-1 RNAi
animals and were increased in tbp-1 RNAi treated worms (Fig. 7B). This is
suggestive that these changes in the expression of lipid biosynthesis genes in
the mafr-1 O/E animals are not simply a response to a global reduction in tRNA
biosynthesis. Importantly, Palian and colleagues have demonstrated that Maf1
occupies the FASN promoter to repress it’s expression (Palian et al. 2014). We
also examined the expression of multiple lipid homeostasis genes that function in
fatty acid oxidation and lipolysis, but could not detect any significant change
when mafr-1 was reduced by RNAi or overexpressed (Fig. 7C and 7D). Taken
together these results reveal that Maf1 regulates intracellular lipids and that an
important mechanism by which this is accomplished is through it’s ability to
regulate lipogenic enzyme gene expression.

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Previous studies on Maf1 function have primarily focused on single cell
models (Boguta, 2013; Johnson et al., 2007; Michels et al., 2010; Rohira et al.,
2013; Shor et al., 2010). The observed changes in lipid homeostasis resulting
from altering the expression of MAFR-1 in the entire organism can result from
cell autonomous and/or cell non-autonomous effects on physiology. The GO
terms enriched in animals with altered mafr-1 expression includes genes
expressed in a variety of tissues, which may act in an autonomous manner.
Because our microarray analysis is a snapshot of expression of all tissues in the
animal combined, we took advantage of the conserved activity of MAFR-1 in
mammalian cells and asked if MAFR-1 could also alter the expression of de novo
lipogenesis genes in this model. We tested whether altered MAFR-1 expression
can modulate expression of lipid biosynthesis genes in human cells. Similar to
our observations for RNA Pol-III targets, 293T cells overexpressing either MAFR-
1 or human Maf1 have lower levels of human ACC1 and FASN mRNAs.
Consistent with these results, Palian et al. have shown that changes in Maf1
expression in human Huh7 hepatocarcinoma cells can alter the levels of
intracellular lipid stores (Palian et al. 2014).

The enrichment for cellular metabolism genes in our microarray analysis
prompted an investigation of the biological relevance of mafr-1 expression on
metabolic homeostasis. Similar to most animals, C. elegans fat homeostasis is
influenced by multiple metabolic processes and pathways (Mak, 2012). As such,
we first examined the levels of stored fat by fixed Nile red and Oil red O staining

65

in worms treated with mafr-1 RNAi and in our single copy mafr-1 overexpression
lines. Overexpression of mafr-1 led to a significant 35% reduction of intracellular
lipids (Fig. 6E and 6F and Fig. 7E), while RNAi of mafr-1 resulted in a striking
94% increase in stored intestinal fat (Fig. 6G and 6H and Fig. 7F). Similar to
previous reports, we found the steady state levels of stored lipids to be lower in
worms fed RNAi bacteria HT115 as compared to OP50, which are the two
standard worm diets (Fig. 6E and 6G and Fig. 7G and 7H). Importantly, the
changes in stored fat were not a result of altered food intake, as measured by
pharyngeal pumping rates (Fig. 7I). These results indicate that organismal lipid
homeostasis is sensitive to even single copy variations in mafr-1 expression.

MAFR-1 is regulated by glucose and insulin signaling.

The measured reduction in stored lipids observed in animals overexpressing
mafr-1 was compelling and led us to test if mafr-1 O/E could counteract diet-
induced obesity in the C. elegans model. To this end, we challenged mafr-1 O/E
animals on a high carbohydrate diet (HCD), which is capable of inducing
increased storage of intracellular lipids (Pang);(Lee et al., 2009; Schulz et al.,
2007) (Fig. 8A and 8B). Consistent with the steady state reduction in stored

66

Fig. 8. Glucose and insulin signaling
pathways regulate the expression of mafr-1.
(A-B) Nile red staining of worms with indicated
genotypes fed OP50 or OP50 plus 2% glucose.
The representative images are shown in (A) and
quantitative data are shown in (B). Dietary glucose reduces the steady state
levels of MAFR-1 protein (C) and mafr-1 transcripts (D). Quantification of Nile
red staining of animals overexpressing mafr-1 (E) or RNAi treated for mafr-1
(F) in daf-18/PTEN or daf-16/FoxO mutant backgrounds. Expression of mafr-1
in daf-16/FoxO RNAi treated (G) or genetic null mutant (H) animals. Data are
presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001 versus respective
controls, (n) number of animals used for statistical analysis in each condition.

67

Fig. 9. Nutrient availability and insulin signaling regulate mafr-1.
(A) Expression of mafr-1 in animals fed the standard OP50 diet compared to the
HT115 RNAi diet. Representative images of Nile red staining of animals
overexpressing mafr-1 (B) or RNAi treated for mafr-1 (C) in daf-18/PTEN or daf-
16/FoxO mutant backgrounds.(D) expression of mafr-1 influenced genes in the
daf-16(mu86) mutant. (E) Measurement of insulin signaling activity through DAF-

68

16/FoxO in the indicated genotypes . (F) ModEncode project ChIPSeq of DAF-
16::GFP animals reveals enrichment at the mafr-1 promoter region. Bioinformatic
analysis of DAF-16 associated elements (DAE) and binding elements (DBE) in a
1.5 kb region upstream of the translational start site of mafr-1.

69

lipids on a normal diet, mafr-1 O/E could partially reduce the accumulation of
lipids on the HCD by ~10%, albeit these animals were still significantly more fat
than animals raised on a normal diet. In fact, because the mafr-1 O/E animals
are less fat than wild type on a regular diet, the percent increase (166%) in
stored lipids when comparing mafr-1 O/E animals are fed a HCD compared to a
regular diet is larger than the percent increase (112%) observed in wild type
animals on similar diets (Fig. 8B).

In the presence of ample dietary sugars, excess carbohydrates are
converted to triglycerides (Hillgartner et al., 1995). The ability of mafr-1 levels to
alter de novo lipogenesis, but not effectively abrogate the total lipid increase on a
HCD suggested mafr-1 itself could be sensitive to nutrient availability. To test
this, we first examined the levels of MAFR-1::GFP in animals fed a HCD. MAFR-
1 protein was reduced in animals raised in the presence of glucose (Fig. 8C).
The reduction in MAFR-1 was in part the result of reduced expression of mafr-1
on the HCD, both in wild type and MAFR-1::GFP expressing animals (Fig. 8D).
This diet-induced repression effectively abrogates the increased expression of
mafr-1 in mafr-1 O/E animals when glucose is present. Previous biochemical
characterization of the E. coli strains OP50 and HT115 have revealed that the
HT115 diet has a higher carbohydrate composition than OP50 (Brooks et al.,
2009). Consistent with these findings, a comparison of the mafr-1 levels in
animals raised on these two diets reveals lower expression of mafr-1 when fed
the HT115 diet (Fig. 9A). These data indicate mafr-1 expression and MAFR-1

70

protein levels are sensitive to the abundance of dietary carbohydrates. In C.
elegans, the presence of glucose represses the FoxO transcription factor DAF-16
(Lee et al., 2009) through a conserved insulin/IGF-like signaling pathway. We
tested whether inhibition of DAF-16/FoxO activity was sufficient to suppress the
effects of mafr-1 expression levels using animals lacking the DAF-16 regulator
daf-18/PTEN. In the absence of daf-18/PTEN, the insulin signaling pathway is
de-repressed and DAF-16 activity is inhibited (Ogg and Ruvkun, 1998). In this
genetic background, the induced changes in stored lipids mediated by mafr-1
overexpression (Fig. 8E, 9B) or RNAi reduction (Fig. 8F, 9C) were abrogated. In
addition, we tested the ability of mafr-1 expression levels to alter intracellular lipid
homeostasis in the absence of daf-16. The ability of altered mafr-1 expression to
change the steady state levels of intracellular lipids was abolished in the absence
of DAF-16/FoxO (Fig. 8E and 8F and Fig. 9B and 9C). These data reveal a
requirement for the insulin-like signaling pathway in MAFR-1 mediated regulation
of stored lipids. This is consistent with the reported role for the insulin receptor
acting down stream of dMaf1 in regulating growth in flies (Rideout et al.).

We further investigated why these daf-16 null-mutants were insensitive to
mafr-1 O/E. The requirement of DAF-16 in the MAFR-1 lipid phenotype could
result from transcriptional regulation of mafr-1. We tested for a genetic role of
DAF-16/FoxO in mafr-1 expression by measuring the levels of mafr-1 transcripts
in animals lacking daf-16 and found a significant 50% reduction in expression as
compared to wild type animals (Fig. 8G and 8H). Next, we looked at the

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requirement of DAF-16 in the induction of tRNAs, vit, and lipid biosynthesis
genes when mafr-1 expression was reduced by RNAi. In the absence of daf-16,
mafr-1 RNAi was still able to induce the expression of most tRNAs, although the
basal expression of multiple tRNAs was lower initially in the daf-16(mu86) null
mutant background as compared to wild type controls. mafr-1 RNAi was similarly
able to increase the expression of pod-2/ACC1, vit-2, -4, and -6, but intriguingly,
not fasn-1 and vit-5 (Fig. 9D).

In flies, inhibition of dMaf1 by RNAi results in the release of insulin-like
peptides and systemic insulin signaling. We tested whether mafr-1 had a similar
effect on insulin-like signaling in C. elegans. We equated activation of insulin-like
signaling by measuring the induction of the DAF-16/FoxO target gene sod-3.
Unlike the effect of the hypomorphic allele of the insulin-like receptor, daf-
2(e1368), which leads to a dramatic ~60-fold increase in the expression of sod-3,
overexpression of mafr-1 led to a small but significant ~2.5-fold increase in sod-3
expression (Fig. 9E). Feedback and feed-forward regulation by endocrine
systems are well established (Murphy and Hu, 2013; Rutter, 1999), and our data
are consistent with previous reports that insulin/IGF-I signaling can potentiate
organismal responses to Maf1 (Rideout et al.). Future dissection of where and
how MAFR-1 integrates into the insulin-like signaling pathway will be of great
interest.

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Our findings reveal that mafr-1 expression is regulated by diet, specifically the
presence of dietary carbohydrates, and this is mediated by DAF-18/PTEN and
DAF-16/FoxO signaling through the insulin/IGF pathway. It is notable that a
similar relationship between PI3K signaling and Maf1 expression is found in the
mouse liver, and that increased Maf1 expression can abrogate diet-mediated
induction of triglycerides (Palian et al. 2014), which is consistent with our finding
that MAFR-1 is a conserved regulator of animal physiology and metabolism
beyond the regulation of RNA Pol-III transcripts. Collectively, our studies have
uncovered novel roles for the conserved protein MAFR-1 in the maintenance of
reproduction and lipid homeostasis, which genetically engage the insulin
signaling effector protein DAF-16/FoxO (Fig. 10).

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Fig. 10. Model of the central role of MAFR-1 in organismal physiology.
MAFR-1 can influence animal physiology in both an insulin/FoxO-dependent
(orange) and independent (blue) manner. Altered mafr-1 levels in the intestine
cell non-autonomously impacts fecundity through changes in the production of
the vitellogenin family of proteins that deregulates lipid transport to developing
oocytes in the germ line. Black arrows and bars indicate genetic interactions.
DAF-16/FoxO may directly regulate the expression of mafr-1 (dashed red arrow).
Other factors that regulate DAF-16/FoxO-dependent and independent regulation
of Maf1 may exist (?). Dashed cell boundary indicates potential auto-, para-, or
endocrine signals resulting from Maf1 activity.

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Discussion

We have identified C. elegans MAFR-1 as the functional ortholog of mammalian
Maf1 and uncovered a novel metabolic role for MAFR-1 in the regulation of
organismal fecundity and lipid homeostasis (Fig. 10). The reduction in tbp-1
expression is not solely responsible for all of the physiological responses we
have documented as tbp-1 RNAi fails to phenocopy the mafr-1 O/E strain. This
finding is intriguing, especially in light of the increased expression of the vit genes
observed in tbp-1 RNAi treated animals.

Our studies uncover the surprising finding that Maf1 regulates intracellular
lipids. The transcriptional and physiological effects of altering mafr-1 abundance
are specific to mafr-1 as we can suppress the observed changes in the mafr-1
O/E strain with RNAi knockdown of mafr-1. Examination of the mechanism by
which this occurs we find that Maf1 represses the expression of lipid biosynthesis
(pod-2 and fasn-1) and lipid transport (vit) genes. We also demonstrate that the
changes observed in lipid biosynthesis genes and vitellogenins are not simply
due to Maf1-mediated changes in RNA pol III transcription as directly altering
RNA pol III-dependent transcription does not change the expression of these
RNA pol II-transcribed genes. These results are further supported by a
complementary study from Deborah Johnson’s group revealing that Maf1 ChIPs
to the FASN promoter in cultured human cells (Palian et al. 2014).

75

Although this doesn’t exclude the possibility that Maf1 may participate in other
pathways to regulate lipid homeostasis, we identify Maf1 as an important
negative regulator of lipid biogenesis.

MAFR-1 is expressed at all life stages with strongest expression in the
hypodermis and intestine. These tissues serve as the primary sites of fat storage
in C. elegans; with the intestine acting as a major endocrine organ, most similar
to the mammalian liver (Barros et al., 2012). We have uncovered a new role for
Maf1 in the regulation of organismal lipid homeostasis. Maf1 represses the
expression of lipogenic enzyme genes that are regulated through PI3K and FoxO
signaling in human cells (Palian et al., 2014). In mammals, these lipogenic
enzymes are aberrantly induced in cancer cells. Thus, Maf1 coregulates these
and other genes transcribed by RNA pol III such as tRNAs that are elevated in
oncogenically transformed cells. Given that mammalian Maf1 plays a role in
suppressing oncogenic transformation and tumorigenesis (Johnson et al 2007;
Palian et al., 2014), our results support the idea that it represents an important
molecular link between metabolic diseases and cancer that are well known to be
epidemiologically associated.

Previous studies have shown a peculiar inverse correlation between
endogenous carbohydrate levels of the bacterial food source and triacylglycerol
stores in the worm (Brooks et al.). Intriguingly, excess dietary carbohydrate
added to the growth medium can increase lipids and decrease survival (Lee et

76

al.). Taken together, these previous studies demonstrate the complex interplay
between diet and adiposity. Although carbohydrates represent just one major
macronutrient difference between the OP50 and HT115 diets, our data reveal a
novel role for MAFR-1 in metabolic homeostasis and that C. elegans lipid
metabolism is sensitive to even small changes in mafr-1 expression: ~50%
reduction by RNAi and ~80% increase in single copy overexpression lines.
Multiple genetic phenotypes have been shown to only manifest on either the
OP50 or HT115 diet. Our understanding of the underlying molecular mechanisms
regulating these differences however is only beginning to emerge (Pang and
Curran; Pang). The resulting lipid storage phenotype induced by altering mafr-1
expression impacts distinct pathways in lipid homeostasis. Under our standard
laboratory growth conditions, the expression of key lipid biosynthesis genes, but
not fatty acid oxidation genes are repressed by mafr-1 and we have found that
endogenous mafr-1 levels are regulated in part by available dietary
carbohydrates. These two findings could be functionally linked as lipid
biosynthesis is also regulated by carbohydrate availability. As such, MAFR-1
could play an integral role in fine-tuning this pathway, which ultimately impacts
steady state levels of stored intracellular lipids. Importantly, these results define
new layers of specificity of the MAFR-1 biologically relevant pathways, as we do
not observe deregulation in the expression of all genes. In fact, only a finite
number of genes are reciprocally responsive to increased and decreased
expression of mafr-1. It is notable that although small changes in expression can
be measured in other genes, that these differences are likely to represent

77

homeostatic transcriptional responses at the organism level; perhaps to the
changes in the expression of de novo lipogenesis and transport genes.

Lipid transport throughout a multicellular organism is facilitated by
lipoproteins that emulsify lipids and allow these fats to move through aqueous
environments. Vitellogenesis is a hormonally controlled and conserved process
in birds, reptiles, fish, and many invertebrates that generate yolk as a nutrient
source for early embryogenesis (Schneider, 1996). We have discovered that
mafr-1 levels influence vitellogenin production. Animals overexpressing mafr-1
have reduced expression of vit-2, -4, -5, and -6 and developing oocytes contain
less VIT-2 protein as measured by a VIT-2::GFP fusion protein. As such, the
change in mafr-1 expression, which leads to a change in the intestinal expression
of vitellogenins exerts a cell non-autonomous influence on oocyte maturation in
the germline.

A second physiological consequence uncovered from altered mafr-1
expression is a change in organismal reproductive capacity. Fecundity is under
both genetic and environmental control and we identify mafr-1 as a central player
in this essential biological process. MAFR-1 levels are continually reduced and
settle to their lowest point at the peak of germ cell development. The reduction of
MAFR-1 levels during the maturation of the reproductive system could be of
functional relevance since animals with increased mafr-1 expression have
reduced egg production. This reduced fecundity correlates with deregulated

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vitellogenin synthesis and localization, which is essential for oocyte maturation.
Previous studies have found that the vit genes are transcribed in excess in the
intestine and can be titrated to cope with reproduction demands (DePina et al.,
2011). As such, increases in yolk synthesis would not necessarily increase
fecundity, as we found, but in the context of low yolk availability, such as in the
mafr-1 overexpression lines, the increased production of vitellogenins following
mafr-1 RNAi in this background can partially restore embryo development. The
use of whole-organism microarray and qPCR analysis of gene expression can
confound the extrapolation of cell autonomous versus cell nonautonomous
effects. Thus, it remains possible that pod-2/ACC1 and fasn-1/FASN expression
in the germline may have important roles in fecundity. Nevertheless, the fact that
the vit family-of-genes are expressed specifically in the intestine when combined
with our finding that intestine specific regulation of mafr-1 levels can alter
reproductive output provides evidence for mafr-1 in the intestine exerting a cell
non-autonomous effect on the reproductive system.

Metabolic homeostasis in a multicellular organism requires the integration
of signals from multiple tissues that coordinate metabolite synthesis, transport,
and utilization. We have identified both cell autonomous and non-autonomous
roles for MAFR-1 in these processes. MAFR-1 expression can act cell
autonomously by changing the expression of de novo lipid biosynthesis. MAFR-1
levels also manifest changes in reproductive success in a cell non-autonomous
manner by altering the biosynthesis of the vitellogenin yolk precursors. Further

79

dissection of the cell non-autonomous mechanisms that mediate Maf1 function
on the organism level will be of future interest. The ability of an organism to
integrate information on available nutrients to coordinate growth and metabolism
is essential for survival (Mair; Pang and Curran; Pang). We find that the levels of
MAFR-1 are influenced by the abundance of nutritional glucose and that mafr-1
expression is lower in animals fed an E. coli K-12 HT115 diet versus the standard
E. coli B OP50 diet. Intriguingly, the observed reduction in expression on these
two diets may involve the insulin-signaling pathway as the expression of mafr-1
in the absence of daf-16/FoxO is similar on either food source.

This finding is correlative with previous characterization of these bacterial
strains, which revealed a 3 to 5-fold higher level of endogenous carbohydrates in
HT115 bacteria (Brooks et al.). We also noted a trend for a slightly larger brood
size in wild type animals fed the HT115 diet, but the inability for mafr-1 RNAi to
further increase this brood. These results are consistent with the lower
expression of mafr-1 on the HT115 diet and suggest that further reduction of
mafr-1 cannot synergistically enhance reproductive capacity, whose maximum is
dictated by spermatogenesis (Hughes et al., 2007; Ward and Carrel, 1979).
Obviously the use of bacteria as a food source increases the complexity of
deciphering diet dependent phenotypes; particularly when additives such of
glucose are utilized, which can alter bacterial growth and physiology. Although
animals feeding on the HT115 diet do not show an increase in stored fat, despite
diminished mafr-1 expression, this could be the result of a variety of metabolic

80

actions initiated by the host in response to this complex diet (Brooks et al.). The
identification of other specific macronutrient triggers will be of particular interest
for future studies to determine the extent to which MAFR-1 integrates into other
metabolic pathways.

The reduction in total cellular fat accumulation when mafr-1 is
overexpressed in worms or in mammals (Palian et al, 2014) is intriguing and
suggests that mafr-1 could be a potential new target for therapies of obesity and
metabolic diseases. Although our worm model of mafr-1 overexpression when
fed a HCD was statistically less fat than wild type animals fed the same diet, the
percent increase in fat as compared to the same animals on a normal diet was
greater for the mafr-1 O/E strain. This could be due to the fact that our
overexpression system is a result of a single additional copy of mafr-1. Future
studies that investigate if higher gene dosages can provide additional protection
from a HCD will be of great interest.

We also identify DAF-16/FoxO as a novel genetic regulator of mafr-1.
However, the strong reduction in MAFR-1 protein levels on the HCD can only
partially be explained by changes in mafr-1 expression. Whether DAF-16 directly
regulates the transcription of mafr-1 remains to be elucidated. A bioinformatic
investigation of the mafr-1 promoter reveals a canonical DAF-16 associated
element (DAE) binding site at -766nt from the translational start site (Murphy et
al., 2003). An unbiased ChIP Seq screen by the modENCODE project identified

81

DAF-16::GFP associated with the mafr-1 locus, near a non-canonical DAF-16
binding element (DBE) (Gerstein et al., 2010) (Fig. 9F), which suggests DAF-
16/FoxO could indeed regulate mafr-1.

Investigating the differences in the regulation of and the physiological
responses to Maf1 across species is of particular interest. Similar to dMaf1,
altering the expression of mafr-1, in a tissue specific manner can have significant
impact at the organism level (Rideout et al.). mafr-1 expression does not appear
to influence developmental timing, as dMaf1 does in flies, although it can
modestly impact organismal growth. While the physiological response from
altering mafr-1 expression does not appear to influence the insulin/IGF-I
signaling pathway as potently as it does in flies, we nevertheless find a partial
requirement for DAF-16/FOXO in mediating these new mafr-1-sensitive
physiological parameters. Moreover, daf-16/FoxO was dispensable for the mafr-
1-dependent changes to tRNA genes. This is consistent with the idea that the
insulin/IGF-I signaling pathway functions downstream of mafr-1, as it has been
described in flies (Figure 10). Our results emphasize the importance of
examining the role of central cellular regulators in the context of a multicellular
system, which have led to the identification of novel physiological roles for Maf1
in reproduction and lipid metabolism. Defining the tissues and cells where insulin
signaling participates, either cell autonomously and/or non-autonomously, in the
mafr-1-dependent phenotypes we have described will be important to further
refine our model.

82

Interestingly, aside from developmental timing there are apparent differences
between the biological role of Maf1 in flies and worms. In flies, only dMaf1 RNAi
can impact the iMet, Ile, and Leu tRNAs expression while overexpression had no
affect (Rideout et al.). In worms we find that increased mafr-1 O/E decreased the
abundance of all tRNA species tested and mafr-1 RNAi led to the increased
expression of 11 out of 13 of these tRNAs (Figure 1 and 2). Second, in flies
dMaf1 and the RNA Pol-III factor Brf1, which controls tRNA biosynthesis, are tied
to the growth and developmental timing phenotypes (Marshall et al.). In worms,
brf-1 and mafr-1 can both regulate tRNA transcript levels, however the lipid
homeostasis phenotypes observed in the mafr-1 O/E strains cannot be
phenocopied by reduction of brf-1 expression. This data implicates a novel
aspect of MAFR-1 function beyond modulating RNA Pol-III transcript levels.
Third, although dMaf1 expression in the fat body was found to be key to it’s
function it remains unknown if dMaf1 can influence fat accumulation or
reproduction, which are two physiological process intimately linked to signaling
from the fly fat body (Arrese and Soulages). While there will certainly be indirect
effects resulting from homeostatic responses to the changes in RNA Pol-III
transcript levels and/or possible positional effects due to the chromosomal
proximity of RNA Pol-III and Pol-II targets (Hull et al., 1994; Kinsey and
Sandmeyer, 1991), defining the molecular mechanisms underlying Maf1 function
will be critical to understanding the physiological differences observed in flies,
worms, and mammals.

83

We have uncovered novel roles for Maf1 in lipid metabolism and
reproduction and identified both cell autonomous and non-autonomous effects of
Maf1 activity in the context of a multicellular organism. Our work identifies Maf1
as a new regulatory node in the maintenance of reproduction and lipid
homeostasis and points to Maf1 as a novel potential target for controlling obesity
and diseases with dysregulated lipid metabolism.

84

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CHAPTER IV: Molecular regulation on MAFR-1

94

Abstract

Cellular growth is inhibited under various stress conditions by suppressing new
protein synthesis. This is partially achieved by the repression of RNA Polymerase
III-dependent transcription, which requires Maf1. It is believed that the
phosphorylation status of Maf1 alters it’s subcellular localization and function,
allowing it to integrate nutrient and stress signaling to cellular growth. It is still
unclear what residues on Maf1 respond to stress, change it’s subcellular location,
and modify it’s activity. Here we have identified a highly conserved but previously
uncharacterized part of the Maf1 protein with residues that possibly alter Maf1
subcellular localization and function. Our results shed more light on the highly
complex regulation of the Maf1 protein and help us understand how stress and
cellular growth are coordinated.

95

Introduction

Maf1 protein domains:
Pluta et al. [1] first identified yeast Maf1 and it’s potential orthologs in humans,
animals, plants, and less complex eukaryotes—but not in prokaryotes. Maf1
proteins in all species shared three regions of high similarity, which were named
A, B and C boxes (Fig 1). The motifs defined in these regions are unique to Maf1
and did not contain sequences linked to any known functions. Pluta et al. did
predict Maf1 to have nuclear targeting signals, but these were characterized later
by Moir et al. [2] and found to be functional as well. Residues in the NLS closer to
the N-terminal, played a more dominant role in redistribution of Maf1 under
repressing conditions; on the other hand, residues in NLS closer to the C-
terminal, seemed to cause aberrant distribution of Maf1 even under normal
conditions. Reina et al. [3] used mutation analysis on human Maf1 to reveal that
the A box is required to interact with the Pol III subunits and the B box is required
for interaction with Brf1, thus defining a more specific role for these highly
conserved regions. Although parts of Maf1 can influence it’s localization and it’s
function, more detail on specific residues and domains in Maf1 is needed to
better define the role of Maf1 in coordinating stress and cellular growth.
.

96

Fig. 1. Maf1 protein contains regions of high similarity. Maf1 proteins
contain highly conserved amino acid residues in A-box, B-box and C-box. RPC1,
RPAC2 are subunits of RNA Pol III complex. A-box has all the annotated kinase
sites for TORC1 (Target of Rapamycin Complex 1). Brf1 is a part of the TFIIIB
complex; A-box and B-box are both required for Maf1 to bind to Brf1.

Acidic Tail
C B A
Maf1
-Binding to RPC1/RPAC2
-TORC1 phosphorylation
sites
Binding to Brf1 (in the Pol III complex)
A
A+B

97

Maf1 as a mediator of pathways that repress protein synthesis:
In yeast, where Maf1 was initially characterized, stress induced by various
conditions has been shown to require Maf1 in order to repress Pol III
transcription. Chemical inducers of cellular stress, capable of activating Maf1
include: tunicamycin (which inhibit’s the secretory signaling pathway),
chlorpromazine (CPZ) (which induces membrane stretching), rapamycin (which
mimics nutrient limitation), methyl mathanesulfonate (MMS) (which induces DNA
damage) Other conditions like carbon source starvation, endoplasmic reticulum
stress, and oxidative stress also required Maf1 to repress RNA Polymerase III
transcription [4]. Human Maf1 has been shown to respond to MMS-induced DNA
damage stress and rapamycin-induced nutrient stress [3].

Regulation of Maf1 activity/subcellular localization:
Maf1 is a serine rich protein--these residues comprise 15.7% of the
protein in yeast, 11.4% in C. elegans, and 12.5% in humans. It has been
biochemically shown that Maf1 exists in different phosphorylation states, and in
response to different stresses, the loss of phosphorylated bands has been
observed [5]. While the dephosphorylation of Maf1 is thought to cause nuclear
localization where it actively function as a transcriptional repressor, at least one
other study has shown that it is possible to alter Maf1 activity without actually
affecting it’s sub-cellular localization [2],[5],[6]. Of the kinases that have been
implicated to phosphorylate Maf1, TORC1 has been identified by multiple groups
to be a Maf1 kinase in yeast [7] drosophila [8] and mammals [9]. Protein kinase A

98

(PKA) sites have been identified on yeast, fly and mammalian Maf1, but shown to
be functionally responsive to PKA only in yeast. Interestingly, on mapping the
kinase sites on Maf1 that have been experimentally shown to be phosphorylated
in vitro, it was found that all identified sites are clustered in the A-box of the
protein. Given the fact that modification on some of these serine residues has
shown to either alter Maf1 localization or Maf1 activity, it is likely that the highly
conserved B-box and C-box also contain serine residues that could regulate
maf1 localization or activity or both.

99

Material and Methods

Standard Growth Conditions
C. elegans were raised on standard 6 cm nematode growth media plates
supplemented with streptomycin and seeded with Escherichia coli OP50. For
RNAi experiments NGM plates containing 5 mM IPTG and 100 ug
ml
−1
carbencillin were seeded with overnight cultures of double-stranded RNAi-
expressing HT115 bacteria. Plates were allowed to induce overnight followed by
transfer of age-synchronous populations of C. elegans. All behavioral and
metabolism analyses of mutant animals are fed E. coli OP50, while all RNAi
based assays are done in animals fed HT115. Animals were fed either food
source for at least two generations before analysis to avoid diet dependent
effects.

Strains use in this study
N2 Bristol (wild type), SPC328[mafr-1p::mafr-1-GFP::mafr-13’UTR(laxIs004)],
CF1038[daf-16(mu86)], mafr-1 ΔC [tm6082].

100

Cell lines and culture conditions
293T cells were cultured in DMEM supplemented with 10% fetal bovine serum.
At 50-70% confluence, cells were transfected with vectors harboring human Maf1
C. elegans mafr-1/ mafr-1 ΔC genes by using Lipofectamine 2000 (Life
Technologies). After twenty-four hours, cells were washed and collected in Trizol
reagent (Invitrogen) for RNA extraction.

Gene Expression
Developmentally synchronous worms of indicated genotype and developmental
stages were collected, washed in M9 buffer and then homogenized in Trizol
reagent (Invitrogen). RNA was extracted according to manufacturer’s protocol.
DNA contamination was digested with DNase I (New England Biolabs) and
subsequently RNA was reverse-transcribed to cDNA by using the SuperScript®
III First-Strand Synthesis System (Life Technologies). Prepared RNA was further
purified using the RNAse easy kit. Samples were hybridized to
Affymetrix C. elegans Gene 1.0 ST Arrays. Data and statistics were analyzed
with Partek Genomics Suite Software version 6.6.
Quantitative PCR was performed by using SYBR Green (BioRad). The
expression of snb-1 was used to normalize C. elegans RNA samples and
GAPDH from human cell culture samples. The efficiencies of all primers used
were within 5% of each other. These genes showed minimal variation after

101

changes in mafr-1/Maf1 expression. All samples were run in triplicate for
quantifying the steady state mRNA levels. Human Pol III primer sequences were
taken from Johnson et al (Johnson et al.)

Immunofluorescence
293T cells were treated with 4% formaldehyde for 15mins at room temperature
then rinsed twice with cold PBS and then treated with 0.25% Triton-X/PBS for
5mins. Excess Triton-X is washed away by rinsing twice with PBS and then cells
are blocked using 10% normal goat serum (NGS) in PBS for 30mins. Primary
antibody (anti-FLAG M2) was added 1:1000 in 10% NGS for 2hours at 37
degrees. After rinsing the cells twice with PBS, the secondary antibody (goat
anti-mouse IgG Alexa fluor 488) was added 1:500.

Statistics
Data presented reflect biological replicates as indicated in each sample’s n.
Sample sizes were determined to reliably reveal the statistic significance given
the magnitude of the changes expected in each experiment. No randomization
was used. For Nile Red staining, strain genotype was blinded prior to staining
and only decoded after quantification of all samples was complete. Data were
presented as mean ± SEM, analyzed by using unpaired student t test. P < 0.05
was considered as significant.

102

Results

MAFR-1 CTD regulates it’s physiological functions
We obtained a mafr-1 deletion mutant that is missing the C-box (mafr-1
ΔC), a highly conserved yet uncharacterized region on the Maf1 protein. As this
allele contains a significantly large deletion of a highly conserved region, we
initially obtained this mutant for use as loss of function allele. However, upon
further investigation we discovered that this mutant did not phenotypically
resemble our mafr-1 RNAi. mafr-1 O/E and mafr-1 ΔC were both compared to
wild type to look for global changes in gene expression. Surprisingly, both
samples had very similar profiles in terms of both downregulated and
upregulated genes when compared to wild type. The overlap in the
downregulated genes between the two mutants can be seen in Fig 2. GO term
analysis of these genes showed enrichment in the same categories as seen in
(Chapter 2, Table 1). Intriguingly, even the genes that didn’t overlap were found
to regulate the same biological processes

The highly similar pattern of gene expression in these two strains drove
the hypothesis that the mafr-1 ΔC strain was behaving like a gain-of-function
mutant. As such, we investigated if this strain also behaved similar to the mafr-1
o/e strain on a physiological level. Our previous work has shown how mafr-1
levels can impact RNA Polymerase III transcription, reproduction and lipid
storage (Khanna et al., 2014).

103

Fig. 2. Gene expression profiles of downregulated genes mafr-1 O/E strain
and mafr-1 ΔC strain when compared to wild type strain N2.

24
380
32
5
mafr-1 O/E ( - 2 fold)
mafr-1 ΔC ( - 2 fold)

104

When the same experiments were performed on the mafr-1 ΔC strain, all three
processes seemed to be negatively impacted in the same way as in the mafr-1
O/E strain (Fig. 3A-C). We concluded that the mafr-1 ΔC strain mimics the mafr-1
O/E strain and is likely a gain–of-function mutant.

MAFR-1 CTD is a regulator of subcellular localization
The RNA Polymerase III repression activity of Maf1 predicts Maf1 to be
predominantly nuclear, where RNA pol III transcription occurs, in response to
stress. Because the mafr-1 ΔC mutant can repress Pol III transcription even in
the absence of stress, we hypothesized that this mutant could have altered
subcellular localization. We generated a C-terminal 3xFLAG tagged wild type
MAFR-1 and MAFR-1 ΔC cDNA expression construct to transfect in 293T cells
and looked the resulting subcellular localization of the MAFR-1 protein.

Immunofluorescence staining with anti-FLAG monospecific antisera in
293T cells transfected with the wild type MAFR-1 revealed staining in both
nucleus and cytoplasm. In contrast, cells transfected with the MAFR-1 ΔC
containing construct, staining was observed to be predominantly nuclear (Fig.
4A). Biochemical analysis using the nuclear and cytoplasmic extracts of these
cells (Fig. 4B) showed ~40% of the wild type MAFR-1 protein was seen in the
nucleus and 60% in the cytoplasm, while more than 90% of MAFR-1 ΔC protein
was seen in the nucleus with very little in the cytoplasm (Fig. 4C). This was an
interesting observation because our results indicate that even in the absence of

105

Fig. 3. MAFR-1 CTD regulates it’s physiological functions. The mafr-1 ΔC
strain mimics the mafr-1 O/E strain in terms of RNA Polymerase III transcription
(A). Total progeny number (B). and Total store lipids (C). Data are presented as
mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001 versus respective controls.

Reproduction Stored lipids Pol III Repression
A. B. C.

106

Fig. 4. MAFR-1 CTD is a regulator of subcellular
localization. The mafr-1 ΔC strain is predominantly
nuclear. Immunofluorescence staining using anti-FLAG
in 293T cells in (A). Biochemical separation of the
nuclear and cytoplasmic extracts after transfecting with
wild type MAFR-1 and MAFR-1ΔC. Western blot in (B). and quantification of the
nuclear/cytoplasmic distribution of MAFR-1 in (C). Green signal is from the FLAG
tag, red signal is from the DAPI nuclear stain.

FLAG
Hsp90
B

Sp1

Nuclear
fraction
Cytoplasmic
fraction
A
.
B
.
C
.
MAFR-1 MAFR-1ΔC

107

stress, this mutant localizes to the nucleus and is active, as we have seen that it
can effectively represses RNA Pol III transcription in C. elegans.

MAFR-1 CTD has conserved residues that alter localization/function
We were curious how this highly conserved region might be altering
subcellular localization. To investigate this, we aligned the deleted residues in the
MAFR-1ΔC with human Maf1 (Fig. 5). These regions on the two proteins have a
higher similarity to each other as compared to the two full proteins (45% similarity
vs 33% on the whole protein). Because Maf1 localization and function are
influenced by phosphorylation we mutagenized the conserved serine residues in
this region to look at how they affect MAFR-1 localization and function. We
mutagenized the conserved serine at 182 to aspartic acid (S182D) and also an
adjacent serine at 185 (S185D) Fig 6.

Next we looked at how these mutants might affect MAFR-1-dependent
cellular functions. To test this, we transfected 293T cells with these mutagenized
constructs and measured the expression of MAFR-1-dependent RNA Pol III
transcripts and select RNA Pol II targets TBP, ACC1, FASN. While wild type
MAFR-1 can suppress all tested RNA Pol III and Pol II targets (Fig. 7 A-B), the
S182D and S185D could not as effectively suppress these same targets.
Moreover, these mutants failed to suppress any MAFR-1 sensitive RNA Pol II
targets (Fig. 7B).

108

Fig. 5. MAFR-1 CTD has high similarity to the human Maf1 Protein.
The deleted region of MAFR-1 ΔC shows 45% identical residues when aligned
with the same region in human Maf1. The top left represents the first amino acid
number where the deletion starts and top right shows where the deletion ends.
Identical residues are shown in boxes and residues of similar chemical nature
are indicated by the same color.

219
21
9

165
21
9

109

Fig. 6. Serine
182
, Serine
185
regulate subcellular localization.
Immunofluorescence of 293T cells transfected with wild type MAFR-1
(A) MAFR-1 S182D (B) and with MAFR-1 S185D (C). Green signal is from the
FLAG tag, red signal is from the DAPI nuclear stain.

MAFR-‐1::FLAG
MAFR-‐1::FLAG DAPI MERGED
S185D::FLAG DAPI MERGED
S182D::FLAG DAPI MERGED
A.
B.
C.

110

Fig. 7. Serine
182
, Serine
185
regulate MAFR-1 function. 293T cells were
transfected with the indicated construct and the abundance of RNA Pol III
transcripts (A) or select RNA Pol II targets (B) was measured by qPCR. Data are
presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P<0.001 versus respective
controls.

A.
B.

111

Discussion

Maf1 proteins contain three regions—A, B, C—that share highly
conserved sequences across all organisms from yeast to mammals. These
regions are unique to Maf1 and do not contain any recognizable motifs that can
be attributed to any known function. The A-box region has been shown to be
necessary for human Maf1 to bind to RPC1 subunit of Pol III, while A and B box
regions together are necessary for binding to Brf1 in TFIIIB (Oficjalska-Pham et
al., 2006). Moreover, previous studies used mass spectrometry to identify
phosphorylated residues on human Maf1 and found several serine residues
located only in A-box. These were subsequently shown to be targets of TORC1
(in yeast and mammals); however, the role of all highly conserved serine
residues in B-box and C-box remains unknown.
Contradicting reports on residues that alter Maf1 localization or Maf1
function have highlighted the complexity of Maf1 regulation. Although some
information is available on the functional importance of the A-box and B-box
domain in Maf1, there is no report highlighting the relevance of the C-box. Given
the fact that these previously characterized serine residues in the A-box have not
fully explained either Maf1 localization or function, it remains to be seen if the
other conserved serine residues in B or C-box might have a role to play in
regulating Maf1 activity. Moreover, the long-term physiologic response to
mutants that lack either of these boxes could be an interesting area to look at.

112

Previous protein interaction screens in yeast have not revealed Maf1 interacting
partners which could be another level at which Maf1 integrates nutritional and
stress signaling to growth. We have identified residues in the C-terminal domain
(CTD) of Maf1, when mutated seem to have reduced capacity to repress Pol III
and Pol II function. This has been an interesting finding as there are no reports
that show any residue in the C-Box to affect Maf1 activity. Intriguingly, our
preliminary data shows no change in the overall phosphorylation status of MAFR-
1 in these mutants, indicated the possibility of a phosphorylation independent
mechanism of regulating Maf1 activity. However, it remains to be seen how the
mutants in the CTD respond to stress under conditions that dephosphorylate
Maf1. Looking at how CTD affects protein stability could help us understand how
the C-box functions to regulate Maf1 activity. Finding proteins that interact with
MAFR-1/Maf1 could help us better understand its regulation.

Our studies of the mafr-1 ΔC strain suggests that the C region represents
a regulatory domain that potentially aids in inhibiting Maf1 function. We have
recently identified novel physiological roles of C. elegans mafr-1 in impacting lipid
homeostasis and reproduction, and interestingly, even in the absence of the
C-box, the MAFR-1 ΔC mutant can still negatively impact both lipid homeostasis
and reproduction. This gain-of-function (gof) mutant mimics mafr-1
overexpression, maintaining it’s canonical function in addition to it’s affect on the
worm physiology. One reason why this mutant mimics a gof mutant could be that
it contains sites that are modified in times when Maf1 activity isn’t required i.e.

113

under no stress and these modifications prevent Maf1 to localize to the nucleus.
As MAFR-1 ΔC actively repressed the abundance of the Pol III transcripts in the
worm even in the absence of stress, we hypothesized that MAFR-1 ΔC must be
active; consequently, we looked at it’s localization after transfecting it in 293T
cells. As compared to the wild type MAFR-1 where only 40% is nuclear, more
than 90% MAFR-1 was nuclear in MAFR-1 ΔC mutant. Previous studies have
indicated that an additional activation step is required after Maf1 is localized to
the nucleus in response to stress and that this step is absolutely necessary for
RNA Pol III repression. Here in MAFR-1 ΔC we find that not only is this mutant
predominantly nuclear but also that it is also active, indicating that it’s deleted
region might be the target of some inhibitory signal that represses Maf1 activity.

We were curious to see how this deleted region could be regulating Maf1
sub cellular localization. This region is highly conserved among all organisms
and contains many conserved residues. We mutated the conserved serine
residues to see how they would affect the subcellular localization of the wild type
protein. We were surprised to find that S182 and S185, when mutated to aspartic
acid, made MAFR-1 very cytoplasmic. Although none of the serine residues in
this region have been previously shown to be phosphorylated, it is possible that
phosphorylation of these residues either affects the binding of other
kinases/phosphatases or the binding of MAFR-1 protein to another protein that
sequesters it in the cytoplasm. As expected, both of these mutants when
overexpressed also had reduced RNA Pol III and Pol II function, indicating a

114

potential role of these residues in regulating Maf1 function and subcellular
localization. At this point, we are not certain what the role of CTD might be. We
are in the process of investigating if this deletion affects the overall
phosphorylation status of Maf1, or is there a different post-translational
modification that happens of the CTD making it inactive or that modifications on
the CTD affect the ability of Maf1 to bind to proteins that could help it mediate
changes in its subcellular localization.

115

References

[1] Pluta K, Lefebvre O, Martin NC, Smagowicz WJ, Stanford DR., “Maf1p, a
negative effector of RNA polymerase III in Saccharomyces cerevisiae.” Mol Cell
Biol. (2001); 21: 5031–5040.

[2] Moir RD, Willis IM., “Regulation of pol III transcription by nutrient and stress
signaling pathways.” Biochim Biophys Acta. (2013); 1829: 361–375.

[3] Michels AA, Robitaille AM, Buczynski-Ruchonnet D, Hodroj W, Reina JH, et
al., “mTORC1 directly phosphorylates and regulates human MAF1.” Mol Cell Biol
(2010); 30: 3749–3757.

[4] Desai N, Lee J, Upadhya R, Chu Y, Moir RD, Willis IM., “Two steps in Maf1-
dependent repression of transcription by RNA polymerase III.” J Biol Chem.
(2005); 280:6455–6462.

[5] Oficjalska-Pham, D., Harismendy, O., Smagowicz, W. J., Gonzalez de
Peredo, A., Boguta, M., Sentenac, A., Lefebvre, O., “General repression of RNA
polymerase III transcription is triggered by protein phosphatase type 2A-
mediated dephosphorylation of Maf1.” Mol. Cell. (2006); 22: 623-632.

116

[6] Roberts, D. N., Wilson, B., Huff, J. T., Stewart, A. J., Cairns, B. R.,
“Dephosphorylation and genome-wide association of Maf1 with Pol III-transcribed
genes during repression.” Mol. Cell. (2006); 22: 633-644.

[7] Wei Y, et al., “Mechanisms of regulation of RNA polymerase III-dependent
transcription by TORC1.” EMBO J. (2009); 28(15):2220-30.

[8] Rideout EJ, Marshall L, Grewal SS., ”Drosophila RNA polymerase III
repressor Maf1 controls body size and developmental timing by modulating
tRNAiMet synthesis and systemic insulin signaling.” Proc Natl Acad Sci. (2012);
109: 1139–1144.

[9] Michels AA, Robitaille AM, Buczynski-Ruchonnet D, Hodroj W, Reina JH, Hall
MN, Hernandez N., ”mTORC1 directly phosphorylates and regulates human
MAF1.” Mol Cell Biol. (2010); 30: 3749–3757.

Abstract (if available)

Abstract Obesity has become markedly more prevalent over the past two decades among developed countries however, the molecular events that connect obesity, lipid deregulation and human diseases are still unclear. Mammalian Maf1 was initially identified as a repressor of RNA Polymerase III-transcribed genes and studies have focused on examining it’s role in repressing RNA pol III-dependent targets. Our work surprisingly revealed that Maf1 is also able to directly repress RNA pol II genes, although little is known regarding these specific gene targets. Furthermore, we have identified a novel and conserved function for Maf1 in the maintenance of intracellular lipid pools in C. elegans and human cell culture. These results are the first to define a specific physiologic role for Maf1 in a multicellular organism. Maf1 negatively regulates lipid accumulation, in part, by repressing the expression of lipid biosynthesis genes. We hypothesize that Maf1 is a central node in the maintenance of organismal lipid homeostasis. These studies define Maf1 as an important new player in lipid metabolism and will be critical towards our future understanding of the roles that Maf1 plays in human diseases such as diabetes, obesity, and cancer, which display prominent lipid dysregulation phenotypes. Understanding the regulation of Maf1 will help us understand how these phenotypes are being impacted by Maf1. The Maf1 protein from yeast to mammals has been shown to have three regions of high similarity, but it’s still unclear how these three regions might regulate the signals that Maf1 responds to or the way Maf1 might regulate reproduction and lipid metabolism. We have found that removing the C-box domain from both Maf1 and MAFR-1, results in a constitutively nuclear protein. Moreover, this MAFR-1 mutant seems to act as a gain of function mutant as it represses the RNA Pol III targets in C. elegans just as the mafr-1 O/E strain. This mutant also negatively impacts reproduction and the lipid phenotypes. This domain is highly conserved with the human Maf1 and we believe it contains important residues that have not been implicated in regulating either Maf1 function or localization or both.

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Asset Metadata

Creator Khanna, Akshat (author)

Core Title Characterizing the physiological roles and regulatory mechanisms of Maf1

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 04/28/2015

Defense Date 03/10/2015

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

Tag OAI-PMH Harvest,obesity,RNA pol III,Transcription

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Curran, Sean P. (committee chair), Ehrenreich, Ian (committee member), Laird-Offringa, Ite A. (committee member), Tower, John G. (committee member)

Creator Email aks20588@gmail.com,akshatkh@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-563262

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