A MOLECULAR ‘SWITCHBOARD’-LYSINE MODIFICATIONS AND THEIR

IMPACT ON TRANSCRIPTION

by

ZHENG GANG

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Thesis advisor: Dr. Yu-Chung Yang

Department of Pharmacology

CASE WESTERN RESERVE UNIVERSITY

January, 2006

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

______GANG ZHENG______

candidate for the Ph.D. degree *.

(signed)______Monica M. Montano______

(chair of the committee)

______Paul N. Macdonald______

______David Danielpour______

______Yu-Chung Yang______

______

(date) ______11/09/05______

*We also certify that written approval has been obtained for any proprietary material contained therein.

I would like to dedicate this dissertation to my parents. Though tens of thousands of miles away, they provided me with support, encouragement and care as they always did.

TABLE OF CONTENTS

Table of Contents 1

List of Figures 2

Acknowledgements 5

List of Abbreviations 7

Abstract 9

Chapter 1 Sumoylation and other lysine-targeted modifications

and statement of purpose 11

Chapter 2 ZNF76, a novel transcriptional repressor targeting TBP, is

modulated by sumoylation 34

Chapter 3 Acetylation and alternative splicing regulate ZNF76-mediated

transcription 91

Chapter 4 Sumoylation and acetylation play opposite roles on the

transactivation of PLAG1 and PLAGL2 122

Chapter 5 Pirh2 is regulated by dimerization 180

Chapter 6 Summary and future directions 202

Bibliography 223

1 LIST OF FIGURES

Figure 1-1 The sumoylation pathway 32

Figure 2-1 ZNF76 is a transcription repressor 56

Figure 2-2 ZNF76 interacts with TBP 63

Figure 2-3 The core domain of TBP interacts with ZNF76 72

Figure 2-4 ZNF76 targets TBP to repress p53-mediated transactivation 74

Figure 2-5 PIAS1 interacts with ZNF76 78

Figure 2-6 PIAS1 sumoylates ZNF76 at Lysine 411 83

Figure 2-7 Sumoylation negatively regulates ZNF76’s transcription 88

repression function

Figure 3-1 ZNF76 is acetylated by p300 103

Figure 3-2 P300 suppresses the sumoylation of ZNF76 and 107

its interaction with TBP

Figure 3-3 HDAC1 deacetylates ZNF76 111

Figure 3-4 Acetylation and sumoylation regulate the transactivation 114

of ZNF76

Figure 3-5 ZNF76 is regulated by alternative splicing 118

Figure 4-1 Characterization of the repression domain in PLAG proteins 140

Figure 4-2 Sumoylation pathway is required for the activity of 143

the PLAGL2 repression domain.

Figure 4-3 PIAS1 promotes sumoylation of PLAGL2 146

Figure 4-4 Sumoylation negatively regulates the transcriptional 153

activation of PLAG1 and PLAGL2

2 Figure 4-5 Sumoylation-deficient PLAGL2 localizes to nucleoli 156

Figure 4-6 PLAG1 and PLAGL2 are regulated by acetylation 158

Figure 4-7 Lysine residues responsible for sumoylation/acetylation are 163

important for the transforming activity of PLAG1 and PLAGL2

Figure 4-S1 PLAG1 has a repression domain 165

Figure 4-S2 PLAGL2(238-387) does not have trans-repression function 165

Figure 4-S3 Alignment of PLAG1 and PLAGL2 sequences 167

Figure 4-S4 Sumoylation pathway is required for the activity of the 169

PLAG1 repression domain

Figure 4-S5 The repression domains of PLAG1 and PLAGL2 172

are activated by DNUbc9

Figure 4-S6 K244, K263 and K353 of PLAG1 are sumoylation targets 174

Figure 4-S7 DNUbc9 activates full-length PLAG1 176

Figure 4-S8 PLAG1K244,263,353R but not PLAG1 concentrates in the nucleolus 176

Figure 4-S9 PLAG1 can be acetylated by p300 178

Figure 4-S10 P300 activates PLAG1 178

Figure 5-1 Pirh2 is ubiquitinated 190

Figure 5-2 Pirh2 forms dimers 192

Figure 5-3 Dimerization of Pirh2 enhances its interaction with p53 196

Figure 5-4 PLAGL2 interacts with Pirh2 dimers to stabilize Pirh2 198

Figure 6-1 Differential expression of ZNF76 in breast cancer cells and 215

normal breast tissues in SAGE

Figure 6-2 RNAi for ZNF76 217

3 Figure 6-3 Schematic representation of the sumoylation-deficient 219

PLAGL2 “Knock-in” strategy

Figure 6-4 Schematic representation of the PLAG1AAA transgene 221

construct used to generate PLAG1AAA transgenic mouse strain

4 ACKNOWLEDGEMENTS

I would sincerely like to thank my family, friends, and colleagues whose support

made my Ph.D thesis work possible. Specifically, I would like to express my deep

gratitude to my advisor, Dr. Yu-Chung Yang, department of Pharmacology, Case

Western Reserve University, for her continuous support, encouragement, patience and

helpful critics.

I also own my sincere gratitude to my committee members, including Dr. Paul N.

MacDonald, Dr. Monica Montano and Dr. David Danielpour for their advices, critics,

guidance and helpful discussions. They gave me a better understanding of the importance

of pursuing biological relevance in basic science research.

My thesis work could not be accomplished without the assistance and generous

help from members of Dr. Yu-Chung Yang’s lab, including Dr. Hui Xiao, Dr. Zhan Yin,

Jennifer Haynie, Jin Chung, Yu-Ting Chou, Dr. DaZhong Zhang, Dr. Keman Zhang,

Shweta Madrekar, James Ellinger, Chad Landsettle, Dr. Bing Xu, Dr. Yu Chen, Mona

Turakhia, Dr. Xiaoling Qu, Dr. Jeena Joseph, Dr. Jinying Ning and Eric Lam, who create

a friendly lab atmosphere. Especially, Dr. Keman Zhang was very helpful in providing

purified p300 proteins and protocols for the in vitro acetylation assay; Mona Turakhia

helped to proofread the dissertation; Dr. Jinying Ning’s collaboration in studying Pirh2

dimerization is greatly appreciated.

5 I would also like to thank all faculties, graduate students, post-docs and other

members in the department of Pharmacology for their daily support, help and friendship, which made my Ph.D study not only possible but also enjoyable.

Finally, the financial support of Case Western Reserve University is gratefully

acknowledged.

6 LIST OF ABBREVIATIONS

ATM: Ataxia Telangiectasia-mutated

CBP: CREB-binding protein

CHIP: chromatin immunoprecipitation

CITED2: CBP/P300 interacting transactivator with ED-rich tail 2

DAPI: 4,6-Diamidino-2-phenylindole

DFO: deferoxamine

DN: dominant negative

E1: ubiquitin activating enzyme

E2: ubiquitin conjugation enzyme

E3: ubiquitin ligase

GAPDH: glyceraldehyde-3-phosphate dehydrogenase

GARD: glutamic acid-rich domain

GST: glutathione S-transferase

HA: hemagglutinin

HDAC: histone deacetylase

IFN-γ: interferon-γ

IGF-П: insulin-like growth factor-2

NEM: N-ethylmaleimide

NOS : Nitric Oxide synthetase

PAGE: polyacrylamide gel electrophoresis

PI: propidium iodide

7 PIRH2: p53-induced protein with a RING-H2 domain

PIAS1: protein inhibitor for activated STAT1

PLAG1: pleomorphic salivary adenoma gene 1

PMSF: phenylmethylsulfonyl fluoride

SAGE: serial analysis of gene expression

STAT1: signal transducer and activator 1

SUMO: small ubiquitin-like modifier

TBP: TATA-binding protein

ZNF76: zinc finger protein 76

8 A “Molecular Switchboard”-Lysine Modifications and Their Impact on

Transcription

Abstract

by

ZHENG GANG

Post-translational modifications greatly increase the complexity of the proteome

in eukaryotic cells. Multi-site modification on a protein resembles a dynamic 'molecular

switchboard' and transduces signals to and from different pathways. We are interested in

the regulation and the functional roles of lysine modifications, especially their impact on

transcription.

ZNF76 (Zinc Finger protein 76) was found to be a general transcriptional

repressor targeting TATA-Binding Protein (TBP). Interaction with ZNF76 prevents TBP from being recruited to the target promoters. Moreover, the interaction between ZNF76 and PIAS1 (Protein Inhibitor for Activated STAT1), an E3 ligase in sumoylation, leads to

sumoylation of ZNF76. Interestingly, a sumoylation site of ZNF76, K411, lies in a

critical region for TBP interaction, and sumoylation negatively regulates ZNF76-TBP interaction. In addition, we found that acetylation and alternative splicing of ZNF76 also

regulate its interaction with TBP, suggesting multiple mechanisms exist to regulate

ZNF76-TBP interaction.

9 PLAG1 (Pleomorphic Adenoma Gene 1) and PLAGL2 (PLAG Like Gene 2) are oncoproteins involved in various malignancies. The regulatory mechanisms of their transactivation remain unknown. We found that sumoylation in the middle regions of

PLAG1 and PLAGL2 represses their transactivation. One of the possible mechanisms for sumoylation-mediated repression is the change in protein subnuclear localization.

Moreover, acetylation activates, while deacetylation represses, transactivation of PLAG1 and PLAGL2. Mutations of the sumoylation sites greatly impair the transforming abilities of PLAG1 and PLAGL2. Taken together, sumoylation and acetylation play opposite roles in regulating the transactivation of PLAG1 and PLAGL2.

Pirh2 (P53 Inducible Ring-H2 protein) is one of the E3 ubiquitin ligases for p53.

It was found to form dimers involving both N- and C-terminus. Furthermore, we

demonstrated that the Pirh2 dimer formation promotes its interaction with both p53 and

PLAGL2. Given the importance of Pirh2 in regulating p53 stability, its dimer may be a

valuable therapeutic target in treating Pirh2-overexpression malignancies.

In summary, we studied the molecular mechanisms by which lysine modifications,

such as sumoylation, acetylation and ubiquitination, regulate transcription factors, including ZNF76, PLAG1, PLAGL2 and p53. Our study may provide novel approaches to modulate their activities in associated diseases, Such as AML (Acute Myeloid

Leukemia) which has PLAG1 or PLAGL2 overexpression, or lung cancer which has

Pirh2 overexpression.

10 CHAPTER 1

SUMOYLATION AND OTHER LYSINE-TARGETED MODIFICATIONS

AND STATEMENT OF PURPOSE

INTRODUCTION

Post-translational modification increases complexity of the proteome in

eukaryotic cells, and regulates substrates by affecting their activity, localization, stability,

and/or interactions with other proteins. From a systematic view, the changes in biological

properties following post-translational modifications are context-dependent: they depend

on specific time, location, and presence of other factors. There are two possible

consequences: “gain of function” and “loss of function”. Adding a modification group to

a protein may create a new surface to recruit new factors, or cause a disruption of the existing interaction surface.

A protein can often be modified at multiple sites. Multisite modification resembles a 'molecular switchboard' which regulates protein functions in responding to various signaling pathways, and thus coordinates different signals for the precise control of protein functions. A well-studied example is p53, a tumor suppressor which is mutated in more than 50% of malignancies. P53 can be phosphorylated (Bode et al, 2004), acetylated (Gu et al, 1997), ubiquitinated (Zhang et al, 2001), sumoylated (Muller et al,

2004) and neddylated (Harper et al, 2004). Different modifications may be independent of each other and have different consequences. There is also interplay between these modifications such that one modification event may promote or inhibit the other, or they

11 may have additive effects, thereby regulating protein functions in a quantitative manner

(Schreiber et al, 2002).

To make it even more complicated, one residue of the same protein could have

different modifications. A good example is lysine residues: acetylation, ubiquitination,

neddylation, isgylation and sumoylation all occur on lysine residues. Among them,

sumoylation is biochemically similar to, but functionally distinct from ubiquitination. In

recent years, sumoylation is recognized as an important post-translational modification that regulates protein functions.

BIOCHEMICAL PATHWAYS OF SUMOYLATION AND OTHER UBLS

(UBIQUITIN-LIKE MODIFIERS) CONJUGATION

SUMO, NEDD8 and ISG15 are all ubiquitin-like modifiers (Ubl). SUMO (Small

Ubiquitin-like Modifier) was first identified in mammals. It shares sequence homology and a similar protein conformation (Bayer et al., 1998) with ubiquitin. However, SUMO

is unique due to its N-terminal extension and the distribution of charged residues on its

surface. These differences may explain why SUMO conjugation has distinct enzymatic

cascades and unique functions.

Ubls are conjugated to target proteins by an enzymatic cascade involving an Ubl activating enzyme (E1), an Ubl conjugating enzyme (E2), and an Ubl ligase (E3). SUMO

conjugation involves similar enzymatic steps. The biochemical pathway of sumoylation

12 is shown in Fig.1. First, free SUMO is generated from SUMO precursor by the cleavage of SUMO-specific proteases (SENPs). Then, SAE1/SAE2, a heterodimer acting as E1 enzyme in sumoylation, catalyzes the formation of adenylated SUMO in an ATP- dependent manner. A covalent bond between SAE2 and SUMO forms after cleavage of the SUMO-AMP linkage (Desterro et al., 1999), which is the final step of SUMO activation. Activated SUMO is then transferred to the E2 conjugating enzyme Ubc9, which is the only known E2 in the pathway. Finally with the help of E3, SUMO is transferred from Ubc9 to a substrate protein through an isopeptide bond between a C- terminal glycine of SUMO with a lysine residue of the substrates. Typically sumoylation occurs in a consensus motif, ψKxE (where ψ is a large hydrophobic residue and x is any random amino acid), which directly interacts with Ubc9 (Rodriguez et al., 2001).

Sumoylation E3s probably enhance specificity and efficiency by interacting with other regions of the substrates. Although it is clear that sumoylation of most substrates takes place within the consensus motif, some substrates are modified on lysine residues where the surrounding sequence does not conform to this consensus. It should also be noted that not all proteins containing the ψKxE sequence are modified by SUMO. This indicates that other factors such as subcellular localization or appropriate surrounding sequences on the substrates may be required for modification. In the following we will discuss the different components in the sumoylation pathway in detail, and compare them with those in the ubiquitination pathway.

SUMOs are around 11kDa, but they appear larger and add almost 20 kDa to most substrates in SDS-PAGE. There are four SUMO proteins in mammals: SUMO-1, SUMO-

13 2, SUMO-3 and SUMO-4 (Bohren et al., 2004; Guo et al., 2004). Among them, SUMO-2

and SUMO-3 are highly homologous to each other, and they appear to modify common

substrates and share similar functions. SUMO-4 was first cloned as a type-І diabetes susceptibility gene, its function as a modifier is still not clear (Guo et al., 2004). However,

SUMO-1 modification may have different effects from modifications by SUMO-2,

SUMO-3 or SUMO-4 (Muller et al., 2001). For example, topoisomerase II is mainly modified by SUMO-2/3, but not SUMO-1 (Saitoh et al., 2000). Currently, the

mechanisms that determine selective modifications by specific SUMO isoforms are not known, and the functional significance of modifications by specific SUMO isoforms also

remains to be determined. Cells have a large pool of free SUMO-2/3, but not SUMO-1,

since majority of SUMO-1 is conjugated to other proteins. Interestingly, SUMO-2/3

conjugation is induced when cells are subjected to stress conditions, such as acute

temperature change (Saitoh et al., 2000). Thus one possible distinct function of SUMO-

2/3 is to provide a pool of free SUMOs for stress response.

The enzymes involved in ubiquitination and sumoylation are different. Ubiquitin

E1 enzyme is a monomer, whereas the SUMO E1 is a heterodimer. SAE1 resembles the

N-terminus of ubiquitin E1, while SAE2 shares similarity with the C-terminus of

ubiquitin E1 (Gill., 2004). Another unique property in sumoylation pathway is that Ubc9

is the only E2 enzyme, whereas there are dozens of E2s in ubiquitination pathway. Ubc9

shares sequence similarity with ubiquitination E2s, and in earlier studies it was shown to

also act as an ubiquitination E2 (Charkrabreti et al., 1999). Another difference between

ubiquitination and sumoylation is the E3s involved. There are hundreds of ubiquitination

14 E3 ligases in the ubiquitination pathway, while only a few E3s have been identified in the

sumoylation pathway (Johnson., 2004). A well-studied E3 is RanBP2, which localizes to

the nuclear pore and promotes the sumoylation of RanGAP1. RanBP2 is responsible for

the localization of SUMO-RanGAP1 to the nuclear pore by interacting with SUMO-

RanGAP1 and Ubc9 (Pichler et al., 2002). In mice and humans, the PIAS proteins

(PIAS1, PIASxα, PIASxβ, PIASγ, and PIAS3), which were first identified as

transcriptional regulators (Liu et al., 1998), are E3 sumoylation ligases. They all contain

the RING domains, which is similar to ubiquitination E3s. Among them, PIAS1 (Protein

Inhibitor for Activated Stat1) was originally shown to be a transcriptional repressor for

Stat1 (Liu et al., 1998) and the first known sumoylation E3 ligase in mammals (Kahyo et

al., 2001). It was shown that the global pattern of sumoylation did not change

significantly in both PIAS1−/− (Liu et al., 2004) and PIASγ−/− mice (Roth et al., 2004),

suggesting that there may be redundancy in sumoylation E3 ligases. PIAS proteins differ in their C-terminal tails, which may contribute to their unique properties and functions.

Unlike E3s in ubiquitination, PIAS proteins lack substrate specificity. In fact,

sumoylation of many substrates can be promoted by different PIAS proteins. S. cerevisiae contains two PIAS family members, Siz1 and Siz2. Siz1 sumoylates septin family cytoskeletal proteins and the replication factor PCNA, while Siz2 sumoylates other proteins (Johnson et al., 2000). Siz1 and Siz2 double knockout mutants have significant growth defects not seen in either single mutant, suggesting that like PIAS proteins, Siz1 and Siz2 have redundant functions (Johnson et al., 2000). HZimp10 is another PIAS-like

SUMO E3 ligase. It colocalizes with AR and SUMO-1 and promotes SUMO modification of AR (Sharma et al., 2003). Polycomb group (PcG) protein Pc2 (Kagey et

15 al., 2003) is another E3 ligase identified. The complexes formed by PcG proteins have

histone methylation activities, and participate in transcriptional repression. The

transcription repressor CtBP associates with Pc2, which stimulates sumoylation of CtBP

in vitro and in vivo. In short, though sumoylation and ubiquitination share similar pathways, the enzymes involved are different.

A unique feature of sumoylation is that it is a highly reversible process. SUMO-

specific proteases (SENPs) generate free SUMO molecules from both target proteins and

SUMO precursors, making the modification reversible and providing free SUMO

molecules for other conjugation processes. Since cellular free SUMO-1 level is low, both

sources of free SUMO are important for SUMO conjugation. Seven genes in mammalian

genome encode potential SENPs. Their N-terminal domains are different, while their C-

terminal domains are conserved and possess cleavage activity (Mossessova et al., 2000).

They localize to different subcellular compartments, which indicates that they may have

different substrate preferences. For example, SENP3 localizes to the nucleolus (Nishida

et al., 2000), while SENP6 (SUSP1) mainly localizes in the cytoplasm (Kim et al., 2000).

Due to the presence of SENPs, it is difficult to detect the sumoylation of target proteins.

In a majority of the literature, researchers have to overexpress SUMO to enhance the

sumoylation signals, and use denaturing buffers or NEM (N-ethylmaleimide) to inhibit

the activities of SENPs when preparing the cell lysates.

For ubiquitination, there are monoubiquitination and polyubiquitination, which

have different consequences. For example, polyubiquitination of p53 mediates its

16 degradation, but monoubiquitination regulates its nuclear export (Li et al., 2003).

Moreover, since multiple lysines in ubiquitin can each serve as a site of ubiquitin attachment, ubiquitination polychains are structurally and functionally diverse. The best known example is lysine 48-linked polyubiquitin chains, which mediates proteasome- mediated degradation. The functions of other forms of ubiquitin polychains are still not clear. Similarly, sumoylation also has mono- and poly-forms. Unlike SUMO-1, which only exists in mono-form in conjugation, the other three SUMO proteins may form poly-

SUMO chains. Although poly-SUMO chains are detected in S. cerevisiae, their precise function remains unknown; yeast cells that cannot form poly-SUMO chains have no detectable phenotypes (Bylebyl et al., 2003).

BIOLOGICAL CONSEQUENCES OF SUMOYLATION

Investigation of the regulation and function of SUMO modification is an exciting field. Although the list of the enzymes and the substrates identified in the sumoylation pathway has grown rapidly, the study of the impact of sumoylation on various biological processes is still in its infancy. The functions of sumoylation are diverse. They include,

but are not limited to, the regulation of subcellular localization, protein-protein interaction,

DNA-binding and/or transactivation function of target proteins. Although both

ubiquitination and sumoylation have important mechanistic similarities and represent

conserved pathways, their physiological consequences are different. Ubiquitination is

generally, but not always, associated with proteasome-mediated protein degradation,

17 whereas sumoylation has diverse biological functions which are highly dependent on individual substrates.

Sumoylation affects cellular growth and division

Sumoylation pathway is required for cell viability in yeast and higher eukaryotes

(Hayashi et al., 2002), and it is involved in the process of mitosis to regulate proper distribution of chromosomes into replicated cells. Misregulation of this process was one of the phenotypes described in SUMO-1 mutants in yeast, with abnormal mitosis and defects in chromosomal segregation (Tanaka et al., 1999; Biggins et al., 2001).

Sumoylation is also involved in the maintenance of the kinetochore, which forms at the centromere and recruits microtubules for anaphase separation. In S. cerevisiae, SUMO-1 has been shown to be a genetic suppressor of yeast homologue of CENP-C (Centromere- specific protein C), a protein which links centromeric DNA to the kinetochore (Meluh et al., 1995). Though these genetic approaches revealed that the sumoylation pathway is important in regulating cell division and viability, the exact mechanisms involved remain to be investigated.

Sumoylation affects protein localization

Sumoylation is most often implicated in regulating cellular localization of target proteins. For example, RanGAP1 localizes to nuclear pore complexes after sumoylation

(Matunis et al., 1998), and nuleus import of Smad4 is linked with its sumoylation (Lin et al., 2003). Another example is NFAT-1, a transcription factor important for regulating the expression of cytokine genes. Sumoylation of NFAT is required for its nuclear

18 translocation (Terui et al., 2004). There are also cases where sumoylation is involved in

protein translocation to nuclear bodies. PML is sumoylated (Kamitani et al., 1998), and

its sumoylation is essential for the formation of PML nuclear bodies. Proteins such as

CBP, Sp100, and Daxx colocalize with wild-type but not sumoylation-deficient PML,

suggesting that sumoylation of PML is required for recruitment of other factors to the

PML nuclear bodies. Interestingly, many proteins in the PML nuclear bodies are also

sumoylation targets, such as p53 (Kwek et al., 2001), LEF1 (Sachdev et al., 2001), Sp100

(Zhong et al., 2000) and Daxx (Jang et al., 2002). There is also evidence that sumoylation

could regulate nuclear export of some substrates. For example, nuclear sumoylation of

Dictyostelium Mek1 is responsible for its movement to the cytoplasm (Sobco et al., 2002).

Mutation of a sumoylation site of the TEL protein leads to increased levels of this protein

in the nucleus, suggesting that sumoylation may be required for its nuclear export (Wood

et al., 2003). Though sumoylation is known to regulate nuclear translocation, nuclear

export, and subnuclear localization, the exact mechanisms involved remain unclear.

Sumoylation affects transcription factors

Most of the sumoylation substrates identified so far are involved in regulating

transcription, including transcriptional activators, repressors, coactivators and

corepressors. Transcription factors that are regulated by SUMO modification include Sp3

(Ross et al., 2002), c-Jun (Schmidt et al., 2002), c-Myb (Bies et al., 2002), IRF-1

(Nakagawa et al., 2002), androgen receptor (AR) (Nishida et al., 2002), STAT1

(Ungureanu et al., 2003), catenin-TCF/LEF (Yamamoto et al., 2003), p300 (Girdwood et

al., 2003), CEBPα (Subramanian et al., 2003), SREBPs (Hirano et al., 2003),

19 progesterone receptor (PR) (Chauchereau et al., 2003), Elk-1 (Yang et al., 2003) and

huntingtin (Johnson et al., 2004; Muller et al., 2004). These transcription factors are involved in various signaling pathways, including those mediated by cytokines and steroid hormones (Muller et al., 2004; Verger et al., 2003).

A majority of sumoylation-targeted transcription factors were shown to be

repressed by sumoylation (Ohshima et al., 2004; Muller et al., 2004). One of the

mechanisms for sumoylation-mediated repression is through “synergy control motifs”,

which were originally identified in the glucocorticoid receptor (GR) as motifs that

negatively regulate GR-dependent transcription (Iniguez-Lluhi et al., 2000). An important

feature of the synergy control motif is that it contains a ψKXE sequence that is

sumoylated, and it only affects promoters which have several transcription factor-binding

elements, suggesting that sumoylation reduces the positive synergistic effect of multiple

receptors on the same promoter. Similarly, the sumoylation motifs of some other

transcription factors, such as Sp3, c-Myb and C/EBP, are located within an inhibitory or

negative regulatory domain, and mutation of the sumoylation sites in these transcription

factors has been shown to increase their transcriptional activity (Iniguez-Lluhi et al.,

2000).

Another possible mechanism of sumoylation-mediated transcriptional repression

is through recruitment of other factors. SUMO itself, when on a promoter, has a

repressive effect on the transcription (Yang et al., 2003), which suggests that other factors

are recruited by SUMO to repress transcription. Some findings suggest that sumoylated

20 substrates may recruit class I and class II HDACs that mediate transcriptional repression.

For example, sumoylation of p300 could mediate repression of gene activities by

recruitment of the corepressor HDAC6 (Girdwood et al., 2003). SiRNA-knockdown of

HDAC6 relieves sumoylation-mediated transcriptional repression by p300. Similarly,

HDAC2 is responsible for the repressive activity of sumoylation on ELK-1 (Yang et al.,

2004). AR interacts with the corepressor SMRT, and is part of a HDAC1-containing complex (Dotzlaw et al., 2003). Mutation of the sumoylation site in AR abolishes SMRT

binding, suggesting that sumoylation of AR is required for its association with SMRT and

HDAC1. Similar data shows that histone H4 sumoylation mediates transcriptional

repression through the recruitment of HDAC1 and HP1 (Shiio et al., 2003). However, for

methyl-transferase 3a (Dnmt3a), sumoylation disrupts its ability to interact with

HDAC1/2, thus abolishes its capacity to repress transcription (Ling et al., 2004). In fact,

the most common function of sumoylation is to change the interactions with other factors.

SUMO itself could interact with other proteins, or both SUMO and the substrate could

contribute to determinants of the interaction interface, or SUMO changes the

conformation of the substrate, thus exposing or hiding the binding site. Though SUMO itself has transcriptional repression function (Shiio et al., 2003), for most of the substrates,

there is a collaboration between both SUMO and the substrate for new interactions.

Although sumoylation of most transcription factors results in repression, SUMO

modification appears to also have positive effects on transcriptional activation.

Sumoylation of HSF1 and HSF2 is correlated with their localization to PML nuclear bodies and increased DNA-binding affinity (Hong et al., 2001; Goodson et al., 2001).

Tcf-4-dependent transcription is activated by PIASy, and the activation is reduced when

21 the sumoylation sites of Tcf-4 are mutated, suggesting that sumoylation is required for the

activation of Tcf-4 (Yamamoto et al., 2003). Another example of the positive effect of

sumoylation on transcription is DJ-1, which positively regulates AR activity and is

modified by SUMO. Mutation of the sumoylation site destroys the capacity of DJ-1 to

positively regulate AR activity (Shinbo et al., 2005). It should be noted that in many cases

the function of sumoylation was studied by mutating the lysine residues that are sumoylation sites. However, these lysine residues may play other roles or have other

modifications. Thus, other amino acids in the sumoylation motifs such as glutamic acid

should be mutated to determine whether the assigned function is specific for sumoylation.

Other functions of sumoylation

There are other biological functions for the sumoylation pathway. For example,

the sumoylation pathway is also involved in repairing DNA and maintaining the genome

stability. In response to DNA topoisomerase inhibitors, both DNA topoisomerases I and

II are sumoylated (Mao et al., 2000). Interestingly, after treating cells with DNA

topoisomerase I inhibitor, wild-type but not sumoylation-deficient DNA topoisomerase I exports from the nucleoli and shows a diffuse pattern in the nucleus (Rallabhandi et al.,

2002), suggesting that sumoylation may regulate its subnuclear localization and activity.

Sumoylation is also involved in regulating the function of viral proteins. Two major immediate-early proteins of the human cytomegalovirus and herpesvirus, IE1 and IE2, have been found to be sumoylated (Xu et al., 2001; Gravel et al., 2004). It is expected that more viral proteins will be identified as sumoylation targets, and sumoylation may affect the life-cycle of viruses and regulate host-virus interactions. Recently, sumoylation was shown to operate at the plasma membrane to control ion channel functions. K2P1 is

22 sumoylated at lysine 274, which silences the K+ channel. Removal of the SUMO

conjugation by SUMO protease activates the channel (Rajan et al., 2005).

A notable feature of sumoylation is that only a small fraction of proteins are

sumoylated. Therefore, an important question is how sumoylation regulates protein

functions by modifying only a small fraction of substrates. One possibility is that

symoylation only targets a functionally distinct population of proteins. For example,

transcription factors can only be sumoylated when bound to certain promoters, or

enzymes can only be sumoylated when bound to the substrates. Another possibility is that

sumoylation is highly dynamic, thus forming the cycle of sumoylation-desumoylation.

Thus, only a small fraction of the total proteins will be sumoylated at a certain time point.

A good example supporting the first possibility is thymine-DNA glycosylase (TDG)

(Hardeland et al., 2002). Unmodified TDG removes thymine or uracil at a mismatched

site, and it is detached from the site after being sumoylated. Then desumoylation occurs,

making it available for binding to another mismatched site. In this way, every molecule is

modified, but only a small fraction will be affected at any given time. This model may

also be true for regulating transcription factors. A concept that has developed over the last several years is that transcription is a dynamic process with continual exchange and turnover of transcription factors and other components of the transcriptional machinery.

DNA-binding transcription factors (such as nuclear receptors) and their coregulators are subjected to rapid modifications (phosphorylation, ubiquitination, acetylation, and methylation) (Freiman et al., 2003). The orderly and sequential recruitment of coactivators with different enzymatic activities has also been recognized. Proteasome degradation is directly linked to transcription. It has been proposed that degradation by

23 the ubiquitin-proteasome pathway is important for regulating the interactions of nuclear receptors with the promoter (Reid et al., 2003). Therefore, it is possible that sumoylation in some cases is also linked with transcription. Sumoylation-desumoylation cycle may only occur when target proteins are present on promoters, and the cycle may be responsible for the rapid exchange and turnover of transcription factors.

How sumoylation is regulated remains largely unknown. More importantly, how other modifications interact with sumoylation is not clear. Since lysine residues are the attachment sites for several modifications, which include ubiquitination, sumoylation, neddylation, isgylation and acetylation, it is possible that these modifications may compete for the same sites, thus regulating each other. In fact, Sp3 contains a lysine residue that can be either acetylated or sumoylated (Braun et al., 2001). Interestingly, though phosphorylation occurs at different sites than those of sumoylation, there are interactions between these two modifications in several substrates. In unstimulated cells, sumoylation represses Elk-1-mediated transcription (Yang et al., 2003). After being phosphorylated in a MAPK-dependent manner, Elk-1 is desumoylated and transcription mediated by Elk-1 is activated.

The use of broad proteomic approaches such as mass spectrometry to identify large numbers of new sumoylation target proteins will promote the already rapid discovery in the field. Key areas requiring further investigation include the molecular and biochemical mechanisms by which this modification plays its critical roles in regulating subcellular localization, transcription, chromosome function, and genomic integrity.

24

ACETYLATION: ENZYMES AND FUNCTIONS

Acetylation of the ε-amino group of lysine residues has recently been recognized

as an important covalent post-translational modification to regulate protein functions. It is

a reversible process whose dynamic equilibrium is controlled by both acetyltransferases

and deacetylases. Acetylation is known to occur in histones, transcription factors, and

other proteins. Similar to protein phosphorylation, it influences a wide range of biological processes.

Dozens of proteins have been identified to possess intrinsic lysine

acetyltransferase activity, which can be classified into at least six groups (Sterner et al.,

2000). The first group is Gcn5/PCAF family, which is composed of Gcn5, PCAF and

related proteins. The second group includes p300/CBP family. The third major group of

acetylatransferase enzymes is MYST family members, which include Esa1, Sas2, MOF,

Tip60, MOZ and MORF. Unlike the enzymes in the first two groups, which mainly

function as transcriptional coactivators, MYST proteins play diverse roles in various

biological processes (Yang et al., 2004). For example, Tip60, one of MYST family

members, which acts as transcription repressors for STAT3 (Xiao et al., 2003), is

involved in cellular response to DNA damage (Kusch et al., 2004), and also regulates

stability of other proteins (Logan et al., 2004). Other groups of lysine acetyltransferase

include nuclear receptor coactivators, TAFП250 and TFШC. Moreover, although most of these enzymes were first identified as histone acetyltransferases, they also have enzyme

activities for other substrates.

25

The p300/CBP family has been extensively studied (Giordano et al., 1999). P300 and CBP form a pair of homologous HATs in mammal and function as key regulators of

RNA polymerase П-mediated transcription. Some viral oncoproteins, such as adenoviral

E1A and SV40 large T antigen, specifically target these proteins. The p300/CBP proteins

participate in many physiological processes, including proliferation, differentiation and

apoptosis. The association of viral oncoproteins with p300/CBP abolishes cell growth

control (Chan et al., 2001), promotes DNA synthesis and inhibits cellular differentiation.

Genetic alterations in the genes encoding p300/CBP and their loss of function have been

linked with human diseases. Recent evidence indicates that p300/CBP genes are altered in

various human tumors (Chan et al., 2001), which is consistent with studies on Cbp+/- mice indicating that CBP possesses tumor suppressor activity in the hematopoietic system

(Kung et al., 2000). Moreover, the human CBP locus is in a chromosomal region which is

implicated in certain subtypes of acute myeloid leukemia (Borrow et al., 1996).

Besides lysine acetyltransferases, HDACs (Histone Deacetylases) are also key

enzymes regulating the acetylation status of the substrates. They can be grouped into

different groups. Class І includes HDAC1, 2, 3, 8, which exclusively localize in the

nucleus. Among them HDAC1 is a major player that regulates chromatin acetylation and

gene expression. Class П includes HDAC 4, 5, 6, 7, 9, 10, which are able to shuttle between nucleus and cytoplasm (De Ruijter et al., 2003). Among them HDAC 4, 5, 7, 9 all contain MEF-2 interacting domains (Verdin et al., 2003), and their cellular localization is regulated by phosphorylation. They are all important regulators of MEF-2,

26 which is a family of transcription factors important for muscle differentiation (Mckinsey

et al., 2002). HDAC6 is a cytoplasmic HDAC that is associated with microtubules and has been shown to regulate aggresome formation in response to misfolded protein stress

(Karvaguchi et al., 2003). Class Ш includes Sir2 and its homologues, which are unique in that they require NAD+ for their enzymatic activity (Denu et al., 2005). Sir2 family members are important regulators of diverse biological processes, such as cell survival, aging, transcription, metabolism and apoptosis (Denu et al., 2005). Class IV only includes HDAC11, which shares low sequence similarity with other classes and mainly localizes in the nucleus (Gao et al., 2002).

An important question is how acetylation affects protein functions. It seems that both “loss of function” and “gain of function” are involved. Acetylation may neutralize the positive charge of lysine residues, thus affecting its interactions with DNA, RNA or proteins. For example, acetylation of histones affects the chromatin conformation since the DNA backbone is negatively charged. An example of “loss of function” is Alba, an archaeal chromatin protein. Lys11 of Alba is involved in forming a hydrogen bond important for oligomerization, and acetylation of this site inhibits oligomerization (Zhao et al., 2003). In another case, the lysine residue that is the acetylation target may be part of the catalytic center for an enzyme, and acetylation inhibits the enzyme activity by affecting catalytic center. Moreover, a lysine residue may be subjected to other

modifications such as methylation and Ubl conjugation. These different modifications are

mutually exclusive, thus leading to their potential competition. It was shown that

acetylation directly competes with ubiquitination for the same lysine residues in Smad7

27 to increase protein stability (Gronroos et al., 2002). Acetylation and methylation of histone H3 at Lys9 have different consequences, the former is associated with transcriptionally active chromatin and the latter is linked to heterochromatin (Nakayama et al., 2001).

Acetylation could also result in “gain of function”. Addition of an acetyl group to a lysine residue may create a novel binding site for protein interaction, thus promoting novel protein-protein interactions. For example, bromodomains recognize acetylated lysine-containing motifs, which is similar to SH2 domains that recognize phosphorylated tyrosines. Several transcription regulators have been shown to use bromodomains to recognize acetylated lysines. One such example is that the bromodomain of the transcriptional coactivator CBP (CREB binding protein) that binds specifically to p53 at the acetylated lysine 382 (Mujtaba et al., 2004). Acetylation can also promote protein-

DNA interactions. For example, DNA damage-dependent acetylation by p300 increases the binding of p53 to its DNA binding elements (Gu et al., 1997). Similarly, acetylation of the erythroid-specific transcription factor GATA-1 enhances its function in erythroblasts (Boyes et al., 1998).

STATEMENT OF PURPOSE

PIAS1 was originally identified as an inhibitor for activated STAT1 (Liu et al.,

1998). Later it was shown to be an E3 sumoylation ligase for many substrates. ZNF76, a zinc finger protein with functions largely unknown, was isolated as a PIAS1 interacting protein in a yeast two-hybrid screen. Subsequently, ZNF76 was found to have strong

28 transcriptional repression effects towards p53, STAT1, and other transcription factors in

reporter assays. Our first goal was to study the mechanisms of ZNF76-mediated

transcriptional repression. Given the general transcriptional repression effects observed,

and that TBP (TATA-Binding Protein) is a target for several known general

transcriptional repressors, we proposed to test if ZNF76 functions by targeting TBP.

Further mapping studies were performed to identify the regions essential for ZNF76-TBP

interaction. Moreover, since ZNF76 interacts with PIAS1, we also proposed to test

whether ZNF76 is a sumoylation substrate, and if so, how sumoylation regulates its

function as a transcriptional repressor.

PLAG1 (Pleomorphic salivary Adenoma Gene 1) and PLAGL2 (PLAG-like 2) are oncogenes involved in various malignancies (Aman et al., 1999; Astrom et al., 1999;

Astrom et al., 2000; Castilla et al., 2004; Declercq et al., 2005; Gisselsson et al., 2001;

Hensen et al., 2002; Hibbard et al., 2000; Kas et al., 1997; Landrette et al., 2004; Voz et al., 1998; Zatkova et al., 2004). Thus studying their regulatory mechanisms is of particular interest, because it may lead to novel approaches to modulate the activities of

PLAG1/PLAGL2 in associated malignancies. One of the mechanisms to regulate the activities of transcription factors is through post-translational modifications. Both PLAG1 and PLAGL2 have repression domains in the middle of coding regions, and three sumoylation motifs are conserved within the repression domain. Our first goal was to test whether PLAG1 and PLAGL2 are sumoylation substrates and whether sumoylation in the repression domain is responsible for the repressive activity. Since sumoylation was shown to regulate protein localization, we proposed to test whether sumoylation may

29 affect the nuclear and subnuclear localization of PLAG1 and PLAGL2. Given the importance of acetylation in regulating transcription factors (Gu et al., 1997), we also

examined whether PLAG1 and PLAGL2 are subject to acetylation, and if so, the effects of acetylation on their transactivation. Finally, we proposed to test whether the transformation potentials of PLAG1 and PLAGL2 are regulated by these modifications.

P53 protein is a central player in coordinating cellular responses to stress. Its stability is tightly regulated by several E3 ubiquitination ligases, including E6-AP (Talis et al., 1998), Mdm2, COP1 (Dornan et al., 2004) and Pirh2 (p53-induced RING-H2)

(Leng et al., 2003). Among them, Pirh2 is induced by and interacts with p53 to promote

the ubiquitination and subsequent degradation of p53. Though Pirh2 functions similarly

to Mdm2, little is known about how its activity is regulated. Given the importance of

Pirh2 in regulating p53, studying its regulatory mechanisms is important since it may identify novel therapeutic targets for malignancies with high expression levels of Pirh2

(Duan et al., 2004). In our laboratory, Pirh2 was first identified as a CITED2 (CBP/p300 interacting transactivator with Glutamic acid (E) and Aspartic acid (D)-rich tail 2)

interacting protein in a yeast two-hybrid screen. It was also found that Pirh2 forms SDS-

resistant dimers. We proposed to further characterize Pirh2 dimers, and test how the dimerization affects Pirh2-p53 interaction.

In short, we uncovered different roles of sumoylation in transcriptional activation and repression. Sumoylation modulates a crucial protein-protein interaction for ZNF76 as a transcription repressor; sumoylation also directly regulates the transactivation of two

30 transcription factors, PLAG1 and PLAGL2. Moreover, by studying the regulatory mechanisms for Pirh2, we show that understanding the interaction between modification enzymes and their substrates may lead to novel approaches to regulate the activities of the substrates.

31

Figure 1. The sumoylation pathway.

First the SUMO precursor is processed by a SUMO specific protease to generate mature

SUMO, which is activated by the E1 enzyme (SAE1/SAE2 in humans) in an ATP- dependent manner. Then SUMO is transferred to E2 conjugation enzyme (Ubc9). Finally with the help of E3 ligase, SUMO is transferred from E2 to the sumoylation substrates.

Sumoylation is reversible; SUMO can be deconjugated from the target protein by the action of SUMO specific proteases.

32 Adapted with permission from Mol Cell. 2005 Apr 1;18(1):1-12.

33 CHAPTER 2

ZNF76, A NOVEL TRANSCRIPTIONAL REPRESSOR TARGETING TBP, IS

MODULATED BY SUMOYLATION

(ZHENG ET AL., 2004)

Introduction

Gene transcription is regulated by positive and negative acting factors. Negative

regulation of transcription is an essential mechanism for precise control of gene

expression. So fundamental are such transcriptional repressors that they are necessary for

the viability of yeast (Davis et al., 1992; Kim et al., 1997). One of the mechanisms

repressors employ is by contacting components of the basal transcription machinery. The

TATA-binding protein (TBP), an important component of the basal transcription

machinery utilized by all three RNA polymerases, is a frequent target for transcriptional

regulators. Several general transcriptional repressors that target TBP have been identified,

such as E1A (Song et al., 1997), p53 (Seto et al., 1992), Even-skipped (Um et al., 1995),

NC2 (Goppelt et al., 1996) and Mot1 (Darst et al., 2003). These inhibitors employ

different mechanisms of action (Lee et al., 1998): Mot1 interferes TATA-TBP complexes

by dissociating TBP from DNA in an ATP-dependent manner (Darst et al., 2003); NC2

does not interfere with the TBP-DNA interaction but prevents association of TBP with

other general transcription factors (Goppelt et al., 1996). Most general transcriptional

repressors were initially identified in yeast. Despite the wealth of data demonstrating interactions of TBP and transcriptional repressors in vitro, there is less experimental data defining how they work in mammalian cells and how their transcriptional repression functions are modulated.

34

Sumoylation is one of the important posttranslational modifications that affect

protein functions. In mammalian cells, the SUMO protein family consists of three

members, SUMO-1, -2, and -3. Sumoylation is biochemically similar to ubiquitination

(Muller et al., 2001). SUMO-1 conjugation utilizes a unique E1-activating enzyme

complex termed SAE1/SAE2 (Aos1/Uba2), an E2-conjugating enzyme, Ubc9, and a group of E3 ligases which were characterized recently, including the PIAS family members (Sachdev et al., 2001; Kahyo et al., 2001), RanBP2 (Kirsh et al., 2002), and

PC2 (Kagey et al., 2003). However, the functional consequences of sumoylation are distinct from ubiquitination. Instead of being marked for degradation by ubiquitination, sumoylation has diverse substrate-specific functions. Since both sumoylation and ubiquitination occur at lysine residues, sumoylation has been shown to inhibit protein ubiquitination and enhance the protein stability, as exemplified by sumoylation of CREB

(Comerford et al., 2003) and the NF-κB inhibitor IκBα (Desterro et al., 1998). Several

transcription factors, including p53 (Rodriguez et al., 1999), the androgen receptor

(Nishida et al., 2002), Sp3 (Sapetschnig et al., 2002; Ross et al., 2002) and c-Myb (Bies

et al., 2002), are sumoylation targets and sumoylation alters their transcriptional activities.

Sumoylation also affects protein localization. A transcriptional repressor, CtBP, can be

recruited to PcG complexes after being sumoylated (Kagey et al., 2003).

ZNF76 is a human homologue of Selenocysteine tRNA gene transcription activating factor (Staf) (Mylinski et al., 1998), which is a Xenopus zinc-finger transcription factor known to regulate genes encoding selenocysteine tRNA (tRNAsec)

35 and small nuclear RNA (snRNA), such as U1 and U6. Staf shows 84% and 64% amino acid sequence identity with human ZNF143 and ZNF76, respectively. Recently, ZNF76 was also shown to activate transcription of a molecular chaperonin subunit Ccta gene

(Kubota et al., 2000), suggesting multiple roles of ZNF76 for transcriptional regulation.

The ZNF76 gene is located at chromosome 6p21 in a region associated with a range of phenotypic abnormalities that affect embryonic development, male fertility and neoplasia

(Tripodis et al., 1998; Nilbert et al., 1990).

In this study, we identified ZNF76 as a TBP interacting protein. Through TBP interaction, ZNF76 inhibits p53-mediated gene expression. We also showed that sumoylation by PIAS1 regulates the transcriptional repression function of ZNF76.

Materials and Methods

Reagents and Antibodies-- Anti-Myc (9E10), anti-TBP (sc-273) and anti-p21 (H-164) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-FLAG (M2) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-HA antibody was from BAbCo (Richmond, CA). Anti-GAPDH was from TREVIGEN, Inc. (Gaithersburg,

MD). Anti-ZNF76, a rabbit polyclonal antibody against purified GST-ZNF76, was prepared by Bio-Synthesis, Inc (Lewisville, TX).

Plasmid construction—pGBKT7-PIAS1 and subfragments of PIAS1 in pGBKT7 vector were constructed by insertion of murine PIAS1 cDNA encoding the full-length, or the corresponding PIAS1 fragments into EcoRI and BamHI sites of pGBKT7 vector

36 (MATCHMAKER GAL4 Two-Hybrid system 3, Clontech, Inc). pcDNA-ZNF76-Myc and other Myc-tagged ZNF76 mutants were constructed by insertion of the corresponding cDNA fragments into EcoRI and BamHI sites of pcDNA3.1-Myc-HisB (Invitrogen).

GAL4 AD fusion construct pGADT7-ZNF76, and other ZNF76 mutants were constructed by insertion of the corresponding cDNA fragments into EcoRI and BamHI sites of pGADT7 vector. PGEX4T-1-ZNF76 was constructed using EcoRI and BamH1 sites of pGEX4T-1. All the site-directed mutant constructs were generated by a two-step

PCR method and were confirmed by sequencing. pCMV5-PIAS1 which encodes Flag- tagged full-length PIAS1 was a gift from Dr. Ke Shuai (Liu et al., 1998). The pMT2-F-

TBP vector was a gift from Dr. Cheng-Ming Chiang (Chiang et al., 1993). The plasmid encoding Flag-tagged SUMO-1 was a gift from Dr. Hideyo Yasuda (Kahyo et al., 2001).

Cell culture and transfection: HEK293 cells, human osteosarcoma U2OS cells and HeLa cells were maintained in DMEM medium with 10% fetal calf serum. 293 cells were transfected by the calcium phosphate precipitation method (Xiao et al., 2003). HeLa cells and U2OS cells were transfected with Fugene6 according to manufacturer’s instructions

(Roche).

Transient transfection, immunoprecipitation and western blot analysis: HEK293 cells were transfected by calcium phosphate precipitation method with various plasmid combinations as indicated. Forty-eight hours later, cells were washed with PBS and 1 ml

37 ice-cold lysis buffer (RIPA) (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM

EGTA, 2 mM Na3VO4, 15 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM

phenylmethylsulfonyl fluoride) was added. Cells were lysed for 30 min at 4°C with

++ occasional vortexing. To test Zn -dependent protein-protein interaction, ZnCl2 was

added to a final concentration of 1.3 μM in the lysis buffer. The lysates were collected

into 1.5 ml tubes and cleared of nuclei by centrifugation for 10 min at 14,000 rpm. The

supernatants (whole cell extracts) were incubated with different antibodies for 16 h at

4°C and protein A-agarose beads were added for the last hour. The beads were washed

five times in TNEN buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5%

NP-40 or 0.5% Brij35 if testing endogenous protein-protein interaction, 10 mM Na3VO4,

1 mM phenylmethylsulfonyl fluoride, 1 mM NaF; plus 1.3 μM Zn++ if testing zinc-

dependent protein-protein interaction). Bound proteins were extracted with SDS-PAGE sample buffer, and analyzed by SDS-PAGE followed by western blot analysis with the

ECL detection system used for protein detection. For sumoylation assays, forty-eight hours after transfection, cells were lysed either in RIPA buffer containing 10 mM N- ethylmaleimide or in a denaturing buffer (2% SDS, 10 mM Tris-HCl (pH 8.0), 150 mM

NaCl), and analyzed by SDS-PAGE followed by western blot analysis.

Luciferase assay: Cells were plated and grown overnight before transfection. The total

amount of DNA transfected was adjusted with pcDNA3. Luciferase assay was performed

according to the manufacturer's instructions (Promega). Renilla luciferase internal control

plasmid was cotransfected with the plasmids as indicated. The relative luciferase units were corrected based on renilla luciferase activity. In the assay for p53-mediated gene

38 expression, a reporter containing a synthetic p53-binding site upstream of the luciferase

reporter gene (pG13-luc) was used.

Yeast two-hybrid screen: The MATCHMAKER GAL4 Two-Hybrid System 3 (Clontech,

Inc) was used in the yeast two-hybrid screen. The bait used was PIAS1(1-390) containing

the N-terminus and the zinc finger domain. After screening 1X106 clones, clones which

interacted with the bait, but not lamin, were considered positive. Positive clones were sequenced and analyzed by the BLAST program. In mapping the interacting domains, the interactions were scored by the expression of β-galactosidase in the yeast two-hybrid

mating assay. Yeast extracts were prepared by urea/SDS method according to

manufacturer’s manual (Clontech, Protocol #PT3024-1). Yeast extracts were separated

on SDS-PAGE and transferred to a PVDF membrane. Protein expression was detected by

western blot with indicated antibodies.

Chromatin Immunoprecipitation (ChIP) Assay: Two days after transfection with

indicated plasmids, HEK293 cells (one 150-mm plate) were cross-linked with 1%

formaldehyde in cell culture medium for 10 min at 37 oC, and cross-linking was quenched

with 1.25 M glycine to a final concentration of 0.125 M. Cells were harvested and rinsed

with PBS, and cell pellets were resuspended in 1 ml of cell lysis buffer (5 mM Pipes, pH

8.0, 85 mM KCl, 0.5% Nonidet P-40, 1 mM DTT, 0.25 mM phenylmethylsulfonyl

fluoride, and 1 µg/ml each of pepstatin, leupeptin, and aprotinin). Nuclei were collected

and resuspended in 500 µl of nuclear lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM

EDTA, 1% SDS, 1 mM DTT, 2.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each

39 of pepstatin, leupeptin, and aprotinin). The chromatin samples were sonicated to yield

DNA fragments between 300 and 2000 bp with an average length of about 500 bp. For each immunoprecipitation, 50 µl of sheared chromatin was diluted to 1 ml with IP

dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH

8.1, and 167 mM NaCl). Prior to chromatin immunoprecipitation, the chromatin solution

was precleared with 80 µl of protein A-Sepharose beads (Sigma) containing 240 µg/ml of

salmon sperm DNA and 240 µg/ml of tRNA at 4 °C for 30 min with rotation. The precleared chromatin was collected and incubated at 4 °C overnight with 5 µg of anti-

Flag, 5 µg anti-HA, 10 µl anti-p53, 10 µl anti-TBP or 10 µl anti-Myc antibody as indicated. Anti-Myc was used as a negative control. The immune complexes were

precipitated with 60 µl of protein A-Sepharose beads (Sigma) at 4 °C for 1 h. The

supernatant in the absence of antibody was used as the total chromatin input for later PCR

analysis. The beads were then washed in the following order: 1 ml of IP dilution buffer,

TSE-500 wash (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1,

500 mM NaCl), LiCl/detergent wash buffer (100 mM Tris-HCl, pH 8.1, 500 mM LiCl,

1% Nonidet P-40, 1% deoxycholic acid), and TE (10 mM Tris-HCl, 1 mM EDTA, pH

8.0), twice in each buffer with a 10-min period between washes. The antibody-protein-

DNA immunocomplexes were eluted twice with 250 µl of elution buffer (1% SDS, 50

mM NaHCO3). The sample was vortexed briefly and incubated at room temperature for

15 min with rotation. Formaldehyde cross-linking was reversed by heating at 65 °C overnight with the addition of 5 M NaCl to a final concentration of 200 mM. All of the

samples were digested at 45 °C for 1 h with 10 µg of RNase A and 20 µg of proteinase K.

The DNA fragments were extracted by phenol-chloroform, ethanol-precipitated, and

40 analyzed by PCR. The DNA agarose gels were visualized with the VersaDoc imaging

system from BioRad. The primer pair (55) for the p21 promoter produced a 111-bp

product. The primer sequences are 5'-CCGCTCGAGCCCTGTCGCAAGGATCC-3' and

5'-GGGAGGAAGGGGATGGTAG-3'.

Results

ZNF76 is a PIAS1 interacting protein

We performed a yeast two-hybrid screen to identify novel PIAS1 interacting

proteins. The bait used in the screening was PIAS1 (1-390) containing the N-terminus

and the zinc finger domain. The first reason to use this fragment as the bait was that full-

length PIAS1 has high background and is not suitable as a bait in the yeast two-hybrid

screen. Secondly, the region we chose contains zinc finger domains and four LXXLL

motifs, which are important domains for mediating protein-protein interactions. After

screening 106 clones, eight positive clones were identified and sequenced. Two of them are the cDNA sequences for Homo sapiens thioredoxin domain containing 7

(NM_005742); two for Homo sapiens translocase of inner mitochondrial membrane 50 homolog (yeast) (TIMM50) (XM_053074); one for Homo sapiens DnaJ (Hsp40) homolog (NM_001539); one for Homo sapiens ankyrin-repeat family A protein 2

(AF314032), one for Homo sapiens heat shock 70kDa protein 5 (NM_005347) (protein disulfide isomerase);. Among them there is a clone whose sequence is identical to that of human ZNF76.

41 ZNF76 acts as a transcriptional repressor

Since PIAS1 is an E3 sumoylation ligase for p53 (Kahyo et al., 2001) and an

activator for p53 (Megidish et al., 2002), we tested whether ZNF76 modulates p53

transcriptional activity. As shown in Fig.1A, we titrated the amount of ZNF76 expressing

plasmid in a luciferase assay. A reporter plasmid pG13-luc that has a p53 binding site was transfected into HEK293 cells along with plasmids expressing HA-p53 and Myc-

ZNF76. P53-mediated reporter gene expression was induced ~10-fold by transfecting 10

ng HA-p53 expressing plasmid. Cotransfection of pcDNA-ZNF76 inhibited p53 induced

luciferase expression in a dose-dependent manner. There is a significant inhibitory effect

when only 5 ng of ZNF76 expression plasmid was transfected, and protein expression

could not be detected by western blotting. This argues against the notion that the

inhibition is an artifact due to gross overexpression of ZNF76. In contrast, as shown in

Fig.1B, overexpression of ZNF76(1-375) or ZNF76(342-570) could not repress the transcriptional activity of p53, suggesting that the structural integrity of ZNF76 is

essential for inhibiting the transcriptional activity of p53. Interestingly, transfection of

ZNF76(342-570) increased basal and p53-induced activity in the reporter assays. The

strong inhibitory effect of ZNF76 on p53 was also observed in various cell lines

including HeLa, U2OS (Fig.1C), MCF-7 and H1299 (data not shown). To test whether

ZNF76 also inhibits p53-dependent expression of endogenous genes, we examined

whether ZNF76 could inhibit p53-mediated expression of the p21 protein. As shown in

Fig.1D, ectopic expression of ZNF76 abolished p21 expression induced by p53, but had

no effect on GAPDH expression. These results suggest that ZNF76 is a transcriptional

repressor, which inhibits p53-mediated gene expression. In order to address whether the

42 repressive effect of ZNF76 is selective for p53, we tested the effect of ZNF76 on two

other transcription activators, Stat1 and Smad3. As shown in Fig.1E, human MCF-7 cells were transfected with a luciferase reporter construct, IRF1-luc, containing three copies of

the Stat1 binding sequence and expression vectors encoding ZNF76-Myc and treated with or without IFN-γ. The Stat1-activated luciferase expression in response to IFN-γ

stimulation was significantly inhibited by ZNF76 in a dose-dependent manner. Similarly,

ZNF76 also strongly inhibited Smad3 mediated transactivation (Fig.1F). These results

suggest that ZNF76 is a general transcriptional repressor.

ZNF76 interacts with TBP

One of the frequent targets for transcriptional repressors is the TATA-binding

protein (TBP) and several viral proteins repress p53 transactivation by interacting with

TBP (Massimi et al., 1997; Qadri et al., 2002) or affecting TBP expression (Mauser et al.,

2002). To test whether ZNF76 and TBP complex in cells, pCMV2-Flag-TBP and

pcDNA-ZNF76-Myc were transfected alone or co-transfected into HEK293 cells. Lysates

of transfected cells were immunoprecipitated with anti-Myc or anti-FLAG antibody.

Immunoblots probed with anti-Myc showed that anti-FLAG pulled down Myc-tagged

ZNF76 only when pCMV2-FLAG-TBP, but not the empty vector was present (Fig.2A),

suggesting TBP complexes with ZNF76 in cells. The endogenous interaction of ZNF76

and TBP was further demonstrated in MCF-7 cells by reciprocal coimmunoprecipitations

using anti-ZNF76 and anti-TBP antibodies (Fig.2B).

To define the regions in ZNF76 essential for interacting with TBP, plasmids

encoding wild type or mutant ZNF76 were co-transfected into HEK293 cells together

43 with pCMV2-Flag-TBP. In contrast to full-length ZNF76, mutant ZNF76(1-375)

interacted weakly with TBP. ZNF76(342-570) but not the zinc finger domain

ZNF76(150-375) also associated with TBP (Fig.2C). To confirm these results, we

employed the yeast two-hybrid assay to map the interacting regions. Both yeast growth

on SD-HTL medium and positive β-gal activity were used as criteria for positive

interactions. As shown in Fig.2D, in agreement with the coimunoprecipitation results,

both the N-terminus and the C-terminus, but not the zinc finger domain of ZNF76,

interacted with TBP. Yeast expression of ZNF76 mutant fusion proteins is shown in

Fig.2E. Since the C-terminus of ZNF76 interacts with TBP and can enhance p53-

mediated transactivation in the reporter assay (Fig.1B), we further mapped the interacting

regions in the C-terminus of ZNF76. After examining the sequence, we found a Glutamic

Acid-Rich Domain (GARD) 83 amino acids in length (aa.362-aa.444) in the C-terminus

of ZNF76 (labeled as a bar in Fig.2I and thereafter). We tested whether GARD itself is

sufficient for interaction with TBP in the yeast two-hybrid assay. As shown in Fig.2I,

both yeast growth on SD-HTL and β-gal assays support the interaction between GARD

and TBP. Fusion protein expression in yeast is shown in Fig.2G. Consistently, we found

that ZNF76(342-570)K400,411R, a C-terminus mutant of ZNF76 with two lysine residues in

GARD changed to arginine, lost its interaction with TBP (Fig.2H), suggesting that

GARD is essential for the C-terminus of ZNF76 to interact with TBP. Fig.2I is a

summary of the mapping results. These data demonstrate that ZNF76 interacts with TBP

through both its N-terminus and GARD in its C-terminus.

44 The 180-amino acid C-terminal domain of TBP is commonly referred to as the

TBP core domain, which is responsible for recognizing the TATA box. The core domain of TBP is highly conserved, while the N-terminal region of TBP is more divergent

(Fig.3A). We performed coimmunoprecipitation experiments to map the regions of TBP responsible for interacting with ZNF76. As shown in Fig.3B, plasmids encoding Flag- tagged wild type or mutant TBP were co-transfected into HEK 293 cells together with

pcDNA-ZNF76-Myc. We found that Flag-tagged TBP(146-339) which includes the core

domain of TBP, but not Flag-tagged TBP(1-159), coimmunoprecipitated with Myc- tagged ZNF76. These results suggest that, like other transcriptional repressors that target

TBP such as NC2 (Kamada et al., 2001) and p53 (Liu et al., 1993), ZNF76 interacts with

the core domain of TBP.

ZNF76 targets TBP to inhibit p53 activity

Since ZNF76 interacts with TBP, we tested whether ZNF76 inhibits p53-mediated

gene expression through interaction with TBP. As shown in Fig.4A, ectopic expression of

TBP reversed the inhibitory effect of ZNF76 on p53-mediated transactivation. Although

ectopic expression of TBP modestly increased both basal and p53-activated reporter

activity, the increase was less significant than the increase in the p53 reporter activity in

the presence of ZNF76. This argues against the possibility that TBP reversed the

inhibitory effect through a nonspecific increase in general transcription. We further tested

whether a correlation exists between the ability of ZNF76 to interact with TBP and

repression of p53-mediated transactivation. As shown in Fig.4B, transfection of a

construct encoding GARD strongly enhanced p53 activity in reporter assays, which is

45 similar to the effect of ZNF76(342-570) shown in Fig.1B. We speculate that this GARD-

containing peptide acts in a dominant negative manner to abolish the strong association

between endogenous ZNF76 and TBP. Since we previously showed that ZNF76(342-

570)K400,411R lost its interaction with TBP, we tested the effect of double mutation

(K400,411R) on p53-mediated transactivation. We found that ZNF76(342-570)K400,411R, unlike ZNF76(342-570), lost its ability to enhance p53 activity (Fig.4C). Likewise,

ZNF76K400,411R lost its inhibitory function on p53-mediated transactivation, which is in

agreement with the observation that C-terminus of ZNF76 is required for its

transcriptional repression function (Fig.1B). The strict correlation between interaction with TBP and an effect on p53-mediated transactivation strongly suggests that ZNF76 targets TBP to mediate its transcriptional repression function.

To test whether ZNF76 is present in the promoter of p53 target genes, we performed chromatin immunoprecipitation. As shown in Fig.4D, HEK293 cells transfected with only pcDNA-HA-p53 showed binding of p53 and TBP to the p21 promoter. However, when Flag-ZNF76 was overexpressed, only p53, but not TBP or

ZNF76, was present in the p21 promoter. We also tested if endogenous ZNF76 occupies the p21 promoter using chromatin immunoprecipitation experiments. As shown in Fig.4E, endogenous p53, but not ZNF76, occupied p21 promoter after MCF-7 cells were treated with 200nM adriamycin, a DNA damage agent, for 4 hours. These results suggest that

ZNF76 modulates the binding of TBP with associated transcription factors, but itself does

not occupy the target promoter.

46

PIAS1 is an E3 ligase for ZNF76 sumoylation

To study the molecular basis for the interaction of PIAS1 and ZNF76, the yeast two-hybrid assay was performed to identify interacting domains. Full-length ZNF76 and various deletion mutants of ZNF76 were tested for their ability to interact with the bait:

PIAS1(1-390). As shown in Fig.5A, ZNF76(160-570), but not ZNF76(1-375), interacted with the bait, suggesting that both the zinc finger domain and the C-terminus of ZNF76 are necessary for interaction with PIAS1. Similarly, a series of PIAS1 mutants were tested for their interaction with ZNF76 (Fig. 5B). A PIAS1 mutant with only the N- terminal 1-277 region can interact with ZNF76. Interestingly, deletion of the first 9 amino acids of PIAS1 abolished this interaction. The expression of the fusion proteins in yeast is shown in Fig.5C and Fig.5D. These results demonstrate that the zinc finger domain and the C-terminus of ZNF76 interact with the N-terminus of PIAS1. Co- immunoprecipitation experiments were next performed to confirm the interaction between ZNF76 and PIAS1. We cotransfected Myc-tagged pCDNA3-ZNF76 and Flag- tagged pCMV5-PIAS1 into HEK 293 cells. After 48 hrs, cells were lysed and immunoprecipitated with anti-Myc. Immunoprecipitates were then washed and analyzed by western blots with anti-Flag antibody. Surprisingly, PIAS1 was not in the immunoprecipitates (Fig.5E, left panel). Since we mapped the zinc finger domain of

ZNF76 to be necessary for the interaction in yeast, we tested whether the interaction of

ZNF76 with PIAS1 is zinc dependent by including 1.3 μM zinc chloride in RIPA buffer and TNEN washing buffer. As shown in Fig.5E (right panel), PIAS1 was present in the

47 anti-Myc immunopreciptates only when Zn++ was added, indicating that zinc is necessary for the interaction between PIAS1 and ZNF76.

Since ZNF76 interacts with PIAS1, a well-characterized E3 ligase for sumoylation, we tested whether ZNF76 can be sumoylated by PIAS1. HEK293 cells were transfected with pcDNA-ZNF76-Myc, a Flag-SUMO-1 expression plasmid, with or without the pCMV5-Flag-PIAS1 plasmid, and lysed after 48 hrs. Cell lysates were separated by SDS-PAGE, and a western blot was probed with anti-Myc to detect unmodified and sumoylated forms of ZNF76. As shown in Fig.6A, overexpression of

Flag-SUMO-1 led to the appearance of multiple weak bands that represent different forms of SUMO-1 conjugated ZNF76. Cotransfection of pCMV5-Flag-PIAS1 strongly enhanced the intensity of these bands. To map the regions of ZNF76 that can be sumoylated, we cotransfected plasmids expressing different deletion mutants of ZNF76 with PIAS1 and SUMO-1. As shown in Fig.6B, all the mutants, except ZNF76(1-165), can be sumoylated. Based on the mapping study, both the zinc finger domain and the C- terminus of ZNF76 have at least one lysine residue that can be sumoylated. In the C- terminus of ZNF76, lysine 411 and surrounding sequences are consistent with a conserved sumoylation motif (Zhang et al., 1996), ψKXE. Notably, Lys411 is within

GARD that interacts with TBP. We therefore mutated lysine 411 to arginine

(ZNF76K411R), and tested sumoylation of ZNF76K411R. As shown in Fig.6C, mutation of

Lys411 diminished sumoylation of ZNF76. On the contrary, mutation of another residue, lysine 30, which is also in the conserved motif, had no effect on ZNF76’s sumoylation

(data not shown). To map the rest of lysine residues which can be sumoylated, we made

48 single mutants ZNF76K156R, ZNF76K226R, ZNF76K237R, ZNF76K364R and ZNF76K400R, and double mutants ZNF7K411,156R, ZNF76411,237R, and ZNF76411,400R to test their sumoylation by PIAS1. No difference was detected in the sumoylation pattern of the single mutants and wild type ZNF76 or the double mutants and ZNF76K411R (data not shown). It is possible that other lysine residues are sumoylated or multiple lysines are responsible for the remaining sumoylation bands. These results suggest that lysine 411 is one of the major sumoylation target residues of ZNF76.

Sumoylation regulates the transcriptional repression activity of ZNF76

Since one of the sumoylation target residues, Lys411, lies in the minimal TBP interaction region, GARD, we tested whether that sumoylation by ZNF76 may regulate its interaction with TBP, and thus modulate its transcriptional repression function. As shown in Fig.7A, overexpression of PIAS1 and SUMO-1 significantly inhibited the association between ZNF76 and TBP in coimmunoprecipitation experiments.

Consistently, we found that sumoylation of ZNF76 suppressed its transcriptional repression activity in reporter assays. As shown in Fig.7B, ZNF76 completely abolished p53 activity in the absence of PIAS1 and SUMO-1 overexpression, but its inhibitory effect was reversed when PIAS1 and SUMO-1 were overexpressed. These results suggest that sumoylation of ZNF76 negatively modulates its interaction with TBP and its repression effect.

49 Discussion

TBP is universally required for eukaryotic transcription which can be positively

or negatively modulated by its interacting proteins. Our data support the conclusion that

ZNF76 is a novel TBP associated factor that interacts with TBP through its N- and C-

termini, leading to repression of p53-mediated transactivation and down regulation of

endogenous p53 target genes. The inhibitory effect of ZNF76 on p53-mediated gene

expression can be relieved by ectopic expression of TBP. We also identified sumoylation

as one of the regulatory mechanisms for the transcriptional repression function of ZNF76.

To our knowledge this is the first demonstration of a transcriptional repressor that targets

TBP and the repression activity is regulated by sumoylation. ZNF76 interacts with the N-

terminal region of PIAS1, which sumoylates ZNF76 at Lys411. Lysine 411 lies in an

essential region of ZNF76 for TBP interaction. Consistent with a model in which

sumoylation negatively regulates interaction between ZNF76 and TBP, overexpression of

PIAS1 and SUMO-1 reverse the repressive effect of ZNF76 on p53-mediated

transactivation in the reporter assay.

Our conclusion that ZNF76 targets TBP to exert its transcriptional repression

function is based on several lines of independent evidence: (i) In reporter assays, we

observed a significant repressive effect on p53-mediated transactivation. (ii) TBP is a

target through which ZNF76 exerts its transcriptional repression effect on p53-mediated

gene expression. (iii) Overexpression of ZNF76(1-375), which lacks the C-terminus of

ZNF76, or ZNF76K400,411R, whose C-terminus loses the interaction with TBP, failed to repress p53 transactivation. (iv) We identified an 83-amino acid region in ZNF76 that

50 activates rather than inhibits p53 transactivation. The dominant-negative effect of the

construct suggests that endogenous ZNF76 may contribute to the transcriptional repression effect.

Consistent with our data, several viral proteins inhibit p53 transactivation by targeting TBP. Hepatitis C virus NS5A and human papilloma virus E7 repress p53

transactivation by binding to and possibly sequestering TBP (Massimi et al., 1997; Qadri

et al., 2002). Epstein-Barr virus immediate-early protein BZLF1 regulates p53 function in

part by suppressing TBP expression (Mauser et al., 2002). Since p53 and TBP can

synergistically activate transcription when p53 is bound to DNA (Farmer et al., 1996), it

is not surprising that TBP can be targeted to affect p53 transactivation. Several general

transcriptional repressors function by interaction with TBP. For example, NC2, a well-

conserved heterodimer, binds to and stablizes TBP/TATA complexes (Mermelstein et

al.,). Interaction of NC2 with TBP precludes association of TBP with other general

transcription factors (Goppelt et al., 1996), such as TFIIA and TFIIB. Proteins such as

E1A and Mot1 function as transcriptional repressors by different mechanisms. E1A

represses transcription by disrupting the interaction between TBP and the TATA box

(Song et al., 1997). In the in vitro experiments, the transcriptional repression effect of

E1A can be reversed by the addition of recombinant TBP (Song et al., 1997), which is

similar to our observation that overexpression of TBP relieves the inhibitory effect of

ZNF76 on p53-mediated transactivation (Fig.4A). Mot1 dissociates TBP from DNA in

part through its ATPase activity. In vitro, TBP monomers rapidly and stably bind to

TATA DNA, which is inconsistent with its recruitment being rate limiting. What impedes

51 TBP from binding TATA DNA in vivo is unclear but could be due to dimerization of

TBP (Jackson-Fisher et al., 1999) or interaction with other inhibitory factors. In

chromatin immunoprecipitation analysis (Fig.4D, Fig.4E), we found that ZNF76 was not

present in the promoter of p53 target genes, suggesting that ZNF76 acts differently from

NC2 which stably binds to TBP/TATA complexes to repress transcription. Instead,

ZNF76 prevents the binding of TBP to the promoter in a way similar to the action of TBP

dimmers.

There are DNA binding proteins which repress transcription by interacting with

TBP. These include p53 (Seto et al., 1992), unliganded thyroid receptor (Fondell et al.,

1996), and Msx1 protein (Zhang et al., 1996). Among them the most extensively studied

is p53. Besides acting as a sequence-specific transcriptional activator, p53 can repress the

expression of many genes that lack p53 response elements. The transcriptional repression

effect of p53 is at least in part due to its interaction with TBP (Seto et al., 1992; Mack et

al., 1993). P53 interacts with TBP in vitro and in vivo (Seto et al., 1992; Mack et al.,

1993; Farmer et al., 1996), and the interaction has been mapped to the N- and C-termini

of p53 (Liu et al., 1993; Horikoshi et al., 1995). Both the N- terminal and C- terminal

domains are necessary for p53 to exert its repression function on RNA polymerase II

directed transcription. It was suggested that the interaction of the N-terminal domain of

p53 with TBP contributes to the transcription activation when bound to promoters

(Horikoshi et al., 1995) However, when p53 is not bound to DNA, its N- and C-terminal

domains are needed to interact with TBP to achieve transcriptional repression. Recently,

it was shown that by targeting TBP, p53 represses RNA polymerase I- and III-mediated

52 transcription (Zhai et al., 2000; Crighton et al., 2003). Notably, interaction of ZNF76

with TBP is very similar to that of p53: the N-terminal activation region and C-terminal

region of ZNF76 interact with TBP, although the interactions are much weaker compared

with the full-length ZNF76. Like p53, both N- and C-terminal regions are required for

ZNF76 to repress transcription. Apparently, the interaction of either N-terminus or C-

terminus of ZNF76 alone with TBP is not sufficient to achieve the repression effect.

ZNF143 shows high sequence similarity with ZNF76 in the N-terminus and the zinc finger domains, but has low similarity in the C-terminus (Myslinski et al., 1998).

Consistently, we did not detect significant transcription repression effect for ZNF143

(data not shown). We speculate that during evolution, the sequence divergence in the C- terminal region renders ZNF76 a novel transcriptional repression function through the interaction of its C-terminus with TBP.

Our studies raise the possibility that the repression effects of sumoylation observed on several transcription factors are due to loss of interaction with the general transcription machinery. Sumoylation of transcription factors has varied effects.

Although sumoylation was shown to be a mechanism that inhibits the transcriptional activity of Sp3 (Sapetschnig et al., 2002; Ross et al., 2002 ), c-Myb (Bies et al., 2002) and the androgen receptor (Nishida et al., 2002), the exact mechanism that mediates repression by sumoylation is unclear. Sumoylation of p53 has been shown to alter its transcriptional activity (Rodriguez et al., 1999; Gostissa et al., 1999), however, the data is controversial in part due to the use of different cell types and/or different reporter constructs by different investigators. In our system, p53 was weakly sumoylated but no

53 significant change in p53-mediated transactivation was detected when PIAS1 and

SUMO-1 were overexpressed (Fig.7B). Nontheless, in this study, sumoylation was identified as a regulatory mechanism for the interaction between ZNF76 and a crucial component of general transcription mechinery, TBP. A major site of sumoylation, K411, is located within a region essential for TBP interaction. Our study indicates that one possible mechanism to achieve the repression by sumoylation is to regulate the interaction between transcription factors and general transcription machinery.

In summary, we have identified ZNF76 as a novel transcriptional repressor that targets TBP. Sumoylation of ZNF76 by PIAS1 occurs in its TBP interaction region and negatively regulates the transcriptional repression function of ZNF76. Since TBP is required for expression of nearly all eucaryotic genes, study of the function of ZNF76 as a TBP inhibitor will provide fundamental knowledge about normal cellular processes and how alteration in these processes can contribute to developmental abnormalities and various disease states.

Acknowledgement

We thank Dr. Ke Shuai for pCMV5-PIAS1; Dr. Cheng-Ming Chiang for pMT2-

F-TBP; Dr. Hideyo Yasuda for the plasmid encoding Flag-tagged SUMO-1; Dr. Philippe

Carbon for the plasmids of ZNF76 and ZNF143; and Drs. David Donner and Cheng-

Ming Chiang for reading the paper and helpful discussions.

54 This study was supported by National Institutes of Health Grants DK50570,

CA78433, and HL48819 (to Y.-C. Y.).

55 Figure 1. ZNF76 is a transcription repressor.

A. ZNF76 represses p53-mediated transactivation in HEK 293 cells. HEK293 cells were

transfected with 25 ng pG13-luc reporter, 10 ng expression plasmid pcDNA-HA-p53 and

various amounts of pcDNA-ZNF76-Myc plasmid alone or in combination as indicated.

B. Structural integrity is required for ZNF76 to repress p53-mediated transactivation.

HEK293 cells were transfected with 25 ng pG13-luc reporter, 10 ng of pcDNA-HA-p53

and 0.2 μg of pcDNA-ZNF76-Myc or mutant plasmids alone or in combination as indicated. 15 μl cell lysates in luciferase assays were subjected to western blot analysis with indicated antibodies.

C. ZNF76 inhibits p53-mediated transactivation in HeLa and U2OS cells. Cells were transfected with 25 ng of pG13-luc, 10 ng of pcDNA-HA-p53 and 0.2 μg of pcDNA-

ZNF76-Myc alone or in combination as indicated.

D. ZNF76 inhibits p53-mediated expression of p21. HEK293 cells were transiently transfected with 50 ng of pcDNA-HA-p53 plasmid with or without 0.5 μg of pcDNA-

ZNF76-Myc plasmid. After 48 hours, cells were lysed for western blot analysis with anti-

HA, anti-Myc, anti-p21 and anti-GAPDH antibody.

E. ZNF76 inhibits Stat1-mediated transactivation. MCF-7 cells were transiently transfected with 0.1 μg of IRF1-Luc and 0.1, 0.3 or 1.0 μg of pcDNA-ZNF76-Myc

plasmid. 24 hours after transfection, cells were treated with IFN-γ at the final concentration of 100 unit/ml for 16 hours before harvesting.

56 F. ZNF76 inhibits Smad3-mediated transactivation. HEK-293 cells were transiently transfected with 0.1 μg of SBE4-luc, 0.1 μg pCMV2-Smad3 plasmid with or without 0.2

μg pcDNA-ZNF76-Myc.

57 0.25 A 0.20 0.15

RLU 0.10 0.05 0.00

ZNF76-Myc (ug) - 0.2 - 0.005 0.01 0.05 0.2 HA-p53 (ug) - - 0.01 0.01 0.01 0.01 0.01

pG13-luc + + + + + + +

Anti-Myc

58 B 3.5 3.0 2.5 2.0 1.5 RLU 1.0 0.5 0.0

HA-p53 - - - - + + + + FL - + - - - + - - ZNF76- Myc (1-375) - - + - - - + -

(342-570) - - - + - - - +

pG13-luc + + + + + + + +

Anti-HA 1 150 375 570

1 150 375

342 570

Anti-Myc

59 C 4.0 3.0 2.0 U2OS RLU 1.0 0.0

0.40

0.30

0.20 HeLa RLU 0.10

0.00

ZNF76-Myc - + - +

HA-p53 - - + +

pG13-luc + + + +

60 HEK293 D

ZNF76-Myc - + - +

HA-p53 - - + +

Anti-HA

Anti-Myc

Anti-GAPDH Anti-GAPDH

Anti-p21

E MCF-7

0.12 0.10 0.08

RLU 0.06 0.04 0.02 0.00 - - - - + + + + IFN-γ

ZNF76(ug) - 0.1 0.3 1.0 - 0.1 0.3 1.0

61 F HEK293

1.6 1.2

RLU 0.8 0.4 0.0

ZNF76-Myc - + - +

Smad3 - - + +

SBE4-Luc + + + +

62 Figure 2. ZNF76 interacts with TBP

A. Coimmunoprecipitation of ZNF76 and TBP in HEK293 cells. 1 µg of plasmids pFLAG-CMV2-TBP and pCDNA-ZNF76-Myc was co-transfected or transfected alone into HEK 293 cells, and cell lysates were immunoprecipitated with anti-FLAG (2 µl) or anti-Myc (10 µl) and immunoblotted with anti-FLAG or anti-Myc.

B. Endogenous association of ZNF76 with TBP in MCF-7 cells. Whole cell lysates were immunoprecipitated with anti-ZNF76, normal rabbit IgG or anti-TBP antibodies and the immunoprecipitates were immunoblotted with anti-TBP and anti- ZNF76.

C. Interaction of ZNF76 mutants with TBP in coimmunoprecipitation experiments.

D. Both N- and the C-termini of ZNF76 interact with TBP in yeast. After yeast mating, both yeast growth on SD-HTL and positive β-gal activity were used as criteria of positive interaction. Shown on the right is the schematic representation of ZNF76 mutants that were expressed in the bait vector as fusions with the Gal4 DNA binding domain. Mutants that were able to interact with the full-length TBP protein are indicated.

E. Expression of GAL4AD-HA-TBP protein and the GAL4DBD-Myc-ZNF76 mutants in yeast. Yeast extracts were separated on SDS-PAGE and transferred to a PVDF membrane.

Protein expression was detected by western blot with indicated antibodies.

F. GARD interacts with TBP. Plasmid pGADT7-TBP encoding GAL4-AD fused TBP was transformed into yeast strain Y187. Plasmid pGBKT-ZNF76(342-570) was transformed into yeast strain AH109 for yeast mating.

G. Expression of Gal4DBD-Myc-ZNF76(362-444) and Gal4-AD-TBP in yeast.

63 H. ZNF76(342-570)K400,411R fails to interact with TBP in coimmunoprepcipitation experiments. 5 µg of plasmids pFLAG-CMV2-TBP and pcDNA-ZNF76(342-570) or other ZNF76 mutants was co-transfected or transfected alone into HEK 293 cells, and cell lysates were immunoprecipitated with anti-Myc (10 µl) and immunoblotted with indicated antibodies.

I. Summary of the interaction between ZNF76 mutants and TBP.

64 A WCL IP: Myc Flag-TBP + + + +

ZNF76-Myc - + - +

Anti-Flag

Anti-Myc

WCL IP: Flag ZNF76-Myc + + + +

Flag-TBP - + - +

Anti-Myc

Anti-Flag

65 B Anti-ZNF76 Anti-TBP Prebleed

IP:

Anti-ZNF76

Anti-TBP

66

C

WCL IP: anti-Myc

c

c

y

y

c

c

M

y

M

- y

-

)

)

M

0

M

-

0

-

) 7

) 7

5 5

5 5

-

7

c

- c

7

2

3

y

2 y

3

- 4

- 4

1 3

M

M

1 3

( (

-

-

( (

A

6 6

6

6

A 6 6

7 7

7

N 7

7 7

N

F F

F

F

F F

D

D

N N

c

N

N

N N

c

p Z Z

Z ZNF76(150-375)-Myc Z

p Z ZNF76(150-375)-Myc Z

Flag-TBP +++ + + + + + + +

Anti-Flag

ZNF76

Anti-Myc ZNF76(1-375)

ZNF76(342-570) ZNF76(150-375)

67 D 0.40

y

t

i

v

i

t 0.30

c

a

l

a

g

- 0.20

β

e

v

i 0.10

t

a

l

e

R 0.00

)

)

)

r

6

5

5

0

o

7

7

7

t

7

F

3

3

c

5

-

-

e

-

N

1

0

2

(

Z

V

5

4

6

1

3

7

(

(

F

6

6

7

7

N

F

F

Z

N

N

Z Z

TBP interaction in yeast Growth on SD-HTL medium ++ ZNF76

+ ZNF76(1-375)

ZNF76(150-375) -

ZNF76(342-570) +

Vector -

68 E ZNF76(150-375) ZNF76 ZNF76(1-375) ZNF76(342-570)

1 150 375 570 93KDa GAL4 1 150 375 GAL4 49KDa 150 375 GAL4 342 570 35KDa GAL4

Anti-Myc

GAL4-AD-TBP Anti-HA

69 F

y 1.4

t

i

v 1.2

i

t

c

a 1.0

l

a 0.8

g - 0.6

β

e 0.4

v

i

t

a 0.2

l e 0.0

R pGADT7 pGAD7-TBP

Bait: ZNF76 (362-444) Growth on SD-HTL medium

pGADT7 -/+

pGADT7-TBP ++

G Vector GAL4AD-HA-TBP GAL4DBD-Myc-ZNF76(362-444) + +

GAL4DBD-Myc-ZNF76(362-444) Anti-Myc

GAL4AD-HA-TBP Anti-HA

70 H

WCL IP:Myc

Flag-TBP + + + + + + ZNF76(342-570)-Myc - + - - + - ZNF76(342-570) - - + - - + K400,411R -Myc Anti-Flag

Anti-Myc

I TBP interaction

ZNF76 +

ZNF76(1-375) +

ZNF76(150-375) -

ZNF76 (342-570) +

ZNF76 (362-444) +

- ZNF76 (342-570) K400,411R

400 KRPRIAYLSEVKEE 413

R R

71 Figure 3. The core domain of TBP interacts with ZNF76.

A. Schematic representation of two domains in TBP.

B. TBP core domain coimmunoprecipitates with ZNF76. pcDNA-ZNF76-Myc (5 µg) was co-transfected with pCMV2-TBP or pCMV2-TBP(146-339) or pCMV2-TBP(1-159) into HEK 293 cells by calcium-phosphate precipitation method. Cell lysates were

subjected to immunoprecipitation with 2 µl of anti-Flag and immunoblotted with

indicated antibodies.

72 in a m A o d n l i a a n m i o m d r e e t r - o N C TBP

TBP(1-159)

TBP(146-339)

B WCL IP:Flag Flag-TBP - + - - - + - - Flag-TBP(146-339) - - - + - - - + Flag-TBP(1-159) - - + - - - + - ZNF76-Myc + + + + + + + +

Anti-Myc

37KDa

Anti-Flag 29KDa

20KDa

73

Figure 4. ZNF76 targets TBP to repress p53-mediated transactivation.

A. Ectopic expression of TBP relieves the repressive effect of ZNF76. HEK293 cells

were transfected with 25 ng of pG13-luc reporter, 10 ng of pcDNA-HA-p53, 0.2 μg of

pcDNA-ZNF76 and 0.5 μg pMT2-F-TBP alone or in combination as indicated.

B. GARD domain enhances p53-mediated transactivation. HEK293 cells were transfected with 25 ng of pG13-luc reporter, 10 ng of pcDNA-HA-p53, 0.2 μg of

pcDNA-ZNF76 or pcDNA-ZNF76(362-444) plasmid alone or in combination as

indicated.

C. ZNF76K400,411R loses its inhibitory function on p53-mediated transactivation.

HEK293 cells were transfected with 25 ng of pG13-luc reporter, 10 ng of pcDNA-HA-

p53, 0.2 μg of pcDNA-ZNF76-Myc or mutant plasmids alone or in combination as

indicated.

D. ZNF76 interferes with the TBP occupancy on the p21 promoter. HEK293 cells (one

150-mm plate) were transfected with pcDNA-HA-p53 (0.2 μg) with or without pCMV2-

Flag-ZNF76 (5 μg). 48 hours after transfection, cells were harvested and chromatin

immunoprecipitation was performed as described in the Materials and Methods. DNA

fragments derived from the immunocomplexes were PCR amplified using primers for

p21waf1.

E. Endogenous ZNF76 is not recruited to the p21 promoter. MCF-7 cells were treated

with 200 nM adriamycin for 24 hours, and cells then were harvested for chromatin

immunoprecipitation experiments.

74 A 2.5 2.0 1.5

RLU 1.0 0.5 0.0

ZNF76-Myc - + - + - + - + HA-p53 - - + + - - + +

Flag-TBP - - - - + + + + pG13-luc + + + + + + + +

B

2.5 2.0 1.5

RLU 1.0 0.5 0.0

HA-p53 - + - + - +

ZNF76-Myc - - + + - -

ZNF76(362-444) -Myc - - - - + +

pG13-luc + + + + + +

75 1.4 1.2 C 1.0 0.8 0.6 RLU 0.4 0.2 0.0 HA-p53 - + - + - + - + - + ZNF76 -Myc - - + + ------ZNF76 K400,411R -Myc - - - - + + - - - - ZNF76 (342-570) -Myc ------+ + - - ZNF76 (342-570) K400,411R -Myc ------+ + pG13-luc + + + + + + + + + +

c y M - R 1 1 4 , c 0 y ) 0 c 4 M y - K M R - ) 1 0 0 1 7 7 4 5 5 , - - c 0 y 2 2 0 4 4 M 4 3 3 - K ( ( 6 6 6 6 7 7 7 7 F F F N N N NF Z Z Z Z

Anti-Myc

76

D

HA-p53 + +

Flag-ZNF76 - + Anti-Myc Anti-TBP Anti-HA Anti-Flag Anti-HA Anti-p53 Anti-p53 Anti-TBP Anti-Flag Input No Ab Anti-Myc Input No Ab

E Anti-TBP Input Anti-Flag Anti-p53 No Ab H4 Anti-Acetylated Prebleed Anti-ZNF76

77 Figure 5. PIAS1 interacts with ZNF76.

A. The zinc finger domain and the C-terminus of ZNF76 are required for interaction with

PIAS1. Plasmid pGADT7-ZNF76 was transformed into yeast strain Y187. PIAS1 deletion mutants in pGBKT7 vector were transformed into yeast strain AH109. After mating, both yeast growth on SD-HTL and positive β-gal activity were indicative of positive interaction.

B.N-terminus of PIAS1 interacts with ZNF76.

C. Expression of Gal4AD-HA-ZNF76 and its mutants in yeast.

D. Expression of Gal4DBD-Myc-PIAS1(1-390) and various mutants in yeast.

E. The interaction between PIAS1 and ZNF76 is zinc dependent. 5 µg of plasmids pFLAG-CMV5-PIAS1 and pcDNA-ZNF76-Myc was co-transfected or transfected alone into HEK 293 cells, and cell lysates were immunoprecipitated with anti-Myc (10 µl) and immunoblotted with anti-FLAG. To detect zinc dependent interaction, 1.3 μM of ZnCl2

was included in both RIPA and TNEN buffer as described in Materials and Methods.

78 A 0.20

y

t i 0.16

v

i

t

c

a

l 0.12

a

g - 0.08 β

e

v

i t 0.04

a

l

e R 0.00 ZNF76(1-375) ZNF76(342-570) ZNF76(160-570) ZNF76

Interaction with PIAS1(1-390)

ZNF76 +

ZNF76(1-375) -

ZNF76(160-570) +

ZNF76(342-570) -

79 B

1.0

y

t

i

v

i 0.8

t

c

a

l

a 0.6

g

-

β

0.4

e

v

i

t

a

l 0.2

e

R 0.0 Lamin I II III IV V

Interaction with ZNF76

I PIAS1(1-390) +

+ II PIAS1(1-334)

III PIAS1(1-277) +

IV PIAS1(10-390) -

V PIAS1(10-130) -

Lamin -

80 C ZNF76(342-570) ZNF76(160-570) ZNF76(1-375) ZNF76

GAL4AD-HA-ZNF76 93KDa

GAL4AD-HA-ZNF76(160-570) GAL4AD-HA-ZNF76(1-375) 49KDa

GAL4AD-HA-ZNF76(342-570) 35KDa

Anti-HA

D

IIIIII IV V Lamin

GAL4DBD-Myc-PIAS1(1-390) GAL4DBD-Myc-PIAS1(10-390) 49KDa GAL4DBD-Myc-PIAS1(1-334) GAL4DBD-Myc-PIAS1(1-277)

GAL4DBD-Myc-Lamin GAL4DBD-Myc-PIAS1(10-130) 35KDa

Anti-Myc

81 E

-Zn++ +Zn++

IP: MycWCL IP: Myc WCL

ZNF76-Myc - + - + - + - + Flag-PIAS1 + + + + + + + + Vector + - + - + - + -

Flag-PIAS1 Anti-Flag

Myc-ZNF76 Anti-Myc

82 Figure 6. PIAS1 sumoylates ZNF76 at Lysine 411.

A. HEK293 cells were transfected with 0.5 μg of pcDNA-ZNF76-Myc, 0.5 μg Flag-

SUMO-1 plasmid and 0.5 μg pCMV5-PIAS1, either alone or in combination as indicated.

48 hours after transfection, cells were lysed with denaturing buffer.

B. Mapping regions of ZNF76 that can be sumoylated. Various deletion mutants of

ZNF76 in pcDNA vector were either transfected alone or cotransfected with Flag-

SUMO-1 and pCMV5-PIAS1. Cell lysates were separated by SDS-PAGE and blotted with anti-Myc.

C. Lysine 411 of ZNF76 is a sumoylation target. Wild type pcDNA-ZNF76-Myc or pcDNA-ZNF76K411R-Myc was transfected alone or cotransfected with pCMV5-PIAS1 and Flag-SUMO-1 expressing plasmid. Cell lysates were separated by SDS-PAGE and blotted with anti-Myc.

83 A Flag-sumo-1 + - + +

ZNF76-Myc - + + +

Flag-PIAS1 - - - +

* * * Anti-Myc ZNF76

Anti-Flag

sumo-1

84 B

ZNF76(1-165)-Myc ZNF76 (1-375)-Myc Flag-sumo-1 - + - + +Flag-PIAS1

Anti-Myc *

Anti-Flag

ZNF76(150-375)-Myc ZNF76(342-570)-Myc Flag-sumo-1 - + - + +Flag-PIAS1 * * Anti-Myc

Anti-Flag

85 B (continued).

sumoylation

- ZNF76(1-165)

ZNF76(1-375) +

ZNF76(150-375) +

ZNF76(342-570) +

86 C

Flag-sumo-1 - + - + ZNF76-Myc + + - - ZNF76K411R-Myc - - + + Flag-PIAS1 - + - +

* * * Anti-Myc

Anti-Flag

ZNF76

VKEE

ZNF76K411R

VREE

87 Figure 7. Sumoylation negatively regulates ZNF76’s transcription repression function.

A. PIAS1 and SUMO-1 overexpression abolishes the interaction between ZNF76 and

TBP. pcDNA-ZNF76-Myc (2 µg) was co-transfected with pCMV2-TBP (2 µg) with or

without pCMV5-Flag-PIAS1 and Flag-SUMO-1 into HEK 293 cells. Cell lysates were

subjected to immunoprecipitation with 10 µl of anti-Myc and immunoblotted with

indicated antibodies.

B. Sumoylation negatively modulates the repression function of ZNF76. HEK293 cells

were transfected with 25 ng of pG13-luc reporter, 10 ng of pcDNA-HA-p53, 0.2 μg of

pcDNA-ZNF76-Myc, and 0.2 μg of pCMV5-PIAS1 and 0.2 μg of Flag-SUMO-1

plasmids alone or in combination as indicated. 15 μl of cell lysates used in luciferase assays were subjected to SDS-PAGE and immunoblotted with indicated antibodies.

88 A

WCL IP:Myc Flag-TBP + + + + + + ZNF76-Myc - + + - + + Flag-PIAS1 - - + - - + Flag-sumo-1 - - + - - +

Flag-PIAS1

Flag-TBP Anti-Flag

Flag-sumo-1

Anti-Myc Myc-ZNF76

89 B

1.0 0.8

RLU 0.6 0.4 0.2 0.0

ZNF76-Myc - + - + - + - +

HA-p53 - - + + - - + +

Flag-PIAS1 - - - - + + + + Flag-sumo-1 - - - - + + + + pG13-luc + + + + + + + +

Anti-HA HA-p53

Anti-Myc Myc-ZNF76

Anti-Flag Flag-sumo-1

Anti-Flag Flag-PIAS1

90 CHAPTER 3

ACETYLATION AND ALTERNATIVE SPLICING REGULATE ZNF76-

MEDIATED TRANSCRIPTION

Introduction

The ZNF76 gene is located at chromosome 6p21 in a region associated with a

range of phenotypic abnormalities that affect embryonic development, male fertility and

neoplasia (Tripodis et al., 1998; Nilbert et al., 1990). ZNF76 is a human homologue of

Selenocysteine tRNA gene transcription activating factor (Staf), which is a Xenopus zinc-

finger transcription factor known to regulate genes encoding selenocysteine tRNA

(tRNAsec) and small nuclear RNA (snRNA) (Schuster et al., 1995), such as U1 and U6.

ZNF76 was also shown to be involved in the transcription of a molecular chaperonin subunit Ccta gene (Saur et al., 2002) and human neuronal nitric-oxide synthase exon 1c gene (Kubota et al., 2000), suggesting multiple roles of ZNF76 for transcriptional regulation. Our previous study identified TATA-binding protein (TBP) as a novel

ZNF76-interacting protein (Zheng et al., 2004), and this interaction leads to transcriptional repression. Mapping studies suggested that a C-terminal glutamic acid rich region of ZNF76 is responsible for the interaction. Moreover, sumoylation of ZNF76 abolishes the interaction with TBP and relieves its transcriptional repression function.

Thus, ZNF76 acts both as a DNA-binding transcription factor (Schuster et al., 1995;

Myslinski et al., 1998; Kubota et al., 2000) and a transcriponal repressor (Zheng et al.,

2004).

91 It was well established that acetylation of nucleosomal histones correlates with transcriptional activation (Pogo et al., 1996). This modification leads to destabilization of local nucleosomal structure and an open chromatin configuration, thus facilitates the access of the transcriptional machinery to promoters. However, it was only in recent years that nonhistone substrates for acetylation were identified (Gu et al., 1997). There are diverse functional consequences for the acetylation of nonhistone proteins. Acetylation has been shown to affect DNA binding, protein stability, and interactions with other proteins (Gu et al., 1997; Barlev et al., 2001; Hung et al., 1999; Li et al., 2002; Martinez-

Balbas et al., 2000; Polesskaya et al., 2000; Chen et al., 2001). These multiple effects have led to the suggestion that acetylation may be broadly involved in the regulation of cellular physiology (Kouzarides et al., 2000). Numerous nuclear histone acetyltransferases (HATs) have so far been identified (Narlikar et al., 2002), Among them, p300 and the closely related CREB-binding protein (CBP) form larger protein complexes including other acetylases and serve as coactivators for transcription factors.

Genome-wide analyses of alternative splicing indicate that 40–60% of human genes have alternative spliced forms (Modrek et al., 2002), suggesting the importance of alternative splicing in the functional complexity of the human genome. One of the challenges we are facing now is the functional characterization of different spliced isoforms. ZNF76 has two alternative spliced isoforms: the longer form has 570 amino acids (NP_003418), the shorter form has only 515 amino acids (A44256). The functional difference between these forms is unknown.

92 In this study, we demonstrate that ZNF76 is regulated by post-translational

modifications and alternative splicing for its ability to interact with TBP, which affect

ZNF76’s functions both as a general transcription repressor and a transcription activator.

Materials and Methods

Reagents and Antibodies Anti-Myc (9E10) was from Santa Cruz Biotechnology Inc.

(Santa Cruz, CA). Anti-Flag (M2) was purchased from Upstate Biotechnology, Inc.

(Lake Placid, NY). Anti-HA antibody was from BAbCo (Richmond, CA). Anti acetylated-lysine was from Cell Signalling (Beverly, MA). pBRN3-ZNF76 was a generous gift from Dr. Philippe Carbon (Myslinsky et al., 1998). HEK-293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.

Transient Transfection, Immunoprecipitation and Western blot analysis HEK-293 cells

were transfected by calcium phosphate precipitation method with various plasmid

combinations as indicated. Forty-eight hours later, cells were washed with PBS and 1 ml

ice-cold lysis buffer (RIPA) (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM

EGTA, 2 mM Na3VO4, 15 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF) was added.

Cells were lysed for 30 min at 4°C with occasional vortexing. The lysates were collected

into 1.5-ml tubes and cleared of nuclei by centrifugation for 10 min at 14,000 rpm. The

supernatants (whole cell extracts) were incubated with different antibodies for 16 h at

4°C and protein A-agarose beads were added for the last hour. The beads were washed

five times in TNEN buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5%

NP-40, 2 mM Na3VO4, 1 mM PMSF, 1 mM NaF.). Bound proteins were extracted with

93 SDS-PAGE sample buffer, and analyzed by SDS-PAGE followed by Western blot

analysis with the ECL detection system. For sumoylation assays, forty-eight hours after transfection, cells were lysed in a denaturing buffer (2% SDS, 10 mM Tris-HCl pH 8.0,

150 mM NaCl), and analyzed by SDS-PAGE followed by Western blot analysis.

Luciferase Assay Cells were plated and grown overnight before transfection. The total amount of DNA transfected was adjusted with pcDNA3. Luciferase assay was performed

according to the manufacturer's instructions (Promega). Renilla luciferase internal control

plasmid was cotransfected with the plasmids as indicated. The relative luciferase units were corrected based on renilla luciferase activity. For GAL4 fusion-driven luciferase reporter gene assays, 0.2 μg/well of reporter (pG5-luc) was cotransfected with 0.1 μg of

GAL4 fusion expression plasmids in 12-well plates.

Plasmid Construction pcDNA-ZNF76-Myc and other Myc-tagged ZNF76 mutants were constructed by insertion of the corresponding cDNA fragments into EcoRI and BamHI sites of pcDNA3.1-Myc-HisB (Invitrogen). PGEX-4T-1-ZNF76 was constructed by insertion of the ZNF76 cDNA fragment into BamH1 and EcoR1 sites. The construct

Flag-TBP was described previously (Zheng et al., 2004).

In Vitro Protein Acetylation Assay P300 protein was purified from bacoluvirus-insect cell expression system. 2-3 µg of the indicated GST fusion protein and 200 ng of purified

p300 protein were incubated in a reaction containing 50 mM Tris-HCl, pH 8.0, 10%

94 glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium butyrate, and 30 μM acetyl-coenzyme A or 1 µl of [14C]acetyl-CoA for 1 h at 30 °C. The

reaction mixture was subjected to SDS-PAGE and analyzed by autoradiography of dried gels.

Results

ZNF76 is acetylated in vitro and in cells by p300

We tested whether ZNF76 is a substrate for a well-established acetyltransferase, p300. Purified GST-ZNF76 fusion protein was incubated with recombinant p300 in the presence of [14C]acetyl-CoA and the incorporation of [14C]acetate was determined by

SDS-PAGE and autoradiography. Recombinant histone proteins, which are acetylated in

vitro by p300, served as the positive control. GST proteins alone served as the negative control. As shown in Fig.1A, histones (lane 1 and 2) and GST-ZNF76 (lane 5 and 6) but

not GST protein (lane 3 and 4) can be acetylated in the presence of p300. To directly

examine the role of p300 in the acetylation of ZNF76 in a cellular context, we transfected

HEK293 cells with ZNF76-Myc in the presence or absence of HA-p300 and examined

the acetylation of ZNF76. Cellular extracts from the transfected cells were

immunoprecipitated with anti-Myc antibody and immunoblotted with either anti-Myc antibody or antibody specific for acetylated lysine (α-AcK). A significant level of

acetylated ZNF76 was observed in the presence of p300 compared with vector alone

(Fig.1B). In order to map specific regions which are acetylated by p300, we tested three

95 deletion constructs of ZNF76 for their abilities to be acetylated. As shown in Fig.1C,

ZNF76(1-375) was strongly acetylated, while the acetylation signals of ZNF76(150-570)

and ZNF76(342-570) were very weak. These results suggest that the N-terminus of

ZNF76 is the major acetylation target for p300. Since p300 acetylates ZNF76 in vitro and

in cells, we tested the interaction between ZNF76 and p300. We transfected HEK293

cells with Flag-ZNF76 alone or in the presence of HA-p300, then cellular extract was

immunoprecipitated with an anti-HA antibody and immunoblotted with either anti-HA

antibody or anti-Flag antibody. As shown in Fig.1D, both p300 and ZNF76 were

immunoprecipitated with the anti-HA antibody, indicating that the two proteins interact in a cellular context.

P300 regulates sumoylation and TBP interaction of ZNF76

Both acetylation and sumoylation occur on lysine residues. Since ZNF76 is regulated by sumoylation (Zheng et al., 2004), we tested whether there is a direct crosstalk between acetylation and symoylation for ZNF76. We examined p300, an enzyme which promotes the acetylation of ZNF76, for its effect on the sumoylation of

ZNF76. We transfected ZNF76-Myc with Flag-SUMO-1 and Flag-PIAS1 in the presence or absence of p300, cells were lysed 48 hours after transfection, and sumoylation of

ZNF76 were analyzed by blotting with anti-Myc antibody. As shown in Fig.2A, SUMO-1 and PIAS1 overexpression caused sumoylation of ZNF76 (lane 3), which was abolished when cells were cotransfected with p300 (lane 5). This result suggests that acetylation and sumoylation may compete with each other for lysine residues. We next tested whether Lysine 411, a sumoylation site of ZNF76, is directly involved in ZNF76-TBP

96 interaction. Flag-tagged TBP was cotransfected with either ZNF76-Myc or ZNF76K411R-

Myc. 48 hours after transfection, the cells were harvested and immunoprecipitated with

anti-Myc antibodies. Although there was a similar level of expression for ZNF76 and

ZNF76K411R, TBP coimmunoprecipitated with ZNF76K411R was much less than that with

wild type ZNF76 (Fig.2B). The effect of the mutation K411R could not be explained by

the loss of sumoylation since we demonstrated that sumoylation represses ZNF76-TBP

interaction (Zheng et al., 2004). Correspondingly, ZNF76K411R is a weaker

transcriptional repressor than the wild-type (Fig.2C). Based on the competition between

modifications and the importance of the sumoylation site K411 in ZNF76-TBP

interaction, we tested the effect of acetylation of ZNF76 on its interaction with TBP. We

cotransfected ZNF76-Myc with Flag-TBP in the presence or absence of HA-p300,

harvested the cells after 48 hours and immonoprecipitated the cell lysates with anti-Myc

antibody. Immunoprecipitates were separated by SDS-PAGE and blotted with anti-Flag

antibody. As shown in Fig. 2D, the amount of Flag-TBP that was immunoprecipitated

with ZNF76 was markedly lower in extracts from cells transfected HA-p300 compared

with vector control. These results suggest that acetylation of ZNF76 inhibits its sumoylation and also regulates its interaction with TBP.

HDAC1 deacetylates ZNF76

Protein acetylation is often a reversible process, which is determined by the interplay of acetyl-group transferases and deacetylases. We tested HDAC1, 3, 4, 5, 7,

which belong to either class І or class П HDACs, for their interaction with ZNF76 through coimmunoprecipitation experiments. Among them, HDAC1 was found to

97 strongly interact with ZNF76. As shown in Fig.3A upper panel, HEK293 cells were transfected with HA-HDAC1 alone or in the presence of ZNF76-Myc. After cell lysates were immunoprecipitated with anti-HA antibody and then blotted with anti-Myc antibody, we found HA-tagged HDAC1 proteins coprecipitated with Myc-tagged ZNF76. The lower panel of Fig.3A shows the reverse coimmunoprecipitation of ZNF76-Myc and HA-

HDAC1. To test whether the interaction results in the deacetylation of ZNF76, we cotransfected ZNF76-Myc with HA-p300 in the presence or absence of HA-HDAC1 and examined the acetylation of ZNF76. P300 transfection caused a strong acetylation of

ZNF76 (lane 2), which was almost abolished by HA-HDAC1 overexpression (lane 3)

(Fig.3B). These results suggest HDAC1 interacts with and deacetylates ZNF76.

Acetylation and sumoylation regulate transactivation of ZNF76

ZNF76 has transactivation function for certain genes, such as chaperonin subunit

Ccta gene and human neuronal nitric-oxide synthase exon 1c (Saur et al., 2002; Kubota et

al., 2000). It consists of a well-established transactivation domain at the N-terminus, a

DNA binding domain in the middle, and a TBP binding region at the C-terminus (Zheng

et al., 2004). To test whether the C-terminal region is directly involved in the

transactivation of ZNF76, we fused the N-terminus, the zinc finger domain and the C-

terminal region of ZNF76 with GAL4-DBD (DNA binding domain) to examine their

transactivation activities (Fig.4A). Surprisingly, C-terminus of ZNF76 showed

transactivation activity as strong as its N-terminus. This result suggests that the C-

terminus of ZNF76 is also important for transactivation. Next we examined the relevance

of K411 in regulating ZNF76’s transactivation, which lies in the C-terminus of ZNF76

98 and is a sumoylation site. As shown in Fig.4B, GAL4-ZNF76K411R has 3-fold higher

transactivation than GAL4-ZNF76, suggesting this lysine residue negatively regulates the

transactivation of ZNF76. Since lysine 411 is important for both sumoylation and TBP

interaction, we determined which property is responsible for the change in transactivation.

In the presence of a sumoylation inhibitor, DNUbc9, the difference between the two constructs was abolished, suggesting sumoylation pathway is required for K411 to regulate ZNF76. Since acetylation antagonizes sumoylation (Fig.2A), we speculated that p300 may also regulate the transactivation of ZNF76. Shown in Fig.4C, p300 significantly activated both pM-ZNF76 and pM-ZNF76K411R, suggesting that acetylation

activates ZNF76 transactivation and the effect is not mediated through K411. Taken

together, sumoylation represses the transcriptional activation of ZNF76 through targeting

K411 in the C-terminus of ZNF76; while acetylation activates ZNF76, which is not

dependent on lysine 411.

Alternative splicing modulates ZNF76’s interaction with TBP

There are two different spliced isoforms of ZNF76. The longer form is 570 amino acids long (NP_003418) which was used in our previous study. The shorter form is 515

amino acids long (A44256). The structure of these two forms is shown in Fig.5A. Though a region of 55 amino acids is lost in ZNF76515 compared with ZNF76570, both forms of

ZNF76 are equally sumoylated by PIAS1 (Fig.5A). To test the functional differences of

the two forms of ZNF76, we compared their ability to interact with TBP. Flag-tagged

TBP was cotransfected with either ZNF76570-Myc or ZNF76515-Myc. 48 hours after

transfection, the cells were harvested and immunoprecipitated with anti-Myc antibodies,

99 Shown in Figure. 5B, though there were similar levels of ZNF76570 and ZNF76515, TBP coprecipitated with ZNF76515 was much less than that with ZNF76570. Correspondingly, in the transcription repression assay (Zheng et al., 2004), ZNF76515 was a weaker repressor compared with ZNF76570 in suppressing p53-mediated transcriptional activation

(Fig.5C). These results suggest that alternative splicing regulates ZNF76’s TBP interaction and transcriptional repression activity

Discussion

In our study, besides sumoylation (Zheng et al., 2004), two additional regulatory mechanisms for ZNF76 were identified: acetylation and alternative splicing. We examined their effects on the dual functions of ZNF76: a TBP interacting transcriptional repressor and a DNA-binding transcriptional activator. We showed that there is a crosstalk between acetylation and sumoylation of ZNF76, both physically and functionally. Interestingly, acetylation is also involved in regulating ZNF76-TBP interaction. Moreover, our data suggested that ZNF76 is also regulated by alternative splicing: two isoforms of ZNF76 vary in their ability to interact with TBP, and to function as transcription repressors. The existence of multiple mechanisms to regulate

ZNF76-TBP interaction suggests that the interaction is important and is tightly controlled.

ZNF76 is a homologue of Staf and ZNF143 with transactivation function

(Myslinsky et al., 1998). To further study the function of ZNF76 as a transcription activator, we mapped the domains responsible for its transcriptional activation.

Interestingly, in addition to the N-terminus of ZNF76, which was well studied and

100 conserved in Staf and ZNF143 (Myslinsky et al., 1998), the C-terminus of ZNF76 also has transactivation function. The C-terminus contains the sumoylation site lysine 411, which lead us to test whether sumoylation may affect the transactivation of ZNF76.

Dominant-negative Ubc9 activates wild type ZNF76, but not ZNF76K411R, suggesting that

sumoylation at lysine 411 represses ZNF76-mediated transcription. However, there is no

correlation between TBP interaction and transactivation: ZNF76 K411R has stronger

transactivation, while it has weaker interaction with TBP than the wild-type ZNF76. Thus

K411 has dual functions: it is involved in both TBP interaction and transactivation of

ZNF76, and both are regulated by sumoylation.

Alternative RNA processing is an important means of increasing proteomic complexity in eukaryotic organisms. Through this process, a majority of human pre- mRNAs generate more than one mRNA molecule, which often leads to production of

multiple polypeptides from a single gene (Black et al., 2000), contributing to functional

complexity of the genome. ZNF76 has at least two alternative spliced forms: the longer

one is 570 amino acids (NP_003418), and the shorter one is 515 amino acids (A44256).

Protein sequence analysis demonstrated that the ZNF76515 isoform results from deletion

of 55 amino acids close to the C-terminus of ZNF76. We investigated the functional

differences between these two different spliced forms of ZNF76. The ability of the

shorter form of ZNF76 to be sumoylated is intact compared with the longer form. In

agreement with our previous study (Zheng et al., 2004), which suggests that the C-

terminus of ZNF76 contributes to the interaction with TBP, we found that ZNF76515 interacts significantly weaker than the longer form. Correspondingly, the transcriptional

101 repression activity of ZNF76515 is also weaker. We speculate that through the regulation

of alternative splicing, different ZNF76 isoforms with different abilities in TBP

interaction and transcriptional repression will be generated. Thus a quantitative but not

“on or off” control for gene expression is accomplished through splicing regulation.

The interaction of different modification pathways has been shown for different

substrates (Gronroons et al., 2002; Zhao et al., 2004; Jin et al., 2004). In the case of

Smad7, acetylation occurs in the same lysine residue as ubiquitination and thus stabilizes

the protein. Sumoylation of IκBα competes with ubiquitination for a lysine residue, thus

stabilizes IκBα through inhibition of ubiquitination. In our study, we found the

competition between acetylation and sumoylation on ZNF76. Interestingly, there is also

functional antagonism between acetylation and sumoylation for ZNF76. Thus the direct

link between physical and functional antagonism will not only facilitate ZNF76 to be

activated by acetylation, but also increase the sensitivity of the signals since unwanted modifications are suppressed.

In summary, our study suggests that multiple mechanisms are involved in a delicate regulatory network to tightly modulate functions of ZNF76 both as a transcriptional activator and a repressor.

102 Figure 1. ZNF76 is acetylated by p300.

A. ZNF76 is acetylated by p300 in vitro. Purified core histones, GST or GST-ZNF76 were incubated with [14C]acetyl-CoA and insect-cell expressed p300 as indicated.

Reaction products were fractionated by SDS-PAGE, and the dried gel was exposed to x-

ray film (Upper panel). Shown in the lower panel is Coomassie Blue staining of the

proteins.

B. ZNF76 is acetylated by p300 in cells. Lysates from HEK-293 cells (in 6-well plates)

transfected with 0.5 μg Myc-tagged ZNF76 with or without 0.5 μg HA-p300 were

immunoprecipitated (IP) with anti-Myc and immunoblotted with indicated antibodies.

C. The N-terminus of ZNF76 is the major acetylation region. Lysates from HEK-293

cells (in 6-well plates) transfected with 0.5 μg Myc-tagged ZNF76 or its mutants with or

without 0.5 μg HA-p300 were immunoprecipitated (IP) with anti-Myc and

immunoblotted with indicated antibodies.

D. Coimmunoprecipitation of ZNF76 and p300 in HEK293 cells. 3 µg of plasmids Flag-

ZNF76 and 3 µg HA-p300 was co-transfected or transfected alone into HEK 293 cells,

and cell lysates were immunoprecipitated with anti-HA (3 µl) and immunoblotted with

anti-Flag or anti-HA.

103 A GST-ZNF76 GST Histone p300 - + - + - + Autoacetylated p300 Acetylated GST-ZNF76 90 kDa

29 kDa Acetylated histones

GST-ZNF76 90 kDa GST 29 kDa

1 2 3 4 5 6

B IP: anti-Myc Myc-ZNF76 + +

HA-p300 - +

Blot: anti-ac-K

Blot: anti-myc

1 2

104 C

IP: anti-Myc

ZNF76-Myc + + - - - ZNF76(1-375)-Myc - - + - - ZNF76(150-570)-Myc - - - + - ZNF76(342-570)-Myc - - - - + HA-p300 - + + + + 90 kDa Acetylated ZNF76

56 kDa Acetylated ZNF76(1-375) 29 kDa

Blot: anti-ac-K

ZNF76-Myc 90 kDa

ZNF76(150-570)-Myc 56 kDa ZNF76(1-375)-Myc 29 kDa ZNF76(342-570)-Myc

Blot: anti-Myc

105 D

IP: HA Flag-ZNF76 + + + + HA-p300 - + - +

Blot: anti-Flag

Blot: anti-HA

106 Figure 2. P300 suppresses the sumoylation of ZNF76 and its interaction with TBP.

A. P300 abolishes the sumoylation of ZNF76. HEK293 cells (in 6-well plates) were

transfected with 0.5 μg of ZNF76-Myc, 0.5 μg Flag-SUMO-1, 0.5 μg pCMV5-PIAS1 and

0.5 μg HA-p300 either alone or in combination as indicated. 48 hours after transfection,

cells were lysed with denaturing buffer, separated by SDS-PAGE and analyzed by

immunoblot with indicated antibodies.

B. Lysine 411 of is involved in its interaction with TBP. ZNF76-Myc or ZNF76K411R-

Myc was co-transfected with Flag-TBP into HEK 293 cells by calcium-phosphate precipitation method. Cell lysates were subjected to immunoprecipitation with 10 µl of anti-Myc and immunoblotted with indicated antibodies.

C. ZNF76K411R has weaker transcription repression activity than wild type ZNF76.

HEK293 cells were transfected with 25 ng of pG13-luc reporter, 10 ng of pcDNA-HA-

p53, 0.2 μg of pcDNA-ZNF76-Myc, or its mutants alone or in combination as indicated.

15 μl of cell lysates used in luciferase assays were subjected to SDS-PAGE and immunoblotted with anti-Myc. Luciferase assays are representatives of at least three independent experiments.

D. P300 inhibits the interaction between ZNF76 and TBP. ZNF76-Myc (2 µg) was co- transfected with Flag-TBP (2 µg) with or without HA-p300 (3 µg) into HEK 293 cells by

the calcium-phosphate precipitation method. Cell lysates were subjected to

immunoprecipitation with 10 µl of anti-Myc and immunoblotted with indicated antibodies.

107 A

ZNF76-Myc + + + + +

Flag-SUMO-1 - + + + + Flag-PIAS1 - - + - + HA-p300 - - - + +

Sumoylated ZNF76 Blot: anti-Myc ZNF76-Myc

Sumoylated proteins Blot: anti-Flag Flag-PIAS1

HA-p300 Blot: anti-HA

1 2 3 4 5

108

WCL IP:Myc B Flag-TBP + + + + + + ZNF76-Myc - + - - + -

ZNF76K411R-Myc - - + - - + Blot: anti-Flag

Blot: anti-Myc

1.2 C 1.0 0.8 0.6 RLU 0.4 0.2 0.0 ZNF76-Myc - + - - - + - - - - + - - - + - ZNF76K411R-Myc - - - + - - - + ZNF76K30R-Myc HA-p53 - - - - + + + + pG13-luc + + + + + + + + -Myc -Myc K30R K411R ZNF76 ZNF76-Myc ZNF76

Blot: anti-Myc

109

D

FLAG-TBP + + + ZNF76-Myc - + + HA-p300 - - + Blot: anti-FLAG

IP: anti-Myc Blot: anti-FLAG Blot: anti-HA

Blot: anti-Myc IP: anti-Myc Blot: anti-Myc

110 Figure 3. HDAC1 deacetylates ZNF76.

A. Coimmunoprecipitation of ZNF76 and HDAC1 in HEK293 cells. 3 µg of plasmids

ZNF76-Myc and 3 µg HA-HDAC1 was co-transfected or transfected alone into HEK

293 cells, and cell lysates were immunoprecipitated with anti-HA (upper panel) or anti-

Flag (lower panel) and immunoblotted with indicated antibodies.

B. HDAC1 deacetylates ZNF76. Lysates from HEK-293 cells (in 6-well plates) transfected with 0.5 μg Myc-tagged ZNF76, 0.5 μg HA-p300 with or without 0.5 μg HA-

HDAC1 were immunoprecipitated (IP) with anti-Myc and immunoblotted with indicated antibodies.

111 A

IP: HA ZNF76-Myc + + + + HA-HDAC1 - + - +

Blot: anti-Myc

Blot: anti-HA

IP: Myc ZNF76-Myc - + - + HA-HDAC1 + + + +

Blot: anti-HA

Blot: anti-Myc

112

B

ZNF76-Myc + + + HA-HDAC1 - - + HA-p300 - + +

Acetylated-ZNF76 IP: Myc Blot: anti-ac-K ZNF76-Myc Blot: anti-Myc

HA-p300 Blot: anti-HA

HA-HDAC1 Blot: anti-HA

1 2 3

113 Figure 4. Acetylation and sumoylation regulate the transactivation of ZNF76.

A. C-terminus of ZNF76 is a novel transactivation domain. HEK293 cells in 12-well

plates were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid

pM-ZNF76 or its mutants. Luciferase assays are representatives of at least three

independent experiments.

B. Sumoylation represses the transactivation of ZNF76. HEK293 cells in 12-well plates

were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid pM-

ZNF76 or pM-ZNF76K411R, in the presence or absence of 0.2 μg DNUbc9 as indicated.

Luciferase assays are representatives of at least three independent experiments.

C. Acetylation activates ZNF76. HEK293 cells in 12-well plates were transfected with

0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid pM-ZNF76 or pM-ZNF76K411R, in the presence or absence of 0.2 μg HA-p300 as indicated. Luciferase assays are representatives of at least three independent experiments.

114 A

RLU 10 0 1 2 3 4 5 6 7 8 9 1342570 GAL4 1 150 375 GAL4 1165 GAL4 1 150 375 570 GAL4

GAL4

115

B

1.4 1.2 1.0 0.8

RLU 0.6 0.4 0.2 0.0 PM-ZNF76 - + - - + - - - + - - + PM-ZNF76K411R DNUbc9 - - - + + +

Anti-GAL4

116 C

0.8 0.7 0.6 0.5 RLU 0.4 0.3 0.2 0.1 0 PM-ZNF76 - + - - + - - - + - - + PM-ZNF76K411R HA-p300 - - - + + +

117 Figure 5. ZNF76 is regulated by alternative splicing.

A. Sumoylation of both isoforms of ZNF76. HEK293 cells (in 6-well plates) were

transfected with 0.5 μg of ZNF76570-Myc or ZNF76515-Myc, 0.5 μg Flag-SUMO-1

plasmid and 0.5 μg pCMV5-PIAS1, either alone or in combination as indicated. 48 hours after transfection, cells were lysed with denaturing buffer. Cell lysates were separated on

SDS-PAGE blotted with the indicated antibodies.

B. The two ZNF76 isoforms interact with TBP differently. 3 µg of plasmids pFLAG-

CMV2-TBP and ZNF76-Myc or ZNF76515-Myc was co-transfected or transfected alone

into HEK 293 cells, and cell lysates were immunoprecipitated with anti-Myc (10 µl).

Immunoblotting was performed with anti-Flag or anti-Myc.

C. Two ZNF76 isoforms have different repressive activities on p53-mediated

transactivation. HEK293 cells were transfected with 25 ng of pG13-luc reporter, 10 ng of

pcDNA-HA-p53 and 0.2 μg of pcDNA-ZNF76-Myc or pcDNA-ZNF76515-Myc alone or

in combination as indicated.

118 A Flag-SUMO-1 - + - + ZNF76-Myc + + - -

ZNF76515 -Myc - - + + Flag-PIAS1 - + - +

Sumoylated ZNF76

ZNF76-Myc Anti-Myc ZNF76515-Myc

Sumoylated proteins Anti-Flag Flag-PIAS1

ZNF76

ZNF76 515

119

B

WCL IP:Myc

c c

y y

M M

c

c

- -

y

y

5 5

1 1

M

M 5 5

-

-

r r 6 6 6

6

o 7 7 7

7

to t

F F F

F

c c

e e

N N N

N

V Z Z V Z

Z Flag-TBP + + + + + +

Blot: anti-Flag

Blot: anti-Myc

120 C

1.8 1.6 1.4 1.2 1.0 RLU 0.8 0.4 0.2 0.0 HA-p53 - - - + + + ZNF76-Myc - + - - + - - - + - - + ZNF76515 -Myc pG13-luc + + + + + +

121 CHAPTER 4

SUMOYLATION AND ACETYLATION PLAY OPPOSITE ROLES IN THE

TRANSACTIVATION OF PLAG1 AND PLAGL2

Introduction

PLAG1 is a developmentally regulated (Kas et al., 1997) transcription factor that

plays an important role in tumorigenesis. Oncogenic activation of the PLAG1 gene on chromosome 8q12 is crucial in the formation of pleomorphic adenomas of the salivary glands (Kas et al., 1997) and lipoblastomas (Hibberd et al., 2000; Gilsselsson et al., 2001;

Astrom et al., 2000). As a result of chromosomal translocation and promoter substitution,

PLAG1 expression is dysregulated. PLAG1 overexpression is also detected in tumors without 8q12 translocation, such as pleomorphic adenomas of the salivary glands with

12q15 translocation or normal karyotype, uterine leiomyomas, leiomyosarcomas and smooth muscle tumors (Astrom et al., 1999). Both PLAG1 and its related molecule,

PLAGL2 (PLAG-like 2), play important roles in the pathogenesis of acute myeloid leukemia in cooperation with Cbfb-MYH11 (Castilla et al., 2004; Landrette et al., 2004).

Both PLAG1 and PLAGL2 consist of an N-terminal zinc finger DNA-binding domain and a C-terminal transactivation domain. The consensus DNA binding site comprises a core sequence (GRGGC) and a G-cluster (RGGK) (Voz et al., 2000).

Although several potential target genes of PLAG1 have been identified (Voz et al., 2004), the regulatory mechanisms of transcriptional activation by PLAG1/PLAGL2 remain unknown. One of the mechanisms to regulate the activity of transcriptional factors is

122 posttranslational modifications, such as phosphorylation (O’shea et al., 2002), acetylation

(Freiman et al., 2003), methylation (Mowen et al., 2001), ubiquitination (Freiman et al.,

2003), isgylation (Malakhova et al., 2003), neddylation (Liu et al., 2002) and sumoylation

(Freiman et al., 2003). Sumoylation is a three-step enzymatic pathway analogous to that

of ubiquitin conjugation which results in the transfer of SUMO from Ubc9 to the target

proteins (Freiman et al., 2003). The functional consequences of sumoylation are distinct from ubiquitination. Instead of being marked for degradation by ubiquitination, sumoylation has diverse substrate-specific functions. Several transcription factors,

including androgen receptor (Nishida et al., 2002), Sp3 (Ross et al., 2002), c-Myb (Bies

et al., 2002) and Elk-1(Yang et al., 2003), are sumoylation targets and sumoylation

represses their transcriptional activities. The exact mechanism of how sumoylation

represses transactivation remains unclear, though SUMO-dependent recruitment of

HDACs has been implicated in the transcriptional repression of p300 (Girdwood et al.,

2003) and Elk-1 (Yang et al., 2004).

In addition to ubiquitination and sumoylation, lysine residues of transcription

factors can be covalently modified by acetylation, a process known to enhance DNA

binding (Soutoglou et al., 2000), change protein-protein interaction (Zhang et al., 2000)

and regulate transactivation (Gu et al., 1997; Zhang et al., 1998). Numerous nuclear

histone acetyltransferases (HATs) have so far been identified. Among them, p300 and the

closely related CREB-binding protein (CBP) are the most potent and versatile of all the

acetyltransferases. Consistent with its role as a global co-activator, p300 acetylates and

regulates various non-histone transcription factors, such as GATA-1 (Boyes et al., 1998),

123 MyoD (Polesskaya et al., 2000), E2F-1 (Martinez et al., 2000) and p53 (Gu et al., 1997).

On the other hand, histone deactylases reverse the acetylation process to maintain the

balance between the acetylated and deacetylated states of chromatin and other non-

histone proteins. Histone deacetylases (HDACs) are divided into four classes: class I

HDACs (HDACs 1, 2, 3, and 8) localize to the nucleus; class II HDACs (HDACs 4, 5, 6,

7, 9, and 10) are found in both the nucleus and cytoplasm; class III HDACs are NAD- dependent enzymes that are similar to the yeast SIR2 proteins; and class IV (HDAC11) is

the smallest of HDAC members (Yang et al., 2005).

In this study, we have identified regulatory mechanisms for transactivation by

PLAG1 and PLAGL2. We show that a region close to the C-terminal transactivation

domain of both PLAG1 and PLAGL2 has repressive activity, which is controlled by

sumoylation. Moreover, PLAG1 and PLAGL2 are acetylated and activated by p300,

while deacetylated and repressed by HDAC7. Sumoylation-deficient PLAGL2 shows

decreased acetylation, suggesting that some lysine residues could be targets for both

modifications. Importantly, mutation of the sumoylation sites greatly reduced

transformation abilities of PLAG1 and PLAGL2, suggesting the important roles of the

modifications for PLAG1 and PLAGL2 as oncoproteins.

Materials and Methods

Reagents and Antibodies Anti-Myc (9E10) and Anti-Ubc9 (Sc-10759) were from Santa

Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-Flag (M2) was purchased from Upstate

Biotechnology, Inc. (Lake Placid, NY). Anti-HA antibody was from BAbCo (Richmond,

124 CA). Anti-acetylated-lysine was from Cell Signalling (Beverly, MA). L8G5-luc and

LexA-VP16 were generous gifts from Dr. Andrew D. Sharrocks (Yang et al., 2003).

PCMV6-SSP3 and pcDNA3-DNUbc9 were obtained from Dr. Ronald T. Hay (Girdwood

et al., 2003). pCG-PLAGL1 and PCG-PLAGL2 were gifts from Dr. Shigeru Taketani

(Furukawa et al., 2001). Human IGF-П-luciferase reporter construct Hup3-luc was

provided by Dr. P. Elly Holthuizen.

Transient Transfection, Immunoprecipitation and Western Blot Analysis HEK-293 cells

were transfected by the calcium phosphate precipitation method with various plasmid combinations as indicated. Forty-eight hours later, cells were washed with phosphate- buffered saline (PBS) and 1 ml ice-cold lysis buffer (RIPA) (50 mM Tris-HCl pH 7.4,

150 mM NaCl, 1% NP-40, 1 mM EGTA, 2 mM Na3VO4, 15 μg/ml aprotinin, 10 μg/ml

leupeptin, 1 mM PMSF) was added. Cells were lysed for 30 min at 4°C with occasional vortexing. The lysates were collected into 1.5-ml tubes and cleared of nuclei by centrifugation for 10 min at 14,000 rpm. The supernatants (whole cell extracts) were incubated with different antibodies for 16 h at 4°C and protein A-agarose beads were added for the last hour. The beads were washed five times in TNEN buffer (20 mM Tris-

HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 2 mM Na3VO4, 1 mM PMSF, 1

mM NaF). Bound proteins were extracted with SDS-PAGE sample buffer, and analyzed

on SDS-PAGE followed by western blot analysis with the ECL detection system. For sumoylation assays, 0.5 μg/well of expression plasmids was cotransfected with 0.5

μg/well of Flag-SUMO-1 plasmid in 6-well plates. Forty-eight hours after transfection,

125 cells were lysed in a denaturing buffer (2% SDS, 10 mM Tris-HCl pH 8.0, 150 mM

NaCl), and analyzed by SDS-PAGE and western blotting.

Luciferase Assay Cells were plated in 12-well plates and grown overnight before transfection. The total amount of DNA transfected was adjusted with pcDNA3.

Luciferase assay was performed according to the manufacturer's instructions (Promega).

Renilla luciferase internal control plasmid was cotransfected with the plasmids as indicated. The relative luciferase units were corrected based on renilla luciferase activity.

For GAL4 fusion-driven luciferase reporter gene assays, 0.1 μg/well of reporter (pG5-luc)

was cotransfected with 0.1 μg of GAL4 fusion expression plasmids in 12-well plates. 0.2

μg/well of DNUbc9 or SSP3 construct was used if indicated. For IGF-П-luciferase reporter assays, 0.05 μg/well of reporter plasmid was cotransfected with effector plamids of indicated amounts in 12-well plates.

Plasmid Construction The PLAG constructs used in mammalian cells were generated by polymerase chain reactions with primers containing restriction sites for cloning. Every

construct was sequenced fully to verify the fidelity of the polymerase chain reaction.

PLAG cDNAs were fused in-frame to the DNA binding domain of GAL4 using the pM vector. PcDNA3-PLAG1-Myc and subfragments of PLAG1 were constructed using XhoІ

and EcoRІ sites. PcDNA3-PLAGL2-Myc and subfragments of PLAGL2 were

constructed using EcoRІ and BamHІ sites. PCMV2-Flag-PLAG1 was constrcted using

EcoRІ and XbaІ sites. PCMV2-Flag-PLAGL2 was constructed using EcoRІ and BamHІ

sites. GFP-PLAG1 or its mutants were constructed by inserting full-length or mutant

126 PLAG1 into XhoІ and EcoRІ sites of pEGFP-C1 (Clontech). GFP-PLAGL2 or its mutants were constructed by inserting wild-type or mutant PLAGL2 into the EcoRІ and

BamHІ sites of pEGFP-C1.

Immunostaining HEK293 cells were seeded in chamber slides (0.5 x 105 cells/ml) and

transfected 24 hours later with 2 µg of respective plasmids by the calcium phosphate

method. 24–48 h later, cells were washed in cold PBS, fixed with 3.7% formaldehyde for

30 min and permeabilized with 0.5% Triton X-100 for 5 min. Cells were then blocked for

30 min in blocking buffer (PBS, 5% BSA, 0.3% triton X-100), incubated with primary

antibody for 1 hour and washed three times with PBS. After incubation with secondary

antibody and washing three times with PBS, cells were examined under confocal

immunofluorescence microscope.

Focus Formation Assay NIH-3T3 cells (in 6-well plates) were transfected with 2 μg of empty vector, expression plasmids for PLAG1, PLAG1 mutant, PLAGL2, PLAGL2 mutants or activated Ha-RAS. The day after transfection, cells were split at 3×104/plate in

10 cm plates and selected in 300 μg/ml G418 and 10% serum in DMEM for 3 days. Then cells were fed once every 4 days with DMEM plus 1% serum and 300 μg/ml G418. After

3 weeks, cells were fixed and stained with methylene blue, and the number of foci was determined.

Results

Identification of repression domains in PLAG1 and PLAGL2

127 In this study, we found PLAG1 and PLAGL2 share similar regulatory

mechanisms. To avoid redundancy, we will only show one set of data for PLAGL2 in the

Results section wherever indicated, and the rest of the data will be included in

Supplemental Data. The structures of PLAG1, PLAGL2 and their GAL4-fusion

derivatives are shown in Fig.1A. Both PLAG proteins have a zinc finger domain in the

N-terminus, and a transactivation domain in the C-terminus. Previous study by Kas et al

(Kas et al., 1998) identified a region in the middle of PLAG1 and PLAGL2 with

transcriptional repression function. As shown in Fig.1B, when PLAGL2(387-496), which

includes only the trans-activation domain, and PLAGL2(238-496), which includes both

the transactivation domain and the middle region were fused with GAL4 DNA binding

domain, there was a 10-fold difference in the transactivation capacity between these two constructs. Similiarly, PLAG1(361-500) exhibited about 10 times more transactivation potential compared with PLAG1(232-500) (Supplemental Fig.S1). These results suggest that PLAG1(232-361) and PLAGL2(238-387) have repressive activity. The repression

domain of Elk-1 was shown to act in trans (17), that is, when bound to a promoter, the

repression domain itself is able to repress the transcription activity of another adjacent

transcription factor. To test whether the repression domains of PLAG proteins also

repress transcription in trans, we tested the repression domains of PLAG1 and PLAGL2

in the transrepression assay in which GAL4 fusion proteins were used to repress the

activity of a Lex-VP16 activated reporter (17). We found that both PLAG1(232-361)

(data not shown) and PLAGL2(238-387) (Supplemental Fig.S2) have no significant

repression activity in the assay, suggesting that the repression domains of PLAG1 and

PLAGL2 function differently from that of Elk-1.

128

Potential SUMO modification sites play a repressive role in PLAG1 and PLAGL2

Since repression activities are conserved in the middle regions of PLAG1 and

PLAGL2, we aligned their sequences to find the possible conserved sites which may be crucial for the repressive activity. We found 3 consensus sumoylation motifs well conserved in both PLAG1 and PLAGL2 (Supplemental Fig.S3). We tested whether they are crucial for the repression domain by introducing triple mutations

K244R/K263R/K353R into PLAG1 and K250R/K269R/K356R into PLAGL2. In the context of a GAL4 fusion protein tethered to an artificial promoter, both GAL4-

PLAG1(232-500)(K244R/K263R/K353R) and GAL4-PLAGL2(238-

496)(K250R/K269R/K356R) exhibited 9-10 fold higher transcriptional activity than their wild-type counterparts (Fig.2A and Supplemental Fig.S4A). Moreover, these mutants became equally active to the transactivation domains alone, suggesting that the activities of the repression domains were abolished by the mutations of the sumoylation motifs.

Western blot analysis showed that the effect of the mutations is not due to higher expression of the mutants (Fig.2B and Supplemental Fig.S4B). These results suggest that these sumoylation motifs are required for the repression domains of PLAG1 and

PLAGL2.

Next we investigated whether sumoylation pathway is involved in the activity of the repression domain of PLAG1 and PLAGL2. We used two approaches to block the sumoylation pathway: a catalytically inactive form of the SUMO conjugating enzyme,

C93S Ubc9 (DNUbc9), which acts as a dominant-negative mutant; and SSP3, which is a

129 SUMO-specific protease. As shown in Fig.2C, transfection of DNUbc9 completely rescued the difference between GAL4-PLAGL2(278-496) and GAL4-PLAGL2(387-496), suggesting that the repressive activity of PLAGL2(278-387) was abolished. Similar observation was made using SSP3 (Fig.2D). When we converted the luciferase activity to fold induction (the ratio between with and without DNUbc9 or SSP3), as shown in

Supplemental Fig.S4C and S4D, both sumoylation pathway inhibitors significantly enhanced the transcriptional activity of PLAG1(232-500), but had much less effect on

PLAG1(361-500) and PLAG1(232-500)(K244R/K263R/K353R). Moreover, the repression domain alone was activated by DNUbc9 (Supplemental Fig.S5). These results suggest that both the sumoylation motifs and the intact sumoylation pathway are essential for the repression domains of PLAG1 and PLAGL2.

PLAG1 and PLAGL2, but not PLAGL1, are modified by sumoylation

To verify that sumoylation of PLAG1/PLAGL2 occurs in cells, we cotransfected

HEK293 cells with PLAGL2-Myc and Flag-SUMO-1 expression plasmids and analyzed sumoylation of PLAGL2 by immunoblotting with anti-Myc antibody. A slower-migrating band reacting with anti-Myc antibody was clearly detected in cells ectopically expressing

Flag-SUMO-1 (Fig.3A). Since PIAS1 is a well-established E3 ligase in sumoylation, we tested whether PIAS1 promotes sumoylation of PLAGL2. HEK293 cells were transfected with PLAGL2-Myc, Flag-SUMO-1, with or without Flag-PIAS1 or Flag-PIAS1C335A, a construct encoding an inactive form of PIAS1. As shown in Fig.3B, cotransfection of

Flag-PIAS1, but not Flag-PIAS1C335A, strongly enhanced the sumoylation of PLAGL2.

130 To demonstrate that the higher-molecular-weight bands correspond to sumoylated

PLAGL2, we included DNUbc9 in the sumoylation assay. Expression of DNUbc9 abolished the appearance of the slower-migrating bands (Fig.3C). To test whether lysine residues in the consensus sumoylation motifs are indeed sumoylation targets, wild type and mutant PLAGL2 were cotransfected with Flag-SUMO-1 with or without Flag-PIAS1.

PLAGL2K250R partially and the triple mutant PLAGL2K250,269,356R completely lost their abilities to be sumoylated (Fig.3D), which correlates with the loss of repression activity

(Fig.2A). We also tested single mutants of PLAGL2. As shown in Fig.3E, each PLAGL2 single mutant still retained the ability to be sumoylated, suggesting that all three lysine residues are sumoylation targets. Similar results were obtained for PLAG1 (Supplemental

Fig.S6). PLAGL1 is another member in the PLAG family. Unlike PLAG1 and PLAGL2,

PLAGL1 is a tumor suppressor gene rather than an oncogene. As shown in Fig.3F, no sumoylation was detected for PLAGL1 even when Flag-SUMO-1 was overexpressed.

These results suggest that both PLAG1 and PLAGL2, but not PLAGL1, can be sumoylated, and lysine residues inside the sumoylation motifs are indeed sumoylation targets.

Sumoylation pathway represses PLAG1/PLAGL2-mediated transcription

Since both intact sumoylation motifs and sumoylation pathway are required for the function of the repression domain (Fig.2), and PLAGL2 can be physically sumoylated at those sites (Fig.3), we next tested in the full-length context, whether sumoylation represses PLAG1/PLAGL2-mediated transcription. We fused full-length PLAGL2 with the GAL4 DNA binding domain, and tested their activity in the presence or absence of

131 DNUbc9. Though DNUbc9 did not change basal reporter activity, it activated PLAGL2

about 15 fold (Fig.4A). Similar observation was made for PLAG1 (Supplemental Fig.S7).

We also tested whether sumoylation regulates PLAG1-induced expression of IGF-П, a well-established target gene of PLAG1 (8). DNUbc9 did not have any effects on the basal

reporter activity, but transfection of as low as 0.02μg of DNUbc9 plasmid enhanced

PLAG1-induced IGF-П promoter activity, and the increase was dose-dependent (Fig.4B).

These results suggest that sumoylation negatively regulates PLAG1/PLAGL2-mediated

transcription.

Sumoylation-deficient PLAG1 and PLAGL2 localize to nucleoli

Since sumoylation regulates protein localization (Pichler et al., 2002), we

compared the cellular localization of wild-type PLAG1/PLAGL2 with that of the

sumoylation-deficient mutants. Both GFP-PLAG1 (Supplemental Fig.S8) and GFP-

PLAGL2 (Fig.5A) showed a diffuse nuclear pattern except for the nucleolus. However,

after lysine residues responsible for sumoylation were mutated, 80% of GFP-

PLAG1K244,263,353R (Supplemental Fig.S8) and 60% of GFP-PLAGL2K250,269,356R (Fig.5A)

exhibited strong and distinct nucleolar localization. PIAS1, which promotes sumoylation

of PLAG1/PLAGL2, colocalized with wild-type but not the mutant PLAGL2 (Fig.5B).

These results suggest that lysine residues responsible for sumoylation of PLAG1 and

PLAGL2 are critical for their proper nuclear localization, and in turn transactivation of

PLAG proteins.

PLAG1 and PLAGL2 are regulated by acetylation

132 Surprisingly, when we compared wild-type and sumoylation-deficient PLAG1 for their ability to activate IGF-П expression in reporter assays, we found that

PLAG1K244,263,353R has lower transactivation activity than wild-type PLAG1 (Fig.6A). We speculated that these lysine residues may play other critical roles in the transactivation.

Since lysine residues can also be acetylated and acetylation has been shown to regulate transactivation of transcription factors, we considered the possibility that one or more of the three lysine residues of PLAGs are also targets for acetylation. To determine whether

PLAG1 and PLAGL2 can be acetylated in cells, GFP-PLAG1 or Flag-PLAGL2 were transiently transfected into HEK293 cells with or without HA-p300. Cell lysates were immunoprecipitated with anti-Flag or anti-GFP antibody, and acetylated forms of

PLAGL2 and PLAG1 were detected by western blotting with anti-acetyl lysine antibody

(Fig.6B and Supplemental Fig.S9). P300 significantly enhanced acetylation of both

PLAG1 and PLAGL2. TSA, which is a histone deacetylase inhibitor, further increased the acetylation level of PLAGL2 (Fig.6C), suggesting that HDACs may regulate the acetylation of PLAG proteins. Indeed, HDAC7, a histone deacetylase, significantly decreased acetylation of PLAGL2 (lane 4 compared with lane 3, Fig.6D). Interestingly, the triple mutant PLAGL2 K246,246,356R, was less acetylated than the wild-type (lane 3

compared with lane 6, Fig.6D), suggesting that these lysine residues may also be the

targets for acetylation, which may explain why the sumoylation-deficient mutant PLAG1

has weaker activity than the wild-type PLAG1 (Fig.6A). To investigate the functional

consequences of acetylation, we tested whether p300 affects the transactivation of

PLAG1 and PLAGL2. When GAL4-fused full-length PLAG1 and PLAGL2 were

transfected alone or with p300, p300 significantly enhanced the transcriptional activation

133 potential of both PLAG proteins (Fig.6E and data not shown), Moreover, transfection of

p300 also enhanced PLAG1-induced IGF-П expression in luciferase reporter assays

(Supplemental Fig.S10). Since acetylation is a reversible process and HDAC7 decreased p300-induced PLAGL2 acetylation (Fig.6C), we also tested the effect of HDAC7 on

PLAG1-mediated transactivation of IGF-П promoter. As shown in Fig.6F, ectopic expression of HDAC7 repressed the PLAG1-induced IGF-П expression in a dose- dependent manner in luciferase reporter assays. Taken together, these results suggest that acetylation activates, while deacetylation represses PLAG1 and PLAGL2.

Lysine residues responsible for sumoylation/acetylation are important for the transforming activity of PLAG1 and PLAGL2

Both PLAG1 and PLAGL2 are oncogenes involved in the pathogenesis of different malignancies. Overexpression of PLAG1 or PLAGL2 transforms NIH-3T3 cells to low-serum growth (31). To evaluate the significance of sumoylation/acetylation in the transforming activity of PLAG1 and PLAGL2, PLAGs or their lysine mutants were transfected into NIH-3T3 cells. One day after transfection, cells were split at the density of 3×104 cells/10cm plate, and grown as a monolayer in the medium containing 10%

serum and G418 (300 μg/ml). After 3 days the medium was changed to 1% serum plus

300 μg/ml G418. The ability of transfected cells to form foci was analyzed 3 weeks following selection. As shown in Fig.7, both activated Ha-RAS oncogene, PLAG1 and

PLAGL2 promoted focus formation in NIH-3T3 cells in low-serum condition. It was

noticed that although RAS group and PLAG1/PLAGL2 groups have similar number of

focus formation, RAS transformed foci were bigger in size than the ones transformed by

134 PLAGs (data not shown), which suggests that though PLAG1 and PLAGL2 can

transform NIH-3T3 cells to grow in low-serum condition, they are weaker oncogenes

than RAS. Importantly, while all the single mutants of PLAGL2 still retained partial

transforming ability, the abilities of PLAG1/PLAGL2 sumoyation-deficient mutants to

transform NIH-3T3 cells were greatly reduced (Fig.7). These results suggest that lysine

residues which are sumoylation/acetylation targets are important for the transforming

ability of PLAG1 and PLAGL2.

Discussion

In our study, we found that PLAG1 and PLAGL2 are modulated by both

sumoylation and acetylation. Sumoylation occurs in a conserved repression domain and is

required for the repression activity. In contrast, acetylation by p300 activates the

transcriptional activity of PLAG1 and PLAGL2. Mutation of the sumoylated lysine

residues severely impairs the transforming ability of PLAG1 and PLAGL2, suggesting

the importance of these modifications on the transforming potential of these proteins.

Sumoylation pathway was shown to be activated under stress situations, such as hypoxia

or genotoxic condition (Shao et al., 2004; Huang et al., 2003; Comerford et al., 2003), and involved in DNA repair and maintenance of genome stability (Stelter et al., 2003).

Several important enzymes involved in DNA repair, such as thymine DNA glycosylase

(Hardeland et al., 2002), DNA topoisomerase І (Rallabhandi et al., 2002) and II (Mao et al., 2000), are regulated by sumoylation. At the same time, sumoylation prevents further cell growth through the repression of transcription factors with mitogenic potential, such as c-jun (Muller et al., 2000), Elk-1 (Yang et al., 2003), androgen receptor (Nishida et al.,

135 2002), Sp3 (Ross et al., 2002) and PLAG1/PLAGL2 in our study. It is tempting to speculate that sumoylation pathway may be one of the mechanisms for cellular stress adaption. That is, sumoylation pathway prevents cellular growth and facilitates cellular adaption to the stress. Consistent with the model, another PLAG family member,

PLAGL1, which is a tumor suppressor rather than an oncogene, can not be sumoylated.

Thus sumoylation may be a regulatory mechanism which differentiates various PLAG family members.

There are several possible mechanisms that could explain the repressive effects exerted by sumoylation. The first one is that sumoylation regulate subcellular localization of the substrates. Early studies have suggested that sumoylation is involved in regulating nucleo-cytoplasmic transport. For example, RanGAP1 is targeted to nuclear pore complex after being sumoylated (Mahajan et al., 1997). Recent studies indicated that sumoylation may play a more important role in regulating subnuclear distribution of proteins. Interestingly, sumoylation is essential for PML to accumulate in the nuclear bodies (Muller et al., 1998), and for Sp3 to localize in the nuclear periphery and nuclear dots (Ross et al., 2002). Our data suggest that sumoylation affects the subnuclear localization of PLAG1 and PLAGL2 (Fig.5), which may contribute to its transcriptional repression effect. Another possible mechanism for sumoylation-mediated transcriptional repression is that sumoylation may abolish some of the crucial protein-protein interactions thus rendering the substrates inactive. One of such examples is that sumoylation of ZNF76 affects its interaction with TATA-binding protein (TBP) (Zheng et al., 2004), which is a critical component for transcription initiation. Finally, it is also

136 possible that sumoylation may cause a gain of function such that sumoylated

PLAG1/PLAGL2 may recruit novel repressors to repress their transcriptional activity. It was shown that SUMO alone in a promoter is sufficient to inhibit promoter activity

(Yang et al., 2003). Since SUMO itself does not have repression activity, it suggests that other factors are recruited by SUMO to repress transcription. HDACs and HP1 have been shown to be recruited to p300 (Girdwood et al., 2003), Elk-1 (Yang et al., 2003) and histones (Shiio et al., 2003) after sumoylation to affect gene expression. However, whether both SUMO and the substrates contribute to the interaction with other factors to mediate repression remains unknown. More importantly, these mechanisms are not mutually exclusive. Although sumoylation affects the subnuclear localization of

PLAG1/PLAGL2, whether changes in protein-protein interactions or recruitment of novel repressors by sumoylation affect PLAG-mediated trans-activation requires further investigation.

Surprisingly, sumoylation-deficient PLAG1 has lower activity than wild type

PLAG1, which is explained by that PLAG1 and PLAGL2 are also regulated by acetylation, and sumoylation-deficient PLAG proteins are partially acetylation-defective.

Acetylation has been shown to modulate activities of a broad range of transcription factors including DNA binding, protein-protein interactions, protein stability, and transcriptional potency. Indeed, we found that PLAG1 and PLAGL2 can be activated by acetylation, and repressed by deacetylation. We propose the function of acetylation may be two-fold: first is to compete lysine residues for sumoylation, second is to activate transcription factors possibly through enhancing DNA binding, or changes in protein-

137 protein interactions such as recruitment of co-activators. Thus, a model emerges whereby

PLAG1/PLAGL2 can respond to activating signals by desumoylation and subsequent acetylation at the same lysine residues, which not only eradicates the function of the

repression domain, but also enhances transcriptional activation.

During the preparation of the current manuscript, Van Dyck et al. (Van Dyck et

al., 2004) published their study on the biochemical characterization of PLAG1

sumoylation. Our study not only agrees with the published data but also provides

additional novel findings: First, our study shows both PLAG1 and PLAGL2 (Landrette et al., 2003; Hensen et al., 2004), but not PLAGL1, which is a tumor suppressor rather than an oncogene, are regulated by sumoylation. Thus regulation by sumoylation may be one of the mechanisms to differentiate the functions of these three PLAG family members.

Second, using GAL4 reporter system, our data clearly demonstrate that sumoylation is required for the function of the repression domain of PLAG1 and PLAGL2 described previously (Kas et al., 1998). Third, our cellular localization studies suggest that sumoylation may play a role in the nuclear localization of PLAG1 and PLAGL2 (Fig.5).

Fourth, we identify acetylation as another posttranslational modification for PLAG1 and

PLAGL2, which has opposite function from sumoylation. Finally, in the transformation assay, our data clearly show that acetylation/sumoylation sites of PLAG1 and PLAGL2 are important for their transforming ability.

Notably, both sumoylation and acetylation are reversible processes, in which

SUMO-specific proteases and deacetylases can desumoylate and deacetylate modified

138 proteins, respectively. Acetylation and sumoylation of PLAG1/PLAGL2 may differentially modulate their affinity for different interacting protein partners, which contribute to different functional consequences of the two modifications: sumoylation causes a switch to a repressed state, while acetylation of PLAGL2/PLAG1 causes a switch to an activated state. Thus, the transactivation potential of PLAG1 and PLAGL2 can be regulated by a dynamic interplay of acetylation, deacetylation, sumoylation and desumoylation in response to various biological signals. In our study, sumoylation and acetylation not only affect PLAG target gene expression, but also affect the transforming ability of PLAG1 and PLAGL2 (Fig.7), suggesting a profound biological effect of these modifications. Given that both PLAG1 and PLAGL2 are oncogenes, involved in the pathogenesis of certain cancers, the enzymes involved in sumoylation and acetylation pathways for PLAG1/PLAGL2 could be considered as potential therapeutic targets to down-regulate the transcription activity of PLAG1/PLAGL2 in PLAG1/PLAGL2 associated malignancies.

Acknowledgement

We thank Dr. Ke Shuai for pCMV5-PIAS1; Dr. Andrew D. Sharrocks for L8G5-luc and

LexA-VP16 constructs; Dr. Ronald T. Hay for pCMV6-SSP3 and pcDNA3-DNUbc9;

Dr.Shigeru Taketani for pCG-PLAGL2 and pCG-PLAGL1; and Dr. P. Elly Holthuizen for human IGF-П-luciferase reporter construct Hup3-luc. This study was supported by

National Institutes of Health Grants DK50570, CA78433, and HL48819 (to Y.-C. Y.).

139 Figure 1. Characterization of the repression domain in PLAG proteins.

A. Structures of PLAG1, PLAGL2 and their GAL4-fusion derivatives.

B. PLAGL2 has a repression domain. HEK293 cells in 12-well plates were transfected

with 0.1 μg of pG5-luc reporter, 2 ng pRV-SV40 (internal control), 0.1 μg of expression plasmid PM-PLAGL2(238-496) or PM-PLAGL2(387-496) as indicated. Luciferase activities were measured 48 hours after transfection. Luciferase assays are representatives of at least three independent experiments.

140 A Repression domain 1 238 387 496 PLAGL2 Zinc finger domain Transactivation domain

387 496 PM-PLAGL2(387-496) GAL-4DBD 238 387 496 PM-PLAGL2(238-496) GAL-4DBD K250,269,356R 238 387 496 PM-PLAGL2(238-496)RRR GAL-4DBD

Repression domain 1 232 361 500 PLAG1 Zinc finger domain Transactivation domain

361 500 PM-PLAG1(361-500) GAL-4DBD 232 361 500 PM-PLAG1(232-500) GAL-4DBD K244,263,353R 232 361 500 PM-PLAG1(232-500)RRR GAL-4DBD

141 B (GAL4)5 E1B Luc

RLU 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

PM-PLAGL2(387-496)

PM-PLAGL2(238-496)

PM

142 Figure 2. Sumoylation pathway is required for the activity of the PLAGL2 repression domain.

A. Lysine residues in the sumoylation motifs are critical for the repression domain of

PLAGL2. HEK293 cells in 12-well plates were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid for GAL4-fusion PLAGL2 derivatives as indicated.

All the data in Figure.2 are representatives of at least three independent experiments.

B. Cell lysates from A were analyzed by western blot with anti-GAL4 antibody to detect various GAL4-PLAGL2 fusion proteins.

C.DNUbc9 abolishes the activity of the repression domain of PLAGL2. HEK293 cells in

12-well plates were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid for GAL4-fusion PLAGL2 derivatives, in the presence or absence of 0.2 μg

DNUbc9 as indicated.

D. SSP3 abolishes the activity of the repression domain of PLAGL2. HEK293 cells in

12-well plates were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid for GAL4-fusion PLAGL2 derivatives, in the presence or absence of 0.2 μg

SSP3 as indicated.

143 A

RLU 0.2 0.4 0.6 0.8 1.0 1.4 1.6 1.8 2.0 0

PM-PLAGL2(387-496)RRR

PM-PLAGL2(387-496)

PM-PLAGL2(238-496)

PM PM-PLAGL2(238-496)RRR B PM-PLAGL2(238-496) PM-PLAGL2(387-496) PM

49kDa

34kDa

28kDa

Anti-GAL4

144 -DNUBC9 C +DNUBC9 PLAGL2 RLU 0 1 2 3 4 5 6

PM-PLAGL2(387-496)RRR

PM-PLAGL2(387-496)

PM-PLAGL2(238-496)

PM

D -SSP3 PLAGL2 +SSP3 RLU 0 1 2 3 4 5 6 7

PM-PLAGL2(387-496)RRR

PM-PLAGL2(387-496)

PM-PLAGL2(238-496)

PM

145 Figure 3. PIAS1 promotes sumoylation of PLAGL2

A. PLAGL2 is sumoylated. HEK293 cells in 6-well plates were transfected with 0.5 μg

of pcDNA-PLAGL2-Myc in the presence or absence of 0.5 μg Flag-SUMO-1 plasmid as indicated. 48 hours after transfection, cells were lysed with denaturing buffer and analyzed with indicated antibodies. All the data in Figure.3 are representatives of at least three independent experiments.

B. PIAS1 promotes sumoylation of PLAGL2. HEK293 cells in 6-well plates were transfected with 0.5 μg of HA-PLAGL2, 0.5 μg Flag-SUMO-1 plasmid and 0.5 μg

pCMV5-PIAS1 or its mutant either alone or in combination as indicated. 48 hours after

transfection, cells were lysed with denaturing buffer and analyzed with indicated

antibodies.

C. DNUbc9 abolishes the sumoylation of PLAGL2. HEK293 cells in 6-well plates were transfected with 0.5 μg of pcDNA-PLAGL2-Myc, 0.5 μg Flag-SUMO-1 plasmid and 0.5

μg DNUbc9 either alone or in combination as indicated. 48 hours after transfection, cells were lysed with denaturing buffer and analyzed with indicated antibodies.

D. K250, K269 and K356 of PLAGL2 are sumoylation targets. HEK293 cells in 6-well plates were transfected with 0.5 μg of pcDNA-PLAGL2-Myc or its mutants, 0.5 μg Flag-

SUMO-1 plasmid and 0.5 μg pCMV5-PIAS1 either alone or in combination as indicated.

48 hours after transfection, cells were lysed with denaturing buffer and analyzed with

indicated antibodies.

E. PLAGL2K250R, PLAGL2K269R and PLAGL2K356R are sumoylated. HEK293 cells in 6-

well plates were transfected with 0.5 μg of pcDNA-PLAGL2-Myc mutants and 0.5 μg

Flag-SUMO-1 plasmid either alone or in combination as indicated. 48 hours after

146 transfection, cells were lysed with denaturing buffer and analyzed with indicated

antibodies.

F. PLAGL1 is not sumoylated. HEK293 cells were transfected with 0.5 μg of HA-

PLAGL1 or HA-PLAGL2 (positive control) with or without 0.5 μg Flag-SUMO-1

plasmid, and cells were lysed with denaturing buffer 48 hours after transfection and analyzed with the indicated antibodies.

147 + + A PLAGL2-Myc Flag-SUMO-1 - + Sumoylated PLAGL2 100kDa

Myc-PLAGL2 60kDa Blot: Anti-Myc

SUMOylated 100kDa proteins

60kDa Blot: Anti-Flag

HA-PLAGL2 + + + +

Flag-SUMO-1 - + + + B Flag-PIAS1 - - + -

Flag-PIAS1C335A - - - + 200kDa Sumoylated PLAGL2 120kDa 100kDa

HA-PLAGL2 60kDa Blot: Anti-HA SUMOylated 200kDa proteins 120KD 100KD Flag-PIAS1/PIAS1C335A

60KD Blot: Anti-Flag

148

C

PLAGL2-Myc + + +

Flag-SUMO-1 - + + DNUbc9 - - +

Sumoylated PLAGL2 WB:Anti-Myc Myc-PLAGL2 Sumoylated proteins

WB:Anti-Flag Flag-SUMO-1

Ubc9 WB:Anti-Ubc9

149

D Flag-PIAS1 - - + - - + - - + Flag-SUMO-1 - + + - + + - + + PLAGL2-Myc + + + ------

PLAGL2 K250R-Myc - - - + + + - - -

PLAGL2 K250,269,356R-Myc ------+ + + 200kDa Sumoylated PLAGL2 120kDa 100kDa

PLAGL2-Myc 60kDa Blot: Anti-Myc 200kDa SUMOylated proteins 120kDa 100kDa Flag-PIAS1

60kDa Blot: Anti-Flag

150

E PLAGL2RRR-Myc PLAGL2K250R-Myc PLAGL2K269R-Myc PLAGL2K356R-Myc Flag-SUMO-1 --++ - + - + Sumoylated 100KD PLAGL2

PLAGL2-Myc 60KD Blot: anti-Myc

SUMOylated 100KD proteins

60KD

Blot: anti-Flag

151

F HA-PLAGL2 - - + + HA-PLAGL1 + + - -

Flag-SUMO-1 - + - +

200KD 120KD

HA-PLAGL1 * 100KD

HA-PLAGL2 60KD

Blot: Anti-HA

200KD SUMOylated 120KD proteins 100KD

60KD Blot: Anti-Flag

* Sumoylated PLAGL2

152 Figure 4. Sumoylation negatively regulates the transcriptional activation of PLAG1 and

PLAGL2.

A. DNUbc9 activates full-length PLAGL2. HEK293 cells in 12-well plates were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid pM-PLAGL2 and 0.2 μg of DNUbc9 alone or in combination as indicated. Cell lysates were analyzed by western blot with the indicated antibodies to detect the PLAGL2 fusion proteins

(indicated by *) and Ubc9 proteins in the lower panels. All the data in Figure.4 are representatives of at least two independent experiments.

B. DNUbc9 enhances PLAG1-induced IGF-П expression. HEK293 cells in 12-well plates were transfected with 50 ng of Hup3-luc reporter, 0.1 μg of expression plasmid pcDNA-PLAG1-Myc and different amounts of DNUbc9 alone or in combination as indicated.

153 A

3.5 3.0 2.5 2.0 RLU 1.5 1.0 0 PM-PLAGL2 - + - + DNUbc9 - - + + pG5-luc + + + +

Anti-GAL4 * *

Anti-Ubc9

154 B

12 10 8 6 RLU 4 2 0

PLAG1 - + + + - - DNUbc9 - - 0.02 0.05 0.02 0.05 Hup3-Luc + + + + + +

155 Figure 5. Sumoylation-deficient PLAGL2 localizes to nucleoli.

A. PLAGL2K250,269,356R but not PLAGL2 concentrates in the nucleolus. HEK293 cells on

chamber slides were transfected with 0.5μg expression vectors for GFP-PLAGL2 (left) or

GFP-PLAGL2K250,269,356R (right) and fixed 48 hours later. Cells were visualized using fluorescence confocal microscopy. All the localization data in Fig.5 are representatives of at least two independent experiments

B. PLAGL2 but not PLAGL2 K250,269,356R colocalizes with PIAS1. HEK293 cells on chamber slides were transfected with 0.5μg GFP-PLAGL2 (top panel) or 0.5μg GFP-

PLAGL2 K250,269,356R (bottom panel) with 0.5μg Flag-PIAS1. Cells were fixed 48 hours

after transfection and treated with anti-Flag antibody and a secondary Alexa Fluor 594

dye-conjugated antibody. Stained cells were visualized using fluorescence confocal

microscopy.

156 A

GFP-PLAGL2 GFP-PLAGL2 K250,269,356R

B

GFP-PLAGL2 Flag-PIAS1 merge

GFP-PLAGL2K250,269,356R Flag-PIAS1 merge

157 Figure 6. PLAG1 and PLAGL2 are regulated by acetylation.

A. Sumoylation-deficient PLAG1 has impaired ability to activate IGF-П expression.

HEK293 cells in 12-well plates were transfected with 50 ng of Hup3-luc reporter and

different amounts of expression plasmid pcDNA-PLAG1-Myc or its mutant as indicated.

All the data in the figure are representatives of at least three independent experiments.

B. PLAGL2 can be acetylated by p300. Lysates from HEK-293 cells (in 6-well plates)

transfected with 0.5 μg Flag-tagged PLAGL2 with or without 0.5 μg HA-p300 were

immunoprecipitated (IP) with anti-Flag and immunoblotted with indicated antibodies.

C. TSA augments PLAGL2 acetylation. Lysates from HEK-293 cells (in 6-well plates)

transfected with 0.5 μg Flag-tagged PLAGL2, 0.5 μg HA-p300 with or without TSA

treatment (330 nM TSA for 12 hours before harvesting) were immunoprecipitated (IP)

with anti-Flag and immunoblotted with indicated antibodies.

D. Acetylation of the sumoylation-deficient mutant of PLAGL2 is impaired. HEK293

cells in 10 cm plates were transfected with 3 μg of pcDNA-PLAGL2-Myc or its mutant,

3 μg HA-p300 and 3 μg HA-HDAC7 alone or in combination as indicated. Anti-Myc

immunoprecipitates from the whole cell extracts were immunoblotted with antibodies

indicated.

E. P300 activates PLAGL2 transcriptional activity. HEK293 cells in 12-well plates were

transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid pM-PLAGL2, in the presence or absence of 0.2 μg p300 as indicated. Luciferase assays are representatives of at least three independent experiments.

F. HDAC7 represses PLAG1-induced IGF-П expression. HEK293 cells in 12-well plates were transfected with 50 ng of Hup3-luc reporter, 0.1 μg of expression plasmid pCMV-

158 Flag-PLAG1 and different amounts of HA-HDAC7 either alone or in combination as indicated. Luciferase assays are representatives of at least three independent experiments.

Total cell lysates were blotted with anti-HA antibody to detect the expression of HDAC7

(bottom panel).

159 A 3.5 3.0 2.5 2.0 1.5 1.0

RLU 0.5 0 PLAG1 - 0.02 0.08 - -

PLAG1K244,263,353R - - - 0.02 0.08

Hup3-Luc + + + + +

B + - + HA-p300 - + + Flag-PLAGL2 IP: anti-Flag Blot: anti-acetylated lysine IP: anti-Flag Blot: anti-Flag

Blot: anti-HA

160 C

- + + HA-p300 - - + TSA + + + Flag-PLAGL2 IP: anti-Flag Blot: anti-acetylated lysine

Blot: anti-HA

IP: anti-Flag Blot: anti-Flag

D

+ - + + - + HA-p300 - + + + - - PLAGL2-Myc - - - + - - HA-HDAC7 - - - - + + PLAGL2 RRR-Myc IP: anti-Myc Blot: anti-acetylated lysine IP: anti-Myc Blot: anti-Myc Blot: anti-HA Blot: anti-HA

1 2 3 4 56

161 E 0.7 0.6 0.5 0.4

RLU 0.3 0.2 0.1 0.0 PM-PLAGL2 - + - + HA-p300 - - + +

pG5-Luc + + + +

F 25 20 15

RLU 10 5 0 Flag-PLAG1 - + + + + + -

HA-HDAC7 - - 0.01 0.03 0.1 0.3 0.3

Hup3-luc + + + + + + +

Blot: anti-HA

162 Figure 7. Lysine residues responsible for sumoylation/acetylation are important for the transforming activity of PLAG1 and PLAGL2. NIH-3T3 cells were transfected with 2 μg of empty vector, expression plasmids for PLAGL2 or its mutants, PLAG1 or its mutants, and Ha-RAS. After selection in medium with 1% serum and 300 μg/ml G418 for 3 weeks, cells were fixed and stained with methylene blue, and the number of transformed foci was determined (standard deviation is shown, n=2). The data shown here is a representative of three independent experiments.

163 30 25

cells 20 4

10 15 × 10 5

Foci/3 0 Vector RAS PLAGL2K250R-Myc PLAGL2K269R-Myc PLAGL2K356R-Myc PLAGL2RRR-Myc PLAGL2-Myc PLAG1RRR PLAG1

164 Supplemental Figures

Figure S1. PLAG1 has a repression domain. HEK293 cells in 12-well plates were

transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid PM-

PLAG1(361-500) or PM-PLAG1(232-500) as indicated. Luciferase assays are representatives of at least three independent experiments.

Figure S2. PLAGL2(238-387) does not have trans-repression function. HEK293 cells in

12-well plates were transfected with 0.1 μg of L8G5-luc reporter (LexA-GAL4-driven

E1A promoter-reporter), 0.2 μg of expression plasmid for Lex-VP16, 0.2 μg of PM-

PLAGL2(238-387) either alone or in combination as indicated. Luciferase assays are

representatives of at least three independent experiments.

165 RLU 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

PM-PLAG1(361-500)

PM-PLAG1(232-500)

PM

Supplemental Fig.S1

0.6 0.5 0.4

RLU 0.3 0.2 0.1 0 PM - - + - LexA-VP16 - + + + PM-PLAGL2(238-387) - - - +

Supplemental Fig.S2

166 Figure S3. Alignment of PLAG1 and PLAGL2 sequences. The repression domains are boxed and three conserved sumoylation motifs in the repression domains are underlined.

Conserved residues are indicated by “*”.

167

168

Figure S4. Sumoylation pathway is required for the activity of the PLAG1 repression

domain.

A. Lysine residues in the sumoylation motifs are critical for the repression domain of

PLAG1. HEK293 cells in 12-well plates were transfected with 0.1 μg of pG5-luc reporter,

0.1 μg of expression plasmid for GAL4-fusion PLAG1 derivatives in combination as indicated. All the data in this figure are representatives of at least three independent experiments.

B. Cell lysates from A were analyzed by western blot with anti-GAL4 antibodies to detect various GAL4-PLAG1 derivatives.

C. DNUbc9 abolishes the activity of the repression domain of PLAG1. HEK293 cells in

12-well plates were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression

plasmid for GAL4-fusion PLAG1 derivatives, in the presence or absence of 0.2 μg

DNUbc9 as indicated. All the luciferase assays in this figure are representatives of at

least three independent experiments.

D. SSP3 abolishes the activity of the repression domain of PLAG1. HEK293 cells in 12-

well plates were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression

plasmid for GAL4-fusion PLAG1 derivatives, in the presence or absence of 0.2 μg SSP3

as indicated.

169 RLU

A 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0

PM-PLAG1(232-500)RRR

PM-PLAG1(361-500)

PM-PLAG1(232-500)

PM PM-PLAG1(232-500)RRR PM-PLAG1(232-500) PM-PLAG1(361-500) B PM

49KD

34KD

28KD

Anti-GAL4

Supplemental Fig.S4

170 C Fold induction 10 12 14 16 0 2 4 6 8

PM-PLAG1(232-500)RRR

PM-PLAG1(361-500)

PM-PLAG1(232-500)

PM

D

Fold induction 10 20 30 40 50 60 70 80 90 0

PM-PLAG1(232-500)RRR

PM-PLAG1(361-500)

PM-PLAG1(232-500)

PM

171 Figure S5. The repression domains of PLAG1 and PLAGL2 are activated by DNUbc9.

HEK293 cells in 12-well plates were transfected with 0.1 μg of pG5-luc reporter, 0.1 μg

of expression plasmid pM-PLAG1(232-361), pM-PLAGL2(238-387) (left panel) or pM-

ZNF76K411R (right panel) and 0.2 μg of DNUbc9 either alone or in combination as

indicated. Luciferase assays are representatives of at least three independent experiments.

172 4.0 3.5 3.0 2.5

RLU 2.0 1.5 1.0 0.5 PM-PLAGL2(238-387) - + + ------+ + - PM-PLAG1(232-361) DNUbc9 - - + - + +

pM + - - - - +

pG5-luc + + + + + +

Supplemental Fig.S5

173 Figure S6. K244, K263 and K353 of PLAG1 are sumoylation targets. HEK293 cells in 6- well plates were transfected with 0.5 μg of GFP-PLAG1 or its mutant, 0.5 μg Flag-

SUMO-1 plasmid and 0.5 μg pCMV5-PIAS1 either alone or in combination as indicated.

48 hours after transfection, cells were lysed with denaturing buffer and analyzed with indicated antibodies. The sumoylation assay data is a representative of at least three independent experiments.

174 Flag-PIAS1 - - + - - + Flag-SUMO-1 - + + - + + GFP-PLAG1 + + + ------+ + + GFP-PLAG1K244,263,353R

SUMOylated PLAG1 120KD

100KD GFP-PLAG1

Blot: Anti-GFP

SUMOylated 120KD proteins 100KD Flag-PIAS1

Blot: Anti-Flag

Supplemental Fig.6

175 Figure S7. DNUbc9 activates full-length PLAG1. HEK293 cells in 12-well plates were

transfected with 0.1 μg of pG5-luc reporter, 0.1 μg of expression plasmid pM-PLAG1

and 0.2 μg of DNUbc9 alone or in combination as indicated. Cell lysates were analyzed

by western blot with the indicated antibodies to detect the PLAG1 fusion proteins

(indicated by *) and Ubc9 proteins in the lower panels. Luciferase assays are representatives of three independent experiments.

Figure S8. PLAG1K244,263,353R but not PLAG1 concentrates in the nucleolus. HEK293

cells on chamber slides were transfected with 0.5μg expression vectors for GFP-PLAGL1

(left) or GFP-PLAG1K244,263,353R (right) and fixed 48 hours later. Cells were visualized using fluorescence confocal microscopy. The data shown in the figure is a representative of at least two independent experiment.

176 12 10 8 6 RLU 4 2 0 PM-PLAG1 - + - +

DNUbc9 - - + + pG5-luc + + + +

Anti-GAL4 **

Anti-Ubc9

Supplemental Fig.7

GFP-PLAG1 GFP-PLAG1K244,263,353R

Supplemental Fig.8

177 Figure S9. PLAG1 can be acetylated by p300. Lysates from HEK-293 cells (in 6-well plates) transfected with 0.5 μg GFP-tagged PLAG1 with or without 0.5 μg HA-p300 were

immunoprecipitated (IP) with anti-GFP and immunoblotted ed with indicated antibodies.

The acetylation assay is a representative of at least three independent experiments.

Figure S10. P300 activates PLAG1 transcriptional activity. HEK293 cells in 12-well

plates were transfected with 50 ng of Hup3-luc reporter, 0.1 μg of expression plasmid

pCMV-Flag-PLAG1, in the presence or absence of 0.2 μg p300 as indicated. Luciferase

assays are representatives of at least three independent experiments.

178 + - + HA-p300 - + + GFP-PLAG1

IP: anti-GFP Blot: anti-acetylated lysine

IP: anti-GFP Blot: anti-GFP

Blot: anti-HA

Supplemental Fig.9

7 6 5 4 3 2

Fold induction 1 0 HA-p300 - +

Hup3-Luc + +

Flag-PLAG1 + +

Supplemental Fig.10

179 CHAPTER 5

PIRH2 IS REGULATED BY DIMERIZATION

Introduction

P53 is important in coordinating cellular responses to stress (Levine et al., 1997), which is mediated through a variety of mechanisms, including cell cycle arrest, apoptosis

and cellular senescence (Lowe et al., 2003). Given its critical role, it is not surprising that

post-translational modifications, including acetylation (Gu et al., 1997), phosphorylation

(Giaccia et al., 1998), sumoylation (Kahyo et al., 2001), neddylation (Harper et al., 2004) and ubiquitination (Brooks et al., 2003) of p53 could regulate its function.

Ubiquitination controls the activities of proteins in numerous cellular processes,

including transcription. It occurs once (monoubiquitination) or in chains

(polyubiquitination) on target proteins, which have different consequences. Attachment of

polyubiquitin chains by ubiquitination E3 ligases to p53 results in destruction of the p53

protein by the 26S proteasome. There are several cellular ubiquitination E3 ligases for

p53. The best studied is Mdm2, which has a RING finger domain (Kubuttat et al., 1997)

and catalyzes the ubiquitination of p53. If activated in certain malignancies, Mdm2 is able to abolish the tumor suppressor function of p53. There are various regulatory mechanisms for Mdm2. Mdm2 enzymatic activity is inhibited by the association with the

p19ARF (Pomenrantz et al., 1998), ribosomal protein L11 (Zhang et al., 2003) or TSG101

(Li et al., 2001), while enhanced by interacting with MTBP (Brady et al., 2005) or YY1

(Sui et al., 2004). Post-translational modifications such as phosphorylation by Ataxia

Telangiectasia-mutated (ATM) in response to DNA damage also regulates Mdm2-p53

180 interaction (Maya et al., 2001). Thus ubiquitination E3 activity is regulated through

protein-protein interactions or post-translational modifications which indirectly control

the ubiquitination and stability of the substrates. Other ubiquitination E3 enzymes for p53

are E6-AP (Scheffner et al., 1993), COP1 (Dornan et al., 2004), ARF-BP1 (Chen et al.,

2005) and Pirh2 (p53-induced RING-H2) (Leng et al., 2003). Among them, Pirh2 is a

target gene of p53; its transcript and protein levels increase in response to UV irradiation

and cisplatin treatment (Leng et al., 2003). Though its function is similar to Mdm2, little

is known about how the activity of Pirh2 is regulated.

PLAGL2 is a homologue of PLAG1 (Pleomorphic salivary Adenoma Gene 1). It is a transcription factor with transforming ability (Hensen et al., 2002) and involved in various malignancies including AML (acute myeloid leukemia) (Landrette et al., 2004).

Though PLAGL2 was proposed to function mainly as a transcription factor (Kas et al.,

1998), how it contributes to the malignancies is still not clear. In our study, we found that Pirh2 forms dimers through both N- and C-terminus. Interestingly, dimerization of

Pirh2 enhances its interaction with p53. We also found that PLAGL2 interacts with Pirh2 dimers and the interaction stabilizes Pirh2. This study suggests that Pirh2 is regulated by dimerization, which may be a potential therapeutic target for Pirh2-overexpressing malignancies.

Materials and Methods

181 Reagents and Antibodies Anti-Myc (9E10) was from Santa Cruz Biotechnology Inc.

(Santa Cruz, CA). Anti-FLAG (M2) was purchased from Upstate Biotechnology, Inc.

(Lake Placid, NY). Anti-HA antibody was from BAbCo (Richmond, CA). HEK293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C and in 5% CO2.

Transient Transfection, Immunoprecipitation and Western blot analysis HEK293 cells

were transfected by calcium phosphate precipitation method with various plasmid

combinations as indicated. Forty-eight hours later, cells were washed with PBS and 1 ml

ice-cold lysis buffer (RIPA) (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM

EGTA, 2 mM Na3VO4, 15 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF) was added.

Cells were lysed for 30 min at 4°C with occasional vortexing. The lysates were collected

into 1.5-ml tubes and cleared of nuclei by centrifugation for 10 min at 14,000 rpm. The

supernatants (whole cell extracts) were incubated with different antibodies for 16 h at

4°C and protein A-agarose beads were added for the last hour. The beads were washed

five times in TNEN buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5%

NP-40, 2 mM Na3VO4, 1 mM PMSF, 1 mM NaF.). Bound proteins were extracted with

SDS-PAGE sample buffer, and analyzed by SDS-PAGE followed by Western blot

analysis with the ECL detection system.

Plasmid Construction pcDNA-Pirh2-Myc and other Myc-tagged Pirh2 mutants were

constructed by insertion of the corresponding cDNA fragments into EcoRI and BamHI

sites of pcDNA3.1-Myc-HisB (Invitrogen). GFP-Pirh2 mutants were constructed by

182 insertion of the corresponding cDNA fragments into EcoRI and BamHI sites of pEGFP-

C1 (Clontech).

Results

Pirh2 is ubiquitinated

Pirh2 was cloned in our laboratory as an interacting protein of Cited2 (CBP/P300

Interacting Transactivator with Glutamic acid (E) and Aspartic acid (D)-rich tail 2) from a yeast two-hybrid screen. Pirh2 has a C2H2 type RING finger domain in the coding region (Fig.1A), which is characteristic of ubiquitination E3 ligases. As ubiquitination E3

ligases are capable of self-ubiquitination, we examined whether Pirh2 is ubiquitinated in

cells. As shown in Fig.1B, HEK293 cells were cotransfectd with Pirh2-Myc or Myc-

tagged Pirh2 RING-deletion mutant with HA-ubiquitin. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody, and the immunoprecipitates were analyzed by Western blot with anti-Myc or anti-HA antibodies. Both Pirh2 and its RING-deletion mutant were ubiquitinated (upper panel, anti-HA blot). Interestingly, the full-length Pirh2 but not the RING-finger deletion mutant can be monoubiquitinated, suggesting that monoubiquitination of Pirh2 depends on the RING-finger domain. These results suggest that Pirh2 is an ubiquitination substrate.

Pirh2 forms dimer

When we ectopically expressed Pirh2-Myc in HEK293 cells, we found that there is always a higher-molecular weight form of Pirh2 through immunoblot with anti-Myc antibody (Fig.2A), both in the whole cell lysates (lane 1) and in the immunoprecipitates

183 (lane 2). Interestingly, when we treated the transfected cells with desferrioxamine (DFO),

the higher-molecular weight form of Pirh2 was abolished (lane 3 and lane 4). These

results suggest that the higher-molecular weight form of Pirh2 may depend on the

availability of iron. Given that the molecular size of Pirh2 is around 30kDa, and the slow

migrating anti-myc reactive band is around 60kDa, we suspected that the slow migrating

band is due to a dimer form of Pirh2. To test whether Pirh2 forms dimers, we generated a

Flag-tagged Pirh2 construct and tested whether Pirh2-Myc interacts with Flag-Pirh2 by coimmunoprecipitation experiment. As shown in Fig.2B, immunoprecipitation with anti-

Myc antibody can pull down Flag-Pirh2 only when Pirh2-Myc was expressed (lane 3 and lane 4), suggesting that Pirh2 forms dimers. Moreover, the higher form of Flag-Pirh2 also coprecipitates with Pirh2-Myc (lane 4, upper panel), which suggest that the higher- molecular weight form of Pirh2 in SDS-PAGE is a dimer. Next we mapped the dimerization region and found both the N- and C-terminus interact with the full-length

Pirh2 (Data not shown). Interestingly, the N-terminus of Pirh2 interacts with itself but not the C-terminus of Pirh2 (Fig.2C), suggesting that N-terminal interaction is involved in the

Pirh2 dimer formation. In short, these results show that Pirh2 forms dimers in cells.

Dimerization of Pirh2 enhances its interaction with p53

Pirh2 interacts and promotes the ubiquitination of p53 (Leng et al., 2003). Since

Pirh2 forms dimers, we next tested whether Pirh2 dimerization affects its interaction with p53. Two possibilities were considered. One is that only Pirh2 monomers interact with p53 and the dimerization of Pirh2 will decrease the available Pirh2 monomers and thus negatively affect the interaction. Another possibility is that Pirh2 dimers interact with p53

184 thus dimerization of Pirh2 is necessary for the interaction. We examined the two possibilities by testing whether Flag-Pirh2 is able to compete with Myc-tagged Pirh2 for p53 binding. The rationale behind is that if only Pirh2 monomers interact with p53, Flag-

Pirh2 will compete with Myc-tagged Pirh2 for p53 interaction. However, if dimers are involved in the interaction, Flag-Pirh2 may have no effects or enhance the interaction between Pirh2-Myc and p53. As shown in Fig.3, we cotransfected 3 μg HA-p53 with 2

μg of Pirh2-Myc in the presence or absence of 6 μg Flag-Pirh2. Cell lysates were immunoprecipitated with anti-Myc antibody, and the interaction between p53 with Pirh2-

Myc was detected by immunoblot with anti-HA antibody. As shown in Fig.3, the presence of Flag-Pirh2 significantly enhanced the amount of HA-p53 proteins coprepcipitated with Myc-tagged Pirh2 (the second panel, comparing lane 2 with lane 3).

The result suggests that Pirh2 dimers are involved in the interaction with p53.

PLAGL2 interacts with Pirh2 dimers to stabilize Pirh2

Pirh2 was isolated as a PLAG1 interacting protein by others (AAK96899). We tested whether another member of PLAG family, PLAGL2, could interact with Pirh2. As shown in Fig.4A. HA-PLAGL2 was cotransfected with Pirh2-Myc or Pirh2ΔRING-Myc.

Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA to detect the interaction between PLAGL2 and Pirh2. HA-PLAGL2 coprecipitated with both Pirh2-Myc and Pirh2ΔRING-Myc, suggesting that the interaction between Pirh2 and PLAGL2 does not depend on its RING finger domain. We further tested whether the Pirh2 dimer is involved in the interaction. As shown in Fig.4B,

HA-PLAGL2 was cotransfected with Pirh2-Myc in the absence or presence of Flag-Pirh2.

185 Cell lysates were immunoprecipitated with anti-Myc and immunoblotted with anti-HA.

With the expression of Flag-Pirh2, the interaction between HA-PLAGL2 and Pirh2-Myc

significantly increased. This result suggests that Pirh2 dimers interact with PLAGL2.

PLAGL2 acts as a transcription factor with the transactivation domain in its C-terminus.

However, Pirh2 did not affect the transactivation of PLAGL2 (Data not shown). The

Pirh2 level is regulated by ubiquitination (Fig.1B), and proteins ubiquitinated are expected to exhibit a relatively short half-life due to rapid turnover by the proteasome.

We next tested whether PLAGL2 regulates the stability of Pirh2 through their interaction.

As shown in Fig.4C, to measure the half-life of Pirh2 in the presence or absence of

PLAGL2, cells were transfected with Flag-Pirh2 alone or cotransfected with HA-

PLAGL2, and treated with cycloheximide for different lengths of time before harvest.

Pirh2 has a short half-life with its level decreased after 1 hour and disappeared after 2

hours. When cotransfected with PLAGL2, Pirh2 expression level was significantly

increased and the half-life became longer with its level started to decrease only after 4

hours. GFP was used as an internal control to monitor transfection efficiency and protein loading. These results suggest that PLAGL2 interacts with Pirh2 dimers to stabilize Pirh2.

Discussion

Pirh2 is one of the E3 ubiquitination enzymes promoting the degradation of p53.

In our study, we found that Pirh2 dimerizes and the dimer form of Pirh2 is involved in

interaction with p53 and PLAGL2. This study not only identifies a molecular mechanism

of how Pirh2 interacts with p53 and PLAGL2, but also suggests Pirh2 dimerization might

be important for its function as an E3 ligase.

186

Dimerization is a common mechanism by which the activities of numerous important biological macromolecules can be regulated. These molecules include receptors for growth hormones, steroid hormones, transcription factors and enzymes.

There are several possibilities for Pirh2 dimer-mediated functions. The first possibility is that the dimer form is important for its interaction with the substrates, which is supported by our data. Pirh2 monomers may only have a weak interaction or no interaction with p53, while Pirh2 dimers have more interaction sites or create novel interaction sites for p53 thus strongly increase the binding affinity. The second possibility is that only the Pirh2 dimer is an active ubiquitination E3 ligase, thus dimerization may regulate its E3 activity.

Examples of such are CHIP (Nikolay et al., 2004) and NOS (Ravi et al., 2004), whose dimerization is required for their enzymatic activities. Another possibility is that dimerization may stabilize Pirh2. Pirh2 itself is regulated by ubiquitination, although the exact ubiquitination sites of Pirh2 are not known at present. It is possible that destabilization of the dimer is an important event for its ubiquitination. We speculate that a degradation signal is present in the dimer interface that is sterically blocked in the stable dimeric state. In support of the model, heterodimerization of MATα2 and MATa1 is known to decrease the ubiquitin-proteasomal degradation of both factors (Johnson et al., 1998), and the stability of NOS is regulated by the dimerization (Bender et al., 2000;

Hattori et al., 2003). Destabilization of dimeric Pirh2 may lead to a relaxation of the structural constraints, rendering the protein more flexible. This relaxation of the protein structure may be sufficient to target the protein for degradation. This possibility is not mutually exclusive to the others discussed above. That is, the dimerization of Pirh2 may

187 be important for both stability and enzymatic activity. It is likely that the functionally

inactive monomeric form of the protein is preferentially ubiquitinated over the enzymatically active homodimers. The structural change, which is related to inactivation and monomerization, serves to expose lysine residues for ubiquitin conjugation.

Dimerization of enzymes is under tight regulation in cells. A well-studied

example is NOS2 (Nitric Oxide Synthase). In stimulated macrophages, inactive NOS2

monomers slowly form active homodimers. Many factors are shown to regulate the

homodimerization of NOS2. Tetrahydrobiopterin, Arginine and heme play critical roles

in promoting the homodimerization of NOS2 (Abu-Soud et al., 1995; Albakri et al., 1996),

while NAP110 interacts with the NOS2 monomer and inhibits its dimerization and

enzymatic activity (Ratovitski et al., 1999). We speculate that dimerization of Pirh2 also has complex regulatory mechanisms; in response to different external signals, Pirh2 dimerization and thus its activity may be regulated by different factors. DFO abolished the SDS-resistant dimers of Pirh2 (Fig.2A) in our study, suggesting that heme may be involved in regulating its dimerization.

PLAGL2 is an oncoprotein involved in the pathogenesis of AML (Acute Myeloid

Leukemia). Though it is suggested to mainly function as a transcription factor, its exact roles in oncogenesis are not clear. In our study, PLAGL2 was found to interact with Pirh2 dimers and regulate the stability of Pirh2. Given that Pirh2 negatively regulates p53,

PLAGL2 may indirectly regulate p53 through its interaction with Pirh2, and partially contribute to its role as an oncoprotein. Interestingly, we observed the monoubiquitinated

188 form of Pirh2 in the ubiquitination assay (Fig.1B), and the monoubiquitinated Pirh2 still

coimmunoprecipitates with PLAGL2 (data not shown). It is likely that the limited

ubiquitination of Pirh2 such as monoubiquitination gives a more stable conjugate whereas the polyubiquitinated forms are rapidly proteolyzed. It is possible that PLAGL2 interacts with monoubiquitinated Pirh2 and prevents further ubiquitination and degradation of Pirh2 by proteasome. Another possible mechanism for PLAGL2-mediated

Pirh2 stabilization is that PLAGL2 may interact with the Pirh2 dimer, and stabilize the dimer form, which in turn stabilizes the protein.

Pirh2 was shown to be overexpressed in lung cancer (Duan et al., 2004). Since it functions similarly to Mdm2 (Leng et al., 2003), it is not surprising that Pirh2 may also act as an oncogene by negatively regulating the p53 level and its stability. Our study identifies the Pirh2 dimer as a novel target to down-regulate Pirh2-mediated p53 degradation. Chemical compounds or small peptides which interfere Pirh2 dimerization may abolish Pirh2’s ability to interact with p53 and to promote the degradation of p53. In cancers with higher expression levels of Pirh2, this strategy is of therapeutic value since it provides an approach to restore p53 activities.

189 Figure 1. Pirh2 is ubiquitinated.

A. Schematic representation of Pirh2 structure.

B. HEK293 cells (in 6-well plates) were transfected with 1 μg Pirh2-Myc or

Pirh2ΔRING-Myc with 1 μg HA-ubiquitin. 48 hours after transfection, cells were harvested and cell lysates were immunoprecipitated with anti-Myc antibody.

Immunoprecipitates were analyzed by SDS-PAGE and subsequent immunoblotting with indicated antibodies.

190 A Pirh2

N C

RING Finger Domain

B IP: anti-Myc HA-ubiquitin + + Pirh2-Myc - + Pirh2 △RING -Myc + -

101kDa

69kDa

44kDa Monoubiquitinated Pirh2-Myc 28kDa

Blot:anti-HA

101kDa

69kDa

Monoubiquitinated 44kDa Pirh2-Myc Pirh2-Myc Pirh2 △RING -Myc 28kDa

Blot:anti-Myc

191 Figure 2. Pirh2 forms dimers.

A. 2 μg Pirh2-Myc was transfected into HEK293 cells using calcium phosphate method.

Transfected cells were either left untreated or treated with DFO (20 μM) for 12 hours before harvesting. Cell lysates were subjected to immunoprecipitation and Western blot with anti-Myc antibody.

B. 3 μg Flag-Pirh2 was cotransfected with or without 3 μg Pirh2-Myc into HEK293 cells using calcium phosphate method. Cell lysates were subjected to immunoprecipitation with anti-Myc and Western blot with indicated antibodies.

C. 3 μg Pirh2(1-135)-Myc was cotransfected with 3 μg GFP-Pirh2(1-135) (upper panel) or GFP-Pirh2(187-261) (lower panel). Cell lysates were subjected to immunoprecipitation with anti-Myc and Western blot with anti-GFP.

192 A

DFO - - + + Pirh2-Myc + + + + IP - Myc - Myc

60kDa

30kDa

1 2 3 4 Blot: anti-Myc

193

B IP:Myc Flag-Pirh2 + + + +

Pirh2-Myc - + - +

60kDa

Blot: Anti-Flag

FLAG-Pirh2 30kDa

60KD

Blot: Anti-Myc

Pirh2-Myc 30KD

1 2 3 4

194 C

IP: anti-Myc GFP-Pirh2(1-135) + + + +

Pirh2(1-135)-Myc - + - +

Blot: Anti-GFP

IP: anti-Myc GFP-Pirh2(187-261) + + + +

Pirh2(1-135)-Myc - + - +

Blot: Anti-GFP

195 Figure 3. Dimerization of Pirh2 enhances its interaction with p53.

3 μg HA-p53 was transfected with 2 μg Pirh2-Myc in the presence or absence of 6 μg

Flag-Pirh2 indicated. Cells were harvested 48 hours after transfection. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody, and Western blot with the antibodies indicated.

196 HA-53 + + + Flag-Pirh2 - - + Pirh2-Myc - + +

Blot: anti-HA

IP: anti-Myc Blot: anti-HA

IP: anti-Myc Blot: anti-Myc

Blot: anti-Flag

IP: anti-Myc Blot: anti-Flag

1 2 3

197 Figure 4. PLAGL2 interacts with Pirh2 dimers to stabilize Pirh2.

A. 3 μg HA-PLAGL2 was cotransfected with 2 μg Pirh2-Myc or 2 μg Pirh2ΔRING-Myc.

48 hours after transfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody, and Western blot with the antibodies indicated.

B. 3 μg HA-PLAGL2 was transfected with 2 μg Pirh2-Myc in the presence or absence of various amounts of Flag-Pirh2 indicated. 48 hours after transfection, cells were harvested.

Cell lysates were subjected to immunoprecipitation with anti-Myc antibody, and Western blot with the antibodies indicated.

C. 0.5 μg Flag-Pirh2 was transfected into HEK-293 cells (6-well plates) in the absence or

presence of 1 μg HA-PLAGL2. 0.2 μg GFP vector was used as an internal control. Cells

were treated with 50 μg/ml cycloheximide for different lengths of time before harvest.

Cell lysates were analyzed by Western blot with indicated antibodies.

198 A

IP: Myc

HA-PLAGL2 + + + + + + Pirh2-Myc - + - - + - Pirh2△RING-Myc - - + - - +

Blot: anti-HA 60kDa

60kDa

Blot: anti-Myc

30kDa

199 B

IP: Myc HA-PLAGL2 + + + +++ + + Pirh2-Myc - + + + - + + + FLAG-Pirh2 - - - - 60kDa Blot:Anti-HA

Blot:Anti-Myc 30kDa

Blot:Anti-FLAG 30kDa

200 C

Cycloheximide (50 μg/ml) 0 1 2 3 4 5 (hour)

Flag-Pirh2+GFP+HA-PLAGL2 Blot anti-Flag

Flag-Pirh2+GFP Blot anti-Flag

Flag-Pirh2+GFP+HA-PLAGL2 Blot anti-GFP

Flag-Pirh2+GFP Blot anti-GFP

201 CHAPTER 6

SUMMARY AND FUTURE DIRECTIONS

The major goal of this work is to understand the biochemical and molecular mechanisms by which sumoylation and acetylation regulate ZNF76, PLAG1 and

PLAGL2. We not only characterized the biochemical processes of these modifications but also studied their biological consequences. We identified the enzymes involved, studied how the enzymes interact with the substrates, and mapped the modification sites.

More importantly, we studied how these modifications affect protein-protein interaction, transactivation and thus the function of the target proteins. Finally, with Pirh2 as an example, we demonstrated that understanding the biochemical mechanisms of how an E3 ligase interacts with the substrates may lead to novel approaches to modulate the activities of the substrates. This chapter will summarize the experimental findings of the current work, and propose future studies which may address some of the unanswered questions.

SUMMARY

ZNF76 interacts with TBP, which leads to its general transcriptional repression function. After mapping the interaction regions, we found that both the N- and C- terminus of ZNF76 interact with the C-terminus of TBP, a region responsible for TATA- box binding and interaction with known general transcription repressors (Goppelt et al.,

1996; Darst et al., 2003). Moreover, ZNF76 also interacts with and can be sumoylated by

PIAS1, an E3 ligase in sumoylation. Interestingly, sumoylation site of ZNF76, K411, is

202 in a critical TBP interaction region, thus sumoylation regulates ZNF76-TBP interaction

and relieves the transcriptional repression function of ZNF76. This study not only

identified ZNF76 as a novel TBP inhibitor, but also for the first time, showed that

sumoylation may block the interaction between a transcription factor and the general

transcription machinery. In the following study, we identified two additional mechanisms

which regulate ZNF76-TBP interaction. ZNF76 is acetylated by p300 both in cells and in

vitro. Moreover, HDAC1 was found to interact with and deacetylate ZNF76, suggesting

that the acetylation of ZNF76 is reversibly regulated. The functional consequences of

acetylation are two fold: First, acetylation of ZNF76 inhibits its sumoylation, suggesting that there may be a competition between the two modifications. Second, acetylation of

ZNF76 inhibits its interaction with TBP. ZNF76 is also regulated at the level of mRNA splicing. Two different alternatively spliced forms of ZNF76 were identified and their

functional differences were studied. We found that they have different abilities to interact

with TBP and thus act differently as transcriptional repressors.

PLAG1 and PLAGL2 are two transcription factors involved in various

malignancies including acute myeloid leukemia. The regulatory mechanisms of

transcriptional activation mediated by these oncoproteins remain unknown. In our study,

we showed that sumoylation and acetylation regulate transactivation and the transforming

potential of PLAG1 and PLAGL2. A conserved transcriptional repression domain exists

in both PLAG1 and PLAGL2, and sumoylation within the repression domain represses

their transcriptional activities. One of the possible mechanisms for the sumoylation-

mediated repression is the change in protein localization: while wild-type PLAG1 and

203 PLAGL2 show diffused nuclear expression pattern, sumoylation-deficient PLAG1 and

PLAGL2 concentrate in the nucleolus. Moreover, PLAG1 and PLAGL2 can be

acetylated and activated by p300, deacetylated and repressed by HDAC7. Sumoylation- deficient mutant of PLAGL2 is acetylated at a lower level than its wild-type counterpart, suggesting that some of the lysine residues may be targets for both sumoylation and acetylation. Finally, mutation of three lysine residues in sumoylation motifs dramatically impairs the transformation abilities of PLAG1 and PLAGL2. Taken together, the activities of PLAG1 and PLAGL2 are tightly modulated by both acetylation and sumoylation, which have opposite effects on their transcriptional activation. Since dysregulated expression of PLAG1 and PLAGL2 is involved in various malignancies, it is tempting to speculate that sumoylation and acetylation pathways may serve as therapeutic targets to downregulate the activities of PLAG1 and PLAGL2 in associated cancers.

P53 plays important roles in coordinating cellular responses to stress. Its stability is tightly regulated by several E3 ubiquitin ligases including Pirh2 (p53-induced RING-

H2). The activities of these E3 ligases are regulated by post-translational modifications or

interactions with other proteins. Pirh2 is a target gene of p53; its transcript and protein

levels increase in response to UV irradiation and cisplatin treatment. Pirh2 physically

interacts with p53 and promotes the ubiquitination and subsequent degradation of p53,

thus forming an autoregulatory negative-feedback loop to control the p53 level in cells.

Though Pirh2 functions similarly to Mdm2, little is known about how its activity is

regulated. In our study, we identified a regulatory mechanism for Pirh2. SDS-resistant

204 dimers of Pirh2 were found to be abolished by treatment with desferrioxamine.

Subsequent coimmunoprecipitation experiments confirmed that Pirh2 proteins with different tags coprecipitated with each other. Mapping study suggested that both the N- and the C-terminus of Pirh2 are involved in the Pirh2 dimer formation. Importantly,

FLAG-tagged Pirh2 enhanced the interaction between Myc-tagged Pirh2 and p53, suggesting dimerization enhances its interaction with p53. Moreover, PLAGL2, an oncoprotein involved in the pathogenesis of AML (acute myeloid leukemia), was found to interact with the Pirh2 dimer and stabilize Pirh2. This study not only identifies a molecular mechanism of how Pirh2 interacts with p53 but also suggests Pirh2 dimerization might be important for its activity as an E3 ligase. Furthermore, Pirh2 dimers may be effective therapeutic targets for malignancies with high expression levels

of Pirh2.

FUTURE DIRECTIONS

1. Study the biological function of ZNF76 by knocking down the expression of

endogenous proteins.

In our study (Zheng et al., 2004), we demonstrated that ZNF76 interacts with TBP, thus acting as a general transcriptional repressor. When overexpressed, ZNF76 represses p53-mediated gene expression in both reporter assays and in endogenous target gene expression. However, the biological roles of endogenous ZNF76 in regulating gene expression are still not clear. Thus one of the future directions is to study the biological

functions of ZNF76 by knocking down endogenous proteins and examining the effects on

gene expression. We will establish two cell lines from MCF-7 human breast cancer cells:

205 one is stably transfected with RNAi construct against ZNF76; another cell line is stably

transfected with a control scrambled RNAi construct, which is generated by introducing a point mutation in the RNAi construct for ZNF76. The reason to choose MCF-7 cells is that with bioinformatics approaches, we found ZNF76 is highly expressed in breast cancer cells compared with normal mammary gland tissues (Figure 1). Our preliminary data showed that the sequence CCACCGCUCAUCACUUAAA is highly efficient in knocking down ZNF76 (Figure 2), thus it can be used to generate ZNF76-knockdown cell lines. In our study, overexpression of ZNF76 represses p53 and STAT1-mediated gene expression. With the established cell lines, we will compare their gene expression caused by different stimuli, such as p21 expression induced by DNA damages, target gene expression induced by IFN-γ, through Northern or Western blot analysis. Given the role of ZNF76 as a general transcriptional repressor, we expect to see an increase in the expression of p21 and IFN-γ target genes in ZNF76-knockdown cells in response to the stimuli. However, TBP is negatively regulated by several factors, including NC2

(Goppelt et al., 1996), Mot1 (Darst et al., 2003), p53 (Crigton et al., 2003), and TBP itself

(Coleman et al., 1997). The redundancy of general transcription repressors may prevent us from observing a significant effect. Other possibilities are that ZNF76 may only affect a subset of genes through targeting TBP, or it may act negatively on some but positively on other promoters. The global effect of ZNF76 can be analyzed by comparing the gene expression profiles through gene microarray analysis. The knockdown experiment will facilitate our study on the roles of endogenous ZNF76 in regulating gene expression.

206 An indication of the biological function of ZNF76 is that it may regulate the

expression of CEBP/β (personal communication with Dr. Daniel Tenen) (Tenen et al.,

2001), which is crucial for myeloid hematopoiesis. ZNF76 also interacts with GATA-1

(personal communication with Dr. Paresh Vyas), which plays critical roles in

erythropoiesis. Thus, ZNF76 may have potential roles in hematopoiesis. Using the above-

mentioned knockdown system in blood cells, we can study the roles of ZNF76 in the

blood cell differentiation, which will be of important biological relevance.

2. Study the in vivo function of sumoylation for ZNF76, PLAG1 and PLAGL2.

We demonstrated sumoylation of ZNF76 modulates its interaction with TBP and

sumoylation of PLAG1/PLAGL2 represses their transcription activities in cells. However,

the in vivo function of the sumoylation of these proteins is still unknown. To examine the roles of sumoylation in the whole organism, we propose to generate sumoylation deficient mice for these proteins by knock-in or transgenic strategy. With these strategies,

we will address the following questions for PLAG proteins: Is sumoylation of PLAG

proteins important during the embryonic development? How is the expression of PLAG

target genes affected by their sumoylation? Is sumoylation involved in PLAG1/PLAGL2- mediated tumorigenesis?

The most important experiment in studying the function of sumoylation for a specific substrate is mutational elimination of the sumoylation sites. This could be done by mutating the targeted lysine residues. Since lysine residues can also serve as target sites for other modifications, the mutations may affect other modifications of the

207 substrate, thus making the results hard to interpret. The functional study of sumoylation will be more convincing if mutations at other positions in the sumoylation motif (ψKXE) show similar effect. In our study, we found that sumoylation-deficient PLAGL2 (lysine residues mutated to arginines) is also acetylation-deficient. Thus mutations which disrupt the sumoylation motifs but keep the lysine residues intact may be necessary to distinguish the function of sumoylation and acetylation. In the “knock-in” experiment, we will mutate the glutamic acid residues in the sumoylation motifs to alanine. Take PLAGL2 for example, as shown in Fig.3, we will introduce E252/271/358A triple mutations at the corresponding positions in the mouse ortholog PLAGL2 gene by homologous recombination. The mouse PLAGL2 gene has two exons, and the sumoylation sites are all in exon 1. First, E252, 271, 358A Knock-in +NEO mice (Fig.3) will be generated using homologous recombination in ES cells to modify the PLAGL2 gene such that the exon 1 contains triple mutations (E252, 271, 358A). To do this, mouse genomic DNA clones will be derived from a BAC library, mutated and cloned into a targeting vector.

Downstream of exon 1, a NEO cassette flanked by LoxP sites will be introduced.

Embryonic stem cells will be electroporated, and clones will be selected for homologous recombination by Southern blot analysis using external probes. Targeted ES cells will be injected into blastocysts to create chimeric animals. F1 progeny will be genotyped for transmission of the mutant allele to obtain transgenic lines for E252, 271, 358A KI

+NEO. Heterozygous E252, 271, 358A KI +NEO mice will be bred with mice of the Cre expression strain to remove the NEO cassette. Germ line transmission will be obtained and transgenic line E252, 271, 358A KI will be established. Homozygous E252, 271,

358A KI and WT littermates will be used for further analysis. With the mutant mice, we

208 can investigate the roles of PLAGL2 sumoylation in vivo. Since targeted disruption of

PLAG1 causes growth retardation and infertility (Hensen et al., 2004), we will examine

whether the mutant mice have developmental defects and study the mechanisms of the

phenotypes. In affected organs, we will examine the expression levels of PLAGL2 target

genes to see whether mutant PLAGL2 is equally capable of activating target genes as the

wild-type. If not, we will examine whether all or only a subset of its target genes are

affected, which can be done by real-time PCR, Northern blot or gene microarray,

depending on whether we compare the specific target genes or global gene expression

profile.

It was shown that PLAG1 has in vivo tumorigenic capacity (Declercq et al., 2005).

Targeted PLAG1 overexpression caused high incidence of salivary gland tumors. We will also examine whether sumoylation of PLAG1/PLAGL2 affects their tumorigenicity. On

the basis of this study, we propose to generate sumoylation-deficient PLAG1 transgenic mice which specifically targets salivary gland. As shown in Fig.4, we will use Cre-LoxP system to generate mouse strains with overexpression of PLAG1 mutant. A stop codon inside the NEO cassette flanked by two LoxP sites will be inserted between CMV promoter and PLAG1 encoding region, thus salivary-specific Cre expression (Wagner et

al., 2001) will cause the cleavage of the NEO cassette and subsequent overexpression of

PLAG1 mutant. We will compare the salivary adenoma incidence in the wide type and

mutant PLAG1 transgenic mice. The information of incidence, morphology and grade of

tumors will uncover the biological function of sumoylation in PLAG1-mediated

tumorigensis. However, it is possible that mutation of glutamic acid in the sumoylation

209 motifs will alter the spatial conformation of proteins, thus the phenotypes observed may not be due to the loss of sumoylation. If this occurs, generation of other sumoylation- deficient mutants will be important to confirm the data. Given that both PLAG1 and

PLAGL2 are oncoproteins inducing acute myeloid leukemia in cooperation with Cbfb-

MYH11 (Landrette et al., 2005), it will be interesting to test whether sumoylation of

PLAG1/PLAGL2 plays any roles in the process with above-mentioned strategies.

3. Study sumoylation-mediated alterations in protein-protein interactions.

With the above-mentioned transgenic techniques, we can test the in vivo function of sumoylation. However, the exact molecular mechanisms of sumoylation-mediated function are still at large. Studying the consequences of sumoylation at the molecular level is particularly difficult since sumoylation is highly dynamic and reversible due to the presence of SUMO specific proteases. Thus it is difficult to detect endogenous cellular sumoylation, let alone study how sumoylation affects the biochemical properties of different proteins. In most cases, the functional consequences of the modification can be explained by altered protein-protein interactions (Zheng et al., 2004; Girdwood et al.,

2003; Yang et al., 2004; Matunis et al., 1998). We propose to study sumoylation- mediated alteration of protein-protein interactions in vitro. Take ZNF76 as an example, purified E1, Ubc9 and SUMO-1 (Girdwood et al., 2003) will be incubated with GST-

ZNF76 and the modified ZNF76 will be recovered on glutathione agarose. 35S labeled in vitro-translated known interacting factors such as TBP will be incubated with glutathione agarose bound GST, unmodified GST-ZNF76 proteins, or the sumo-modified GST-

ZNF76 proteins to compare their interaction with TBP. Alternatively, we will incubate

210 the unmodified and modified ZNF76 with total cell lysates. After washes and elution, we will use mass spectrometry to identify proteins which interact with modified but not unmodified ZNF76, or vice versa. Some potential targets for sumoylated PLAG1 and

PLAGL2 are HDACs and HP1 (Girdwood et al., 2003; Shiio et al., 2003), which may be involved in sumoylation-mediated transcriptional repression of PLAG1 and PLAGL2.

Finally, to have a better understanding of sumoylation-mediated function, we could study the effects of sumoylation on protein structure. With NMR or crystallography, we can examine sumoylation-mediated structural changes, which may explain the functional consequences of sumoylation.

4. Investigate whether sumoylation or acetylation of PLAG1 and PLAGL2 can be therapeutic targets for associated malignancies.

Both PLAG1 and PLAGL2 are involved in the pathogenesis of various malignancies. Since sumoylation represses, while acetylation activates the transcription mediated by PLAG1 and PLAGL2, we wonder whether these modifications could be targeted in therapeutic settings. One could activate the sumoylation pathway by increasing the expression of specific SUMO E3 ligases such as PIAS1, or inhibit the activity of sumo-specific enzymes. Both will promote the sumoylation of

PLAG1/PLAGL2, and in turn down-regulate the activities of PLAG1/PLAGL2. Another approach is to inhibit the acetylation of PLAG1 and PLAGL2. In malignancies associated with PLAG1/PLAGL2 overexpression, inhibition of the interaction between p300 and

PLAG1/PLAGL2 or enhancement of the expression of HDAC7 could repress the activity

211 of PLAG proteins. Therapeutic strategies specific for PLAG proteins should be pursued

with caution to avoid nonspecific effects due to global changes imposed on these modification pathways and corresponding substrates. Take PLAG1 as an example, we could screen chemical compounds which specifically inhibit the interactions between

PLAG1 and SUMO-specific proteases, thus only inhibiting the desumoylation process for

PLAG1 but not other sumoylation substrates. The compounds could be used to enhance

the sumoylation of PLAG1 and thus repress its activity in associated malignancies.

5. Study the biological relevance of Pirh2 dimerization.

In our study, we found that Pirh2 forms homodimers and dimerization enhances

its interaction with p53 and PLAGL2, which suggests that Pirh2 dimers may be the

functional form in cells. To confirm the existence of Pirh2 dimers in cells, we will

develop antibodies against Pirh2 and use gel-filtration to examine endogenous Pirh2

dimer formation. With purified Pirh2 proteins, we can also test Pirh2 dimer formation in

vitro by gel-filtration or cross-linking with 0.025% glutaraldehyde (Nikolay et al., 2004).

It should be noted that the physiological concentration of Pirh2 is likely to be less than 1

μM, therefore in vitro experiments will be carried out under similar conditions.

Since Pirh2 is an E3 ubiquitin ligase for p53 (Leng et al., 2003), we will examine

the effects of Pirh2 dimers on the p53 level. We will use lung cancer cell lines which

have high expression levels of Pirh2 (Duan et al., 2004). We will generate two stably

transfected cell lines. One cell line will be stably transfected with a control vector, the

other one with a vector expressing the N-terminus of Pirh2, which will interact with the

212 full-length Pirh2 and disrupt the dimer formation of Pirh2. With these two cell lines, we

will compare the stability and levels of p53. Our hypothesis is that dimerization of Pirh2

is required for its interaction with p53. If the dimer is disrupted by the N-terminus of

Pirh2, the interaction between Pirh2 and p53 will be abolished, resulting in p53 stabilization. However, we still can’t distinguish the possibilities that p53 stabilization is due to the loss of Pirh2-p53 interaction or the loss of the E3 activity of Pirh2.

Dimerization is required for the activities of certain enzymes (Nikoley et al., 2004;

Venema et al., 1997). For example, dimerization of CHIP is required for its ubiquitin E3 activity. Thus it is possible that the E3 activity of Pirh2 depends on the association of two inactive monomers. If the dimerization of Pirh2 is crucial for its activity, any treatment that disrupts the dimer is expected to result in inactivation. To test the hypothesis, E3 ligase activity of Pirh2 will be tested in the in vitro assay, which will include E1, E2, ubiquitin, ATP and Pirh2 alone or preincubated with Pirh2(1-135). A potential difficulty to this approach is that Pirh2 dimers may be very stable thermodynamically and kinetically, thus the formation of the heterodimer between Pirh2 and Pirh2(1-135) and disruption of Pirh2 dimers may be extremely slow. An alternative approach is that we will first generate Pirh2 monomers using detergents, then mix Pirh2 monomers with

Pirh2(1-135) at the ratio of 1:10 and dialyzed the sample against PBS. If Pirh2 dimer formation is important for its E3 activity, we expect to see a decrease in its E3 activity in the presence of the N-terminus of Pirh2. In an alternative approach, we will first map the amino acids which are critical for dimerization of Pirh2 by coimmunoprecipitation or other assays for protein-protein ineteraction, then mutate these amino acids and test the mutant’s ability as an E3 enzyme in vitro.

213

Dimerization of proteins has also been shown to affect protein stability. Thus another possible function for Pirh2 dimerization is to stabilize Pirh2. We will test the hypothesis by examining whether the N-terminus Pirh2 which disrupts the dimer affects the stability of Pirh2. Notably all these possibilities are not mutually exclusive. Through dimerization, Pirh2 may stabilize itself, interact with the substrates and act as an ubiquitin

E3 ligase.

In summary, one of the disadvantages in our study is that majority of the experiments were carried out by overexpression, which has limitations since overexpression may cause physiological irrelevant effects. It is very important to use the above-mentioned complementary approaches to have a better understanding of the biological significance of our findings from in vitro systems to physiological settings.

214 Figure 1. Differential expression of ZNF76 in breast cancer cells and normal breast tissues in SAGE.

SAGE data of breast cancer cells and normal mammary gland tissues from genome.ucsc.edu were compared against each other (Use t-test in statistical analysis,

P<0.001).

215 30

25

20

n

o

i

l l

i 15

m

/

s g

a 10 T

5

0 Normal Cancer Normal Cancer

GAPDH ZNF76

216 Figure 2. RNAi for ZNF76.

HEK293 cells were transfected with the calcium phosphate method. In 6-well plates, 1μg

ZNF76-Myc and 1 μg Pirh2-Myc (as an internal control) were transfected with or without

ZNF76RNAia and ZNF76 RNAib ( 200 pM per well). 48 hours after transfection, cells were harvested and analyzed by immunoblot with anti-Myc antibody.

The RNAi oligoes were ordered from Dharmacon.

ZNF76 RNAia: CCACCGCTCATCACTTAAA

ZNF76 RNAib: CCAGCGCCACCAACTATAA

217 ZNF76-Myc + + + Pirh2-Myc + + + ZNF76RNAia - + - ZNF76RNAib - - +

ZNF76-Myc Blot: anti-Myc

Pirh2-Myc Blot: anti-Myc

218 Figure 3. Schematic representation of the sumoylation-deficient PLAGL2 “Knock-in” strategy.

Shown are a part of genomic structure of the wild type PLAGL2 allele, targeting vector, predicted structure after homologous recombination (E252, 271, 358A +NEO allele), and after Cre-mediated deletion of the NEO-cassette (E252, 271, 358A). LoxP sites are indicated by triangles.

219 Wild type allele Exon1 Exon2

AAA

Targeting vector Exon1 NEO Exon2

AAA

E252, 271, 358A KI+NEO allele Exon1 NEO Exon2

AAA

E252, 271, 358A KI Exon1 Exon2

220 Figure 4. Schematic representation of the PLAG1AAA transgene construct used to

generate PLAG1AAA transgenic mouse strain.

A stop cassette is inserted between the CMV promoter and the PLAG1 mutant coding

sequences. The stop cassette consists of the NEO gene, which is flanked on both sides by loxP sites. Expression of mutant PLAG1 is therefore dependent on Cre-mediated removal of the stop cassette.

221 AAA

CMV promoter NEO PLAG1 ORF

Stop

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