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Title: A key function for the PP1 interactor NIPP1 in cancer cell proliferation and gene expression
Other Titles: De PP1 regulator NIPP1 speelt een cruciale rol in genexpressie en in de proliferatie van kankercellen
Authors: Van Dessel, Nele
Issue Date: 28-Jun-2012
Abstract: Polycomb Group (PcG) proteins are repressive chromatin modifiers that are important in maintaining stem cell pluripotency, establishing cell fate and cancer development. One of the key events in PcG-mediated gene silencing is the trimethylation of Lysine 27 on histone H3 (H3K27) by the methyltransferase EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2) complex. NIPP1, an interactor of protein Ser/Thr phosphatase PP1, is a ubiquitously expressed nuclear protein that interacts with the two PRC2 components EZH2 and EED. Furthermore, NIPP1 associates with a subset of PcG target genes, where it is essential for the trimethylation of H3K27. In addition, the loss of NIP1 is associated with a defective proliferation and embryonic lethality.In the first part of my thesis, we demonstrated that NIPP1 is an essential regulator of EZH2 recruitment in a PP1-dependent manner. We found that the depletion of either NIPP1 or PP1 inhibits the EZH2 occupancy on NIPP1-associated PcG target genes whereas ectopically expressed NIPP1 induces a redistribution of EZH2 from fully repressed to partially active PcG target genes. Conversely, the expression of a PP1-binding mutant of NIPP1 (NIPP1-RATA) has no effect on the chromatin targeting of EZH2. Although NIPP1-RATA still interacts with EED and EZH2, we demonstrated via DamID that it is no longer associated with PcG target genes. In the second part of my thesis, we explored the role of NIPP1 and associated PP1 in cancer cell proliferation. The cellular concentration of NIPP1 needs to be strictly regulated in cancer cells since both the depletion and the stable expression of NIPP1-WT show inhibitory effects on tumor growth. Furthermore, the expression of a NIPP1 mutant that is associated with constitutively active PP1 (NIPP1-&#8710;C) abrogates tumor growth, whereas the expression NIPP1-RATA only has limited effects, indicating that NIPP1-mediated growth inhibition is dependent on PP1. Moreover, the expression of NIPP1-WT and NIPP1-&#8710;C, but not of NIPP1-RATA, induces the expression of smooth muscle-specific genes in HeLa cells and increases cell contraction upon KCl-induced depolarization. The smooth muscle-like phenotype is lost upon EZH2 depletion, indicating that NIPP1 transdifferentiates HeLa cells in an EZH2- and PP1-dependent manner.In conclusion, we have demonstrated that the NIPP1-PP1 holoenzyme is a key regulator of PRC2 chromatin targeting but also plays a role in PcG-mediated differentiation processes. In addition, we have shown that NIPP1 is strictly regulated in cancer cells, making it a potential therapeutic target. <w:latentstyles <br semihidden="false" priority="0" locked="false" <w:latentstyles role="" target="" semihidden="false" priority="0" locked="false" <w:lsdexception="" defunhidewhenused="true" deflockedstate="false" target.< therapeutic="" potential="" making="" cells,="" shown="" processes.="" differentiation="" plays="" also="" holoenzyme="" nipp1-pp1="" have="" conclusion,="" manner.in="" ezh2-="" transdifferentiates="" depletion,="" lost="" phenotype="" muscle-like="" depolarization.="" kcl-induced="" upon="" contraction="" increases="" hela="" muscle-specific="" smooth="" nipp1-rata,="" not="" but="" nipp1-?c,="" moreover,="" pp1.="" dependent="" inhibition="" growth="" nipp1-mediated="" indicating="" effects,="" limited="" only="" growth,="" abrogates="" (nipp1-?c)="" constitutively="" growth.="" tumor="" effects="" inhibitory="" show="" nipp1-wt="" stable="" both="" since="" cells="" regulated="" strictly="" be="" needs="" concentration="" cellular="" proliferation.="" explored="" second="" genes. in="" longer="" damid="" via="" eed="" still="" nipp1-rata="" although="" ezh2.="" targeting="" effect="" no="" has="" (nipp1-rata)="" mutant="" pp1-binding="" expression="" conversely,="" genes.="" active="" partially="" to="" repressed="" fully="" from="" redistribution="" induces="" ectopically="" whereas="" genes="" nipp1-associated="" occupancy="" inhibits="" pp1="" or="" either="" depletion="" found="" manner.="" pp1-dependent="" recruitment="" regulator="" demonstrated="" we="" thesis,="" my="" part="" first="" lethality.in="" embryonic="" proliferation="" defective="" associated="" nip1="" loss="" addition,="" h3k27.="" for="" essential="" it="" where="" genes,="" pcg="" subset="" associates="" nipp1="" furthermore,="" eed. ="" ezh2="" components="" prc2="" two="" with="" interacts="" nuclear="" expressed="" ubiquitously="" pp1,="" phosphatase="" thr="" ser="" protein="" interactor="" an="" nipp1,="" complex.="" (prc2)="" 2="" complex="" component="" core="" a="" ezh2,="" methyltransferase="" by="" (h3k27)="" h3="" histone="" on="" 27="" lysine="" trimethylation="" is="" silencing="" gene="" pcg-mediated="" events="" key="" the="" of="" one="" development.="" cancer="" and="" fate="" establishing="" pluripotency,="" cell="" stem="" maintaining="" in="" important="" that="" modifiers="" chromatin="" repressive="" are="" proteins="" (pcg)="" group="" polycomb=""
Table of Contents: Table of contents

Dankwoord V
Table of content VII
List of abbreviations XI
List of publications XIII

Chapter I: Introduction 1

I Epigenetics 1
I.A DNA methylation 1
I.B Histone modifications 2
II Polycomb-mediated gene silencing 4
II.A The molecular composition of PcG complexes 4
II.A.1 The PRC2 complex 4
II.A.2 The PRC1 complex 8
II.B Genome-wide PcG distribution and targeting 10
II.B.1 Genome-wide mapping of the PcG binding sites 10
II.B.2 Targeting PcG complexes in Drosophila 10
II.B.3 Targeting PcG complexes in mammalian cells 11
II.C Regulation of H3K27 trimethylation 16
II.C.1 Spreading of the H3K27me3 mark 16
II.C.2 Demethylation of H3K27 16
II.C.3 PRC2-antagonizing marks 17
II.D The physiological function of PcG signaling 19
II.D.1 PcG silencing, stem cell regulation and differentiation 19
II.D.2 PcG silencing, X-inactivation and imprinting 23
II.D.3 PcG silencing and cell cycle 24
III Cancer epigenetics 26
III.A DNA methylation 26
III.A.1 Hypomethylation 26
III.A.2 Hypermethylation 27
III.B PcG silencing 28
III.B.1 EZH2 in cancer proliferation 28
III.C Epigenetic markers and therapy 30
IV NIPP1 is a multifunctional scaffold protein 32
IV.A NIPP1 structure and interactome 32
IV.B NIPP1-regulated cellular processes 33
IV.B.1 NIPP1 is involved in cellular signaling 33
IV.B.2 NIPP1 is essential player in PcG-mediated gene silencing 34
IV.B.3 NIPP1 is involved in pre-mRNA splicing 35
IV.C NIPP1 is essential for proliferation and development 35

Chapter II: Aims and scopes 37

Chapter III: The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets 39
I Abstract 39
II Introduction 40
III Material and methods 42
IV Results 46
IV.A Identification of a chromatin-associated complex of NIPP1, PP1 and PRC2 46
IV.B The loss of NIPP1 or PP1 reduces the targeting of EZH2 47
IV.C The overexpression of NIPP1 redistributes EZH2 48
IV.D The association of NIPP1 with PcG target genes depends on PP1 53
IV.E Mapping of NIPP1 chromatin binding sites 56
IV.F PP1 is not required for the assembly of the NIPP1–PRC2 complex 58
V Discussion 59
VI References 62
VII Supplementary materials and methods 65
VIII Supplementary figures 68

Chapter IV: Transdifferentiation of HeLa cells into smooth muscle-like cells by NIPP1, a regulator of PP1 and EZH2 71

I Abstract 71
II Introduction 72
III Material and Methods 74
IV Results 77
IV.A The stable ectopic expression of NIPP1 hampers cancer cell proliferation and tumor growth in a PP1-dependent manner 77
IV.B The ectopic expression of NIPP1 induces the expression of differentiation genes 81
IV.C NIPP1 transdifferentiates HeLa cells into a smooth muscle-like phenotype 85
IV.D The depletion of NIPP1 reduces cancer cell proliferation, colony formation and tumor growth 87
V Discussion 91
VI References 93
VII Supplementary material and methods 95
VIII Supplementary figures 96

Chapter V: Discussion and perspectives 101

I NIPP1 is a PP1-dependent regulator of PRC2 recruitment 101
II NIPP1 is a putative differentiation factor 103
III NIPP1 as a therapeutic target 104

Summary 107
Samenvatting 109
References 111
ISBN: 978 94 6165 051 1
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Biosignaling & Therapeutics

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