Genome-wide promotor binding profiling of Protein phosphatase 1 and its major nuclear targeting subunits

Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. In the first part of the thesis, we show that the alfa, ß and gamma isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter-binding pattern. PP1ß emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. In the second part of the thesis, we further found that PP1 does not affect the promoter-targeting of RepoMan, but enhances the binding affinity of PNUTS and alters the binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms, and reveal that PP1 guides the chromatin targeting of some of its own interactors. In the third part of the thesis we identified the PP1ß-PNUTS holoenzyme as overlapping with the elongating Pol II-pS2 complex both bioinformatically and in vivo, and further link this complex to two major gene clusters, namely histone and small nucleolar RNA genes. This revealed a potential link between the PP1ß-PNUTS holoenzyme and the 3’ end processing of these two gene clusters.<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph;text-indent:36.0pt;line-height:150%"><span style="font-size:10.0pt;line-height:150%;font-family:Arial;color:black"><!-- /* Font Definitions */@font-face {font-family:Arial; panose-1:2 11 6 4 2 2 2 2 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;}@font-face {font-family:"&#65325;&#65331; &#26126;&#26397;"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:128; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:fixed; mso-font-signature:1 134676480 16 0 131072 0;}@font-face {font-family:"&#65325;&#65331; &#26126;&#26397;"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:128; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:fixed; mso-font-signature:1 134676480 16 0 131072 0;}@font-face {font-family:Cambria; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;} /* Style Definitions */p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"&#65325;&#65331; &#26126;&#26397;"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}@page WordSection1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;}div.WordSection1 {page:WordSection1;}-Protein phosphatase-1 (PP1) is a key regulator oftranscription and is targeted to promoter regions via associated proteins.However, the chromatin binding sites of PP1 have never been studied in asystematic and genome-wide manner. Methylation-based DamID profiling in HeLacells has enabled us to map hundreds of promoter binding sites of PP1 and threeof its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS<span style="font-size:10.0pt;font-family:Arial;mso-fareast-font-family:&quot;&#65325;&#65331; &#26126;&#26397;&quot;;mso-fareast-theme-font:minor-fareast;mso-bidi-font-family:&quot;Times New Roman&quot;;mso-bidi-theme-font:minor-bidi;color:black;mso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">-->ACKNOWLEDGEMENTS I TABLE OF CONTENTS III LIST OF ABBREVIATIONS VII CHAPTER I: INTRODUCTION 1 1. TRANSCRIPTIONAL REGULATION OF EUKARYOTIC GENES 1 1.1. Chromatin and nucleosome dynamics during transcription 1 1.1.1. Transcription and chromatin 1 1.1.2. The nucleosome barrier 1 1.1.2.1 Nucleosome positioning 1 1.1.2.2. Chromatin remodeling complexes 2 1.1.3 The histone code 3 1.1.3.1. Transcription-coupled histone variants 4 1.1.3.2 Histone acetylation 4 1.1.3.3. Histone methylation 5 1.1.3.4. Histone phosphorylation 5 1.1.3.5. Histone ubiquitination 6 1.1.3.6. Cross-talk of histone modifications 6 1.2. Transcription factors 7 1.2.1. Gene-specific transcription factors 7 1.2.2. General transcription factors 7 1.3. Pre-initiation complex assembly and initiation of transcription 8 1.3.1. Core promoter elements 8 1.3.2. The pre-initiation complex assembly pathway 9 1.4. Transcriptional elongation 10 1.4.1. Pausing 10 1.4.2. 5’ Capping of mRNA 10 1.4.3. Pre-mRNA splicing 11 1.5. Transcriptional termination 11 1.5.1. Nascent mRNA 3’ end processing 11 1.5.2. RNA Polymerase II Termination 12 2. REGULATION OF TRANSCRIPTION BY REVERSIBLE PROTEIN PHOSPHORYLATION 13 2.1. Importance of histone (de)phosphorylation in transcription 13 2.1.1. Histone phosphorylation is essential for transcription 13 2.1.2. Binary switches 13 2.1.2.1. “Methyl/Phos” switches 14 2.1.2.2. “Phos/methyl-acetyl” switch 15 2.2. The RNA Polymerase-II CTD phosphorylation cycle 15 2.2.1. Transcriptional Pausing 16 2.2.2. Nascent mRNA 5’-end Capping 16 2.2.3. Splicing of mRNA 17 2.2.4. mRNA 3’-end Processing 18 3. PROTEIN SER/THR PHOSPHATASE I 18 3.1. The catalytic Subunit 18 3.1.1. Isoform diversity 18 3.1.2. Structure and catalytic mechanism 19 3.2. PP1-binding Motifs 20 3.2.1. RVxF Motif 21 3.2.2. SILK Motif 22 3.2.3. MyPhoNE 22 3.2.4. Other PP1 binding motifs 22 3.3. PP1-interacting Proteins 23 3.3.1. PNUTS 23 3.3.1.1. Modulation of the PI3K/AKT Pathway 24 3.3.1.2. p53 Signalling pathway 24 3.3.1.3. pRb dephosphorylation upon hypoxia 25 3.3.1.4. DNA Damage 25 3.3.1.5. RNA Polymerase-II dephosphorylation 26 3.3.2. NIPP1 26 3.3.2.1. NIPP1 as part of the PRC2 complex 27 3.3.2.2. NIPP1 as a PP1 inhibitor 27 3.3.2.3. NIPP1 in pre-mRNA Splicing 28 3.3.3 RepoMan 28 3.3.3.1. Regulation of mitotic H3T3 phosphorylation 28 3.3.3.2. Regulation of chromosome architecture and nuclear envelope 29 3.3.3.3. Contribution to the DNA damage response 30 4. CHROMATIN-INTERACTING PROTEINS 31 4.1. Importance of identifying chromatin-bound proteins 31 4.2. Major techniques 31 4.2.1. Determination of chromatin Interaction 31 4.2.1.1. Cellular fractionation and immunoprecipitation 31 4.2.1.2. Co-localization microscopy 32 4.2.2. Fluorescence recovery after photo-bleaching (FRAP) 32 4.2.3. Mapping of chromatin interaction sites 33 4.2.3.1. Chromatin immunoprecipitation (ChIP) 33 4.2.3.2. DNA-adenine methyltransferase identification (DamID) 34 CHAPTER II. AIMS AND STRATEGIES 37 CHAPTER: III. MATERIALS AND METHODS 39 1. MATERIALS 39 1.1. Antibodies 39 1.2. DNA Constructs 39 1.3. Chemicals and Reagents 40 1.4. Buffers 40 2. METHODS 40 2.1. Cell culture 40 2.2. Cellular Fractionation 41 2.3. Co-immunoprecipitation 42 2.4. Immunoblotting and immunostaining 43 2.5. Confocal co-localization microscopy 44 2.6. Chromatin immunoprecipitation 44 2.7. Quantitative-PCR 45 2.8. DNA adenine methyltransferase identification (DamID) 45 2.9. Computational analysis 47 2.9.1. Promoter Tiling array analysis 47 2.9.2. Signal profile analysis 48 2.9.3. Genome Structure Correction and Co-association analysis 49 2.9.4. Correlation coefficient analysis 49 2.9.5. Gene Ontology Analysis 50 CHAPTER: IV. RESULTS AND DISCUSSION 51 1. CELLULAR FRACTIONATION OF PP1 ISOFORMS 51 1.1. PP1 isoforms co-fractionate with chromatin 51 1.2. Discussion 52 2. PROMOTER-WIDE ANALYSIS OF PP1 INTERACTION SITES 52 2.1. Identification of PP1 isoform-specific interaction on promoter sites 52 2.1.1. ChIP analysis 52 2.1.2. DamID analysis 53 2.2. PP1 isoforms binding profile across TSS 54 2.2.1. PP1 isoforms have distinct binding profiles 54 2.2.2. Proximal promoter region-specific PP1 redundancy 55 2.2.3. PP1 isoforms are linked to “active” chromatin 57 2.2.4. Gene ontology of PP1-linked genes 58 2.3. Discussion 59 2.3.1. PP1 binding sites: ChIP vs. DamID 59 2.3.2. PP1 exhibits isoform-specific promoter binding 60 2.3.3. PP1 isoforms control different subsets of genes 61 3. PROMOTER-WIDE ANALYSIS OF PIP INTERACTION SITES 61 3.1. Identification of PIP specific interactions on promoter sites 61 3.2. PIPs have distinct promoter-binding profiles 62 3.3. The promoter binding of PNUTS and NIPP1 is regulated by PP1 64 3.3.1. Comparative analysis of genomic binding sites of WT and M PIPs 64 3.3.2. Co-association and correlation analysis 65 3.3.3. PIPs are linked to “active chromatin” 66 3.4. Discussion 67 3.4.1. PNUTS is the major promoter-interacting PIP 67 3.4.2. PNUTS and RepoMan target to chromatin independent of PP1 68 4. IDENTIFICATION OF PROMOTER-ASSOCIATED PP1 HOLOENZYMES 69 4.1. Shedding light on PP1 holoenzymes through co-association analysis 69 4.2. Promoter-interacting PP1 holoenzymes 70 4.3. Discussion 72 4.3.1. Promoter-bound PP1 holoenzymes 72 4.3.2. The identification of the PP1β-PNUTS holoenzyme 73 4.3.3. Unidentified promoter-bound PIPs 74 5. RNA POLYMERASE II ASSOCIATED PP1Β-PNUTS 74 5.1. Characterization of the PP1β-PNUTS holoenzyme 74 5.1.1. PP1β-PNUTS is associated with RNA Polymerase-II transcribed genes 74 5.1.2. PNUTS associates with RNAPII in vivo 77 5.2. Characterization of RNA Polymerase II associated PP1β-PNUTS 77 5.2.1. Characterization of PP1β-PNUTS that is co-associated with Pol II-pS2 77 5.2.2. RNA Polymerase II associated PP1β-PNUTS linked genes 78 5.3. Discussion 78 5.3.1. PP1β-PNUTS associates with Pol II-pS2 78 5.3.2. Possible link between PP1β-PNUTS and mRNA 3’ end Processing 79 CHAPTER V: CONCLUSIONS AND PERSPECTIVES 85 1. PP1Β AND PNUTS ARE LINKED TO PROMOTER-PROXIMAL REGIONS 85 2. PNUTS AS A LINK BETWEEN POL II-PS2 AND PP1Β 86 3. THE EXISTENCE OF ADDITIONAL PROMOTER-BOUND PIPS 87 4. PROPOSED MODEL FOR PIP RECRUITMENT TO PROMOTER 88 5. INVOLVEMENT OF PP1 IN TRANSCRIPTION-RELATED PROCESSES 89 CHAPTER: VI. SUMMARY 93 CHAPTER: VII. SAMENVATTING 95 CHAPTER: VIII. REFERENCES 97 CHAPTER: IX. APPENDIX 119 CURRICULUM VITAE 121 LIST OF PUBLICATIONS 123status: publishe