Epigenetic signature of pericentromeric heterochromatin

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Table of contents

Abbreviations
Table of contents
Figures
Tables
Introduction
CHAPTER I Structure and function of centromeric regions Usage of the terms
I. 1. Determining the centromeric region
1.1. First description of the centromere
1. 2. Organisation of the centromeric region
1.3. DNA at the centromeric and pericentromeric region
1.3.1 Repetitive DNA
1.3.2. Diverse functions of repetitive DNA
1.3.3. Repetitive DNA at the centromeric region
1.3.4. Variation of centromeric sequences between species
1.4. Neocentromeres and dicentrics
I. 2. Chromatin organisation at the centromere
2.1. Histone modifications and the underlying chromatin state
2.2. The centromere core
2.2.1. Histone H3 variant CENP-A as the determinant of a functional centromere
2.2.2. Centromere associated proteins
2.2.3. Centrochromatin – the chromatin forming at the centromeres
2.3. Pericentromeric region
2.3.1. Epigenetic signature of pericentromeric heterochromatin
2.3.2. The interaction network which allows the establishment and maintenance of pericentromeric heterochromatin
2.3.3. Heterochromatin assembly in S pombe requires RNA interference
2.3.4. Role of pericentromeric heterochromatin
2.4. Centromeric and pericentromeric regions in mouse cells
2.4.1. Organisation of mouse centromeric and pericentromeric region
2.4.2. Association of pericentromeric regions
CHAPTER II. Non-coding RNA from centromeric and pericentromeric regions
II.1 Non-coding RNA
1.1. The RNA world, old and new
1.1.1. A new perspective on RNA
1.1.2. Non-coding RNA as a key to complexity
1.2. Emerging role for ncRNAs
1.2.1. Short ncRNA
1.2.2. Long ncRNA
II.2. Expression of repetitive sequences
2.1. Evidence for transcription from centromeres and pericentromeres
2.2. Centromeric transcripts as integral components of centromeric chromatin
2.2.1. Centromeric RNAs regulate the kinetochore activity
2.2.2. Centromeric transcription stabilise CENP-C binding to the centromeres
2.3. Transcription from the pericentromeric region
2.3.1. Pericentromeric transcription during stress
2.3.2. Pericentromeric transcripts participate to heterochromatin reorganisation during development and differentiation
2.3.3 Non-coding RNA in heterochromatin formation: lessons from fission yeast .
2.3.4. Non-coding RNA as a component of pericentromeric heterochromatin
II.3. Regulation of pericentromeric transcription
3.1. Chromatin modifications and transcription
3.1.1.Histone modifications
3.1.2. DNA methylation
3.2. Transcription factors
3.3. Cell cycle
Chapter III Tools for study of repetitive sequences
I.1. Oligonucleotides for detection of nucleic acids
1.1. Hybridization properties of nucleic acids
1.2. Oligonucleotides
1.2.1 A brief history of oligonucleotides
1.2.2. Oligos with chemical modifications: 2’-O-Me and LNA
1.3. Locked nucleic acids
1.3.1 Thermodynamic properties of LNA
1.3.2. Use of LNA oligonucleotides
1.4. LNA probes for the study of repetitive sequences
I. 2. Tools for (epi)genetic engineering
2.1. Tools for targeted genome manipulation
2.1.1. Targeting specific DNA loci in living cells
2.1.2. TALE as a novel DNA binding domain
2.2.3. Designer TALEs
2.2. Epigenetic engineering for studying the functions of chromatin modifications
2.2.1. Targeting chromatin modifications in living cells
Objectives
Materials and methods
I.1. Methods
1.1. Cell culture
1.2. Northern blot
1.3. Radioactive labelling
1.4. RNA extraction
1.5. RT PCR
1.6. Major satellite DNA probe preparation
1.7. In vitro transcription
1.8. Small RNA separation
1.9. Cell transfection
1.10. Immunofluorescence
1.12. Microscopy
1.13. TANGO analysis
I.2. Materials
2.1. Oligonucleotides
2.2. Primers
Results
CHAPTER I Characterization of major satellite transcripts
I.I. Characterization of major satellite transcripts using LNA oligonucleotides
1.1. Probes for detection of major satellite repeats
1.2. Characterisation of major satellite transcription by northern blotting of total RNA from mouse cells
1.2.1. Transcriptional profile of major satellites in growing mouse cells
1.2.2. Detailed characterization of northern hybridization signals
1.2.3. Comparison of transcriptional profile revealed by isosequential LNA and 2’- O-Me oligo
1.2.4. High stringency washing
1.2.5. RNA and DNA probes
2.2. Strand specific RT PCR confirms transcription from major satellites
I.2. Expression and regulation mechanisms implicated in major satellite transcription
2.1. Influence of the inhibitors of chromatin modifiers on major satellite transcription
2.2. Changes in major satellite transcription upon thermal stress
2.3. Influence of different RNA polymerase inhibitors on major satellite transcription
I.3. Sequence characterization
3.1. Technical details/methods for major satellite sequence characterization
I. 4. Conclusion and discussion
CHAPTER II TALE fused to histone demethylase mJMJD2D for epigenetic engineering at pericentromeric regions of mouse cells
II.1. Targeting mouse major satellites using TALE fused to histone demethylase mJMJD2D
1.1. Context of the study
1.2. Visualisation of the specificity of TALE recruitment on major satellites
1.3. Loss of H3K9me3 upon transfection with TALE fused to histone demethylase mJMJD2D
II.2. Analysis of the effect of histone demethylation upon transfection with TALEs fused to histone demethylase mJMJD2D
2.1. Tools for Analysis of Nuclear Genome Organisation (TANGO)
2.2. Quantitative analysis of the demethylation of H3K9me3 at the major satellite foci upon transfection with the TALE N212-mJMJD2D
2.3. Effect of the demethylation of H3K9me3 on major satellite foci
II. 3. Conclusion and discussion
General conclusion and perspectives
Appendix
Bibliography