Three-dimensional chromatin structure and its role in cellular function Pubblico

Abstract

Cellular function is controlled by a complex interplay between genomic sequence and its surrounding context. A large force in establishing genomic context is the physical partitioning of the genome into defined neighborhoods that allows coordination of transcriptional activity, DNA interactions with RNA and proteins, and chemical modifications. Spatial organization is also involved in X-inactivation, cell fate determination, and senescence. Recent high-throughput resequencing technologies have allowed investigation of chromatin architecture on a scale and resolution overcoming the previous limits of microscopy and inference at individual loci from less direct assessments. It is now possible to create a genome-wide map of DNA fragment interactions or investigate a protein-specific DNA interaction network. These approaches have revealed a complex hierarchical organization ranging from whole chromosomes down to short-range associations between adjacent features. Because these technologies generate large amounts of data representing a complex system, pose significant computational and analytical challenges. We developed HiFive, a framework for analyzing HiC and 5C data, to address these challenges. HiFive allows handling of large amounts of data in an efficient manner and easy access to subsets of data for downstream analysis and plotting. We have also included an approximation approach to normalization that allows processing of data for a fraction of the computational cost and time. Compared to other available methodologies, HiFive performs as well or better across a variety of measures. To further validate the approaches used in HiFive, we also present downstream analyses locating significant structural signatures and analyzing gene spatial arrangements. We are able to increase sensitivity to detection of subdomain structures and their associated features. We also present a new approach to three-dimensional modeling that reveals a spatial partitioning of genes organized around transcriptional activity. Our results are consistent with our current understanding of chromatin architecture and suggest exciting possible avenues for future exploration.

Table of Contents

Chapter 1 - Introduction 1

Organization of the animal nucleus 1

Function of the nucleus 1

Chromosome territories 2

The fractal globule 4

Topological domains 4

Organization of the nucleus 6

The nucleolus 7

Polycomb bodies 8

Lamina associated domains 9

RNA polymerase II-dependent transcriptional foci 11

Assaying chromatin structure 14

Microscopic detection of chromatin structures 14

Chromatin conformation capture 15

3C-derived approaches 18

PMLA data analysis 20

Chapter 2 - Determining chromatin conformation from interaction frequency data using a probabilistic modeling approach 22

Introduction 22

Material and Methods 25

Mapping of interaction data 25

Software Implementation 26

Data Filtering 28

Estimation of Distance-Dependent Signal 33

HiFive Data Normalization 37

HiFive-Express Iterative Approximation for Bias Correction 40

Neighboring Fend Correlations 42

Results 43

HiC Unit of Interaction 43

HiFive's 5C Normalization Performance 45

HiFive's HiC Normalization Performance 49

Discussion 54

Chapter 3 - Validation of HiFive through method comparisons and biological findings 57

Introduction 57

Materials and methods 59

Acquiring and Mapping Data 59

5C Data Normalization with HiFive 61

5C Data Normalization with Alternate Methods 62

HiC Data Normalization with HiFive 63

HiC Data Normalization with Alternate Methods 63

Annotation Data Processing 65

Dynamic Binning 65

5C Data Correlations with HiC Data 67

HiC Inter-Dataset Correlations 68

Calculating the boundary index 68

Boundary index comparison to the directionality index 71

Three-dimensional chromatin modeling 72

Calculating gene spatial arrangements 75

Results 76

5C Method Comparison 76

HiC Method Comparison 78

The boundary index captures more significant features than the directionality index 79

Three dimensional chromatin models 81

Spatial partitioning of genes by transcriptional activity 88

Discussion 91

Chapter 4 - Discussion 95

Explaining nuclear organization 95

Areas of future inquiry 98

Delineating the conservation of boundaries 98

Defining boundary types and elements 100

Deciphering between targeted and stochastic association 102

Applications for chromatin structural understanding 106

Associations between chromatin structure and disease 106

Synthetic biology 108

Conclusions 110

Chapter 5 - References 111

Chapter 6 - Non-printed sources 128

Chapter 7 - Abbreviations 130

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Population Biology, Ecology & Evolution
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Bioinformatics Biology, Cell
Parola chiave	5C HiC topology normalization chromatin conformation
Committee Chair / Thesis Advisor	Taylor, James, Johns Hopkins University
Committee Members	Kelly, William, Emory University Corces, Victor, Emory University Boss, Jeremy, Emory University Waller, Lance, Emory University

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