Examination of Brain Chromatin Accessibility in the Prairie Vole (Microtus ochrogaster) Public

Abstract

Oxytocin (OXT) is a small and highly conserved neuropeptide that is known to modulate the neural circuitry that is associated with the processing of social information. Dysfunctions in OXT signaling are considered relevant to a handful of psychiatric conditions, including schizophrenia and autism spectrum disorder. Many scientific efforts to understand the biological mechanisms of the OXT system have involved research of the OXT receptor, OXTR, and the gene that encodes the receptor, OXTR. In humans, variation in the OXTR sequence has been linked to a wide variety of traits that relate to social cognition and behavior. In animals, behavioral paradigms combined with DNA sequencing and imaging of OXTR in the brain have revealed strong correlations between Oxtr single nucleotide polymorphisms (SNPs), OXTR distribution in reward and sensory processing networks, and pro-social phenotypes. Although much has been studied regarding the function of OXT in the brain, and associations between OXTR/Oxtr SNPs and behavior have been identified, less is known about underlying molecular genetic factors that contribute to variation in gene expression. To gain insight into potential mechanisms of OXTR/Oxtr regulation, we established a workflow for examining chromatin accessibility in brain tissue of the socially monogamous and highly affiliative prairie vole, Microtus ochrogaster. At its core, the Assay for Transposase Accessible Chromatin and DNA sequencing (ATAC-Seq) allows detection of open regions of DNA throughout the genome. We applied this method in brain areas that are known to differentially express Oxtr from previous studies, with the striatum being of specific interest due to a critical role in reward processing. Here, we demonstrate validation of the ATAC-Seq method with prairie vole brain tissue samples, and we perform tests for differential enrichment between the striatum, insular cortex, and inferior colliculus (n=11). Our findings present genes and genetic loci that have the strongest signals of striatum-specific chromatin accessibility. We also provide evidence that sex differences are a predominant factor contributing to variation in chromatin accessibility. We then analyze the prairie vole ATAC-Seq data at Oxtr, where we perform variant calling to compare SNP accessibility frequencies between each brain region. Interestingly, we show different sets of SNPs with high accessibility for the striatum compared to insular cortex, each of which we refer to as differentially accessible alleles. These SNPs may serve as candidates for genetic modification of Oxtr to alter its transcription in a region-specific manner. Additionally, the ATAC-Seq data presented here provides hundreds of thousands of accessible sites in the prairie vole genome that can be explored in further research. This dissertation is a unique resource that provides some of the first functional characteristics of the prairie vole genome, as well as experimental and analytical tools for further development of chromatin-based assays in the brain.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION................................................................................................ 1

GENERAL INTRODUCTION: FROM BRAIN EXTRACTS TO DNA ACCESSIBILITY ..... 2

OXYTOCIN, VASOPRESSIN, AND SOCIAL BEHAVIOR.................................................... 4

OXYTOCIN IN THE BRAIN AND SOCIAL COGNITION ..................................................... 8

Lessons from Rodent and Animal Models............................................................................... 8

OXTR Polymorphisms in Clinical Research........................................................................... 9

Towards Molecular Mechanisms of OXTR Regulation ........................................................ 12

PRAIRIE VOLES AS A MODEL FOR UNDERSTANDING OXYTOCIN FUNCTION ...... 14

CONCLUSIONS ...................................................................................................................... 19

CHAPTER 2: CHROMATIN ACCESSIBILITY IN THE PRAIRIE VOLE BRAIN ........ 22

ABSTRACT.............................................................................................................................. 23

INTRODUCTION .................................................................................................................... 24

METHODS ............................................................................................................................... 27

Animals ................................................................................................................................. 27

Dissection and Pooling ......................................................................................................... 27

Cell Lysis and Nuclei Quantification.................................................................................... 28

Transposition and Library Amplification ............................................................................. 28

Quality Control, Sequencing and Alignment ........................................................................ 29

Data Analysis ........................................................................................................................ 29

RESULTS ................................................................................................................................. 31

Prairie Vole ATAC Workflow ............................................................................................... 31

Coverage, Validation, and Peak Calling .............................................................................. 33

Housekeeping Regions for Prairie Vole Brain Tissue .......................................................... 37

Sex Explains Most Variation in Brain Chromatin Accessibility........................................... 37

Differentially Enriched Genes in Striatum are Associated with DNA binding..................... 41

DISCUSSION ........................................................................................................................... 45

CHAPTER 3: OXTR CHROMATIN ACCESSIBILITY IN THE PRAIRIE VOLE BRAIN .................... 53

ABSTRACT.............................................................................................................................. 54

INTRODUCTION .................................................................................................................... 55

METHODS ............................................................................................................................... 57

Prairie Vole ATAC-Seq Data and Visualization................................................................... 57

OXTR Variant Calling .......................................................................................................... 58

RESULTS ................................................................................................................................. 58

Individual Variability of Prairie Vole ATAC-Seq Oxtr Signal ............................................. 58

Establishing a Pipeline for Variant Calling of Prairie Vole ATAC-seq data....................... 60

Differentially Accessible SNPs at Prairie Vole Oxtr ............................................................ 62

DISCUSSION ........................................................................................................................... 65

CHAPTER 4: DISCUSSION ..................................................................................................... 70

ABSTRACT.............................................................................................................................. 71

INTRODUCTION .................................................................................................................... 73

MOLECULAR TECHNIQUES FOR STUDYING GENE REGULATION IN THE BRAIN ................74

Expanding ATAC-Seq Techniques ........................................................................................ 74

RNA-Seq................................................................................................................................ 75

ChIP-Seq ............................................................................................................................... 77

DISCUSSION; TOWARDS MOLECULAR MECHANISMS OF OXTR REGULATION..... 78

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Bioinformatics Biology, Neuroscience Biology, Genetics
Mot-clé	Sex Differences Prairie Vole Brain ATAC-Seq OXTR
Committee Chair / Thesis Advisor	Larry Young, Emory University

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