Chromatin Accessibility and Transcription Dynamics During In Vitro Astrocyte Differentiation of Huntington’s Disease Monkey Pluripotent Stem Cells Open Access

Goodnight, Alexandra (Fall 2018)

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Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion, producing a mutant huntingtin (mHTT) protein with an extended polyglutamine tract. Evidence exists showing transcriptional dysregulation across neurodevelopment contributes to HD pathogenesis by disrupting many cellular processes. Additionally, aberrant epigenomic profiles have been reported in HD, but their impact on transcription remains unclear. Most HD research has focused on understanding the mechanisms of neuron degeneration, but neurons require astrocytes for normal development, function and survival. Recent evidence suggests that astrocytes not only contribute to, but are sufficient to trigger, neuronal dysfunction and HD pathogenesis, highlighting their pathogenic role, as well as potential therapeutic value in HD. However, transcriptional and epigenomic dysregulation in HD astrocytes has not been fully examined. Using pluripotent stem cells (PSCs) from transgenic HD non-human primates (NHP), we characterized global transcription and chromatin accessibility dynamics during in vitro astrocyte differentiation. We show genomic alterations in accessibility and transcription at all stages; however, trends observed throughout differentiation are established in neural progenitor cells (NPCs), after commitment to a neural lineage. Promoter-proximal accessibility is not associated with transcriptional changes. Differential distal accessibility, including a subset of NHP putative brain enhancers, show trends across astrogenesis. We also found p53 signaling and cell cycle dysregulation across HD differentiation, with observed down regulation of cell cycle genes in NPCs and aberrant cell cycle reentry and apoptosis in astrocytes. Interestingly, E2F target genes (ETGs) show this inverse expression between HD NPCs and astrocytes. While this coincides with differential E2F motif enrichment at promoters genome-wide, ETG promoter accessibility did not reflect differential expression patterns. Closer examination revealed that ETG expression shows stronger association with accessibility at nearby putative enhancers, suggesting interactions between regulatory elements, possibly enhancers, and promoters may drive aberrant transcription profiles across HD differentiation, highlighting the complex interplay of epigenetic mechanisms contributing to the HD transcriptome. Taken together, these results show altered chromatin accessibility and transcription throughout in vitro HD astrocyte differentiation and suggest E2F dysregulation may cause alterations in cell cycle and apoptosis pathways as HD NPCs differentiate towards astrocytes. 

Table of Contents

Chapter 1: General Introduction 

1.1 Huntington’s disease 

1.2 Neurodevelopmental dysregulation in Huntington’s disease 

1.3 Epigenetics of Huntington’s disease 

1.4 Study proposal  

Chapter 2: Chromatin Accessibility and Transcription Dynamics During In Vitro Astrocyte Differentiation of Huntington’s Disease Monkey Pluripotent Stem Cells 

2.1 Author’s Contribution and Acknowledgement of Reproduction 

2.2 Abstract 

2.3 Introduction

2.4 Methods 

2.4.1 NHP PSC cultures

2.4.2 NHP NPC culture maintenance

2.4.3 In vitro astrocyte differentiation

2.4.4 Quantitative reverse transcription PCR (qRT-PCR)

2.4.5 RNA-seq experiments

2.4.6 Assay for transposase-accessible chromatin using sequencing (ATAC-seq)

2.4.7 Analysis of RNA-seq and ATAC-seq data  

2.4.8 Differential Heatmaps 

2.4.9 ANOVA 

2.4.10 Motif Analysis 

2.4.11 Gene Set Enrichment Analysis (GSEA) 

2.5 Results 

2.5.1 In vitro astrocyte differentiation of HD and WT NHP cells

2.5.2 Aberrant transcriptional profiles are present during in vitro HD astrocyte differentiation

2.5.3 Promoter-proximal chromatin accessibility dynamics during HD astrocyte differentiation

2.5.4 Distal THSS profiles are widely altered during in vitro HD astrocyte differentiation 

2.5.5 Enhancer accessibility is altered during in vitro HD astrocyte differentiation 

2.5.6 De novo motif analysis at differentially accessible enhancers 

2.5.7 Gene Ontology (GO) analysis of differentially expressed (DE) genes across HD astrocyte differentiation 

2.5.8 Progressive up regulation of p53 signaling genes occurs during HD astrogenesis  

2.5.9 Cellular pathways related to the cell cycle are altered during HD astrocyte differentiation 

2.5.10 E2F dysregulation during HD astrocyte differentiation  

2.6 Discussion 

Chapter 3: General Discussion and Future Directions 

3.1 Summary 

3.2 Discussion and Future Directions

3.3 Conclusions


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