Defining the cis-regulatory sequence elements regulated by BAF complexes containing different ARID1 variants Pubblico
Flano, Sara (Summer 2023)
Abstract
BRG1/BRM-associated factor (BAF) complexes regulate gene expression by repositioning the histone proteins that package DNA inside the cell’s nucleus. This process, known as chromatin remodeling, regulates gene expression by controlling DNA accessibility to regulatory proteins known as transcription factors (TFs). TFs bind to specific DNA sequences, which are often grouped into larger cis-regulatory sequence elements (cREs) where the binding of multiple TFs influences the transcription of one or more target genes. One subunit of BAF complexes is an AT-rich interaction domain 1 (ARID1) protein. Mammalian genomes encode for two different ARID1 proteins, known as ARID1A and ARID1B. Mutations in genes that encode BAF complex subunits can cause a variety of diseases. Interestingly, mutations in both ARID1A and ARID1B cause disease in humans, but the diseases that are associated with each variant differ: ARID1A is the most frequently mutated BAF subunit in cancer, whereas ARID1B is the BAF subunit most frequently mutated in neurodevelopmental disorders. While the importance of BAF complexes and their ARID1 subunits in human health has been well established, the specific cREs and target genes they regulate remain undefined. Based on the difference in disease involvement between ARID1A and ARID1B, I hypothesize that BAF complexes containing different ARID1 subunits regulate chromatin accessibility at different sets of cREs, which in turn regulate the expression of different target genes. To identify the regulatory targets of BAF complexes containing ARID1A/B, I assessed changes in chromatin accessibility in Neuro-2A cells treated with an ATPase inhibitor or Arid1a-targeting DsiRNAs. My results produce novel and foundational data that pave the way for further characterization of the cREs and target genes that may be differentially regulated by ARID1A- and ARID1B-containing BAF complexes. These investigations will likely contribute to a better understanding of associated neurodevelopmental disorders, and to exploring potential therapeutic targets for associated cancers.
Table of Contents
CHAPTER 1: INTRODUCTION 1
1.1: GENE EXPRESSION REGULATION THROUGH CHROMATIN REMODELING. 1
1.2: BAF COMPLEXES IN GENE REGULATION AND DISEASE 2
1.3: ARID SUBUNITS OF THE CBAF COMPLEX 3
CHAPTER 2: METHODS 7
2.1: CELL CULTURE 7
2.2: ATPASE INHIBITOR TREATMENT 7
2.3: DSIRNA TRANSFECTION 8
2.3A: DsiRNA Selection 8
2.3B: DsiRNA Treatment 8
2.3C: RNA Isolation and cDNA Synthesis 9
2.3D: TaqMan qPCR 9
2.4: ATAC-SEQ LIBRARY PREPARATION AND SEQUENCING ANALYSIS 10
2.5: BUFFERS AND SOLUTIONS 11
CHAPTER 3: RESULTS 13
3.1: IDENTIFYING THE OPTIMAL SEEDING DENSITY OF N2A CELLS FOR DRUG TREATMENT AND SIRNA KNOCKDOWN EXPERIMENTS. 13
3.2: CHARACTERIZING REGIONS OF THE GENOME WHERE BAF IS REQUIRED TO MAINTAIN CHROMATIN ACCESSIBILITY. 14
3.2: CHARACTERIZING REGIONS OF THE GENOME WHERE ARID1A AND/OR ARID1B ARE REQUIRED TO MAINTAIN CHROMATIN ACCESSIBILITY. 17
3.2A: Optimizing siRNA transfections 17
3.2B: Establishing TaqMan Assays to measure Arid1a and Arid1b expression. 19
3.2C: Confirming knockdown of positive control gene: Hprt1. 20
3.2D: Assessing siRNA knockdown of Arid1b expression. 21
3.2E: Assessing siRNA knockdown of Arid1a expression. 22
3.2F: Characterizing changes in chromatin accessibility after Arid1a Knockdown. 24
CHAPTER 4: CONCLUSIONS 28
WORKS CITED 30
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