ncBAF function in iPSCs: Investigating a feedback loop between BICRA and BICRAL Público
Wang, Jianing (Spring 2025)
Abstract
Chromatin accessibility is regulated by a group of proteins called chromatin remodelers. The BRG1/BRM-Associated Factor (BAF) family of chromatin remodeling complexes play pivotal roles in regulating gene expression by repositioning nucleosomes at non-coding cis-regulatory elements. Recently, a new subtype of BAF complexes was discovered and are referred as non-canonical BAF (ncBAF) complexes. These ncBAF complexes contain some subunits not found in other BAF subtypes, including the proteins BICRA and BICRAL. Importantly, loss-of-function mutations in BICRA cause a rare syndromic form of intellectual disability, which suggests that BICRA and the ncBAF complexes that contain it play an important role in brain development. To further investigate the role of BICRA in brain development, I helped a graduate student in the lab, Ziben Zhou, engineer human induced pluripotent stem cells (hiPSC) with loss-of-function mutations in BICRA, which could then be differentiated to neurons in vitro. In the process of characterizing these cells, I discovered that BICRA null hiPSC show increased protein levels of BICRA’s paralog, called BICRAL. In my thesis research, I found that BICRAL protein and mRNA levels are increased in BICRA null hiPSCs, suggesting a possible transcriptional feedback loop. I further discovered that this feedback loop is not reciprocal. I found that BICRA protein and mRNA levels are unchanged in hiPSCs with BICRAL mutations. Together, the data suggest the existence of a previously-unknown feedback loop in which BICRAL mRNA expression level is responsive to BICRA abundance. In the future, I plan to investigate the mechanism of BICRAL mRNA upregulation and whether it is conserved in different cell types.
Table of Contents
List of Figures
Chapter 1: Background and Introduction
1.1 Eukaryotic chromosome organization
1.2 Chromatin accessibility at cREs
1.3 BAF family of chromatin remodelers
1.4 The non-canonical BAF contains BICRA and BICRAL
1.5 Research is needed for understanding the functional relatedness of BICRA and BICRAL
Chapter 2: Evidence of a feedback loop between BICRA and BICRAL in iPSCs
2.1 Overview
2.2 Generation of BICRA and BICRAL knockout iPSCs
2.3 BICRA and BICRAL KO genotyping
2.4 BICRA and BICRAL KO Western Blot
2.5 Conclusion: BICRAL protein level is increased in BICRA KO iPSCs
Chapter 3: BICRAL is transcriptionally upregulated in BICRA knockouts
3.1 Overview
3.2 Testing BICRAL probe specificity
3.3 BICRAL is transcriptionally upregulated in BICRA KO iPSCs
Chapter 4: Materials and Methods
4.1 iPSC Cell Culture
4.2 CRISPR-Cas9 and genotyping
4.3 RNA extraction, quality validation, and reverse transcription
4.4 RT-qPCR
4.5 Western Blot
Chapter 5: Discussion and conclusions
5.1 Summary
5. 2 Potential Mechanism for BICRAL upregulation in BICRA knockout iPSCs
Chapter 1: Background and Introduction
5. 3 Future Directions
Works Cited
About this Honors Thesis
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