All ETDs from UAB

Advisory Committee Chair

Quamarul Hassan

Advisory Committee Members

Amjad Javed

Mary Macdougall

Thomas Ryan

Xinyang Zhao

Document Type

Dissertation

Date of Award

2019

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Bone loss is a worldwide problem resulting in increased risk of fracture. Osteoblasts are responsible for bone synthesis; therefore, treatments promoting osteoblast differentiation and/or activity would result in increased bone formation. The regulation of DNA accessibility is a key mechanism controlling gene expression and cellular differentiation. BAF (BRG1 Associated Factor) mediated chromatin remodeling increases DNA accessibility by sliding or ejecting nucleosomes. This process can occur in a cell type specific manner based on the composition of BAF. In many tissue types, a unique combination of BAF subunits has been identified to be responsible for the maintenance or differentiation of that cell type. To date, an osteoblast specific BAF complex has yet to be identified. It has been recently found in our studies that BAF45A, a potential member of the BAF complex, plays an essential role in osteoblast function. The central hypothesis of this work is that BAF45A is critical to remodel and activate chromatin at osteoblast specific genes promoting the synthesis and maintenance of bone. In order to address this hypothesis, the study has been divided into two specific aims. 1) Define the pheno-type of BAF45A osteoblast-specific knockout mice: We have developed an osteoblast-specific knockout of Baf45a. The physical phenotype of this mouse was analyzed by whole skeletal staining, microCT, histology/histomorphometry, double calcein labeling, and a three-point bend test. Results showed unchanged bone at 1 week, decreased bone in 2-month males and increased bone marrow adiposity in 6-month-old mice. The molecular mechanisms were assessed using RT-qPCR for in-vivo osteoblastic gene expression analysis, chromatin openness was investigated using ATAC sequencing and RNA sequencing in order to understand the corresponding expression pattern. It was found the Baf45a deletion results in altered osteoblastic chromatin and expression patterns. 2) Identify stage-specific BAF complex members during osteoblast differentiation: To uncover the composition of the osteoblast-specific BAF complex, immunoprecipitation mass spectrometry, epigenetic status and mRNA expression patterns were used to identify an osteoblast-specific composition of BAF. Understanding the physiological role and molecular mechanism of Baf45a as well as the composition of BAF in osteoblasts will reveal novel insights into osteoblast development and function.

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