All ETDs from UAB

Advisory Committee Chair

Quamarul Hassan

Advisory Committee Members

Quamarul Hassan

Amjad Javed

Alecia Gross

Rosa Serra

Xinyang Zhao

Document Type

Dissertation

Date of Award

2020

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Differentiation of pre-osteoblasts is critical to controlling in-vivo development and growth of bone. Recent studies highlight the importance of epigenetic regulation in directing osteoblast commitment and function. Here we show that the microRNA-23a cluster (miR-23a, 27a, and 24-2) controls bone mass in-vivo through a previously unknown epigenetic mechanism. MiR-23a cluster knockdown increased the intensity of Alkaline Phosphatase staining in MC3T3-E1 cells. Additionally, it upregulated mRNA expression of osteogenic marker genes such as Runx2 and Osteocalcin. Micro-CT analysis of 2-month and 6-month femurs showed that trabecular bone volume and trabecular number significantly increased in miR-23aClZIP mice as compared to controls. Histological analysis revealed increased osteoblast and osteocyte numbers along with increased bone formation and 2 and 6 months of age. 3 point bend testing showed enhanced femoral strength in both males and females at 2 months of age. Supporting this increased bone mass, Runx2 expression levels were significantly upregulated along with the osteoblast transcription program. In contrast, the levels of a potent epigenetic repressor Ezh2 and the Polycomb Repressive Complex (PRC2) were significantly reduced in whole bone RNA sequenced from miR-23aClZIP mice. Mechanistically, we found that the miR-23a cluster inhibits RUNX2 translation by binding to the 3’ UTR of Runx2 mRNA transcripts. Furthermore, ChIP assays revealed RUNX2 binds to the Ezh2 promoter inhibiting transcription in MC3T3-E1 cells. Additional ChIP experiments in miR-23aClZIP mouse primary calvarial pre-osteoblasts showed that miR-23a cluster knockdown results in decreased binding of the epigenetic repressor EZH2 to osteogenic gene promoters such as Osteocalcin and Runx2, resulting in a more osteogenic transcription program. Our findings show that the microRNA-23a cluster coordinates an epigenetic axis that maintains bone mass In-vivo. The miRNA-23a cluster negatively regulates Runx2, master transcription factor of the osteogenic transcription program. We have also demonstrated that Runx2 negatively regulates Ezh2 by occupying its promotor and inhibiting transcription. Additionally, our data show that EZH2 epigenetically represses osteoblast-specific genes by adding the trimethylation mark to H3K27. Taken together, the microRNA-23a cluster regulates the osteoblast differentiation through a Runx2-Ezh2 epigenetic axis to maintain bone mass In-vivo.

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