Epigenetic regulation of osteoblast differentiation
by Zeynab Najafova
Date of Examination:2016-08-09
Date of issue:2017-06-07
Advisor:Prof. Dr. Steven Johnsen
Referee:Prof. Dr. Steven Johnsen
Referee:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Gregor Bucher
Persistent Address:
http://dx.doi.org/10.53846/goediss-6333
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Abstract
English
Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. However, epigenetic mechanisms involved in control of bone-specific transcriptional programs remain largely unexplored. In this study we focused on functional characterization of two epigenetic regulators – Bromodomain-containing Protein-4 (BRD4) and Ring Finger Protein 40 (RNF40) in development and maintenance of osteoblast fate. BRD4 was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses show that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated during differentiation and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4. RNF40 forms with RNF20 the obligate E3 complex and mediates monoubiquitination of lysine 120 of histone H2B (H2Bub1). Despite its global correlation with active gene transcription, we previously demonstrated a specific role for H2Bub1 in controlling osteoblast (OB)-specific gene expression during OB differentiation of mesenchymal stem cells (Karpiuk et al., 2012, Mol Cell). To address the significance of H2Bub1 for bone development in vivo, we generated OB-specific Rnf40 conditional knockout (KO) models. Given the crucial role of RUNX2 in osteoblast lineage commitment and chondrocyte hypertrophy, the Rnf40Runx2-Cre knockout mice developed achondroplastic-like phenotype with shorter bone sizes. Surprisingly, micro computed tomography (µCT) analysis showed increased bone mineral density (BMD) in these mice, a finding that could be associated with decreased osteoclast function and hence osteopetrosis-like phenotype. Remarkably, the Rnf40Bglap-Cre KO mice displayed only osteopetrotic-like phenotype with significant increase in bone mineral density without any further alterations in bone formation and apposition rate. This suggested the importance of H2Bub1 at the earlier stages of osteoblast commitment. Strikingly, the number of osteoclasts was substantially reduced in these mice suggesting the increased BMD to be associated with reduced bone resorption. Ex vivo differentiation studies confirmed a role for Rnf40 and H2Bub1 in OB differentiation as revealed by decreased alkaline phosphotase activity/expression, mineralization and expression of Runx2 and Sp7 in KO OBs. Consistently, Rnf40-deficient OBs showed significantly reduced Rankl expression and triggered less osteoclast formation as revealed by osteoblast and osteoclast co-culture experiments. Remarkably, the reverse effect in modulating osteoblast specific H2Bub1 levels in Ubiquitin-Specific Protease 22 (Usp22)Runx2-Cre KO mice did not have any impact on bone in mice. Altogether, these data indicate significance of H2Bub1 signaling in regulating the osteoblast lineage commitment. Moreover, H2Bub1 appears to act as defining epigenetic regulator of osteoblast-osteoclast “communication“ in vivo which provides a basis for future studies to investigate the potential of its regulatory pathway for the treatment of conditions such as aging-related osteoporosis.
Keywords: Bone remodeling; Enhancers; Epigenetic; Transcription factors; H2B monoubiquitination; Histone modifications; Bone remodeling; BRD4; Osteoblast
