Identification of an Osteoblast-Specific BAF Chromatin Remodeling Complex

Objectives: The Brg1 Associated Factors (BAF) complex plays an indispensable role in the formation and maintenance of cell-type-specific patterns of accessible DNA, ultimately controlling cellular phenotypic fate. BAF is an 11 subunit complex encoded by 22 genes, where different combinations of BAF provide cell-type specificity. We have identified Baf45a to be a key osteogenic BAF factor required in osteoblasts. The objective of this work is to define the osteoblast BAF complex composition and the specific role of Baf45a in osteoblasts.
Methods: The osteoblast-specific BAF complex composition was investigated in differentiating MC3T3-E1 cells using anti-BRG1 Immuno-precipitation (IP) mass spectrometry (MS). Osteogenic responsive factors were determined by RT-qPCR following BMP-2 and TGFβ treatment of MC3T3-E1 cells. BAF45A-RUNX2 interaction was probed by co-IP in HEK293 cells. A Baf45a floxed allele (Baf45a^fl/fl) osteoblast-specific (osteocalcin-Cre) knockout mouse model was analyzed by whole skeletal staining, µCT, and histomorphometry. Calvarial osteoblasts from Baf45a^fl/fl mice were treated with lenti-viral Cre to investigate chromatin accessibility (ATAC-sequencing) and modification (ChIP).
Results: IP-MS and RT-PCR suggest a unique osteoblast-specific BAF combination. Treatment with BMP2 resulted in a >2.5 fold increase in Baf45a expression, while TGFβ decreased expression by 50%. BAF45A was found to interact with osteoblast master transcription factor RUNX2. Deletion of Baf45a in osteoblast resulted in disrupted skeletogenesis (1 week), decreased bone formation (2 months), and altered bone maintenance (6 months). Additionally, 6-month-old mice were found to have high bone marrow adiposity. Analysis of chromatin accessibility revealed open chromatin at adipocyte-specific genes with the deletion of Baf45a. Finally, deletion of Baf45a resulted in drastic decreases in activating histone modification H3K27ac at osteoblast-specific genes.
Conclusions: These data reveal the osteoblastic BAF composition, as well as the osteogenic role of Baf45a. By understanding these epigenetic mechanisms regulating osteoblasts and bone formation, we hope to uncover potential therapeutic targets to aid in the treatment of bone-diseases.