Background/Purpose

Osteoclasts are bone resorbing cells which differentiate from myeloid precursors. The crosstalk between osteoblasts and osteoclasts tightly regulates the dynamic and continuous process of bone remodeling. Deregulation of this delicate balance is implicated in osteoporosis pathology. A variety of transcription factors including PU.1, MITF, NFATc1, and cFOS are essential for osteoclast differentiation. However, the interplay between these factors is not fully defined. We have previously shown that the transcription factors MITF and PU.1 act as a complex to regulate multiple genes required for osteoclast function. This study was designed to understand the global transcriptional regulatory processes involved in osteoclast differentiation.

Methods

We examined transcriptional regulation of target genes by MITF and PU.1 in myeloid precursors and osteoclasts using ChIP-Seq to map the genome-wide binding of these factors. In parallel, microarray analysis was performed to monitor target gene expression changes over the course of differentiation. We also queried the sequences jointly bound by PU.1 and MITF to search for conserved binding sequences potentially indicating novel co-partners in osteoclasts. We used micro-computed tomography and histological analysis to examine the effects of myeloid lineage-specific and osteoclast-specific deletion of PU.1 and its copartners in vivo and in vitro. 

Results

ChIP-Seq and microarray profiling revealed that MITF and PU.1 jointly regulate the transcription of over 1000 genes in developing osteoclasts. Most of the MITF/ PU.1 co-bound regions were found in distal enhancer-like elements at the same sites in both myeloid precursors and osteoclasts. Overlap of our ChIP-Seq and microarray data sets utilizing Gene Set Enrichment Analysis (GSEA) revealed that transcription factor genes were the only subset significantly enriched in genes with PU.1/ MITF co-bound regions. These transcription factors include NFATc1, c-FOS, and NFKB, which are already known to be essential for osteoclast differentiation. Additionally, 38% of genomic regions jointly bound by PU.1 and MITF in developing osteoclasts contained the binding motif of the T-box transcription factor EOMES. Conventional ChIP assays validated binding of EOMES to MITF/PU.1 bound regions. Micro-computed topography analysis of murine bone has demonstrated that the loss of PU.1 or EOMES in osteoclasts and their myeloid precursors leads to a severe osteopetrotic phenotype in neonatal mice due to highly deficient osteoclast differentiation and function.

Conclusion

Our results demonstrate that MITF and PU.1 set up a transcription factor regulatory network in myeloid precursors which triggers osteoclast differentiation in response to cues from the bone microenvironment. Ablation of PU.1 or its novel copartner EOMES results in disruption of this transcription factor network and therefore halts the differentiation program.

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« Back to 2014 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/pu-1-mitf-and-their-novel-co-partner-eomes-set-up-a-transcription-factor-network-that-is-critical-for-osteoclast-differentiation/