Hox-Regulated MSCs in Skeletal Development, Growth and Fracture Healing

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PROJECT SUMMARY/ABSTRACT Hox genes are a family of evolutionarily conserved transcription factors that are essential for the proper development of the axial and appendicular skeleton during embryogenesis. Posterior Hox genes (Hox9-13) are critical for patterning the skeletal elements of the limb along the proximodistal axis. Specifically, Hox11 paralogous genes pattern the zeugopod, which is comprised of the radius/ulna and the tibia/fibula in the forelimb and hindlimb, respectively. Recent studies have demonstrated that Hox11 genes are expressed in skeletal stem/progenitor cell population that self-renews and gives rise to osteoblasts, chondrocytes, and adipocytes of the zeugopod throughout life. Loss of Hox11 function in skeletal stem progenitor cells impairs their capacity to differentiate into osteoblasts; subsequently affecting bone remodeling and repair. Further, Hoxa11eGFP reporter expression demonstrates that Hox11 mutant stem/progenitor cells turn off Hox expression, leave the stem/progenitor pool and initiate differentiation but fail to differentiate into mature osteoblasts. These findings are consistent with a model in which Hox11 function is required to establish the differentiation potential during lineage commitment. Despite well understood genetic contributions to limb development, maintenance and repair, the mechanisms by which Hox11 regulates skeletal stem cell differentiation remain unknown. My current work in the Wellik laboratory focuses on identifying Hox11-bound genomic loci along with RNA-Seq analyses to identify potential downstream target genes that regulate stem/progenitor cell differentiation. Taking advantage of two newly generated epitope-tagged mouse alleles (Hoxa11-3XFLAG and Hoxd11-3XFLAG) and using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assays, I have identified hundreds of Hox11 binding sites in stem/progenitor cells. However, how Hox11 transcriptional functions regulate the chromatin state and accessibility of these sites remains unclear. This proposal seeks to understand the epigenomic landscape regulated by Hox11 during osteochondral differentiation. In the proposed experiments (Aim 1), I will utilize a novel chromatin profiling method, Multiple Target Identification by Tagmentation (MulTI-Tag) in embryonic control and Hox11 mutant stem progenitor cells to define their chromatin status by probing for several histone modifications. Further, I will investigate how Hox11 function regulates chromatin accessibility during osteochondral differentiation, using ATAC-Seq in embryonic control and Hox11 mutant stem progenitor cells (Aim 2). Taken together, the proposed experiments will elucidate the mechanism by which Hox11 function regulates skeletal stem/progenitor cell differentiation in the development and regeneration of the skeleton.