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

Hox 调节的 MSC 在骨骼发育、生长和骨折愈合中的作用

• 批准号: 10840553
• 负责人: Deneen M Wellik
• 金额: $ 10.82万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-08 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10840553
• 关键词: ATAC-seq; Adipocytes; Adult; Affect; Alleles; Binding; Binding Sites; Biological Assay; Bone remodeling; Chondrocytes; Chromatin; Development; Development Plans; Elements; Embryo; Embryonic Development; Epitopes; Family; Forelimb; Genes; Genetic; Genetic Transcription; Growth; Hindlimb; Homeobox Genes; Impairment; Laboratories; Life; Limb Development; Limb structure; Maintenance; Mediating; Mentors; Methods; Modeling; Mus; Natural regeneration; Osteoblasts; Pattern; Population; Radial; Reporter; Research; Site; Skeletal Development; Skeleton; Work; bone fracture repair; bone repair; career development; epigenomics; experimental study; fibula; genomic locus; histone modification; mutant; novel; nuclease; osteochondral tissue; paralogous gene; progenitor; repaired; self-renewal; skeletal; skeletal stem cell; stem; stem cell differentiation; stem cell population; stem cells; tibia; transcription factor; transcriptome sequencing; ulna

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.

项目总结/摘要 Hox基因是一个进化上保守的转录因子家族，其对于适当的转录调控是必需的。 在胚胎发生过程中轴骨和无骨骨骼的发育。后Hox基因（Hox 9 -13）是 这对于沿着近远轴沿着图案化肢体的骨骼元素至关重要。Hox11 旁系同源基因模式的zeugopod，这是由桡骨/尺骨和胫骨/腓骨在 前肢和后肢。最近的研究表明，Hox 11基因表达于 骨骼干/祖细胞群，其自我更新并产生成骨细胞、软骨细胞和 脂肪细胞的脂肪细胞。Hox 11在骨骼干/祖细胞中的功能缺失 它们分化成成骨细胞的能力;随后影响骨重建和修复。此外，本发明还 Hoxa 11 eGFP报告基因表达证实Hox 11突变体干/祖细胞关闭Hox 表达，离开干细胞/祖细胞库并启动分化，但不能分化成成熟的 成骨细胞这些发现与需要Hox 11功能来建立 分化潜力在血统承诺。尽管对肢体的遗传贡献已经很好地理解， 发育、维持和修复，Hox 11调节骨骼干细胞的机制 分化仍然未知。我目前在Wellik实验室的工作重点是识别Hox 11结合的 基因组位点沿着RNA-Seq分析，以鉴定潜在的下游靶基因， 干/祖细胞分化。利用两个新产生的表位标记的小鼠等位基因 （Hoxa 11 -3XFLAG和Hoxd 11 -3XFLAG）和使用靶下切割和使用核酸酶释放 (CUT&RUN）分析，我已经确定了干/祖细胞中的数百个Hox 11结合位点。但如何 Hox 11转录功能调节染色质状态，这些位点的可及性仍不清楚。 该提案旨在了解骨软骨形成过程中Hox 11调控的表观基因组景观 分化在拟议的实验（目标1）中，我将利用一种新的染色质分析方法，多重 在胚胎对照和Hox 11突变体干祖细胞中通过标签化（Multi-Tag）进行靶标鉴定 细胞通过探测几种组蛋白修饰来确定它们的染色质状态。此外，我将研究如何 Hox 11功能调节骨软骨细胞分化过程中染色质的可及性，使用ATAC-Seq在 胚胎对照和Hox 11突变体干祖细胞（Aim 2）。综上所述，拟议的实验 将阐明Hox 11功能调节骨骼干/祖细胞分化的机制， 骨骼的发育和再生。