Analysis of environmentally-sensitive epigenetic machinery during osteogenic differentiation

在美国，每4.5分钟就有一个婴儿出生时就有先天缺陷。骨性骨骼的骨性缺陷 与子宫内环境中的化学物质接触有关。我们的实验室已经证明这是正确的 成骨细胞是骨形成细胞，它的发育依赖于骨特异性基因的严格调控。我们 开发了一种人类胚胎干细胞(HESC)系统，其中毒物诱导差异基因表达 干扰了成骨细胞的分化。MicroRNAs(MiRNAs)是表观遗传的非编码小RNA 调节基因表达，是骨骼发育的参与者。目前尚不清楚miRNAs是否 对环境因素的负面反应会导致骨骼畸形。在这个提案中，我假设 毒物诱导的miRNA改变在骨骼出生缺陷的表现中起着关键作用。MiRNA 分化hESCs的图谱先前发现了来自化学物质下调的10个miRNAs 暴露抑制了成骨细胞的分化。我建议1)验证候选人的职能角色 成骨过程中的miRNAs(K99)；2)研究候选miRNAs的功能效应是否 体内复制(K99)；3)确定受候选基因影响的mRNA靶基因和信号通路 MiRNAs(K99)；以及4)研究miRNA失调是否通过表观遗传机制促进 抑制miRNA表达(R00)。拟议的目标将显著影响对 骨骼发育过程中环境诱发的表观遗传毒性。导师马丁·里科马诺博士 和Nicole Zur Nieden(hESCs、miRNAs和基因组工程) 将帮助我成功完成拟议的目标并成为一名独立校长的培训 调查员。其他支持将来自马丁·加西亚-卡斯特罗博士(分子细胞遗传学技术)和 帕特里克·阿拉德(表观遗传毒理学)。职业发展计划包括加强我的研究的培训 技能，包括下一代测序、生物信息学、宫内电穿孔和活体骨骼 分析以及学术技能和专业发展，包括但不限于科学 沟通、写作、指导和管理技能。计划中的培训和研究将有助于 过渡到独立并成功地成为一名首席研究员。

Every 4.5 minutes, a baby is born with a birth defect in the United States. Skeletal defects of the bony skeleton have been associated with environmental chemical exposure in utero. Our lab has shown that proper development of osteoblasts, the bone forming cells, depends on tight regulation of bone-specific genes. We developed a human embryonic stem cell (hESC) system where toxicant-induced differential gene expression perturbed osteoblast differentiation. MicroRNAs (miRNAs) are small non-coding RNAs that epigenetically regulate gene expression and are actors of skeletal development. There is no knowledge whether miRNAs adversely respond to environmental agents to result in skeletal malformations. In this proposal, I hypothesize that toxicant-induced miRNA changes play a critical role in the manifestation of skeletal birth defects. MiRNA profiling on differentiating hESCs previously identified 10 miRNAs downregulated stemming from chemical exposure that repressed osteoblast differentiation. I propose to 1) validate the functional role of candidate miRNAs during osteogenesis (K99); 2) investigate whether the functional effects of the candidate miRNAs are replicated in vivo (K99); 3) determine the mRNA target genes and signaling pathways affected by the candidate miRNAs (K99); and 4) investigate whether miRNA dysregulation is facilitated through epigenetic machinery that represses miRNA expression (R00). The proposed aims will significantly impact the understanding of environmentally induced epigenetic toxicity during skeletal development. Mentors Drs. Martin Riccomagno (survival surgeries and genome engineering) and Nicole zur Nieden (hESCs, miRNAs and genome engineering) will aid in my training for successful completion of the proposed aims and becoming an independent principal investigator. Additional support will come from Drs. Martin Garcia-Castro (molecular cytogenetic techniques) and Patrick Allard (epigenetic toxicology). The career development plan includes training to enhance my research skills, including Next Generation Sequencing, bioinformatics, in utero electroporation, and in vivo skeletal analysis, as well as scholarly skills and professional development, including but not limited to science communication, writing, mentoring, and management skills. The planned training and research will facilitate the transition to independency and success as a principal investigator.