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有先天缺陷。骨骼缺损 与子宫内环境化学品暴露有关。我们的实验室已经证明， 成骨细胞（骨形成细胞）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，其中毒素诱导的差异基因表达 扰乱成骨细胞分化。microRNAs（miRNAs）是一类小的非编码RNA， 调节基因表达和骨骼发育的演员。目前还不清楚miRNAs是否 对环境因素产生不利反应，导致骨骼畸形。在这个提议中，我假设 毒物诱导的miRNA变化在骨骼出生缺陷的表现中起关键作用。miRNA 对分化的hESC进行分析，先前鉴定了10种来自化学物质的下调的miRNA。 暴露抑制成骨细胞分化。我建议1）验证候选人的职能作用 骨形成过程中的miRNAs（K99）; 2）研究候选miRNAs的功能效应是否与骨形成过程中的miRNAs（K99）相关。 在体内复制（K99）; 3）确定受候选物影响的mRNA靶基因和信号通路 miRNA（K99）;和4）研究miRNA失调是否通过表观遗传机制促进， 抑制miRNA表达（R 00）。所提出的目标将大大影响对 在骨骼发育过程中环境引起的表观遗传毒性。导师Martin Riccomagno博士 （生存手术和基因组工程）和Nicole zur Nieden（hESC，miRNA和基因组工程） 这将有助于我成功完成拟议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、子宫内电穿孔和体内骨骼 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促进 过渡到独立和成功的主要研究者。

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.