Regulation of motor neuron enhancers by the transcription factor Nkx2.2

转录因子 Nkx2.2 对运动神经元增强子的调节

• 批准号: 8981087
• 负责人: Elena Abarinov
• 金额: $ 4.31万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8981087
• 关键词: Address; Architecture; Binding; Binding Sites; Biochemical; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Electroporation; Elements; Enhancers; Event; Exhibits; Gene Expression; Gene Silencing; Gene Targeting; Generations; Genes; Genetic; Genetic Enhancer Element; Genetic Programming; Genome; Genomic Segment; In Vitro; Interneurons; Maps; Mediating; Methods; Modification; Motor Neurons; Multipotent Stem Cells; Mutate; Mutation; Nerve Degeneration; Neural tube; Neuraxis; Neuronal Differentiation; Neurons; Population; Process; Protocols documentation; Recruitment Activity; Regenerative Medicine; Regulation; Regulator Genes; Regulatory Element; Repression; Site-Directed Mutagenesis; Spinal Cord; Staging; Testing; Therapeutic; Transcription Coactivator; Transcription Repressor; cell type; chromatin modification; clinically relevant; density; embryonic stem cell; genome-wide; histone modification; homeodomain; insight; nerve stem cell; novel strategies; progenitor; programs; promoter; prospective; public health relevance; research study; selective expression; stem cells; transcription factor; transcriptome sequencing

 DESCRIPTION (provided by applicant): The use of embryonic stem cells for therapeutic purposes holds great promise in the field of regenerative medicine. Transcriptional programming is of utmost importance for this objective but remains inefficient due to our lack of understanding of the obstacles which must be overcome in order to redirect cell-fate. During development, the differentiation of multipotent progenitor cells into postmitotic cells is accompanied by the progressive restriction of cell-fate potential. At the biochemical level, this is achieved through he silencing of enhancer and promoter elements regulating genes whose expression is incompatible with the final fate of the developing cell type. While many studies have examined the mechanism by which transcriptional activators program cell identity, little is known regarding the mechanisms by which transcriptional repressors silence their target genes. Gaining a deeper understanding of these mechanisms is pivotal to identifying and creating novel strategies for surmounting the current barriers of reprogramming protocols. To address these questions, I have performed a detailed examination of the transcription factor (TF) Nkx2.2. In the developing spinal cord, Nkx2.2 is transiently expressed in motor neuron (MN) progenitors, where it acts to suppress the MN developmental program in order to establish V3 interneuron identity. While it is well known that Nkx2.2 is a repressor of MN cell fate, the direct targets of Nkx2.2 are unknown, and its effects on the chromatin architecture remain to be elucidated. More generally, it is unclear whether TFs transiently expressed in progenitor cells can stably silence the genetic programs of postmitotic, mature neurons. Interestingly, genome binding analysis of Nkx2.2 expressed in MN progenitors has revealed that Nkx2.2 binds not only to enhancer elements that control MN progenitor identity but also to enhancers of postmitotic MN genes, which become activated much later in development. These findings raise the intriguing possibility that Nkx2.2 represses MN identity in part through the stable and prospective silencing of genes that regulate postmitotic MN identity. To define the effects of Nkx2.2 on MN differentiation, I will: i) determine the impact of Nkx2.2 binding on the chromatin landscape of MN progenitor and postmitotic genes and regulatory elements and ii) examine whether Nkx2.2 directly and stably represses the postmitotic MN program. These experiments will elucidate the still poorly understood mechanisms by which transiently expressed TFs control genetic regulatory networks. The findings from this study may inform more efficient strategies for transcriptional reprogramming of cellular identity, a process that will undoubtedly be useful for the generation of clinically relevant cell types in neurodegenerative treatments.

 描述（由申请人提供）：将胚胎干细胞用于治疗目的在再生医学领域具有巨大的前景。转录编程对于这一目标至关重要，但由于我们缺乏理解，转录编程仍然效率低下 为了改变细胞的命运而必须克服的障碍。在发育过程中，多能祖细胞分化为有丝分裂后细胞伴随着细胞命运潜力的逐渐限制。在生化水平上，这是通过沉默调节基因的增强子和启动子元件来实现的，这些基因的表达与发育细胞类型的最终命运不相容。虽然许多研究已经研究了转录激活因子编程细胞身份的机制，但对于转录抑制因子沉默其靶基因的机制知之甚少。更深入地了解这些机制对于识别和创建克服重编程协议当前障碍的新策略至关重要。为了解决这些问题，我对转录因子 (TF) Nkx2.2 进行了详细检查。在发育中的脊髓中，Nkx2.2 在运动神经元 (MN) 祖细胞中短暂表达，其作用是抑制 MN 发育程序，以建立 V3 中间神经元身份。虽然众所周知 Nkx2.2 是 MN 细胞命运的抑制因子，但 Nkx2.2 的直接靶点尚不清楚，其对染色质结构的影响仍有待阐明。更一般地说，目前尚不清楚在祖细胞中瞬时表达的转录因子是否可以稳定地沉默有丝分裂后成熟神经元的遗传程序。有趣的是，对 MN 祖细胞中表达的 Nkx2.2 的基因组结合分析表明，Nkx2.2 不仅与控制 MN 祖细胞身份的增强子元件结合，而且还与有丝分裂后 MN 基因的增强子结合，后者在发育后期才被激活。这些发现提出了一个有趣的可能性，即 Nkx2.2 部分通过稳定和前瞻性地沉默调节有丝分裂后 MN 身份的基因来抑制 MN 身份。为了确定 Nkx2.2 对 MN 分化的影响，我将：i) 确定 Nkx2.2 结合对 MN 祖细胞和有丝分裂后基因及调控元件染色质景观的影响，ii) 检查 Nkx2.2 是否直接且稳定地抑制有丝分裂后 MN 程序。这些实验将阐明暂时表达的转录因子控制基因调控网络的机制，目前人们还知之甚少。这项研究的结果可能会为细胞身份的转录重编程提供更有效的策略，这一过程无疑对于神经退行性治疗中临床相关细胞类型的生成有用。