Stable silencing of spinal motor neuron enhancers by transiently expressed Nkx2.2

 描述(申请人提供)：多细胞生物体发育的标志是多能细胞协调分化为不同的有丝分裂后细胞类型。这一过程 依赖于转录程序，以协调的方式激活末端效应器基因的表达，并抑制在其他谱系中表达的基因。虽然在识别转录激活物和建立新的细胞身份的机制方面取得了进展，但目前对渐进抑制替代发育计划的过程知之甚少。具体地说，许多控制神经细胞分化的转录抑制因子只在神经上皮形成的早期短暂表达，目前还不确定这些抑制因子是否只作用于神经前体细胞中表达的基因，或者它们是否能够持续地沉默增强子，从而即使在基因表达下调后也能预期地改变基因的表达。在这里，我们将检测瞬时表达的运动神经元抑制因子Nkx2.2对其结合的增强子染色质修饰的影响，以及对小鼠运动神经元前体细胞基因表达模式的影响(同时Nkx2.2是异位表达的)及其下调。Nkx2.2在稳定沉默的增强子中的功能将通过删除Nkx2.2结合基序进行实验测试。总之，这些研究将揭示在多细胞生物体的细胞终末分化过程中稳定沉默稳定或活性增强剂的基本机制。由于沉默的增强子在细胞命运重编程过程中需要恢复到活跃或稳定的状态，我们的发现可能有助于开发更有效的诊断和治疗目的的患者细胞重编程策略。

 DESCRIPTION (provided by applicant): The hallmark of multicellular organism development is concerted differentiation of pluripotent cells into diverse postmitotic cell types. This process relies on transcriptional programs that in a coordinated manner activate expression of terminal effector genes and repress genes expressed in other lineages. While progress has been made in identifying transcriptional activators and mechanisms through which they establish new cell identities, little is currently known about the process underlying progressive repression of alternative developmental programs. Specifically, many transcriptional repressors that control differentiation of neural cells are expressed only transiently during early patterning of neuroepithelium and it has not been determined whether these repressors act solely on genes expressed in neural progenitors or whether they can silence enhancers in a persistent fashion to prospectively modify gene expression even following their downregualtion. Here we will examine effects of a transiently expressed motor neuron repressor Nkx2.2 on chromatin modifications of enhancers it binds and on patterns of gene expression in mouse motor neuron progenitors (while Nkx2.2 is ectopically expressed) and following its downregulation. Function of Nkx2.2 in enhancers that are stably silenced will be experimentally tested by deletion of Nkx2.2 binding motif. Together these studies will reveal fundamental mechanisms underlying stable silencing of poised or active enhancers during terminal differentiation of cells in multicellular organisms. As silenced enhancers need to be reverted into an active or poised state during cell fate reprogramming, our findings might be instrumental for the development of more efficient patient cell reprogramming strategies for diagnostic and therapeutic purposes.