The role of cis-regulatory elements in the inheritance of transcriptional memory through mitosis.

项目总结 这项建议的目标是确定转录程序所定义的细胞身份是如何 通过系统地评估特定的顺式调节元件和反式调节元件的作用而通过有丝分裂遗传的 作用因素。中心假设是基因启动子上的顺式调节元件维持转录 通过有丝分裂的能力，而与增强子的反式相互作用调节适时和适当的水平 有丝分裂退出过程中的基因表达。这项提议将利用潜在的DNA序列如何决定 启动子和细胞类型特异性增强子的多方面相互作用确保细胞忠实地 建立适当的转录程序。这将在具有稳定标识的单元格中进行评估，并扩展到 理解在早期发育的背景下，当细胞经历不断的命运时，这是如何被调节的 决定。虽然以前认为有丝分裂过程中的染色质凝聚排除了大多数蛋白质 因此与转录相关的过程，最近的研究最初来自Zaret实验室，现在 表明转录在低水平上是活跃的，启动子(但不是大多数增强子)是可获得的，并且 维持积极的组蛋白修饰[1-7]；有丝分裂染色质臂保留动态染色质区域[8]。 尽管组蛋白修饰、染色质可及性和染色质的全基因组测量 组织建议在各种顺式调控元件上发生有丝分裂变化，它们无法获得高水平的 关于有丝分裂记忆如何保留在启动子上并在有丝分裂过程中起作用的解决机制的洞察力 退出，由增强器完成。虽然参与转录的成分，如TATA结合蛋白TBP， 检测到延长形式的RNAP2和转录因子(TF)，如GATA1、FOXA1和Esrrb 有丝分裂染色质；这些转录因子中的许多没有保持与它们的间期增强子靶标结合，因此如何 这些因子在特定的位置通过有丝分裂传递转录记忆的功能仍然难以捉摸[5-7，9- 13]。因此，这项提议旨在确定如何通过首先通过细胞分裂来保存细胞命运 评价有丝分裂转录中启动子的功能需求及其机制 肝脏特异性转录因子在有丝分裂退出时调节增强子的激活。这将是 在肝脏来源的Huh7人细胞系中进行评估，这对有丝分裂同步化具有高度的适应性 方法，并扩展以确定已确定的因子在多能性和内胚层分化中的作用 人类胚胎干细胞。这一提议将扩大我们对分子机制的理解 维持细胞特性和细胞命运规范，从而提高我们靶向和调节干细胞的能力 用于治疗的价值。

PROJECT SUMMARY The goal of this proposal is to determine how cell identity, as defined by a transcriptional program, is inherited through mitosis by systematically evaluating the role of specific cis-regulatory elements and trans- acting factors. The central hypothesis is that cis-regulatory elements at gene promoters maintain transcriptional competency through mitosis while trans interactions with enhancers mediate the timely and appropriate level of gene expression during mitotic exit. This proposal will leverage how the underlying DNA sequence determines the multifaceted interactions at promoters and cell-type specific enhancers that ensure cells faithfully re- establish proper transcriptional programs. This will be evaluated in cells with a stable identity and expanded to understand how this is mediated in the context of early development, when cells undergo constant fate decisions. While it was previously thought that chromatin condensation during mitosis excluded most proteins and thereby transcription-related processes, recent studies originally emanating from the Zaret lab now indicate that transcription is active at a low level and promoters, but not most enhancers, are accessible and maintain active histone modifications [1-7]; and mitotic chromatin arms retain areas of dynamic chromatin [8]. Although genome-wide measurements of histone modifications, chromatin accessibility and chromatin organization suggest mitotic changes at various cis-regulatory elements, they are unable to garner high- resolution mechanistic insight into how mitotic memory is retained at promoters and acted upon, during mitotic exit, by enhancers. While components involved in transcription such as the TATA-binding protein TBP, the elongating form of RNAP2, and transcription factors (TFs) such as GATA1, FOXA1, and ESRRB are detected on mitotic chromatin; many of these TFs do not remain bound to their interphase enhancer targets, thus how these factors function at specific loci to transmit transcription memory through mitosis remains elusive [5-7, 9- 13]. Therefore, this proposal aims to determine how cell fate is preserved through cell division by first evaluating the functional requirement for promoters in mitotic transcription, and then the mechanisms employed by liver-specific transcription factors to mediate enhancer activation upon mitotic exit. This will be assessed in the liver-derived HUH7 human cell line, which is highly amenable to mitotic synchronization methods, and expanded to determine the role of identified factors in pluripotent and endoderm-differentiated human embryonic stem cells. This proposal will expand our understanding of the molecular mechanisms that maintain cell identity and cell fate specification, thereby improving our ability to target and modulate stem cells for therapeutic value.