Genomic reprogramming in the early embryo

项目总结 我们的长期目标是阐明重组基因组的分子机制，以允许 从特定的细胞类型过渡到多能状态。在胚胎发育的最初阶段， 特定生殖细胞的基因组被快速而有效地重新编程，以产生多能细胞 早期胚胎。在早期胚胎中发生的有效的重新编程之间有许多共同的特征 以及在培养中发生的效率低得多的重新编程。产生多能干细胞的能力 在疾病的建模和治疗方面具有巨大的潜力。对基础分子的理解 驱动重新编程的机制将对我们快速和可重复地 诱导出多能状态。无论是在培养中还是在早期胚胎中，重新编程都需要 专门的转录因子，称为先锋因子。先锋因素的独特之处在于，与其他因素不同 转录因子，它们可以在核小体的背景下与DNA结合。此功能使它们具有独一无二的 获得基因组，并有助于重新定义染色质的可及性。尽管如此，它仍然 不清楚驱动重新编程需要先锋因素的哪些特定功能，以及障碍是什么 开创了因子介导的重新编程的先河。在果蝇中，转录因子塞尔达是最初需要的 合子基因组的转录激活。我们已经证明了塞尔达拥有基本的特征 这种活性在合子基因组的整个过程中都是必需的 激活。然而，塞尔达不太可能独立工作，我们已经牵连到另一位开拓者 Gaga因子，与塞尔达共同作用，在早期定义可访问的染色质结构域 胚胎。通过结合我们在早期开发的询问转录因子功能的新工具 胚胎凭借经过充分研究的Fly系统的优势，我们处于独特的地位，可以确定必要的 重编程的特点。我们将使用遗传、基因组、生化和成像策略来1)定义 先锋因子合作对合子基因组进行重新编程，2)识别先锋因子的染色质障碍- 介导的重编程和3)确定内在无序区域在建立染色质中的作用 早期胚胎中的结构域。我们提出的研究具有重要意义，因为通过定义基本特征 对于开创性转录因子，我们将确定高效重新编程所需的统一原则 指定的细胞具有多能性。

PROJECT SUMMARY Our long-term goal is to elucidate the molecular mechanisms that restructure the genome to allow for the transition from a specified cell type to a pluripotent state. During the initial stages of embryonic development, the genomes of the specified germ cells are rapidly and efficiently reprogrammed to generate the pluripotent cells of the early embryo. Many features are shared between the efficient reprogramming that occurs in the early embryo and the much less efficient reprogramming that occurs in culture. The capacity to generate pluripotent stem cells has great potential in the modelling and treatment of disease. Understanding the fundamental molecular mechanisms that drive reprogramming will have important implications in our ability to rapidly and reproducibly induce the pluripotent state. Both in culture and in the early embryo, reprogramming requires the activity of specialized transcription factors, termed pioneer factors. Pioneer factors are distinctive in that, unlike other transcription factors, they can bind to DNA in the context of nucleosomes. This feature allows them unique access to the genome and helps to redefine the chromatin accessibility landscape. Nonetheless, it remains unclear what specific functions of pioneer factors are required to drive reprogramming and what the barriers are to pioneer factor-mediated reprogramming. In Drosophila the transcription factor Zelda is required for the initial transcriptional activation of the zygotic genome. We have demonstrated that Zelda possesses essential features of pioneer transcription factors and that this activity is required throughout the process of zygotic genome activation. However, Zelda is unlikely to be working independently, and we have implicated another pioneering factor, GAGA factor, as functioning together with Zelda to define accessible chromatin domains in the early embryo. By combining our development of novel tools to interrogate transcription factor function in the early embryo with the strengths of the well-studied fly system, we are uniquely positioned to determine essential features of reprogramming. We will use genetic, genomic, biochemical, and imaging strategies to 1) define how pioneering factors cooperate to reprogram the zygotic genome, 2) identify chromatin barriers to pioneer factor- mediated reprogramming and 3) determine the role of intrinsically disordered regions in establishing chromatin domains in the early embryo. Our proposed research is significant because by defining essential characteristics of pioneering transcription factors we will identify unifying principles required for efficient reprogramming of specified cells to pluripotency.