Pioneer factor activity in transcription and DNA replication

项目总结 受精后不久，基因组和转录组发生了戏剧性的重新编程，使 胚胎迅速而健壮地发育。这一点在果蝇胚胎中尤为明显，它在一个 两个小时的时间经历了13个有丝分裂周期，使胚层细胞化，形成身体计划，并做好准备 用于原肠发育。虽然这些过程背后的基因网络已经被很好地研究了，但还不清楚是如何进行的 它们是集体启动的，这个过程被称为合子基因组激活。此外，尽管它是 观察到DNA复制发生在转录之前，控制这一时机的机制尚不清楚。 在此之前，我们证明了一个单一的因子，塞尔达，在全球范围内激活早期表达的基因。我们 研究发现，Zelda与基因组上的CAGGTAG位点结合可以降低增强子的核小体屏障， 从而促进其他关键转录因子的结合。塞尔达和这些其他因素出现在 亚核“中心”(离散的焦点)，可在增强子上共同定位。我们的目标是使用高分辨率 显微镜下观察塞尔达的哪些结构特征参与了轮毂的形成。此外，我们正在 对控制转录和DNA复制时间的调控机制感兴趣 避免了两个过程之间的冲突。我们认为塞尔达扮演着双重先锋的角色：1)开启 用于装载起始识别复合体并随后形成复制前复合体的染色质， 2)打开新形成的染色质，以便 开始转录。我们的目标是首先定义早期胚胎的复制起源，并表征 塞尔达在原产地许可和转录启动中的作用。我们的研究将有助于了解这两个基本原理 DNA复制和转录的过程在基因组激活过程中是协调的。

PROJECT SUMMARY Shortly after fertilization, a dramatic reprogramming of the genome and transcriptome occurs, enabling the embryo to develop quickly and robustly. This is especially exemplified in the Drosophila embryo, which within a two-hour period undergoes 13 mitotic cycles, cellularizes the blastoderm, patterns the body plan, and gets ready for gastrulation. While the gene networks underlying these processes have been well studied, it is not clear how they are collectively initiated, a process referred to as zygotic genome activation. Moreover, although it was observed that DNA replication occurs before transcription, the mechanisms that control this timing are not known. Previously, we demonstrated that a single factor, Zelda, acts globally to activate early-expressed genes. We found that Zelda binding to CAGGTAG sites across the genome lowers nucleosome barriers at enhancers, thereby facilitating the binding of other key transcription factors. Zelda and these other factors are present in sub-nuclear “hubs” (discrete foci) that were seen to colocalize at enhancers. We aim to use high-resolution microscopy to investigate the which structural features of Zelda mediate hub formation. In addition, we are interested in the regulatory mechanisms that control the timing of transcription and DNA replication so that conflicts between the two processes are avoided. We propose that Zelda plays a dual pioneering role: 1) to open chromatin for the Origin Recognition Complex to load and subsequent formation of the Pre-replication Complex, which occludes the transcriptional machinery until origins have fired, and 2) to open newly formed chromatin for transcription to initiate. Our goal is to first define origins of replication in early embryos, and to characterize Zelda's role in origin licensing and transcriptional initiation. Our studies will lend into how these two fundamental processes of DNA replication and transcription are coordinated during genome activation.