Transcriptional interference through LUTI mRNA expression in development

项目摘要 细胞分化由基因表达的变化驱动。从一个 当细胞从一种类型向另一种类型转化时，细胞必须同时激活促进分化的基因， 抑制基因对抗忠实的进展。基因抑制是如何在 广泛的转录激活尚未完全了解。最近描述的基因模式 抑制依赖于整合的转录和翻译干扰， 转录激活和基因抑制的协调。在这种形式的基因调控中， 长未解码转录本同种型（LUTI）从基因远端启动子转录，导致 在基因近端启动子上的抑制性共转录染色质修饰4，6，34.在 与来自近端启动子的有效翻译的典型转录物相反， LUTI在其5'延伸序列中含有上游开放阅读框， 翻译的蛋白质编码序列4，5，34. 首次观察到芽殖酵母配子发生-所需的分化程序 产生性细胞-后来在酵母未折叠蛋白反应和人类细胞中12，36， 基于LUTI的调节允许细胞在抑制和编码转录本之间切换 整个分化。此外，LUTI依赖性干扰近端启动子 是可调的和可逆的，允许动态发育基因表达。而 已经描述了由LUTI转录诱导的染色质修饰， LUTI依赖性核小体定位变化所需的基因组学尚未被发现。这 一项提案试图利用酵母中的正向遗传学来揭示这些因素，并描述其特征。 在基因发育调控中的作用这些方法的初步工作表明， Swi/Snf染色质重塑复合物在基于LUTI的转录干扰中的作用 目的1中提出的实验将研究Swi/Snf复合物在LUTI中的作用。 基于酵母配子发生过程中的调控，首先通过测定模型LUTI基因的调控 NDC 80，然后在全球范围内。来自aim 2的结果将揭示LUTI转录是否 从酵母到人类，干扰机制是保守的。Aim 2还试图揭示 LUTI基因抑制在人胚胎干细胞分化中的作用 细胞到内胚层的命运总之，这些实验将阐明 基于LUTI的基因抑制和LUTI转录在发育中的功能意义。

PROJECT SUMMARY Cellular differentiation is driven by changes in gene expression. To transition from one type of cell to another, cells must simultaneously activate genes that promote differentiation while repressing genes that antagonize faithful progression. How gene repression is achieved amidst widespread transcriptional activation is not fully understood. A recently described mode of gene repression, which relies on integrated transcriptional and translational interference, exemplifies the coordination of transcriptional activation and gene repression. In this form of gene regulation, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, leading to repressive co-transcriptional chromatin modifications over the gene-proximal promoter4, 6, 34. In contrast to the efficiently translated, canonical transcript derived from the proximal promoter, the LUTI contains upstream open reading frames in its 5’ extended sequence which prevent translation of the protein coding sequence4, 5, 34. First observed in budding yeast gametogenesis - the differentiation program required to produce sex cells - and later during the yeast unfolded protein response and in human cells12, 36, LUTI-based regulation allows cells to toggle between a repressive and coding transcript throughout differentiation. Furthermore, LUTI-dependent interference over the proximal promoter is tunable and reversible, permitting dynamic developmental gene expression. While the chromatin modifications induced by LUTI transcription have been described, trans-acting factors required for LUTI-dependent nucleosome positioning changes have yet to be uncovered. This proposal seeks to uncover these factors using forward genetics in yeast and to characterize their role in developmental gene regulation. Preliminary work from these approaches implicates the Swi/Snf chromatin remodeling complex in LUTI-based transcriptional interference. Experiments proposed in aim 1 will investigate the role of the Swi/Snf complex in LUTI- based regulation during yeast gametogenesis, first by assaying regulation of the model LUTI gene NDC80 and then on a global scale. Results from aim 2 will reveal whether the LUTI transcriptional interference mechanism is conserved from yeast to humans. Aim 2 also seeks to uncover a functional role for LUTI-based gene repression during differentiation of human embryonic stem cells to the endoderm fate. Together, these experiments will illuminate the molecular requirements for LUTI-based gene repression and functional significance of LUTI transcription in development.