Transcriptional interference through LUTI mRNA expression in development

发育过程中 LUTI mRNA 表达的转录干扰

• 批准号: 10520056
• 负责人: Kaitlin Morse
• 金额: $ 4.02万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10520056
• 关键词: Affect; Biological Assay; Biological Process; Cells; Chemicals; Chromatin; Chromatin Remodeling Factor; Code; Complement; Defect; Development; Developmental Gene; Disease; Distal; Endoderm; Enzymes; Eukaryota; Exhibits; Gametogenesis; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Germ Cells; Histones; Human; Human Cell Line; Human Development; Investigation; Lysine; MDM2 gene; Meiosis; Messenger RNA; Methylation; Methyltransferase; Modeling; Modification; Molecular; Mutation; Nucleic Acid Regulatory Sequences; Nucleosomes; Oncogenes; Open Reading Frames; Orthologous Gene; Play; Positioning Attribute; Protein Biosynthesis; Protein Isoforms; Proteins; Regulation; Repression; Role; SWI/SNF Family Complex; Saccharomyces cerevisiae; Saccharomycetales; TP53 gene; Time; Trans-Activators; Transcript; Transcription Coactivator; Transcription Initiation; Transcription Repressor; Transcriptional Activation; Transcriptional Regulation; Translating; Translational Repression; Translations; Western Blotting; Work; Yeasts; cell type; chromatin modification; chromatin remodeling; experimental study; forward genetics; gene regulatory network; gene repression; genome-wide analysis; histone methylation; histone methyltransferase; human embryonic stem cell; mRNA Expression; mutant; novel; prevent; programs; promoter; response; transcription factor; ubiquitin-protein ligase

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.

项目总结 细胞分化是由基因表达的变化驱动的。从一个人过渡到 不同类型的细胞之间，细胞必须同时激活促进分化的基因，同时 抑制了对抗忠实前进的基因。基因抑制是如何在 广泛的转录激活尚不完全清楚。最近描述的一种基因模式 依赖于转录和翻译综合干预的抑制，例证了 转录激活和基因抑制的协调。在这种形式的基因调控中， 一个长的未解码的转录异构体(Luti)是从基因末端的启动子转录而来的，导致 抑制共转录染色质修饰对基因近端启动子4，6，34的影响。在……里面 与来自近端启动子的高效翻译的规范转录本相比， LUTI在其5‘端延伸序列中包含上游开放阅读框，这阻止了 蛋白质编码序列4、5、34的翻译。 首次在发芽酵母配子发生中观察到-分化程序需要 产生性细胞--随后在酵母未折叠蛋白反应和人类细胞12、36、 基于LUTI的调控允许细胞在抑制转录和编码转录之间切换 整个分化过程中。此外，对近端启动子的Luti依赖干扰 是可调节和可逆的，允许动态发育基因表达。而当 已经描述了由Luti转录引起的染色质修饰，反式作用因子 LUTI依赖的核小体定位所需的改变尚未被发现。这 一项提案试图利用酵母中的正向遗传学来发现这些因素，并描述它们的特征 在发育基因调控中的作用。这些方法的初步工作表明， SWI/SNF染色质重塑复合体在基于Luti的转录干扰中的作用。 目标1中提出的实验将研究Swi/SNF复合体在LUT-1中的作用。 酵母配子发生过程中的基础调控，首先通过分析模型Luti基因的调控 NDC80，然后在全球范围内。AIM 2的结果将揭示Luti转录 干扰机制从酵母到人类都是保守的。Aim 2还试图揭示一个 基于LUTI的基因抑制在人胚胎干细胞分化中的作用 细胞对内胚层的命运。总之，这些实验将阐明分子需求 针对基于Luti的基因抑制和Luti转录在发育中的功能意义。