A Novel Post-transcriptional Regulatory Mechanism Mediated by Zhx2

Zhx2介导的新型转录后调控机制

• 批准号: 1158234
• 负责人: Martha Peterson
• 金额: $ 60.49万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158234&HistoricalAwards=false
• 关键词: Novel; Post; transcriptional; Regulatory; Mechanism

Intellectual Merit: It has become increasingly clear that transcriptional and post-transcriptional steps of gene expression are coupled, interdependent and potentially co-regulated. Much of the evidence for regulating mRNA processing through coupling with transcription is derived from artificial systems. While these models demonstrate the capacity for regulating post-transcriptional steps of gene expression through interactions with transcriptional machinery, there are few natural examples of where this occurs in vivo. Recently, the Zinc fingers and homeoboxes 2 (Zhx2) gene was cloned and shown to be responsible, in part, for repressing the expression of alpha-fetoprotein (AFP) and other genes in the liver after birth. Published studies have shown that the AFP promoter is sufficient to confer Zhx2 regulation on a heterologous reporter gene. However, preliminary results have shown that the transcription rate across the AFP gene is the same in the presence or absence of Zhx2, even though AFP mRNA accumulation is repressed. Moreover, this repression occurs because splicing of multiple AFP introns is inhibited. Because Zhx2 functions in a promoter-dependent manner but acts at a post-transcriptional level, this system provides a unique opportunity to understand a biologically relevant and novel mechanism that couples transcriptional to post-transcriptional gene regulation. Based on preliminary studies, it is hypothesized that Zhx2 acts through the promoter of its target genes to decrease the splicing efficiency of the nascent RNA and thus reduce fully processed mRNA levels. To test this hypothesis and investigate mechanistic details, mice that express a FLAG-tagged Zhx2 transgene, but low levels of endogenous Zhx2, have been generated. Using these mice, this project will determine whether Zhx2 is a direct or indirect regulator of AFP expression and whether it associates with target promoters or along the whole gene by using chromatin immunoprecipitation (ChIP) assays. Combining ChIP with DNA sequencing will identify other Zhx2-regulated genes. The mechanism of AFP RNA splicing repression will be investigated by comparing the pol II complex in the presence or absence of Zhx2 by ChIP. Whether splicing repression occurs co-transcriptionally will be determined by measuring splicing of nascent RNA. Finally, this project will identify proteins that interact with Zhx2, which will provide essential information about Zhx2 function. The components of FLAG-Zhx2-containing protein complexes from adult mouse liver will be identified by mass spectrophotometry. The long-term goal of the project is to understand Zhx2-mediated regulation of AFP because it is a novel regulatory mechanism that appears to couple transcription to post-transcriptional events in the liver. Details of this mechanism will lead to a better understanding of gene regulation at the step of RNA biogenesis.Broader impacts: Graduate, undergraduate, and, potentially, high school students will be involved in the research, building on the laboratory's strong history of student training. Results of the research will be shared with the broader scientific community through national meeting presentations and publications. Scientifically, the research has the potential to uncover novel insights into a poorly understood mechanism of gene regulation.

智力优点：越来越清楚的是，基因表达的转录和转录后步骤是耦合的，相互依存的，并有潜在的共同调节。通过与转录耦合来调节mRNA处理的许多证据来自人工系统。尽管这些模型证明了通过与转录机械相互作用来调节基因表达的转录后步骤的能力，但在体内发生这种情况的自然示例很少。最近，克隆了锌的手指和同源蛋白毒素2（ZHX2）基因，部分原因是抑制出生后肝脏中α-五蛋白（AFP）和其他基因的表达。 已发表的研究表明，AFP启动子足以在异源报告基因上授予ZHX2调节。 但是，初步结果表明，即使AFP mRNA积累受到抑制，在存在或不存在ZHX2的情况下，AFP基因的转录速率是相同的。 此外，由于抑制了多个AFP内含子的剪接，因此发生这种抑制作用。 由于ZHX2以启动子的方式起作用，但在转录后层面起作用，因此该系统提供了一个独特的机会，可以理解一种与生物学相关和新颖的机制，该机制将转录与转录后基因调节相结合。 基于初步研究，假设ZHX2通过其靶基因的启动子起作用，以降低新生RNA的剪接效率，从而降低完全处理的mRNA水平。为了检验这一假设并研究机理细节，已经产生了表达标记为标记的ZHX2转基因但内源性ZHX2较低水平的小鼠。使用这些小鼠，该项目将通过使用染色质免疫沉淀（CHIP）测定法确定ZHX2是AFP表达的直接或间接调节剂，以及它是否与目标启动子或沿整个基因相关联。将芯片与DNA测序结合使用将鉴定其他ZHX2调节的基因。 AFP RNA剪接抑制的机理将通过在芯片存在或不存在ZHX2的情况下比较POL II复合物。是否会通过测量新生RNA的剪接来确定剪接抑制是否共转录。最后，该项目将识别与ZHX2相互作用的蛋白质，该蛋白将提供有关ZHX2功能的基本信息。 来自成年小鼠肝脏的含有FLAG-ZHX2的蛋白质复合物的成分将通过质量分光光度法鉴定。 该项目的长期目标是了解AFP的ZHX2介导的调节，因为它是一种新型的调节机制，似乎将转录与肝脏的转录后事件进行了息息。 这种机制的详细信息将在RNA生物学的步骤中更好地理解基因调节。Boader的影响：毕业生，本科生，并可能会参与研究，这是基于实验室强大的学生培训历史的基础。这项研究的结果将通过全国会议演讲和出版物与更广泛的科学界分享。 从科学上讲，这项研究有可能发现对较知之不书的基因调节机制的新见解。