A Novel Post-transcriptional Regulatory Mechanism Mediated by Zhx2

智力优势：越来越清楚的是，基因表达的转录和转录后步骤是耦合的，相互依赖的，并可能共同调节。通过转录偶联调节mRNA加工的证据大多来自人工系统。虽然这些模型证明了通过与转录机制的相互作用调节基因表达的转录后步骤的能力，但很少有在体内发生这种情况的自然例子。最近，锌指和同源盒2 （Zhx2）基因被克隆出来，并被证明在一定程度上抑制了出生后肝脏中甲胎蛋白（AFP）和其他基因的表达。已发表的研究表明，AFP启动子足以使Zhx2调控异源报告基因。然而，初步结果表明，在存在或不存在Zhx2的情况下，AFP基因的转录率是相同的，即使AFP mRNA的积累受到抑制。此外，这种抑制发生是因为多个AFP内含子的剪接受到抑制。由于Zhx2以启动子依赖的方式发挥作用，但在转录后水平起作用，因此该系统提供了一个独特的机会来理解生物学相关的新机制，将转录与转录后基因调控结合起来。根据初步研究，我们假设Zhx2通过其靶基因的启动子降低新生RNA的剪接效率，从而降低全加工mRNA的水平。为了验证这一假设并研究机制细节，我们培育了表达flag标记的Zhx2转基因的小鼠，但内源性的Zhx2水平较低。利用这些小鼠，本项目将通过染色质免疫沉淀（ChIP）测定确定Zhx2是AFP表达的直接或间接调节因子，以及它是与靶启动子相关还是沿整个基因相关。结合ChIP和DNA测序将鉴定其他zhx2调控基因。我们将通过ChIP比较Zhx2存在或不存在时pol II复合物对AFP RNA剪接抑制的机制。剪接抑制是否发生共转录将通过测量新生RNA剪接来确定。最后，本项目将确定与Zhx2相互作用的蛋白质，这将提供关于Zhx2功能的基本信息。用质谱法鉴定成年小鼠肝脏中含有flag - zhx2蛋白复合物的组分。该项目的长期目标是了解zhx2介导的AFP调控，因为它是一种新的调控机制，似乎将转录与肝脏中的转录后事件偶联。这一机制的细节将有助于更好地理解RNA生物发生阶段的基因调控。更广泛的影响：研究生，本科生，以及潜在的高中生将参与研究，建立在实验室强大的学生培训历史的基础上。研究结果将通过国家会议报告和出版物与更广泛的科学界分享。从科学上讲，这项研究有可能揭示对基因调控机制知之甚少的新见解。

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.