A novel post-transcriptional regulatory mechanism mediated by Zhx2

Intellectual Merit It has become increasingly clear that transcriptional and post-transcriptional steps of gene expression are coupled and interdependent. Furthermore, this coupling is a point at which gene expression can be regulated. Much of the evidence for regulating mRNA processing through coupling with transcription is derived from artificial systems. While these models demonstrate the potential 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. The BALB/cJ mouse strain, which expresses low Zhx2 levels due to a natural hypomorphic mutation, accumulates ~20-fold more AFP mRNA in the adult liver than other mouse strains. Using transgenic mice, the AFP promoter was shown to be sufficient to confer Zhx2 regulation on a heterologous reporter gene. However, the transcription rate across the AFP gene is not altered in the presence of Zhx2, but rather, AFP mRNA accumulation is repressed 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 the 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 in the BALB/cJ mouse strain have been generated. Using these mice, this novel gene regulatory mechanism will be investigated through the following aims: (1) Determine whether Zhx2 is a direct or indirect regulator of AFP expression by using chromatin immunoprecipitation (ChIP) assays. Whether Zhx2 binds the AFP and other target gene promoters or associates along the whole gene will be determined and other Zhx2-regulated genes will be identified by combining ChIP with deep sequencing. (2) Examine the mechanism of AFP RNA splicing repression. The differences in the pol II complex on AFP and other Zhx2-regulated genes in the presence or absence of Zhx2, will be identified using ChIP. Whether splicing repression occurs co-transcriptionally will be determined by measuring splicing of nascent RNA. (3) Identify proteins that interact with Zhx2. FLAG-Zhx2-containing protein complexes will be isolated from adult mouse liver and interacting factors will be identified by mass spectrophotometry. The identity of interacting proteins will provide essential information about Zhx2 function. The long-term goal of the proposed work is to understand Zhx2-mediated regulation because it is a novel regulatory mechanism that appears to couple transcription to post-transcriptional events in the liver. Details of this mechanism should lead to a better understanding of gene regulation at this step of RNA biogenesis. Broader Impacts Graduate, undergraduate, and, potentially, high school students will be involved in the research; the majority of students associated with my lab have been female. The PI, post-doc and students will participate in sharing the results of this research with the broader scientific community through national meeting presentations and publications. The PI of this grant continues to support science in the broader community by participating as a science fair judge, visiting high school classrooms and, as a part-time Associate Vice President for Research at the University of Kentucky and member of the KY Academy of Sciences, supports research in institutions across the state of KY.