It is generally believed that genes are under the control of positive-acting factors and, therefore, silent genes are those that simply have yet to be activated. However, there is substantial evidence that this lack-of-activation hypothesis is too simplistic to explain gene control. In this project, the phenomenon of gene silencing will be analyzed using whole-genome microarray analysis. Genome-wide gene expression in liver-derived cells will be compared with that observed in cell types that have silenced liver gene expression (cell hybrids and hepatoma variant cells). Validation of data will be carried out by analysis of gene expression of potentially interesting genes using cell-based read-out assays to screen for restoration or repression of liver gene function. The goal is to identify genes involved in gene silencing in cell hybrids and hepatoma variants. Specifically, genes that show at least 5-fold differences in expression and encode proteins that contain signature motifs associated with transcriptional regulatory proteins will be analyzed. These analyses will include placement of genes into classes depending on DNA sequence similarities to genes of known function. Finally, candidate regulatory genes will be tested for either activation or repression of target genes.
Broader Impact Undergraduate college students will play an essential role in each aspect of this research. Microarray data sets obtained will be used in two aspects of undergraduate teaching and training. First, this data will be used as a tool in an existing molecular biology lab course (required for Biology majors) to understand the scope of tissue-specific gene expression and ask open-ended, broad questions about the link between gene expression and cellular identity. Using a team approach, students will also be able to identify and classify candidate genes based on homologies to genes of known function through BLAST searches, thus gaining valuable experience in assessing gene function. Secondly, several undergraduate students will carry out research projects in the PI's lab to screen candidate genes and even test candidate genes for functional rescue of phenotypes. Data obtained will be compiled into poster presentations at regional meetings as well as in publications. This will provide valuable experience in modern molecular biology and prepare students for graduate or professional schools.
Regulation of tissue specific gene expression in eukaryotic organisms involves complex regulatory networks that work in combination to produce distinct cell types. Cultured cell culture models have proven to be robust tools by which to understand these regulatory networks in normal development and maintenance of tissue specificity. We used two cell model systems that have contributed to our understanding of liver funtion regulation: hepatoma x fibroblast cell hybrids (which show complete silencing of hepatic gene expression) and hepatoma variant cells (which have silenced large numbers of liver genes). In both of these cellular systems, profound effects are observed silencing of hundreds of liver genes. This proposal attempted to answer fundamental questions regarding how genetic networks establish and maintain distinct tissue identity in mammals. The goal of this project was to analyze the regulatory circuitry controlling liver phenotype using liver-derived cells. Cell culture model systems combined with whole-genome microarray analysis and bioinformatics techniques were utilized. Using this strategy we successfully identified novel pathways that interact with well-known networks to dictate the liver phenotype and mapped the interplay of these genetic pathways (both activated and repressed) in the rat liver. Undergraduate college students played an essential role in each aspect of this project. Microarray data sets obtained were be used in two aspects of undergraduate teaching and training. First, data was used as a tool in an existing molecular biology lab course (required for Biology majors) to understand the scope of tissue-specific gene expression and ask open-ended, broad questions about the link between gene expression and cellular identity. Students were able to, using a team approach, identify and classify candidate genes based on homologies to genes of known function through BLAST searches, thus gaining valuable experience in assessing gene function. Secondly, several undergraduate students carried out research projects in the lab to screen candidate genes and even test candidate genes for functional rescue of liver Data obtained was compiled into several poster presentations at regional meetings as well as in two publications with several undergraduate co-authors. These experiences also provided for experience in modern molecular biology and prepared students for graduate school or professional schools. This enhanced the department’s goal of integrating course experiences into current research efforts.
