The transition of human cells from the quiescent stage to proliferation is a hallmark of normal human biology, but also underlies diseases like cancer. This transition is accompanied by global gene expression changes that are mediated in part by oncogenic transcription factors (TFs) binding to target promoters and activating or repressing gene expression. Some targets of TFs can themselves be TFs, that can go on to regulate targets at deeper levels of regulation, forming transcriptional regulatory networks. We have recently found that some oncogenic TFs like c-Myc and E2F4 occupy transcriptional start sites (TSS), enabling them to potentially regulate a very broad set of transcriptional targets. Recently it has also been found that some targets of such oncogenic TFs are microRNAs (miRNAs), a different class of regulators of gene expression. Interestingly, miRNAs can regulate TFs. The motivating hypothesis for this project is that gene regulatory networks mediating the global gene expression programs underlying the transition of human cells from quiescence to proliferation involve TFs, miRNAs and regulatory interactions between them. The overall objective of this project is to reconstruct such global transcriptional regulatory networks when quiescent primary cells are stimulated to proliferate, through the following aims. First, we will identify the direct and functional transcriptional targets of immediate-early, oncogenic TFs that are active during this transition. We will use chromatin immunoprecipitation combined with either microarrays (ChIP-chip) or high-throughput sequencing (ChIP-seq) to identify targets genome wide. We will use siRNA knockdown of TFs in combination with expression profiling microarrays to identify genes that are functionally regulated by the TFs. Second, we will identify miRNAs that are likely to be relevant during the quiescence to proliferation transition. We will do this by profiling the expression of miRNAs during this transition, and determining which miRNAs functionally affect this process using proliferation assays. We will also determine which miRNAs are regulated by key immediate early TFs, and identify the target genes for those miRNAs. Third, we will combine the information from the above two aims to reconstruct transcriptional regulatory networks which incorporates the regulation by TFs and miRNAs of other mRNAs including TF genes. We will identify sequence motifs that explain the binding of TF to their experimentally defined target promoters. We will test aspects of this regulatory network by removing key regulatory nodes through the use of siRNAs against TFs, and miRNA duplexes and anti-miRs in combination, and experimentally verifying whether predicted sub-networks are affected as expected.
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