Gene regulatory networks during the transition from quiescence to proliferation

描述（由申请人提供）：人类细胞从静止期到增殖的转变是正常人类生物学的标志，但也是癌症等疾病的基础。这种转变伴随着全局基因表达的变化，其部分由致癌转录因子（TF）与靶启动子结合并激活或抑制基因表达介导。转录因子的一些靶点本身也可以是转录因子，它们可以在更深层次上调节靶点，形成转录调控网络。我们最近发现，一些致癌的转录因子，如c-Myc和E2 F4占据转录起始位点（TSS），使他们能够潜在地调节一组非常广泛的转录靶点。最近还发现，这类药物的一些靶点 致癌TF是microRNA（miRNAs），一种不同类型的基因表达调节因子。有趣的是，miRNAs可以调节TF。该项目的动机假设是，基因调控网络介导的全球基因表达程序的人类细胞从静止到增殖的过渡涉及TF，miRNA和它们之间的相互作用。该项目的总体目标是通过以下目标，在刺激静止的原代细胞增殖时重建这种全球转录调控网络。首先，我们将确定直接的和功能性的转录靶点的即时早期，致癌的TF是活跃在这个过渡。我们将使用染色质免疫沉淀结合微阵列（ChIP-chip）或高通量测序（ChIP-seq）来识别全基因组靶点。我们将使用siRNA敲除TF结合表达谱微阵列来鉴定功能上受TF调控的基因。其次，我们将鉴定可能在静止期向增殖期过渡期间相关的miRNA。我们将通过分析这一转变过程中miRNA的表达，并使用增殖测定来确定哪些miRNA在功能上影响这一过程。我们还将确定哪些miRNAs受关键的即刻早期转录因子调控，并确定这些miRNAs的靶基因。第三，我们将联合收割机从以上两个目的中获得的信息结合起来，构建包含转录因子和miRNA调控的转录调控网络 包括TF基因在内的其他mRNA。我们将确定序列基序，解释TF的实验定义的目标启动子的结合。我们将通过使用针对TF的siRNA以及miRNA双链体和抗miR的组合来去除关键调控节点，并通过实验验证预测的子网络是否如预期的那样受到影响，来测试该调控网络的各个方面。

DESCRIPTION (provided by applicant): 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.