Gene regulatory networks during the transition from quiescence to proliferation

描述(申请人提供)：人类细胞从静止阶段向增殖阶段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。这种转变伴随着全球基因表达的变化，这种变化部分是由致癌转录因子与靶启动子结合并激活或抑制基因表达而介导的。一些转录因子的靶标本身可以是因子，它们可以在更深层次的调控下对靶标进行调控，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E2F4，占据了转录起始位点(TS)，使它们能够潜在地调节一系列非常广泛的转录靶点。最近还发现，一些这样的目标 致癌因子是microRNAs(MiRNAs)，是一类不同的基因表达调节因子。有趣的是，miRNAs可以调节转录因子。该项目的激励假设是，介导人类细胞从静止到增殖的全球基因表达程序的基因调控网络涉及转录因子、miRNAs以及它们之间的调控相互作用。这个项目的总体目标是在静止的原代细胞被刺激增殖时重建这样的全球转录调控网络，通过以下目标。首先，我们将确定在这一转变过程中活跃的即刻早期致癌转录因子的直接和功能转录靶点。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)来识别全基因组的靶标。我们将使用转录因子的siRNA敲除结合表达谱微阵列来识别受转录因子功能调控的基因。其次，我们将确定在从静止到增殖的转变过程中可能相关的miRNAs。我们将通过分析这一转变过程中miRNAs的表达来实现这一点，并使用增殖分析来确定哪些miRNAs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，我们将结合以上两个目标的信息来重建包含转录因子和miRNAs调控的转录调控网络 包括转铁蛋白基因在内的其他mRNAs。我们将确定解释TF与其实验定义的目标启动子结合的序列基序。我们将测试这个调控网络的各个方面，方法是通过使用针对TF的siRNAs以及miRNA双链和抗miRs来移除关键调控节点，并通过实验验证预测的子网络是否受到预期的影响。

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.