The Antisense Transcriptome and the Epigenetics of Cancer.

描述（由申请人提供）：最近的发现涉及与其他非编码RNA不同的大的反义RNA，这促使我们提出存在一种新的内源性转录调节系统。我们假设反义转录介导的转录沉默和激活的染色质修饰复合物的招聘特定的遗传位点。我们设想过量的反义转录物来发出转录沉默的信号，而低水平的反义转录物来激活转录。在本申请中，我们建议将此假设应用于癌症中观察到的差异的表观遗传学确定的转录。我们将描述在基因表达中表现出表观遗传变化的癌细胞的反义转录组。我们也将进行全基因组分析反义转录对细胞的差异，由一个单一的癌基因的表达。然后，我们将通过双免疫沉淀芯片测定和深度测序来鉴定这些细胞中参与染色质重塑的遗传位点，并将这些数据与反义谱相关联，指定显示与染色质修饰偶联的显著反义转录的基因。从该列表中，我们将选择癌症相关基因并通过靶向RT和3'RACE表征其反义转录物。特定反义转录物的转录调节活性将通过过表达和通过使用RNAi构建体干扰反义RNA的功能来确定。我们期望获得决定性的数据，支持或反驳我们的核心假设，并独立于这一结果，以产生全面的信息反义相关的转录变化，与特定的癌基因的行动和基因，显示表观遗传改变转录在癌症中。非编码RNA介导的转录调控是长期的，与转录激活蛋白和转录抑制因子的常规调控相反。一种新的细胞内源性转录调控系统的应用，涉及反义RNA介导染色质重塑的挑战，目前的转录调控范式。这样一个系统的验证将对我们对转录的理解产生深刻的影响。它还将揭示涉及转录表观遗传变化的癌症、慢性感染和发育障碍的新治疗策略。

DESCRIPTION (provided by applicant): Recent discoveries involving large, antisense RNAs that are distinct from other non-coding RNAs have prompted us to propose the existence of a novel, endogenous system of transcriptional regulation. We hypothesize that antisense transcripts mediate transcriptional silencing and activation by the recruitment of chromatin-modifying complexes to specific genetic loci. We envisage an excess of antisense transcripts to signal transcriptional silencing and low levels of antisense transcripts to activate transcription. In this application, we propose to investigate this hypothesis as applied to differential, epigenetically determined transcription seen in cancer. We will characterize the antisense transcriptome of cancer cells that show epigenetic changes in gene expression. We will also carry out genome-wide profiling of antisense transcripts on isogenic pairs of cells that differ by the expression of a single oncogene. We will then identify genetic loci involved in chromatin remodeling in these cells by double immunoprecipitation ChiP assays and deep sequencing and correlate these data with the antisense profiles, specifying genes that show significant antisense transcription coupled to chromatin modification. From that list, we will select cancer-relevant genes and characterize their antisense transcripts by targeted RT and 3' RACE. The transcriptional regulatory activity of specific antisense transcripts will be determined by overexpression and by interference with the function of antisense RNA using RNAi constructs. We expect to obtain decisive data that support or refute our core hypothesis and, independent of this outcome, to generate comprehensive information on antisense-associated transcriptional changes that are tied to the action of specific oncogenes and to genes that show epigenetically altered transcription in cancer. The transcriptional regulation mediated by non-coding RNAs is long-term, in contrast to the conventional regulation by transcriptional activator proteins and transcriptional repressors. The application of a new cell-endogenous system of transcriptional controls involving antisense RNAs that mediate chromatin remodeling challenges the current paradigm of transcriptional regulation. The validation of such a system would have a deep impact on our understanding of transcription. It would also reveal new therapeutic strategies for cancer, chronic infections and developmental disorders that involve epigenetic changes of transcription.