The Antisense Transcriptome and the Epigenetics of Cancer.

描述(由申请人提供)：最近涉及不同于其他非编码RNA的大的反义RNA的发现促使我们提出了一种新的内源性转录调控系统的存在。我们假设，反义转录通过将染色质修饰复合体招募到特定的遗传位点来调节转录沉默和激活。我们预计会有过多的反义转录产物发出转录沉默的信号，而有低水平的反义转录产物激活转录。在这一应用中，我们建议将这一假设应用于癌症中所见的差异、表观遗传决定的转录。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。我们还将在不同于单个癌基因表达的同基因细胞对上进行反义转录的全基因组图谱。然后，我们将通过双重免疫沉淀芯片分析和深度测序来确定参与这些细胞染色质重塑的遗传位点，并将这些数据与反义图谱相关联，指定显示显著反义转录与染色质修饰相耦合的基因。从这个列表中，我们将选择与癌症相关的基因，并通过靶向RT和3‘RACE来鉴定它们的反义转录本。特定反义转录物的转录调节活性将通过过度表达和使用RNAi构建物对反义RNA功能的干扰来确定。我们希望获得支持或驳斥我们核心假设的决定性数据，并独立于这一结果，产生与特定癌基因的作用有关的反义相关转录变化的全面信息，以及与癌症中显示表观遗传变化的基因有关的信息。非编码RNA介导的转录调控是长期的，与传统的转录激活蛋白和转录抑制因子的调控不同。一种新的细胞-内源性转录调控系统的应用，涉及到介导染色质重塑的反义RNA，挑战了目前的转录调控范式。对这样一个系统的验证将对我们对转录的理解产生深远的影响。它还将揭示癌症、慢性感染和涉及转录表观遗传变化的发育障碍的新治疗策略。 与公共卫生相关：大量基因产生反义转录本。越来越多的证据表明，这些非编码RNA可以通过诱导染色质的表观遗传变化来调节转录。目前的应用将在基因组范围内检查反义依赖的转录调控。

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. PUBLIC HEALTH RELEVANCE: Numerous genes generate antisense transcripts. There is emerging evidence that these non-coding RNAs can regulate transcription by inducing epigenetic changes of the chromatin. The present application will examine antisense-dependent transcriptional regulation on a genome wide scale.