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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Special Emphasis Panel (ZCA1-SRLB-R (M1))
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Mietz, Judy
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Scripps Research Institute
La Jolla
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