Functional characterization of long noncoding RNAs involved in the activation of genes regulated by androgens

Abstract

In the past decade a number of studies have shown that a significant portion of the human transcribed messages is comprised of noncoding RNAs (ncRNAs). A subclass of these RNAs, called long ncRNAs (lncRNAs) maps to intronic and intergenic regions,and has been demonstrated to have a regulatory role in gene expression both in normal and pathological conditions. Overall, lncRNA regulatory function involves interactions with protein complexes, especially chromatin remodeling complex. In this project, we intend to functionally characterize the possible involvement of lncRNAs in activation of gene expression by androgen hormone stimulus through epigenetic mechanisms. It was reported in the literature that demethylases JMJD2C and LSD1 interact together with androgen receptor, forming a LSD1/JMJD2C/AR complex that activates transcription of androgen-dependent genes by demethylation of histone 3 lysine 9. Initially we plan to characterize an intergenic lncRNA (lincRNA), upstream to the PSA gene named lincPSA. This lincRNA was identified by our group as expressed in LNCaP and is regulated by androgen, however it still needs to be characterized. We will perform a detailed study, using chromatin immunoprecipitation, of the possible involvement of lincPSA in the recruitment of JMJD2C/LSD1/AR complex to its target (PSA promoter and enhancer region), which would promote activation of PSA transcription. In parallel PAR-CLIP (photoactivatable-Ribonucleoside-enhanced Crosslinking and immunoprecipitation) will be used to map the interactions of lincPSA with its protein targets to discover binding motif sequences. In addition, we intend to identify possible new lncRNAs involved in this pathway of epigenetic regulation by androgen, using RNA immunoprecipitation and sequencing. Together the proposed set of experiments intends to provide new information about the molecular mechanisms of gene expression regulation involving lncRNAs, androgen and chromatin remodeling complex.