5' extended protein-coding INO80E transcript regulates expression of two head-to-head overlapping genes: INO80E and HIRIP3

Abstract

The INO80E gene encodes the protein involved in the chromatin remodeling processes as a part of the multi-subunit INO80-chromatin remodeling complex. The INO80E gene is located on chromosome 16 and overlaps head-to-head with the HIRIP3 gene encoding protein, which binds H2B and H3 core histones and HIRA protein and regulates the chromatin and histone metabolism. Antisense transcription of head-to-head overlapping PC genes may have several consequences and none of them have been comprehensively investigated and explained. Here, we determined that INO80E-201, which overlaps the HIRIP3 gene transcripts, forms an R-loop at its 5 end. We also demonstrated that overlapping transcripts of INO80E and HIRIP3 form an RNA:RNA duplex that has the stabilizing effect on the involved mRNAs. Our results confirmed that this stabilization could be mediated by the ELAVL1 protein. We additionally introduced de novo methylation using the CRISPR/Cas system into the promoter sequence of INO80E gene. As a result of the introduced changes, reduced expression of HIRIP3 and INO80E gene transcripts was observed. It was determined that methylated cytosines were located in the binding sites for four transcription factors including RARG, which was further confirmed to be important in the transcription of both studied genes. Our results strongly suggest that the formation of an RNA:RNA duplex is necessary for stable simultaneous expression of both genes. Lack of this dsRNA structure results in a loss of a wider DNA opening and in consequence transcriptional interference. We also concluded that forming R-loops probably plays only a supplementary role and is not required for proper expression of HIRIP3 and INO80E gene transcripts.