Intragenic Locus in Human PIWIL2 Gene Shares Promoter and Enhancer Functions

Abstract

Recently, more evidence supporting common nature of promoters and enhancers has been accumulated. In this work, we present data on chromatin modifications and non-polyadenylated transcription characteristic for enhancers as well as results of in vitro luciferase reporter assays suggesting that PIWIL2 alternative promoter in exon 7 also functions as an enhancer for gene PHYHIP located 60Kb upstream. This finding of an intragenic enhancer serving as a promoter for a shorter protein isoform implies broader impact on understanding enhancer-promoter networks in regulation of gene expression.

Fig 1. Chromatin modifications around PIWIL2 canonical and alternative promoters.

Relative level of H3K4me1 (Histone 3 lysine 4 monomethylated, enhancer mark), H3K4me3 (Histone 3 lysine 4 trimethylated, active promoter mark), H3K27ac (Histone 3 lysine 27 acetylated, active regulatory element mark) and H3K27me3 (Histone 3 lysine 27 trimethylated, facultative heterochromatin mark) histone modifications assessed by ChIP-PCR with two sets of primer pairs around PIWIL2 canonical promoter in exon 1 and alternative promoters in exons 5 and 7. Results are are shown for four cell lines: TERA1 and NT2D1 –embryonal carcinoma, TCam2 –seminoma, and A549 –lung carcinoma. The negative controls are PIWIL2 intron 8 and an intergenic locus on chromosome 1, the positive control is GAPDH promoter. P-value summary of Mann-Whitney non-paired U test is presented for some peaks (ns–non-significant, *—p-value<0.05, **—p-value<0.01).

Fig 2. Non-polyadenylated transcription across exon-exon junctions of PIWIL2 gene.

qRT-PCR was used to assess the level of total RNA and its polyA+ fraction and the ratio of total RNA to polyA+ fraction was calculated. Seminoma and nonseminoma testicular cancer samples as well as adjacent normal testis tissues were assayed. P-value summary of Mann-Whitney non-paired U test is presented for 4–5 and 8–9 exon-exon junction peaks (ns–non-significant, ***—p-value<0.001).

Fig 3. Structure of luciferase reporter vectors used in the assays.

pGL4.10 plasmid was used to construct vectors with luc gene expression driven by various promoters (upper part in brackets) and a candidate enhancer in either orientation (lower part). Because the candidate enhancers also possess promoter activity, to discern between those, the vectors were linearized by cutting at the sites shown with red arrows. Note that after such linearization the candidate enhancer will be located either upstream (reverse orientation) or downstream (forward orientation) of the luc gene and its promoter.

Fig 4. PIWIL2 alternative promoter in exon 7 acts as an enhancer in luciferase reporter constructs.

Relative promoter activity of SV40 and CMV promoters was assessed in luciferase reporter vectors from Fig 3 in two cell lines: TERA1 and NT2D1 (embryonal carcinoma, panel A). PPP3CC and PHYHIP (panel B) promoters were assessed in luciferase reporter vectors from Fig 3 in four cell lines: TERA1 and NT2D1 –embryonal carcinoma, Tcam2 –seminoma, and A549 –lung carcinoma. P-value summary of Mann-Whitney non-paired U test is presented for peaks showing more than two-fold increase (above horizontal dashed lines) for both forward and reverse orientation of the candidate enhancer compared to “no enhancer” controls (*—p-value<0.05, **—p-value<0.01).