The human genome, like other mammalian genomes, encodes numerous natural antisense transcripts (NATs) that have been classified into head-to-head, tail-to-tail, or fully overlapped categories in reference to their sense transcripts. Evidence for NAT-mediated epigenetic silencing of sense transcription remains scanty. The DHRS4 gene encodes a metabolic enzyme and forms a gene cluster with its two immediately downstream homologous genes, DHRS4L2 and DHRS4L1 , generated by gene duplication. We identified a head-to-head NAT of DHRS4 , designated AS1DHRS4, which markedly regulates the expression of these three genes in the DHRS4 gene cluster. By pairing with ongoing sense transcripts, AS1DHRS4 not only mediates deacetylation of histone H3 and demethylation of H3K4 in cis for the DHRS4 gene, but also interacts physically in trans with the epigenetic modifiers H3K9- and H3K27-specific histone methyltransferases G9a and EZH2, targeting the promoters of the downstream DHRS4L2 and DHRS4L1 genes to induce local repressive H3K9me2 and H3K27me3 histone modifications. Furthermore, AS1DHRS4 induces DNA methylation in the promoter regions of DHRS4L2 by recruiting DNA methyltransferases. This study demonstrates that AS1DHRS4, as a long noncoding RNA, simultaneously controls the chromatin state of each gene within the DHRS4 gene cluster in a discriminative manner. This finding provides an example of transcriptional control over the multiple and highly homologous genes in a tight gene cluster, and may help explain the role of antisense RNAs in the regulation of duplicated genes as the result of genomic evolution.

The human DHRS4 gene cluster consists of DHRS4 and two immediately downstream homologous genes, DHRS4L2 and DHRS4L1, generated by evolutionarily gene-duplication events. We previously demonstrated that a head-to-head natural antisense transcript (NAT) of DHRS4, denoted DHRS4-AS1, regulates all three genes of the DHRS4 gene cluster. However, it is puzzling that DHRS4L2 and DHRS4L1 did not evolve their own specific NATs to regulate themselves, as it seems both have retained sequences highly homologous to DHRS4-AS1. In a search of the DHRS4-AS1 region for nearby enhancers, we identified an enhancer located 13.8 kb downstream of the DHRS4-AS1 transcriptional start site. We further showed, by using a chromosome conformation capture (3C) assay, that this enhancer is capable of physically interacting with the DHRS4-AS1 promoter through chromosomal looping. The enhancer produced an eRNA, termed AS1eRNA, that enhanced DHRS4-AS1 transcription by mediating the spatial interactions of the enhancer and DHRS4-AS1 promoter in cooperation with RNA polymerase II and p300/CBP. Moreover, the distributions of activating acetyl-H3 and H3K4me3 modifications were found to be greater at the DHRS4-AS1 promoter than at the homologous duplicated regions. We propose that AS1eRNA-driven DNA looping and activating histone modifications promote the expression of DHRS4-AS1 to economically control the DHRS4 gene cluster.

Brain injury after intracranial hemorrhage (ICH) results in significant morbidity and mortality. Blood brain barrier (BBB) disruption is a hallmark of ICH-induced brain injury; however, data mirroring BBB disruption in human ICH are scarce. The aim of this study was to assess the significance of circulating biomarkers in evaluating BBB disruption after ICH. Twenty-two patients with ICH were recruited in this study. Concentrations of the tight junction proteins (TJs) Claudin-5 (CLDN5), Occludin (OCLN), and zonula occludens 1 (ZO-1) and vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were measured by using enzyme-linked immunosorbent assay in serum and cerebrospinal fluid (CSF) samples obtained from patients with ICH. The white blood cell (WBC) count in blood and CSF, albumin (ALB) levels in the CSF (ALBCSF), and the BBB ratio were significantly higher in the ICH than in controls (p < 0.05). Significantly higher levels of CLDN5, OCLN, ZO-1, MMP-9, and VEGF in CSF were observed in the ICH group; these biomarkers were also positively associated with BBB ratio (p < 0.05). Our data revealed that circulating TJs could be considered the potential biomarkers reflecting the integrity of the BBB in ICH.

Background High expression of secreted matricellular protein cysteine-rich 61 ( CYR61 ) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation has been shown to correlate with expression of key genes involved in cancer progression. However, such mechanisms in CYR61 transcription regulation remain unexplored. Methods Expression of CYR61 was determined by immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and western blotting (WB) in CRC patients paraffin specimens and colon cell lines. ChIP-seq data of enhancer-characteristic histone modifications, in CRC tissues from the Gene Expression Omnibus (GEO) database, were reanalyzed to search for putative enhancers of CYR61 . Dual-luciferase reporter assay was used to detected enhancer activity. Physical interactions between putative enhancers and CYR61 promoter were detected by chromosome conformation capture (3C) assay. Histone modification and transcription factors (TFs) enrichment were detected by ChIP-qPCR. Additionally, biological function of enhancers was investigated by transwell migration assays. Results CRC tissues and cell lines expressed higher level of CYR61 than normal colon mucosa. Three putative enhancers located downstream of CYR61 were found in CRC tissues by ChIP-seq data reanalysis. Consistent with the ChIP-seq analysis results in the GEO database, the normal colon mucosal epithelial cell line NCM460 possessed no active CYR61 enhancers, whereas colon cancer cells exhibited different patterns of active CYR61 enhancers. HCT116 cells had an active Enhancer3, whereas RKO cells had both Enhancer1 and Enhancer3 active. Pioneer factor FOXA1 promoted CYR61 expression by recruiting CBP histone acetyltransferase binding and increasing promoter-enhancer looping frequencies and enhancer activity. CBP knockdown attenuated H3K27ac enrichment, promoter-enhancer looping frequencies, and enhancer activity. Small molecule compound 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment, which stimulated CYR61 expression, and verteporfin (VP) treatment, which inhibited CYR61 expression, confirmed that the enhancers regulated CYR61 expression. Knockdown and ectopic expression of CYR61 rescued cell migration changes induced by over-expressing and knockdown of FOXA1, respectively. Conclusions CYR61 enhancer activation, mediated by FOXA1 and CBP, occurs during CRC progression to up-regulate CYR61 expression and promote cell migration in CRC, suggesting inhibition of recruitment of FOXA1 and/or CBP to CYR61 enhancers may have therapeutic imp...

HMGB3 belongs to the high-mobility group box subfamily and has been found to be overexpressed in gastric cancer. However, the expression and the role of HMGB3 in human hepatocellular carcinoma remain unknown. Here, we report that HMGB3, which is suppressed by miR-200b, contributes to cell proliferation and migration in human hepatocellular carcinoma. After analyzing The Cancer Genome Atlas data of 371 patients with hepatocellular carcinoma, we identified HMGB3 to be upregulated in human hepatocellular carcinoma tissue. Knockdown of HMGB3 in the hepatocellular carcinoma cell line suppressed cell proliferation and migration. TargetScan analysis showed miR-200b to be a possible regulator for HMGB3. Subsequent luciferase assays indicated that HMGB3 was a direct target of miR-200b. In addition, upregulation of miR-200b inhibited hepatocellular carcinoma cell growth and migration. HMGB3 overexpression or miR-200b downregulation was associated with poor prognosis. Our findings suggest HMGB3 may serve as an important oncoprotein whose expression is negatively regulated by miR-200b in hepatocellular carcinoma.