The Association Between H3K4me3 and Antisense Transcription

Histone H3 lysine 4 trimethylation （H3K4me3） is well known to occur in the promoter region of genes for transcription activation. How- ever, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3＇ end of actively transcribed （sense） genes, named as 3＇-H3K4me3. 3＇-H3K4me3 is associated with ~15% of pro- tein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II （RNAPII） binding sites and Y-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3＇-H3K4me3 is associated with the ini- tiation of antisense transcription. Furthermore, 3＇-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3＇-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3＇ end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3＇-H3K4me3 modification. More importantly, we observed the 3＇-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3＇-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in ver- tebrate. Therefore, we speculate that these 3＇-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3＇-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.

Antisense transcription regulates the expression of sense gene via alternative polyadenylation

Natural antisense transcripts （NAT） and alternative polyadenylation （APA） of messenger RNA （mRNA） are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense （S-AS） gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA （pA） site in sense gene RNASEH2C, which generated longer 3＇ untranslated region （3＇UTR） and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene＇s protein level in RNASEH2C.KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.

HOX transcript antisense intergenic RNA represses E-cadherin expression by binding to EZH2 in gastric cancer

AIM To clarify the mechanisms of HOX transcript antisense intergenic RNA(HOTAIR) in gastric cancer(GC) migration and invasion.METHODS Quantitative real-time polymerase chain reaction(qp CR) was used to detect the expression level of HOTAIR in GC tissues. The correlation of its expression with clinicopathological features was analyzed. Area under receiver operating characteristic curve(AUCROC) was constructed to evaluate the diagnostic value of HOTAIR. Wound-healing assay and Transwell assay were performed to detect the biological effects of HOTAIR in GC cells. qp CR,western blot and immunohistochemistry were used to evaluate the m RNA and protein expression of E-cadherin. RNAbinding protein immunoprecipitation was used for the analysis of EZH2 interactions with HOTAIR. Chromatin immunoprecipitation assay was performed to investigate direct interactions between EZH2 and E-cadherin.RESULTS The expression of HOTAIR was up-regulated in GC tumorous tissues compared with the para-tumorous tissues(p < 0.001). Its over-expression was correlated with tumor-node-metastasis(TNM) stage(p = 0.024),tumor invasion(p = 0.018),lymph node metastasis(p = 0.023),and poor prognosis(p < 0.001). Multivariate Cox regression analysis confirmed expression of HOTAIR as an independent predictor of overall survival(p = 0.033),together with TNM stage(p = 0.002) and lymph node metastasis(p = 0.002). The AUCROC was up to 0.709(95%CI: 0.623-0.785,p < 0.001). Knockdown of HOTAIR by si RNA in GC cells suppressed the migration and invasion of GC cells. Significantly negative correlation between HOTAIR and E-cadherin was found in GC tissues and cell lines,and HOTAIR contributed to the regulation of E-cadherin through binding to EZH2 with the E-cadherin promoter. CONCLUSION HOTAIR may play a pivotal role in tumor cell migration and invasion. It can be used as a potential diagnostic and prognostic biomarker for GC.

A Novel ATM Antisense Transcript ATM-AS Positively Regulates ATM Expression in Normal and Breast Cancer Cells

Objective:The ataxia telangiectasia mutated(ATM)gene is a master regulator in cellular DNA damage response.The dysregulation of ATM expression is frequent in breast cancer,and is known to be involved in the carcinogenesis and prognosis of cancer.However,the underlying mechanism remains unclear.The bioinformatic analysis predicted a potential antisense transcript ATM-antisense(AS)from the opposite strand of the ATM gene.The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.Methods:Single strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus.qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples.Luciferase reporter gene assays,biological mass spectrometry,ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression.Immunofluorescence and host-cell reactivation(HCR)assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair.Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.Results:The ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter.The reduced ATM-AS level led to the abnormal downregulation of ATM expression,and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro.The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples,and the patient prognosis.Conclusion:The present study demonstrated that ATM-AS,an antisense transcript located within the ATM gene body,is an essential positive regulator of ATM expression,and functions by mediating the binding of KAT5 to the ATM promoter.These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in breast cancer,and enrich our understanding of how an antisense transcript regulates its host gene.

Abrupt decrease of c-myc expression by antisense transcripts induces terminal differentiation and apoptosis in human promyelocytic leukemia HL-60 cells

This study was designed using c-myc antisense transcripts to evaluate how alteration of c-myc expression in human myeloid leukemic HL-60 cells could influence the myelomonocytic differentiation and induction of apoptosis. The recombinant plasmid pDACx expressing antisense transcripts to c-myc fragment containing a part of intron 1 and 137 nt exon 2 was constructed. pDACx was transfected into HL-60 cell line by lipofectin reagent.Cytochemical stainings including NBT reduction, peroxidase and α -NAE as well as detection of CD13 and CD33 antigens by flow cytometric analysis indicated occurrence of myelomonocytic differentiation in cells expressing antisense transcripts to c-myc. DNA degradation measured by DNA gel electrophoresis and typical morphological changes observed under electron microscope proved the switchon of apoptosis in terminally differentiating HL-60 cells.

Association between homeobox protein transcript antisense intergenic ribonucleic acid genetic polymorphisms and cholangiocarcinoma

BACKGROUND Cholangiocarcinoma(CCA)represents a rare but highly aggressive malignancy that is often challenging to diagnose,especially in early stages.The role of existing tumor biomarkers for CCA diagnosis,remains controversial due to their low sensitivity and specificity.Increasing evidence has implicated long non-coding ribonucleic acid polymorphisms with cancer susceptibility in a variety of tumor types.The association between long non-coding ribonucleic acid homeobox protein transcript antisense intergenic ribonucleic acid(HOTAIR)polymorphisms and CCA risk has not been reported yet.AIM To investigate the influence of HOTAIR variants on the risk of CCA development.METHODS We conducted a case-control study in which three HOTAIR single nucleotide polymorphisms(rs920778,rs4759314 and rs7958904)were genotyped in a Greek cohort.Our study population included 122 CCA patients(80 males and 42 females)and 165 healthy controls.The polymorphisms under investigation were examined in peripheral blood samples.RESULTS HOTAIR rs4759314 AG and GG genotypes were associated with a significantly increased CCA risk[P=0.004,odds ratio:3.13;95%confidence interval:1.65-5.91 and P=0.005,odds ratio:12.31;95%confidence interval:1.48-101.87,respectively].However,no significant associations of HOTAIR rs920778,and rs7958904 were detected.Similarly,we found no significant associations between rs4759314 AA genotype and CCA susceptibility.CONCLUSION HOTAIR rs4759314 AG and GG genotypes may be implicated with CCA development and may serve as a potential diagnostic biomarker.

An efficient and rapid method to detect and verify natural antisense transcripts of animal genes

High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA（long non-coding RNA）, can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix（Xist antisense RNA）, chicken LXN（latexin） and GFM1（Gelongation factor, mitochondrial 1）, were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.

The non-coding RNA SVALKA locus produces a cis-natural antisense transcript that negatively regulates the expression of CBF1 and biomass production at normal temperatures

Non-coding transcription is present in all eukaryotic genomes,but we lack fundamental knowledge about its importance for an organism’s ability to develop properly.In plants,emerging evidence highlights the essential biological role of non-coding transcription in the regulation of coding transcription.However,we have few molecular insights into this regulation.Here,we show that a long isoform of the long noncoding RNA SVALKA-L(SVK-L)forms a natural antisense transcript to the host gene CBF1 and negatively regulates CBF1 mRNA levels at normal temperatures in the model plant Arabidopsis thaliana.Furthermore,we show detailed evidence for the specific mode of action of SVK-L.This pathway includes the formation of double-stranded RNA that is recognized by the DICER proteins and subsequent downregulation of CBF1 mRNA levels.Thus,the CBF1-SVK regulatory circuit is not only important for its previously known role in cold temperature acclimation but also for biomass production at normal temperatures.Our study characterizes the developmental role of SVK-L and offers mechanistic insight into how biologically important overlapping natural antisense transcripts can act on and fine-tune the steady-state levels of their host gene’s mRNA.

Sequence signatures of genes with accompanying antisense transcripts in Saccharomyces cerevisiae

Recent studies have found many antisense non-coding transcripts at the opposite strand of some protein-coding genes.In yeast,it was reported that such antisense transcripts play regulatory roles for their partner genes by forming a feedback loop with the protein-coding genes.Since not all coding genes have accompanying antisense transcripts,it would be interesting to know whether there are sequence signatures in a coding gene that are decisive or associated with the existence of such antisense partners.We collected all the annotated antisense transcripts in the yeast Saccharomyces cerevisiae,analyzed sequence motifs around the genes with antisense partners,and classified genes with and without accompanying antisense transcripts by using machine learning methods.Some weak but statistically significant sequence features are detected,which indicates that there are sequence signatures around the protein-coding genes that may be decisive or indicative for the existence of accompanying antisense transcripts.

Expression and function of natural antisense transcripts in mouse embryonic stem cells

Non-coding RNAs(nc RNAs),such as micro RNAs and large intergenic non-coding RNAs,have been shown to play essential roles in regulating pluripotency.Yet,it is not clear the role of natural antisense transcripts(NATs),also belonging to nc RNAs,in embryonic stem cells.However,the role of NATs in embryonic stem cells remains unknown.We further confirmed the expression of the NATs of three key pluripotency genes,Oct4,Nanog and Sox2.Moreover,overexpression of Sox2-NAT reduces the expression of Sox2 protein,and slightly enhances the Sox2 m RNA level.Altogether,our data indicated that like other nc RNAs,NATs might be involved in pluripotency maintenance.