Transcriptional Profiling of Mouse Uterus at Pre-implantation Stage under VEGF Repression
ABSTRACT: In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at G2.5 (gestation day 2.5) by Solexa/ Illumina’s digital gene expression (DGE) system. 831 uterus-specific and 2398 VEGF regulated genes were identified. Gene ontology analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF regulated genes, up-regulated genes were associated with RNA polymerase III activity while down-regulated genes were strongly associated with muscle development.Comparable numbers of antisense RNA transcripts were also identified. Expression levels of the antisense RNAs were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers. The antisense RNAs with exceptionally high or low expression levels and the antisense RNAs under VEGF regulation were also identified. These transcripts may be important candidates in regulation of uterus development. This study provides a global survey on genes and antisense transcripts regulated by VEGF in the pre-implantation stage. Results will contribute to the further studies of candidate genes and pathways in regulating implantation process and related diseases. Uteri mRNA profiles of G2.5 VEGF-normal (Dox+) and VEGF-repression (Dox-) were generated by Solexa/ Illumina’s digital gene expression (DGE) system.
Project description:Uterus development during pre-implantation stage affects implantation process and embryo growth. Aberrant uterus development is associated with many human reproductive diseases. Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells. VEGF mediation in reproduction has been broadly studied, but VEGF-induced transcriptional machinery during implantation window has not been systematically studied. In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at gestation 2.5 (G2.5) by Solexa/Illumina's digital gene expression (DGE) system. A number of 831 uterus-specific and 2398 VEGF-regulated genes were identified. Gene ontology (GO) analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development. Comparable numbers of antisense transcripts were identified. Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers. The antisense transcripts with exceptionally high or low expression levels and the antisense transcripts under VEGF regulation were also identified. These transcripts may be important candidates in regulation of uterus development. This study provides a global survey on genes and antisense transcripts regulated by VEGF in the pre-implantation stage. Results will contribute to further study the candidate genes and pathways in regulating implantation process and related diseases.
Project description:The precise nature of antisense transcripts in eukaryotes such as Saccharomyces cerevisiae remains elusive. Here we show that the 3' regions of genes possess a promoter architecture, including a pre-initiation complex (PIC), which mirrors that at the 5' region and which is much more pronounced at genes with a defined antisense transcript. Remarkably, for genes with an antisense transcript, average levels of PIC components at the 3' region are ?60% of those at the 5' region. Moreover, at these genes, average levels of nascent antisense transcription are ?45% of sense transcription. We find that this 3' promoter architecture persists for highly transcribed antisense transcripts where there are only low levels of transcription in the divergent sense direction, suggesting that the 3' regions of genes can drive antisense transcription independent of divergent sense transcription. To validate this, we insert short 3' regions into the middle of other genes and find that they are capable of both initiating antisense transcripts and terminating sense transcripts. Our results suggest that antisense transcription can be regulated independently of divergent sense transcription in a PIC-dependent manner and we propose that regulated production of antisense transcripts represents a fundamental and widespread component of gene regulation.
Project description:A large number of antisense transcripts have been detected in diverse microbial genomes and considerable effort has been devoted to elucidating the functional role of antisense transcription. In this study, we reanalysed extensive RNA sequencing data from the opportunistic pathogen Pseudomonas aeruginosa and found that the majority of genes have a propensity for antisense transcription. Although antisense transcripts were found in more than 80?% of the genes of the P. aeruginosa genome, the majority of sequencing reads were mapping sense and only a minority (<2?%) were mapping antisense to genes. Similarly to the sense expression levels, the antisense expression levels varied under different environmental conditions, with the sense and antisense expression levels often being inversely regulated and modulated by the activity of alternative sigma factors. Environment-modulated antisense transcription showed a bias towards being antisense to genes within regions of genomic plasticity and to those encoding small regulatory RNAs. In the future, the validation and functional characterization of antisense transcripts, and novel transcripts that are antisense to small regulatory RNAs in particular, have the potential to contribute to our understanding of the various levels of transcriptional regulation and its dynamics in the bacterial pathogen P. aeruginosa.
Project description:A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.
Project description:Alu RNAs are present at elevated levels in stress conditions and, consequently, Alu repeats are increasingly being associated with the physiological stress response. Alu repeats are known to harbor transcription factor binding sites that modulate RNA pol II transcription and Alu RNAs act as transcriptional co-repressors through pol II binding in the promoter regions of heat shock responsive genes. An observation of a putative heat shock factor (HSF) binding site in Alu led us to explore whether, through HSF binding, these elements could further contribute to the heat shock response repertoire.Alu density was significantly enriched in transcripts that are down-regulated following heat shock recovery in HeLa cells. ChIP analysis confirmed HSF binding to a consensus motif exhibiting positional conservation across various Alu subfamilies, and reporter constructs demonstrated a sequence-specific two-fold induction of these sites in response to heat shock. These motifs were over-represented in the genic regions of down-regulated transcripts in antisense oriented Alus. Affymetrix Exon arrays detected antisense signals in a significant fraction of the down-regulated transcripts, 50% of which harbored HSF sites within 5 kb. siRNA knockdown of the selected antisense transcripts led to the over-expression, following heat shock, of their corresponding down-regulated transcripts. The antisense transcripts were significantly enriched in processes related to RNA pol III transcription and the TFIIIC complex.We demonstrate a non-random presence of Alu repeats harboring HSF sites in heat shock responsive transcripts. This presence underlies an antisense-mediated mechanism that represents a novel component of Alu and HSF involvement in the heat shock response.
Project description:Antisense transcription of protein-coding genes has been increasingly recognized as an important regulatory mechanism of gene expression. However, less is known about the extent and importance of antisense transcription of noncoding genes. Here, we investigate the breadth and dynamics of antisense transcription of miRNAs, a class of important noncoding RNAs. Because the antisense transcript of a miRNA is likely to form a hairpin suitable as the substrate of ADARs, which convert adenosine to inosine in double-stranded RNAs, we used A-to-I RNA editing as ultrasensitive readout for antisense transcription of the miRNAs. Through examining the unstranded targeted RNA-seq libraries covering all miRNA loci in 25 types of human tissues, we identified 7275 editing events located in 81% of the antisense strand of the miRNA loci, thus uncovering the previously unknown prevalent antisense transcription of the miRNAs. We found that antisense transcripts are tightly regulated, and a substantial fraction of miRNAs and their antisense transcripts are coexpressed. Sense miRNAs have been shown to down-regulate the coexpressed antisense transcripts, whereas the act of antisense transcription, rather than the transcripts themselves, regulates the expression of sense miRNAs. RNA editing tends to decrease the miRNA accessibility of the antisense transcripts, therefore protecting them from being degraded by the sense-mature miRNAs. Altogether, our study reveals the landscape of antisense transcription and editing of miRNAs, as well as a previously unknown reciprocal regulatory circuit of sense-antisense miRNA pairs.
Project description:The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3' end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs.
Project description:Natural antisense transcripts are endogenous transcripts that are complementary to the sense-strand of DNA. These transcripts have been identified in various eukaryotic species and are involved in a broad range of regulatory events and biological processes. However, their general biological functions, expression characteristics and regulatory mechanisms are still unclear. In this study, 497 liver and 586 muscle samples were harvested from a White Duroc×Erhualian F(2) resource population. The expression profiles of sense and antisense transcripts were determined by tag-based RNA sequencing. We identified 33.7% and 20.4% of transcripts having both sense and antisense expression, and 12.5% and 6.1% of transcripts only expressing antisense transcripts in liver and muscle, respectively. More than 32.2% of imprinting or predicted imprinting genes in the geneimprint database were detected with both sense and antisense expression. The correlations between sense and antisense expression in sense-antisense pairs were diverse in both liver and muscle, showing positive, negative or absent correlation. Antisense expression increases gene expression variability. More interestingly, compared to eQTL mapping of sense transcripts in which more than one eQTL was mapped for a transcript, only one eQTL was identified for each antisense transcript, and the percentage of cis-eQTL in antisense eQTL was higher than that in sense eQTL. This suggests that the expressions of antisense transcripts tend to be cis-regulated by a single genomic locus. To our knowledge, this study is the first systematical investigation of antisense transcription in pigs. The findings improve our understanding of the complexity of porcine transcriptome.
Project description:Mitogen inducible gene 6 (Mig-6) is an important mediator of progesterone (P4) signaling to inhibit estrogen (E2) signaling in the uterus. Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and E2-induced endometrial cancer. To identify the molecular pathways regulated by Mig-6, we performed microarray analysis on the uterus of ovariectomized Mig-6(f/f) and PGR(cre/+)Mig-6(f/f) (Mig-6(d/d)) mice treated with vehicle or P4 for 6 h. The results revealed that 772 transcripts were significantly regulated in the Mig-6(d/d) uterus treated with vehicle as compared with Mig-6(f/f) mice. The pathway analysis showed that Mig-6 suppressed the expression of gene-related cell cycle regulation in the absence of ovarian steroid hormone. The epithelium of Mig-6(d/d) mice showed a significant increase in the number of proliferative cells compared to Mig-6(f/f) mice. This microarray analysis also revealed that 324 genes are regulated by P4 as well as Mig-6. Cited2, the developmentally important transcription factor, was identified as being regulated by the P4-Mig-6 axis. To determine the role of Cited2 in the uterus, we used the mice with Cited2 that were conditionally ablated in progesterone receptor-positive cells (PGR(cre/+)Cited2(f/f); Cited2(d/d)). Ablation of Cited2 in the uterus resulted in a significant reduction in the ability of the uterus to undergo a hormonally induced decidual reaction. Identification and analysis of these responsive genes will help define the role of P4 as well as Mig-6 in regulating uterine biology.
Project description:Natural antisense transcripts (NATs) exist ubiquitously in mammalian genomes and play roles in the regulation of gene expression. However, both the existence of bidirectional antisense RNA regulation and the possibility of protein-coding genes that function as antisense RNAs remain speculative. Here, we found that the protein-coding gene, deoxyhypusine synthase (DHPS), as the NAT of WDR83, concordantly regulated the expression of WDR83 mRNA and protein. Conversely, WDR83 also regulated DHPS by antisense pairing in a concordant manner. WDR83 and DHPS were capable of forming an RNA duplex at overlapping 3' untranslated regions and this duplex increased their mutual stability, which was required for the bidirectional regulation. As a pair of protein-coding cis-sense/antisense transcripts, WDR83 and DHPS were upregulated simultaneously and correlated positively in gastric cancer (GC), driving GC pathophysiology by promoting cell proliferation. Furthermore, the positive relationship between WDR83 and DHPS was also observed in other cancers. The bidirectional regulatory relationship between WDR83 and DHPS not only enriches our understanding of antisense regulation, but also provides a more complete understanding of their functions in tumor development.