Enhancer-gene specificity in development and disease.
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ABSTRACT:
SUBMITTER: Pachano T
PROVIDER: S-EPMC7612879 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Enhancers and antisense RNAs play key roles in transcriptional regulation through differing mechanisms. Recent studies have demonstrated that enhancers are often associated with non-coding RNAs (ncRNAs), yet the functional role of these enhancer:ncRNA associations is unclear. Using RNA-Sequencing to interrogate the transcriptomes of undifferentiated mouse embryonic stem cells (mESCs) and their derived neural precursor cells (NPs), we identified two novel enhancer-associated antisense transcripts that appear to control isoform-specific expression of their overlapping protein-coding genes. In each case, an enhancer internal to a protein-coding gene drives an antisense RNA in mESCs but not in NPs. Expression of the antisense RNA is correlated with expression of a shorter isoform of the associated sense gene that is not present when the antisense RNA is not expressed. We demonstrate that expression of the antisense transcripts as well as expression of the short sense isoforms correlates with enhancer activity at these two loci. Further, overexpression and knockdown experiments suggest the antisense transcripts regulate expression of their associated sense genes via cis-acting mechanisms. Interestingly, the protein-coding genes involved in these two examples, Zmynd8 and Brd1, share many functional domains, yet their antisense ncRNAs show no homology to each other and are not present in non-murine mammalian lineages, such as the primate lineage. The lack of homology in the antisense ncRNAs indicates they have evolved independently of each other and suggests that this mode of lineage-specific transcriptional regulation may be more widespread in other cell types and organisms. Our findings present a new view of enhancer action wherein enhancers may direct isoform-specific expression of genes through ncRNA intermediates.
| S-EPMC3427357 | biostudies-literature
Project description:Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. Here we show that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters--one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor--and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, we identify two transcription factors--Dref and Trl--that bind and activate housekeeping versus developmental enhancers, respectively. Our results provide evidence for a sequence-encoded enhancer-core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.
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Project description:DNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe the development of a 5hmC tissue map by characterizing the genomic distributions of 5hmC in 19 human tissues derived from ten organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separates samples by tissue type. Further comparison of the 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC is preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, the results provide an extensive 5hmC map across diverse human tissue types that suggests a potential role of 5hmC in tissue-specific development; as well as a resource to facilitate future studies of DNA demethylation in pathogenesis and the development of 5hmC as biomarkers.
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Project description:The transcription activity of the tumor-specific promoter may be increased using specific DNA sequences such as simian virus 40 (SV40). Human heparanase (HPSE) gene promoter is also considered a tumor-specific promoter. However, whether or not the SV40 enhancer affects the tumor specificity of HPSE remains to be determined. The SV40 enhancer sequence, 237 bp in length, was amplified and correctly inserted into the assigned multiple clone sites (MCS) of the eukaryotic expression vector pEGFP-Hp, which was constructed in advance. The recombinant plasmid pEGFP-Hp-SV40e was consistent with the anticipated Genbank data and transfected into human umbilical vein endothelial cell (ECV) and tumor cell lines, including hepatoma carcinoma (HepG2), laryngeal carcinoma (Hep2) and chronic myelogenous leukemia cell lines (K562) using lipofectamine, respectively. The expression of the reporter gene, green fluorescent protein (GFP), was detected using fluorescence microscopy and flow cytometry. The length of the amplified SV40 enchancer was 237 bp and the sequence was in accordance with the GenBank data. The recombinant plasmid pEGFP-Hp-SV40 was consistent with the anticipated results. Fluorimetric analysis showed that the fluorescence of pEGFP-Hp-SV40e in ECV cells was as dim as pEGFP-Hp, and obviously weaker than pEGFPN1. In tumor cells including HepG2, Hep2 and K562 cells, the fluorescence of pEGFP-Hp-SV40e was similar to that of pEGFP-N1, which was clearly brighter than pEGFP-Hp. The average transfecion rates in the 4 types of cells were 4.1, 17.2, 8.8 and 6.4% in the pEGFP-Hp; 18.3, 29.3, 17.0 and 13.0% in the pEGFP-Nl and 4.3, 28.8, 16.4 and 11.7% in the pEGFP-Hp-SV40e groups, respectively. The ratio of pEGFP-Hp-SV40e to pEGFP-Hp in all cells was 1.05, 1.67, 1.86 and 1.83, respectively. In conclusion, the inserted SV40 enhancer sequence is able to improve the transcriptional activity of the human HPSE gene promoter, but does not affect its tumor specificity.
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Project description:IntroductionTrophoblast invasion establishes adequate blood flow between mother and fetus in early placental development. However, little is known about the cis-regulatory mechanisms underlying this important process. We aimed to identify enhancer elements that are active during trophoblast invasion, and build a trophoblast invasion gene-enhancer network.MethodsWe carried out ChIP-Seq for an enhancer-associated mark (H3k27Ac) at two time points during early placental development in mouse. One time point when invasion is at its peak (e7.5) and another time point shortly afterwards (e9.5). We use computational analysis to identify putative enhancers, as well as the transcription factor binding sites within them, that are specific to the time point of trophoblast invasion.ResultsWe compared read profiles at e7.5 and e9.5 to identify 1,977 e7.5-specific enhancers. Within a subset of e7.5-specific enhancers, we discovered a cell migration associated regulatory code, consisting of three transcription factor motifs: AP1, Ets, and Tcfap2. To validate differential expression of the transcription factors that bind these motifs, we performed RNA-Seq in the same context. Finally, we integrated these data with publicly available protein-protein interaction data and constructed a trophoblast invasion gene-enhancer network.DiscussionThe data we generated and analysis we carried out improves our understanding of the regulatory mechanisms of trophoblast invasion, by suggesting a transcriptional code exists in the enhancers of cell migration genes. Furthermore, the network we constructed highlights novel candidate genes that may be critical for trophoblast invasion.
| S-EPMC4707081 | biostudies-literature
Project description:Each language has standard books describing that language's grammatical rules. Biologists have searched for similar, albeit more complex, principles relating enhancer sequence to gene expression. Here, we review the literature on enhancer grammar. We introduce dependency grammar, a model where enhancers encode information based on dependencies between enhancer features shaped by mechanistic, evolutionary, and biological constraints. Classifying enhancers based on the types of dependencies may identify unifying principles relating enhancer sequence to gene expression. Such rules would allow us to read the instructions for development within genomes and pinpoint causal enhancer variants underlying disease and evolutionary changes.
| S-EPMC8462829 | biostudies-literature
Project description:Premature ventricular contractions (PVCs) are common in the general population, and frequent PVCs may result in the poor quality of life or even the damage of cardiac function. We examined the efficacy and safety of a traditional Chinese medicine Wenxin Keli for the treatment of frequent PVCs among a relatively large Chinese cohort.We performed a randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. A total of 1200 eligible participants were randomly assigned in a ratio of 1:1 to receive Wenxin Keli or the placebo for 4 weeks. The primary and secondary endpoint was the change of PVC numbers and PVC-related symptoms after a 4-week treatment compared with baseline, respectively. In addition, vital signs, laboratory values, and electrocardiographic parameters were assessed in a safety analysis.At the initial evaluation, no significant differences in the baseline characteristics were observed between the Wenxin Keli group and the placebo group. A smaller number of PVCs was observed after the 4-week treatment than at baseline, in both the Wenxin Keli group (5686 ± 5940 vs. 15,138 ± 7597 beats/d, P < 0.001) and the placebo group (10,592 ± 8009 vs. 14,529 ± 5929 beats/d, P < 0.001); moreover, the Wenxin Keli group demonstrated a significantly greater reduction in the frequency of PVCs than the placebo group (P < 0.001). In a full analysis set, patients in the Wenxin Keli group exhibited significantly higher total effective responses in the reduction of PVCs compared to those in the placebo group (83.8% vs. 43.5%,P < 0.001). The per-protocol analysis yielded similar results (83.0% vs. 39.3%,P < 0.001). Treatment with Wenxin Keli also demonstrated superior performance compared to the placebo with respect to PVC-related symptoms. No severe adverse effects attributable to Wenxin Keli were reported.Wenxin Keli treatment effectively reduced the overall number of PVCs and alleviated PVC-related symptoms in patients without structural heart diseases and had no severe side effects.
| S-EPMC4736860 | biostudies-literature
Project description:The regulatory logic of time- and tissue-specific gene expression has mostly been dissected in the context of the smallest DNA fragments that, when isolated, recapitulate native expression in reporter assays. It is not known if the genomic sequences surrounding such fragments, often evolutionarily conserved, have any biological function or not. Using an enhancer of the even-skipped gene of Drosophila as a model, we investigate the functional significance of the genomic sequences surrounding empirically identified enhancers. A 480 bp long "minimal stripe element" is able to drive even-skipped expression in the second of seven stripes but is embedded in a larger region of 800 bp containing evolutionarily conserved binding sites for required transcription factors. To assess the overall fitness contribution made by these binding sites in the native genomic context, we employed a gene-replacement strategy in which whole-locus transgenes, capable of rescuing even-skipped(-) lethality to adulthood, were substituted for the native gene. The molecular phenotypes were characterized by tagging Even-skipped with a fluorescent protein and monitoring gene expression dynamics in living embryos. We used recombineering to excise the sequences surrounding the minimal enhancer and site-specific transgenesis to create co-isogenic strains differing only in their stripe 2 sequences. Remarkably, the flanking sequences were dispensable for viability, proving the sufficiency of the minimal element for biological function under normal conditions. These sequences are required for robustness to genetic and environmental perturbation instead. The mutant enhancers had measurable sex- and dose-dependent effects on viability. At the molecular level, the mutants showed a destabilization of stripe placement and improper activation of downstream genes. Finally, we demonstrate through live measurements that the peripheral sequences are required for temperature compensation. These results imply that seemingly redundant regulatory sequences beyond the minimal enhancer are necessary for robust gene expression and that "robustness" itself must be an evolved characteristic of the wild-type enhancer.
| S-EPMC3213169 | biostudies-literature
Project description:The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.
| S-EPMC4445080 | biostudies-literature
Project description:Enhancers are often studied as noncoding regulatory elements that modulate the precise spatiotemporal expression of genes in a highly tissue-specific manner. This paradigm has been challenged by recent evidence of individual enhancers acting in multiple tissues or developmental contexts. However, the frequency of these enhancers with high degrees of "pleiotropy" out of all putative enhancers is not well understood. Consequently, it is unclear how the variation of enhancer pleiotropy corresponds to the variation in expression breadth of target genes. Here, we use multi-tissue chromatin maps from diverse human tissues to investigate the enhancer-gene interaction architecture while accounting for 1) the distribution of enhancer pleiotropy, 2) the variations of regulatory links from enhancers to target genes, and 3) the expression breadth of target genes. We show that most enhancers are tissue-specific and that highly pleiotropy enhancers account for <1% of all putative regulatory sequences in the human genome. Notably, several genomic features are indicative of increasing enhancer pleiotropy, including longer sequence length, greater number of links to genes, increasing abundance and diversity of encoded transcription factor motifs, and stronger evolutionary conservation. Intriguingly, the number of enhancers per gene remains remarkably consistent for all genes (∼14). However, enhancer pleiotropy does not directly translate to the expression breadth of target genes. We further present a series of Gaussian Mixture Models to represent this organization architecture. Consequently, we demonstrate that a modest trend of more pleiotropic enhancers targeting more broadly expressed genes can generate the observed diversity of expression breadths in the human genome.
| S-EPMC8383896 | biostudies-literature