Search

DNA regulatory elements intricately control when, where, and how genes are activated. Therefore, understanding the function of these elements could unveil the complexity of the genetic regulation network. Genome-wide significant variants are predominantly found in non-coding regions of DNA, so comprehending the predicted functional regulatory elements is crucial for understanding the biological context of these genomic markers, which can be incorporated into breeding programs. The emergence of CRISPR technology has provided a powerful tool for studying non-coding regulatory elements in genomes. In this study, we leveraged epigenetic data from the Functional Annotation of Animal Genomes project to identify promoter and putative enhancer regions associated with three genes (HBBA, IRF7, and PPARG) in the chicken genome. To identify the enhancer regions, we designed guide RNAs targeting the promoter and candidate enhancer regions and utilized CRISPR activation (CRISPRa) with dCas9-p300 and dCas9-VPR as transcriptional activators in chicken DF-1 cells. By comparing the expression levels of target genes between the promoter activation and the co-activation of the promoter and putative enhancers, we were able to identify functional enhancers that exhibited augmented upregulation. In conclusion, our findings demonstrate the remarkable efficiency of CRISPRa in precisely manipulating the expression of endogenous genes by targeting regulatory elements in the chicken genome, highlighting its potential for functional validation of non-coding regions.

Background: How transcription factors (TFs) down-regulate gene expression remains ill-understood, especially when they bind to multiple enhancers contacting the same gene promoter. In particular, it is not known whether they exert similar or significantly different molecular effects at these enhancers. Results: To address this issue, we used a particularly well-suited study model consisting of the down-regulation of the TGFB2 gene by the TF Fra-1 in Fra-1-overexpressing cancer cells, as Fra-1 binds to multiple enhancers interacting with the TGFB2 promoter. We show that Fra-1 does not repress TGFB2 transcription via reducing RNA Pol II recruitment at the gene promoter but by decreasing the formation of its transcription-initiating form. This is associated with complex long-range chromatin interactions implicating multiple molecularly and functionally heterogeneous Fra-1-bound transcriptional enhancers distal to the TGFB2 transcriptional start site. In particular, the latter display differential requirements upon the presence and the activity of the lysine acetyltransferase p300/CBP. Furthermore, the final transcriptional output of the TGFB2 gene seems to depend on a balance between the positive and negative effects of Fra-1 at these enhancers. Conclusion: Our work unveils complex molecular mechanisms underlying the repressive actions of Fra-1 on TGFB2 gene expression. This has consequences for our general understanding of the functioning of the ubiquitous transcriptional complex AP-1, of which Fra-1 is the most documented component for prooncogenic activities. In addition, it raises the general question of the heterogeneity of the molecular functions of TFs binding to different enhancers regulating the same gene. [ABSTRACT FROM AUTHOR]

Simple Summary: Enhancers serve as logic gates of the regulatory mechanism of gene expression, and their malfunction is associated with numerous diseases. Therefore, the functional validation of enhancer elements is of great importance in genomics research. Recent technological advancements have enabled the perturbation of enhancers and the examination of their impact on the expression of nearby genes. Here, we review the progress made in experimental and computational methods, which have equipped researchers with a promising arsenal to uncover relationships between enhancers and phenotypes, providing mechanistic insights into diseases. Higher eukaryotic enhancers, as a major class of regulatory elements, play a crucial role in the regulation of gene expression. Over the last decade, the development of sequencing technologies has flooded researchers with transcriptome-phenotype data alongside emerging candidate regulatory elements. Since most methods can only provide hints about enhancer function, there have been attempts to develop experimental and computational approaches that can bridge the gap in the causal relationship between regulatory regions and phenotypes. The coupling of two state-of-the-art technologies, also referred to as crisprQTL, has emerged as a promising high-throughput toolkit for addressing this question. This review provides an overview of the importance of studying enhancers, the core molecular foundation of crisprQTL, and recent studies utilizing crisprQTL to interrogate enhancer-phenotype correlations. Additionally, we discuss computational methods currently employed for crisprQTL data analysis. We conclude by pointing out common challenges, making recommendations, and looking at future prospects, with the aim of providing researchers with an overview of crisprQTL as an important toolkit for studying enhancers. [ABSTRACT FROM AUTHOR]

The establishment and stable inheritance of individual patterns of gene expression in different cell types are required for the development of multicellular organisms. The important epigenetic regulators are the Polycomb group (PcG) and Trithorax group (TrxG) proteins, which control the silenced and active states of genes, respectively. In Drosophila, the PcG/TrxG group proteins are recruited to the DNA regulatory sequences termed the Polycomb response elements (PREs). The PREs are composed of the binding sites for different DNA-binding proteins, the so-called PcG recruiters. Currently, the role of the PcG recruiters in the targeting of the PcG proteins to PREs is well documented. However, there are examples where the PcG recruiters are also implicated in the active transcription and in the TrxG function. In addition, there is increasing evidence that the genome-wide PcG recruiters interact with the chromatin outside of the PREs and overlap with the proteins of differing regulatory classes. Recent studies of the interactomes of the PcG recruiters significantly expanded our understanding that they have numerous interactors besides the PcG proteins and that their functions extend beyond the regulation of the PRE repressive activity. Here, we summarize current data about the functions of the PcG recruiters. [ABSTRACT FROM AUTHOR]

Extensive morphological variation found in mammals reflects the wide spectrum of their ecological adaptations. The highest morphological diversity is present in the craniofacial region, where geometry is mainly dictated by the bony skull. Mammalian craniofacial development represents complex multistep processes governed by numerous conserved genes that require precise spatio-temporal control. A central question in contemporary evolutionary biology is how a defined set of conserved genes can orchestrate formation of fundamentally different structures, and therefore how morphological variability arises. In principle, differential gene expression patterns during development are the source of morphological variation. With the emergence of multicellular organisms, precise regulation of gene expression in time and space is attributed to cis-regulatory elements. These elements contribute to higher-order chromatin structure and together with trans-acting factors control transcriptional landscapes that underlie intricate morphogenetic processes. Consequently, divergence in cis-regulation is believed to rewire existing gene regulatory networks and form the core of morphological evolution. This review outlines the fundamental principles of the genetic code and genomic regulation interplay during development. Recent work that deepened our comprehension of cis-regulatory element origin, divergence and function is presented here to illustrate the state-of-the-art research that uncovered the principles of morphological novelty. This article is part of the theme issue 'The mammalian skull: development, structure and function'. [ABSTRACT FROM AUTHOR]

Deoxyribonucleic acid (DNA) methylation (DNAm) is an important epigenetic mechanism that plays a role in chromatin structure and transcriptional regulation. Elucidating the relationship between DNAm and gene expression is of great importance for understanding its role in transcriptional regulation. The conventional approach is to construct machine-learning-based methods to predict gene expression based on mean methylation signals in promoter regions. However, this type of strategy only explains about 25% of gene expression variation, and hence is inadequate in elucidating the relationship between DNAm and transcriptional activity. In addition, using mean methylation as input features neglects the heterogeneity of cell populations that can be reflected by DNAm haplotypes. We here developed TRAmaHap, a novel deep-learning framework that predicts gene expression by utilizing the characteristics of DNAm haplotypes in proximal promoters and distal enhancers. Using benchmark data of human and mouse normal tissues, TRAmHap shows much higher accuracy than existing machine-learning based methods, by explaining 60~80% of gene expression variation across tissue types and disease conditions. Our model demonstrated that gene expression can be accurately predicted by DNAm patterns in promoters and long-range enhancers as far as 25 kb away from transcription start site, especially in the presence of intra-gene chromatin interactions. [ABSTRACT FROM AUTHOR]

Existing models restrict transcription factors (TFs) to individual functional categories, and these apply across all contexts within a cell type. Recent results suggest that, instead of having a single, fixed role, TFs can play varied functions across different settings, which may give rise to diversity in phenotypic cell states. We refer to this as the 'variegated model of TF function'. This variation in function may be mediated by direct or indirect TF–TF crosstalk, or by interplay between TFs and chromatin state. Descriptions of the many ways TFs can affect transcription emphasize that classic bind–activate (or bind–repress) scenarios vastly oversimply TF interplay and quantitative effects. Alternatives to TF knockouts, such as natural cis -genetic variation, acute TF degradation, or continuous tuning of TF concentrations, may better capture direct TF targets. New measurement tools and computational methods will be required to develop a quantitative and predictive understanding of TF-mediated gene regulatory networks in immunity. Unlike previous models of TF function in immunity, which assigned a single role to each TF across all contexts within a cell type, recent results suggest that TFs flexibly have different roles across distinct cell states, with implications for an understanding and control of immunocyte gene regulatory networks. Specific combinations of transcription factors (TFs) control the gene expression programs that underlie specialized immune responses. Previous models of TF function in immunocytes had restricted each TF to a single functional categorization [e.g., lineage-defining (LDTFs) vs. signal-dependent TFs (SDTFs)] within one cell type. Synthesizing recent results, we instead propose a variegated model of immunological TF function, whereby many TFs have flexible and different roles across distinct cell states, contributing to cell phenotypic diversity. We discuss evidence in support of this variegated model, describe contextual inputs that enable TF diversification, and look to the future to imagine warranted experimental and computational tools to build quantitative and predictive models of immunocyte gene regulatory networks. [ABSTRACT FROM AUTHOR]

Objective To explore and identify H3K27ac enriched non-conserved enhancers near human NPTX1, improve the annotation of NPTX1-related regulatory regions, and provide a corresponding theoretical basis for the study of NPTX1-related diseases and mutations in non-coding regions. Methods A total of 39 H3K27ac ChIP-seq data involving 7 tissues were downloaded from the CistromeDB database and analyzed. Peaks in the range of 100 kb on both sides of NPTX1 were selected. Fragments’ sequence conservation among human, mouse and zebrafish was analyzed by ECR browser. The fragments were inserted into a dual luciferase validation vector, and dual luciferase assays were performed in human neural-derived cell line SH-SY5Y and human kidney-derived cell line 293T to detect enhancers’ activity. Results We obtained 5 H3K27ac-enriched fragments. The DNA sequences of each fragment were not conserved among human, mouse and zebrafish. The ratio of dual luciferase activity of NPTX1-E1-P was significantly higher than that of the negative control in SH-SY5Y, but it was about 40% lower in 293T cells. Conclusion Among the H3K27ac-enriched fragments near NPTX1, NPTX1-E1-P has enhancer activity in human neurogenic cell line SH-SY5Y, and low sequence conservation among species. Compared with the human neurogenic cell line SH-SY5Y, its enhancer activity is decreased in human kidney-derived cell line 293T. [ABSTRACT FROM AUTHOR]

Enhancers are key regulatory elements that exert crucial roles in diverse biological processes, including tumorigenesis and cancer development. Active enhancers could produce transcripts termed enhancer RNAs (eRNAs), which could be used as an index of enhancer activity. Here, we present a versatile data portal, enhancer activity quantitative trait loci database (eaQTLdb; http://www.bioailab.com:3838/eaQTLdb), for exploring the effects of genetic variants on enhancer activity and prioritizing candidate variants across different cancer types. By leveraging the accumulated multiomics data, we systematically identified genetic variants which influence enhancer activity in different cancer types, termed as eaQTLs. We have linked the eaQTLs to hallmarks of cancer and patients' overall survival to illustrate their potential biological roles in cancer development and progression. Notably, eaQTLs associated with the infiltration abundance of 24 different immune cell types were identified and incorporated into eaQTLdb. In addition, we applied colocalization analyses to examine 59 complex diseases and traits to identify eaQTLs colocalized with diseases/traits GWAS signals. Overall, eaQTLdb, incorporating a rich resource for exploration of eaQTLs in different cancer types, will not only benefit users in prioritizing candidate genetic variants and enhancers, but also help researchers decipher the roles of eaQTLs in the dysregulated pathways of cancer and tumor immune microenvironment, opening new diagnostic and therapeutic avenues in precise medicine. [ABSTRACT FROM AUTHOR]

Eukaryotic RNA polymerase II (Pol II) contains a tail-like, intrinsically disordered carboxy-terminal domain (CTD) comprised of heptad-repeats, that functions in coordination of the transcription cycle and in coupling transcription to co-transcriptional processes. The CTD repeat number varies between species and generally increases with genome size, but the reasons for this are unclear. Here, we show that shortening the CTD in human cells to half of its length does not generally change pre-mRNA synthesis or processing in cells. However, CTD shortening decreases the duration of promoter-proximal Pol II pausing, alters transcription of putative enhancer elements, and delays transcription activation after stimulation of the MAP kinase pathway. We suggest that a long CTD is required for efficient enhancer-dependent recruitment of Pol II to target genes for their rapid activation.

Genetic factors underlying coronary artery disease (CAD) have been widely studied using genome-wide association studies (GWASs). However, the functional understanding of the CAD loci has been limited by the fact that a majority of GWAS variants are located within non-coding regions with no functional role. High cholesterol and dysregulation of the liver metabolism such as non-alcoholic fatty liver disease confer an increased risk of CAD. Here, we studied the function of non-coding single-nucleotide polymorphisms in CAD GWAS loci located within liver-specific enhancer elements by identifying their potential target genes using liver cis-eQTL analysis and promoter Capture Hi-C in HepG2 cells. Altogether, 734 target genes were identified of which 121 exhibited correlations to liver-related traits. To identify potentially causal regulatory SNPs, the allele-specific enhancer activity was analyzed by (1) sequence-based computational predictions, (2) quantification of allele-specific transcription factor binding, and (3) STARR-seq massively parallel reporter assay. Altogether, our analysis identified 1,277 unique SNPs that display allele-specific regulatory activity. Among these, susceptibility enhancers near important cholesterol homeostasis genes (APOB, APOC1, APOE, and LIPA) were identified, suggesting that altered gene regulatory activity could represent another way by which genetic variation regulates serum lipoprotein levels. Using CRISPR-based perturbation, we demonstrate how the deletion/activation of a single enhancer leads to changes in the expression of many target genes located in a shared chromatin interaction domain. Our integrative genomics approach represents a comprehensive effort in identifying putative causal regulatory regions and target genes that could predispose to clinical manifestation of CAD by affecting liver function.

BackgroundCo-localized combinations of histone modifications ("chromatin states") have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points ("chromatin state trajectories") have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs.ResultsWe present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories. We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation. The code of the framework is available at https://github.com/henriettemiko/TimelessFlex .ConclusionsTimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.

Abstract Background The largest sequence-based models of transcription control to date are obtained by predicting genome-wide gene regulatory assays across the human genome. This setting is fundamentally correlative, as those models are exposed during training solely to the sequence variation between human genes that arose through evolution, questioning the extent to which those models capture genuine causal signals. Results Here we confront predictions of state-of-the-art models of transcription regulation against data from two large-scale observational studies and five deep perturbation assays. The most advanced of these sequence-based models, Enformer, by and large, captures causal determinants of human promoters. However, models fail to capture the causal effects of enhancers on expression, notably in medium to long distances and particularly for highly expressed promoters. More generally, the predicted impact of distal elements on gene expression predictions is small and the ability to correctly integrate long-range information is significantly more limited than the receptive fields of the models suggest. This is likely caused by the escalating class imbalance between actual and candidate regulatory elements as distance increases. Conclusions Our results suggest that sequence-based models have advanced to the point that in silico study of promoter regions and promoter variants can provide meaningful insights and we provide practical guidance on how to use them. Moreover, we foresee that it will require significantly more and particularly new kinds of data to train models accurately accounting for distal elements.

Objective To profile the genome-wide distribution of H3K27ac-marked enhancers in gastric intestinal metaplasia (IM) tissues and to investigate the potential regulatory mechanism of enhancers on IM. Methods IM tissues from 41 patients and normal gastric mucosa from 21 healthy individuals were collected for analysis using Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing and RNA sequencing. The number and signal intensity of H3K27ac modifications in the 2 tissues were compared to reveal the reprogramming characteristics of the enhancers in the IM tissues. Meanwhile, the underlying regulatory mechanism of enhancer-associated transcription factors on the expression of IM-related genes were investigated. Results As compared with normal gastric mucosa, the number and signal intensity of H3K27ac modifications were significantly elevated in the IM tissues, and the reprogramming areas were mainly located in the enhancers. The enhancers with greatly increased activity accounted for 92.4% of all variant enhancer loci in IM, which improved the expression of many intestinal epithelium-related genes, leading to altered phenotypes and biological properties of metaplastic mucous cells. The binding motifs of 14 transcription factors including CDX2 were identified by enrichment analysis in the enhancer loci. These transcription factors were up-regulated in the IM tissues and thus constituted a transcriptional regulatory network, and may play an important role in enhancer activating IM-related genes. Conclusion Through integration analysis of epigenetic and transcriptomic sequencing, enhancer reprogramming is identified as a critical epigenetic modification feature in gastric IM. Multiple transcription factors like CDX2 might be recruited to the enhancer loci, and then generate a regulatory network to synergistically up-regulate the expression of IM-related genes.

Abstract Background Enhancers are essential in defining cell fates through the control of cell-type-specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers including the monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27. Results Here we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early differentiation of mouse embryonic stem cells. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1 but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. However, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of these chromatin events from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers. Conclusions Collectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription.

Abstract Background Transcriptional enhancers are essential for gene regulation, but how these regulatory elements are best defined remains a significant unresolved question. Traditional definitions rely on activity-based criteria such as reporter gene assays, while more recently, biochemical assays based on chromatin-level phenomena such as chromatin accessibility, histone modifications, and localized RNA transcription have gained prominence. Results We examine here whether these two types of definitions, activity-based and chromatin-based, effectively identify the same sets of sequences. We find that, concerningly, the overlap between the two groups is strikingly limited. Few of the data sets we compared displayed statistically significant overlap, and even for those, the degree of overlap was typically small (below 40% of sequences). Moreover, a substantial batch effect was observed in which experiment set rather than experimental method was a primary driver of whether or not chromatin-defined enhancers showed a strong overlap with reporter gene-defined enhancers. Conclusions Our results raise important questions as to the appropriateness of both old and new enhancer definitions, and suggest that new approaches are required to reconcile the poor agreement among existing methods for defining enhancers.

Enhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene's endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. We hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using the Drosophila melanogaster eve n-skipped stripe 2 enhancer as a case study, we tested the effect of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. We found that, in contrast to the misexpression caused by the gt-2 binding site deletion in the minimal enhancer, the same gt-2 binding site deletion in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Deleting the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. Our results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context.

Accumulating evidence suggests that dysregulation of transcriptional enhancers plays a significant role in cancer pathogenesis. Herein, we performed a genome-wide discovery of enhancer elements in colorectal cancer (CRC). We identified PVT1 locus as a previously unrecognized transcriptional regulator in CRC with a significantly high enhancer activity, which ultimately was responsible for regulating the expression of MYC oncogene. High expression of the PVT1 long-non-coding RNA (lncRNA) transcribed from the PVT1 locus was associated with poor survival among patients with stage II and III CRCs (p

Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

Hox genes encode transcription factors whose roles in patterning animal body plans during embryonic development are well-documented. Multiple studies demonstrate that Hox genes continue to act in adult cells, in normal differentiation, in regenerative processes, and, with abnormal expression, in diverse types of cancers. However, surprisingly little is known about the regulatory mechanisms that govern Hox gene expression in specific cell types, as they differentiate during late embryonic development, and in the adult organism. The murine Hoxc8 gene determines the identity of multiple skeletal elements in the lower thoracic and lumbar region and continues to play a role in the proliferation and differentiation of cells in cartilage as the skeleton matures. This study was undertaken to identify regulatory elements in the Hoxc8 gene that control transcriptional activity, specifically in cartilage-producing chondrocytes. We report that an enhancer comprising two 416 and 224 bps long interacting DNA elements produces reporter gene activity when assayed on a heterologous transcriptional promoter in transgenic mice. This enhancer is distinct in spatial, temporal, and molecular regulation from previously identified regulatory sequences in the Hoxc8 gene that control its expression in early development. The identification of a tissue-specific Hox gene regulatory element now allows mechanistic investigations into Hox transcription factor expression and function in differentiating cell types and adult tissues and to specifically target these cells during repair processes and regeneration.

Abstract Lung cancer is the leading cause of cancer‐related deaths worldwide. DNA methylation has been recognized as a potential biomarker for lung cancer diagnosis. Most reported DNA methylation biomarkers focus on promoter regions, leaving enhancer regions largely unexplored. Here, we employed multiomics data to identify DNA methylation biomarkers for non‐small‐cell lung cancer diagnosis. Especially, we linked enhancers to their target genes using long‐range chromatin interactions for biomarker prediction. We discovered two sets of DNA methylation biomarkers: one in promoter regions and another in enhancer regions. Both achieved extremely high sensitivity and specificity in five independent validation data sets. Compared with three other reported biomarker sets, both groups in our study demonstrated better and more robust classification performance in validation data sets. These novel DNA methylation biomarkers may improve lung cancer screening and ultimately contribute to improved clinical outcomes for patients. To our best knowledge, this is the first time to introduce chromatin interactions to link enhancers to their targets for biomarker study, highlighting the potential of enhancer methylation as a complement to current promoter‐based DNA methylation biomarkers. Our approach may have potential applications for other cancer types and could be a valuable direction for future research in the field of cancer biomarker discovery.

C-reactive protein (CRP) is an evolutionary highly conserved protein. Like humans, CRP acts as a major acute phase protein in pigs. While CRP regulatory mechanisms have been extensively studied in humans, little is known about the molecular mechanisms that control pig CRP gene expression. The main goal of the present work was to study the regulatory mechanisms and identify functional genetic variants regulating CRP gene expression and CRP blood levels in pigs. The characterization of the porcine CRP proximal promoter region revealed a high level of conservation with both cow and human promoters, sharing binding sites for transcription factors required for CRP expression. Through genome-wide association studies and fine mapping, the most associated variants with both mRNA and protein CRP levels were localized in a genomic region 39.3 kb upstream of CRP. Further study of the region revealed a highly conserved putative enhancer that contains binding sites for several transcriptional regulators such as STAT3, NF-kB or C/EBP-β. Luciferase reporter assays showed the necessity of this enhancer-promoter interaction for the acute phase induction of CRP expression in liver, where differences in the enhancer sequences significantly modified CRP activity. The associated polymorphisms disrupted the putative binding sites for HNF4α and FOXA2 transcription factors. The high correlation between HNF4α and CRP expression levels suggest the participation of HNF4α in the regulatory mechanism of porcine CRP expression through the modification of its binding site in liver. Our findings determine, for the first time, the relevance of a distal regulatory element essential for the acute phase induction of porcine CRP in liver and identify functional polymorphisms that can be included in pig breeding programs to improve immunocompetence.

Abstract Enhancers are essential cis-acting regulatory elements that determine cell identity and tumor progression. Enhancer function is dependent on the physical interaction between the enhancer and its target promoter inside its local chromatin environment. Enhancer reprogramming is an important mechanism in cancer pathogenesis and can be driven by both cis and trans factors. Super enhancers are acquired at oncogenes in numerous cancer types and represent potential targets for cancer treatment. BET and CDK inhibitors act through mechanisms of enhancer function and have shown promising results in therapy for various types of cancer. Genome editing is another way to reprogram enhancers in cancer treatment. The relationship between enhancers and cancer has been revised by several authors in the past few years, which mainly focuses on the mechanisms by which enhancers can impact cancer. Here, we emphasize SE’s role in cancer pathogenesis and the new therapies involving epigenetic regulators (BETi and CDKi). We suggest that understanding mechanisms of activity would aid clinical success for these anti-cancer agents.

The nutmeg seed oil contains myristicin which can absorb UV rays. Nanoemulsion with isopropyl myristate can be used to increase the activity of sunscreen. This study aimed to determine the physical characteristics and effectivity of nutmeg oil (NMO) nanoemulsion with isopropyl myristate as an enhancer. The nanoemulsion was made with 6.4% NMO and variations of isopropyl myristate 1% (FI), 3% (FII), and 5% (FIII). The nanoemulsions were evaluated for physical characteristics such as appearance, pH, viscosity, transmittance percentage, particle size, and polydispersity index (PI). The in vitro SPF value was tested using a spectrophotometer, and sunscreen effectivity was determined by the Minimum Erythemal Dose (MED) value. Data obtained were analyzed using one-way ANOVA. This study showed that the colour of the NMO nanoemulsion was light yellow and clear, with a transmittance percentage of 97.5-97.9%. The pH FI, FII, and FIII results were 6.79±0.03, 6.84±0.04, and 7.02±0.03. The viscosity was 1.63 ± 0.81 to 1.82 ± 0.85 d.Pas with Newtonian rheology. The particle size was 14.3 ± 1.41 nm to 16.0 ± 2.13 nm, with PI less than 0.5. SPF value were 16.34 ± 5.50 (FI); 16.70 ± 5.20 (FII) and 17.80 ± 3.20 (FIII). MED values were 220.5 ± 6.34 (FI), 225.4 ±5.41 (FII), and 240.2 ±3.45 (FIII) minutes. The MED value showed that FIII was significantly different from FI and FII. Isopropyl myristate at 5% in nanoemulsion increases the effectivity of sunscreen.

IFNß is a single-copy gene without an intron. Under normal circumstances, it shows low or no expression in cells. It is upregulated only when the body needs it or is stimulated. Stimuli bind to the pattern recognition receptors (PRRs) and pass via various signaling pathways to several basic transcriptional regulators, such as IRFs, NF-κB, and AP-1. Subsequently, the transcriptional regulators enter the nucleus and bind to regulatory elements of the IFNß promoter. After various modifications, the position of the nucleosome is altered and the complex is assembled to activate the IFNß expression. However, IFNß regulation involves a complex network. For the study of immunity and diseases, it is important to understand how transcription factors bind to regulatory elements through specific forms, which elements in cells are involved in regulation, what regulation occurs during the assembly of enhancers and transcription complexes, and the possible regulatory mechanisms after transcription. Thus, this review focuses on the various regulatory mechanisms and elements involved in the activation of IFNß expression. In addition, we discuss the impact of this regulation in biology. [ABSTRACT FROM AUTHOR]

Chromatin regulation is an important gene expression/regulation system, but little is known about how it affects nitrogen metabolism in Saccharomyces cerevisiae. A previous study demonstrated the regulatory role of the chromatin regulator Ahc1p on multiple key genes of nitrogen metabolism in S. cerevisiae , but the regulatory mechanism remains unknown. In this study, multiple key nitrogen metabolism genes directly regulated by Ahc1p were identified, and the transcription factors interacting with Ahc1p were analyzed. It was ultimately found that Ahc1p may regulate some key nitrogen metabolism genes in two ways. First, Ahc1p acts as a co-factor and is recruited with transcription factors such as Rtg3p or Gcr1p to facilitate transcription complex binding to target gene core promoters and promote transcription initiation. Second, Ahc1p binds at enhancers to promote the transcription of target genes in concert with transcription factors. This study furthers the understanding of the regulatory network of nitrogen metabolism in S. cerevisiae from an epigenetic perspective. • Ahc1p regulates multiple nitrogen metabolism genes, but the mechanism is unclear. • Ahc1p directly regulates nitrogen-related genes by binding to their promoters. • Ahc1p acts as a transcription cofactor to promote gene transcription initiation. • Ahc1p may binds at an enhancer to enhance target gene transcription. • This study furthers the understanding of nitrogen metabolism in S. cerevisiae. [ABSTRACT FROM AUTHOR]

Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain (IgH) locus under control of its 3’ cis-regulatory region (3’RR). The 3’RR is itself transcribed and can undergo “locus suicide recombination” (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined. Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3’RR. Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies. Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro, this study suggests that LSR may contribute to the elimination of self-reactive B cells. [ABSTRACT FROM AUTHOR]

Background: Glioma stem cells (GSCs) are responsible for glioma recurrence and drug resistance, yet the mechanisms underlying their maintenance remains unclear. This study aimed to identify enhancer-controlled genes involved in GSCs maintenance and elucidate the mechanisms underlying their regulation. Methods: We analyzed RNA-seq data and H3K27ac ChIP-seq data from GSE119776 to identify differentially expressed genes and enhancers, respectively. Gene Ontology analysis was performed for functional enrichment. Transcription factors were predicted using the Toolkit for Cistrome Data Browser. Prognostic analysis and gene expression correlation was conducted using the Chinese Glioma Genome Atlas (CGGA) data. Two GSC cell lines, GSC-A172 and GSC-U138MG, were isolated from A172 and U138MG cell lines. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to detect H3K27ac of enhancers, and binding of E2F4 to target gene enhancers. Western blot was used to analyze protein levels of p-ATR and γH2AX. Sphere formation, limiting dilution and cell growth assays were used to analyze GSCs growth and self-renewal. Results: We found that upregulated genes in GSCs were associated with ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) pathway activation, and that seven enhancer-controlled genes related to ATR pathway activation (LIN9, MCM8, CEP72, POLA1, DBF4, NDE1, and CDKN2C) were identified. Expression of these genes corresponded to poor prognosis in glioma patients. E2F4 was identified as a transcription factor that regulates enhancer-controlled genes related to the ATR pathway activation, with MCM8 having the highest hazard ratio among genes positively correlated with E2F4 expression. E2F4 bound to MCM8 enhancers to promote its transcription. Overexpression of MCM8 partially restored the inhibition of GSCs self-renewal, cell growth, and the ATR pathway activation caused by E2F4 knockdown. Conclusion: Our study demonstrated that E2F4-mediated enhancer activation of MCM8 promotes the ATR pathway activation and GSCs characteristics. These findings offer promising targets for the development of new therapies for gliomas. [ABSTRACT FROM AUTHOR]

Simple Summary: Carbonic anhydrases (CAs) play a crucial role in numerous physiological and pathological processes in animals. Specifically, carbonic anhydrase VB (CA5B) is integral to both gluconeogenesis and lipogenesis. Structural variations, particularly retrotransposon insertion polymorphisms (RIPs), can contribute significantly to these processes. When an RIP is inserted into a genome region containing functional elements (such as promoters, enhancers, and insulators), it can have an impact on both the expression and phenotypic changes of target genes. In this study, we identified an RIP located in the first intron of the CA5B gene, inserted by the SINE element, which enhanced the core promoter activity of the target gene. Through experimental analysis and growth data provided by the pig breeding farm (Anhui academy of agriculture sciences), we found that the SINE insertion improved the expression of the porcine CA5B gene in adipose tissue. Additionally, the number of (ATTT) repeats within the SINE insertion at this site was inconsistent across breeds and individuals. This study provides insight into the role RIPs play in genetic variation of CA5B genes and its phenotypic expression in pigs. Transposons are genetic elements that are present in mammalian genomes and occupy a large proportion of the pig genome, with retrotransposons being the most abundant. In a previous study, it was found that a SINE retrotransposon was inserted in the 1st intron of the CA5B gene in pigs, and the present study aimed to investigate the SINE insertion polymorphism in this gene in different pig breeds. Polymerase chain reaction (PCR) was used to confirm the polymorphism in 11 pig breeds and wild boars), and it was found that there was moderate polymorphism information content in 9 of the breeds. Further investigation in cell experiments revealed that the 330 bp SINE insertion in the RIP-CA5B site promoted expression activity in the weak promoter region of this site. Additionally, an enhancer verification vector experiment showed that the 330 bp SINE sequence acted as an enhancer on the core promoter region upstream of the CA5B gene region. The expression of CA5B in adipose tissue (back fat and leaf fat) in individuals with the (SINE+/+) genotype was significantly higher than those with (SINE+/−) and (SINE−/−) genotypes. The association analysis revealed that the (SINE+/+) genotype was significantly associated with a higher back fat thickness than the (SINE−/−) genotype. Moreover, it was observed that the insertion of SINE at the RIP-CA5B site carried ATTT repeats, and three types of (ATTT) repeats were identified among different individuals/breeds (i.e., (ATTT)4, (ATTT)6 and (ATTT)9). Overall, the study provides insights into the genetic basis of adipose tissue development in pigs and highlights the role of a SINE insertion in the CA5B gene in this process. [ABSTRACT FROM AUTHOR]

Background: High expression of inhibitor of DNA binding 1 (ID1) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation in regulating ID1 transcription is limited. Methods: Immunohistochemistry (IHC), quantitative RT-PCR (RT-qPCR) and Western blotting (WB) were used to determine the expression of ID1. CRISPR-Cas9 was used to generate ID1 or enhancer E1 knockout cell lines. Dual-luciferase reporter assay, chromosome conformation capture assay and ChIP-qPCR were used to determine the active enhancers of ID1. Cell Counting Kit 8, colony-forming, transwell assays and tumorigenicity in nude mice were used to investigate the biological functions of ID1 and enhancer E1. Results: Human CRC tissues and cell lines expressed a higher level of ID1 than normal controls. ID1 promoted CRC cell proliferation and colony formation. Enhancer E1 actively regulated ID1 promoter activity. Signal transducer and activator of transcription 3 (STAT3) bound to ID1 promoter and enhancer E1 to regulate their activity. The inhibitor of STAT3 Stattic attenuated ID1 promoter and enhancer E1 activity and the expression of ID1. Enhancer E1 knockout down-regulated ID1 expression level and cell proliferation in vitro and in vivo. Conclusions: Enhancer E1 is positively regulated by STAT3 and contributes to the regulation of ID1 to promote CRC cell progression and might be a potential target for anti-CRC drug studies. [ABSTRACT FROM AUTHOR]

The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of the MGMT promoter. MGMT promoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation between MGMT promoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status of MGMT enhancers is associated with MGMT promoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenic MGMT enhancers. For CpGs in an enhancer located 560 kb upstream of the MGMT promoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated with MGMT promoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01–03 and CpGs 09–13 was associated with the longer OS of the GBM patients. Our findings indicate an association between MGMT enhancer methylation and MGMT promoter methylation, MGMT protein expression, and/or OS. [ABSTRACT FROM AUTHOR]

Recently, the myelin proteolipid protein gene (Plp1) was shown to be expressed in the glia of the enteric nervous system (ENS) in mouse. However, beyond this, notmuch is known about its expression in the intestine. To address thismatter, we investigated Plp1 expression at themRNA and protein levels in the intestine of mice at different ages (postnatal days 2, 9, 21, and 88). In this study, we show that Plp1 expression preferentially occurs during early postnatal development, primarily as the DM20 isoform. Western blot analysis indicated that DM20 migrated according to its formula weight when isolated fromthe intestine. However, mobilities of both PLP and DM20 were faster than expected when procured from the brain. The 6.2hPLP(+)Z/FL transgene, which uses the first half of the human PLP1 gene to drive expression of a lacZ reporter gene, recapitulated the developmental pattern observed with the native gene in the intestine, indicating that it can be used as a proxy for Plp1 gene expression. As such, the relative levels of b-galactosidase (b-gal) activity emanating from the 6.2hPLP(+)Z/FL transgene suggest that Plp1 expression is highest in the duodenum, and decreases successively along the segments, toward the colon. Moreover, removal of the wmN1 enhancer region from the transgene (located within Plp1 intron 1) resulted in a dramatic reduction in both transgene mRNA levels and b-gal activity in the intestine, throughout development, suggesting that this region contains a regulatory element crucial for Plp1 expression. This is consistent with earlier studies in both the central and peripheral nervous systems, indicating that it may be a common (if not universal) means by which Plp1 gene expression is governed. [ABSTRACT FROM AUTHOR]

Background: As parts of the cis-regulatory mechanism of the human genome, interactions between distal enhancers and proximal promoters play a crucial role. Enhancers, promoters, and enhancer-promoter interactions (EPIs) can be detected using many sequencing technologies and computation models. However, a systematic review that summarizes these EPI identification methods and that can help researchers apply and optimize them is still needed. Results: In this review, we first emphasize the role of EPIs in regulating gene expression and describe a generic framework for predicting enhancer-promoter interaction. Next, we review prediction methods for enhancers, promoters, loops, and enhancer-promoter interactions using different data features that have emerged since 2010, and we summarize the websites available for obtaining enhancers, promoters, and enhancer-promoter interaction datasets. Finally, we review the application of the methods for identifying EPIs in diseases such as cancer. Conclusions: The advance of computer technology has allowed traditional machine learning, and deep learning methods to be used to predict enhancer, promoter, and EPIs from genetic, genomic, and epigenomic features. In the past decade, models based on deep learning, especially transfer learning, have been proposed for directly predicting enhancer-promoter interactions from DNA sequences, and these models can reduce the parameter training time required of bioinformatics researchers. We believe this review can provide detailed research frameworks for researchers who are beginning to study enhancers, promoters, and their interactions. [ABSTRACT FROM AUTHOR]

Programmed DNA double-strand break (DSB) formation is a crucial step in meiotic recombination, yet techniques for high-efficiency and precise mapping of the 3′ ends of DSBs are still in their infancy. Here, we report a novel technique, named DNA End tailing and sequencing (DEtail-seq), which can directly and ultra-efficiently characterize the 3′ ends of meiotic DSBs with near single-nucleotide resolution in a variety of species, including yeast, mouse, and human. We find that the 3′ ends of meiotic DSBs are stable without significant resection in budding yeast. Meiotic DSBs are strongly enriched in de novo H3K4me3 peaks in the mouse genome at leptotene stage. We also profile meiotic DSBs in human and find DSB hotspots are enriched near the common fragile sites during human meiosis, especially at CCCTC-binding factor (CTCF)-associated enhancers. Therefore, DEtail-seq provides a powerful method to detect DSB ends in various species, and our results provide new insights into the distribution and regulation of meiotic DSB hotspots. [ABSTRACT FROM AUTHOR]

Breast cancer biology varies markedly among patients. Basal-like breast cancer is one of the most challenging subtypes to treat because it lacks effective therapeutic targets. Despite numerous studies on potential targetablemolecules in this subtype, few targets have shown promise. However, the present study revealed that FOXD1, a transcription factor that functions in both normal development andmalignancy, is associated with poor prognosis in basal-like breast cancer. We analyzed publicly available RNA sequencing data and conducted FOXD1-knockdown experiments, finding that FOXD1 maintains gene expression programs that contribute to tumor progression. We first conducted survival analysis of patients grouped via a Gaussian mixturemodel based on gene expression in basal-like tumors, finding that FOXD1 is a prognostic factor specific to this subtype. Then, our RNA sequencing and chromatin immunoprecipitation sequencing experiments using the basal-like breast cancer cell lines BT549 and Hs578T with FOXD1 knockdown revealed that FOXD1 regulates enhancer-gene programs related to tumor progression. These findings suggest that FOXD1 plays an important role in basal-like breast cancer progression and may represent a promising therapeutic target. [ABSTRACT FROM AUTHOR]

Rapid enhancer and slow promoter evolution have been demonstrated through comparative genomics. However, it is not clear how this information is encoded genetically and if this can be used to place evolution in a predictive context. Part of the challenge is that our understanding of the potential for regulatory evolution is biased primarily toward natural variation or limited experimental perturbations. Here, to explore the evolutionary capacity of promoter variation, we surveyed an unbiased mutation library for three promoters in Drosophila melanogaster. We found that mutations in promoters had limited to no effect on spatial patterns of gene expression. Compared to developmental enhancers, promoters are more robust to mutations and have more access to mutations that can increase gene expression, suggesting that their low activity might be a result of selection. Consistent with these observations, increasing the promoter activity at the endogenous locus of shavenbaby led to increased transcription yet limited phenotypic changes. Taken together, developmental promoters may encode robust transcriptional outputs allowing evolvability through the integration of diverse developmental enhancers. This article is part of the theme issue 'Interdisciplinary approaches to predicting evolutionary biology'. [ABSTRACT FROM AUTHOR]

Eukaryotic non-coding regulatory features contribute significantly to cellular plasticity which on aberration leads to cellular malignancy. Enhancers are cis-regulatory elements that contribute to the development of resistance to endocrine therapy in estrogen receptor (ER)-positive breast cancer leading to poor clinical outcome. ER is vital for therapeutic targets in ER-positive breast cancer. Here, we review and report the different regulatory features present on ER with the objective to delineate potential mechanisms which may contribute to development of resistance. The UCSC Genome Browser, data mining, and bioinformatics tools were used to review enhancers, transcription factors (TFs), histone marks, long non-coding RNAs (lncRNAs), and variants residing in the non-coding region of the ER gene. We report 7 enhancers, 3 of which were rich in TF-binding sites and histone marks in a cell line-specific manner. Furthermore, some enhancers contain estrogen resistance variants and sites for lncRNA. Our review speculates putative models suggesting potential aberrations in gene regulation and expression if these regulatory landscapes and assemblies are altered. This review gives an interesting perspective in designing integrated in vitro studies including non-coding elements to study development of endocrine resistance in ER-positive breast cancer. [ABSTRACT FROM AUTHOR]

Regulatory T (Treg) cells that infiltrate human tumors exhibit stronger immunosuppressive activity compared to peripheral blood Treg cells (PBTRs), thus hindering the induction of effective antitumor immunity. Previous transcriptome studies have identified a set of genes that are conserved in tumor-infiltrating Treg cells (TITRs). However, epigenetic profiles of TITRs have not yet been completely deciphered. Here, we employed ATAC-seq and CUT&Tag assays to integrate transcriptome profiles and identify functional regulatory elements in TITRs. We observed a global difference in chromatin accessibility and enhancer landscapes between TITRs and PBTRs. We identified two types of active enhancer formation in TITRs. The H3K4me1-predetermined enhancers are poised to be activated in response to tumor microenvironmental stimuli. We found that AP-1 family motifs are enriched at the enhancer regions of TITRs. Finally, we validated that c-Jun binds at regulatory regions to regulate signature genes of TITRs and AP-1 is required for Treg cells activation in vitro. High c-Jun expression is correlated with poor survival in human HCC. Overall, our results provide insights into the mechanism of AP-1-mediated activation of TITRs and can hopefully be used to develop new therapeutic strategies targeting TITRs in liver cancer treatment. [ABSTRACT FROM AUTHOR]

Genome-wide association studies (GWASs) have repeatedly reported multiple non-coding single-nucleotide polymorphisms (SNPs) at 2p14 associated with rheumatoid arthritis (RA), but their functional roles in the pathological mechanisms of RA remain to be explored. In this study, we integrated a series of bioinformatics and functional experiments and identified three intronic RA SNPs (rs1876518, rs268131, and rs2576923) within active enhancers that can regulate the expression of SPRED2 directly. At the same time, SPRED2 and ACTR2 influence each other as a positive feedback signal amplifier to strengthen the protective role in RA by inhibiting the migration and invasion of rheumatoid fibroblast-like synoviocytes (FLSs). In particular, the transcription factor CEBPB preferentially binds to the rs1876518-T allele to increase the expression of SPRED2 in FLSs. Our findings decipher the molecular mechanisms behind the GWAS signals at 2p14 for RA and emphasize SPRED2 as a potential candidate gene for RA, providing a potential target and direction for precise treatment of RA. [Display omitted] Our findings reveal that enhancer variants on chromosome 2p14 regulating SPRED2 and ACTR2 act as a signal amplifier to protect against rheumatoid arthritis, providing a potential target for precise therapy of rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Atypical teratoid rhabdoid tumor (ATRT) is a rare, highly malignant central nervous system cancer arising in infants and younger children, historically considered to be homogeneous, monogenic, and incurable. Recent use of intensified therapies has modestly improved survival for ATRT; however, a majority of patients will still succumb to their disease. While ATRTs almost universally exhibit loss of SMARCB1 (BAF47/INI1/SNF5), recent whole genome, transcriptome, and epigenomic analyses of large cohorts reveal previously underappreciated molecular heterogeneity. These discoveries provide novel insights into how SMARCB1 loss drives oncogenesis and confer specific therapeutic vulnerabilities, raising exciting prospects for molecularly stratified treatment for patients with ATRT.

Aim: To provide a comprehensive understanding of gene regulatory networks in the developing human brain and a foundation for interpreting pathogenic deregulation. Materials & methods: We generated reference epigenomes and transcriptomes of dissected brain regions and primary neural progenitor cells (NPCs) derived from cortical and ganglionic eminence tissues of four normal human fetuses. Results: Integration of these data across developmental stages revealed a directional increase in active regulatory states, transcription factor activities and gene transcription with developmental stage. Consistent with differences in their biology, NPCs derived from cortical and ganglionic eminence regions contained common, region specific, and gestational week specific regulatory states. Conclusion: We provide a high-resolution regulatory network for NPCs from different brain regions as a comprehensive reference for future studies.

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

BackgroundWilson disease (WD) is an autosomal recessive disease caused by mutations in ATP7B encoding a copper transporter. Consequent copper accumulation results in a variable WD clinical phenotype involving hepatic, neurologic, and psychiatric symptoms, without clear genotype-phenotype correlations. The goal of this study was to analyze alterations in DNA methylation at the whole-genome level in liver and blood from patients with WD to investigate epigenomic alterations associated with WD diagnosis and phenotype. We used whole-genome bisulfite sequencing (WGBS) to examine distinct cohorts of WD subjects to determine whether DNA methylation could differentiate patients from healthy subjects and subjects with other liver diseases and distinguish between different WD phenotypes.ResultsWGBS analyses in liver identified 969 hypermethylated and 871 hypomethylated differentially methylated regions (DMRs) specifically identifying patients with WD, including 18 regions with genome-wide significance. WD-specific liver DMRs were associated with genes enriched for functions in folate and lipid metabolism and acute inflammatory response and could differentiate early from advanced fibrosis in WD patients. Functional annotation revealed that WD-hypermethylated liver DMRs were enriched in liver-specific enhancers, flanking active liver promoters, and binding sites of liver developmental transcription factors, including Hepatocyte Nuclear Factor 4 alpha (HNF4A), Retinoid X Receptor alpha (RXRA), Forkhead Box A1 (FOXA1), and FOXA2. DMRs associated with WD progression were also identified, including 15 with genome-wide significance. However, WD DMRs in liver were not related to large-scale changes in proportions of liver cell types. DMRs detected in blood differentiated WD patients from healthy and disease control subjects, and distinguished between patients with hepatic and neurologic WD manifestations. WD phenotype DMRs corresponded to genes enriched for functions in mental deterioration, abnormal B cell physiology, and as members of the polycomb repressive complex 1 (PRC1). 44 DMRs associated with WD phenotype tested in a small validation cohort had a predictive value of 0.9.ConclusionsWe identified a disease-mechanism relevant epigenomic signature of WD that reveals new insights into potential biomarkers and treatments for this complex monogenic disease.

Epigenomic regulation and lineage-specific gene expression act in concert to drive cellular differentiation, but the temporal interplay between these processes is largely unknown. Using neural induction from human pluripotent stem cells (hPSCs) as a paradigm, we interrogated these dynamics by performing RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and assay for transposase accessible chromatin using sequencing (ATAC-seq) at seven time points during early neural differentiation. We found that changes in DNA accessibility precede H3K27ac, which is followed by gene expression changes. Using massively parallel reporter assays (MPRAs) to test the activity of 2,464 candidate regulatory sequences at all seven time points, we show that many of these sequences have temporal activity patterns that correlate with their respective cell-endogenous gene expression and chromatin changes. A prioritization method incorporating all genomic and MPRA data further identified key transcription factors involved in driving neural fate. These results provide a comprehensive resource of genes and regulatory elements that orchestrate neural induction and illuminate temporal frameworks during differentiation.

Abstract Enhancers are small regions of DNA that bind to proteins, which enhance the transcription of genes. The enhancer may be located upstream or downstream of the gene. It is not necessarily close to the gene to be acted on, because the entanglement structure of chromatin allows the positions far apart in the sequence to have the opportunity to contact each other. Therefore, identifying enhancers and their strength is a complex and challenging task. In this article, a new prediction method based on deep learning is proposed to identify enhancers and enhancer strength, called iEnhancer-DCLA. Firstly, we use word2vec to convert k-mers into number vectors to construct an input matrix. Secondly, we use convolutional neural network and bidirectional long short-term memory network to extract sequence features, and finally use the attention mechanism to extract relatively important features. In the task of predicting enhancers and their strengths, this method has improved to a certain extent in most evaluation indexes. In summary, we believe that this method provides new ideas in the analysis of enhancers.

Aberrant DNA methylation is an epigenetic hallmark of malignant tumours. The DNA methylation level is regulated by not only DNA methyltransferases (DNMTs) but also Ten-Eleven Translocation (TET) family proteins. However, the exact role of TET genes in breast cancer remains controversial. Here, we uncover that the ERα-positive breast cancer patients with high TET2 mRNA expression had better overall survival rates. Consistently, knockout of TET2 promotes the tumorigenesis of ERα-positive MCF7 breast cancer cells. Mechanistically, TET2 loss leads to aberrant DNA methylation (gain of 5mC) at a large proportion of enhancers, accompanied by significant reduction in H3K4me1 and H3K27ac enrichment. By analysing the epigenetically reprogrammed enhancers, we identify oestrogen responsive element (ERE) as one of the enriched motifs of transcriptional factors. Importantly, TET2 loss impairs 17beta-oestradiol (E2)-induced transcription of the epigenetically reprogrammed EREs-associated genes through attenuating the binding of ERα. Taken together, these findings shed light on our understanding of the epigenetic mechanisms underlying the enhancer reprogramming during breast cancer pathogenesis.