Copper-binding proteins and exonic splicing enhancers and silencers.

Eukaryotic DNA codes not only for proteins but contains a wealth of information required for accurate splicing of messenger RNA precursors and inclusion of constitutively or alternatively spliced exons in mature transcripts. This "auxiliary" splicing code has been characterized as exonic splicing enhancers and silencers (ESE and ESS). The exact interplay between protein and splicing codes is, however, poorly understood. Here, we show that exons encoding copper-coordinating amino acids in human cuproproteins lack ESEs and/or have an excess of ESSs, yet RNA sequencing and expressed sequence tags data show that they are more efficiently included in mature transcripts by the splicing machinery than average exons. Their largely constitutive inclusion in messenger RNA is facilitated by stronger splice sites, including polypyrimidine tracts, consistent with an important role of the surrounding intron architecture in ensuring high expression of metal-binding residues during evolution. ESE/ESS profiles of codons and entire exons that code for copper-coordinating residues were very similar to those encoding residues that coordinate zinc but markedly different from those that coordinate calcium. Together, these results reveal how the traditional and auxiliary splicing motifs responded to constraints of metal coordination in proteins.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Pervasive structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs.

Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.

Super enhancer-associated circRNA-circLrch3 regulates hypoxia-induced pulmonary arterial smooth muscle cells pyroptosis by formation of R-loop with host gene.

BACKGROUND: Pulmonary hypertension (PH) is a complex vascular disorder, characterized by pulmonary vessel remodeling and perivascular inflammation. Pulmonary arterial smooth muscle cells (PASMCs) pyroptosis is a novel pathological mechanism implicated of pulmonary vessel remodeling. However, the involvement of circRNAs in the process of pyroptosis and the underlying regulatory mechanisms remain inadequately understood. METHODS: Western blotting, PI staining and LDH release were used to explore the role of circLrch3 in PASMCs pyroptosis. Moreover, S9.6 dot blot and DRIP-PCR were used to assess the formation of R-loop between circLrch3 and its host gene Lrch3. Chip-qPCR were used to evaluate the mechanism of super enhancer-associated circLrh3, which is transcriptionally activated by the transcription factor Tbx2. RESULTS: CircLrch3 was markedly upregulated in hypoxic PASMCs. CircLrch3 knockdown inhibited hypoxia induced PASMCs pyroptosis in vivo and in vitro. Mechanistically, circLrch3 can form R-loop with host gene to upregulate the protein and mRNA expression of Lrch3. Furthermore, super enhancer interacted with the Tbx2 at the Lrch3 promoter locus, mediating the augmented transcription of circLrch3. CONCLUSION: Our findings clarify the role of a super enhancer-associated circLrch3 in the formation of R-loop with the host gene Lrch3 to modulate pyroptosis in PASMCs, ultimately promoting the development of PH.

Super enhancer lncRNAs: a novel hallmark in cancer.

Super enhancers (SEs) consist of clusters of enhancers, harboring an unusually high density of transcription factors, mediator coactivators and epigenetic modifications. SEs play a crucial role in the maintenance of cancer cell identity and promoting oncogenic transcription. Super enhancer lncRNAs (SE-lncRNAs) refer to either transcript from SEs locus or interact with SEs, whose transcriptional activity is highly dependent on SEs. Moreover, these SE-lncRNAs can interact with their associated enhancer regions in cis and modulate the expression of oncogenes or key signal pathways in cancers. Inhibition of SEs would be a promising therapy for cancer. In this review, we summarize the research of SE-lncRNAs in different kinds of cancers so far and decode the mechanism of SE-lncRNAs in carcinogenesis to provide novel ideas for the cancer therapy.

The dynamic landscape of enhancer-derived RNA during mouse early embryo development.

Enhancer-derived RNAs (eRNAs) play critical roles in diverse biological processes by facilitating their target gene expression. However, the abundance and function of eRNAs in early embryos are not clear. Here, we present a comprehensive eRNA atlas by systematically integrating publicly available datasets of mouse early embryos. We characterize the transcriptional and regulatory network of eRNAs and show that different embryo developmental stages have distinct eRNA expression and regulatory profiles. Paternal eRNAs are activated asymmetrically during zygotic genome activation (ZGA). Moreover, we identify an eRNA, MZGAe1, which plays an important function in regulating mouse ZGA and early embryo development. MZGAe1 knockdown leads to a developmental block from 2-cell embryo to blastocyst. We create an online data portal, M2ED2, to query and visualize eRNA expression and regulation. Our study thus provides a systematic landscape of eRNA and reveals the important role of eRNAs in regulating mouse early embryo development.

Differential Regulation of Male-Hormones-Related Enhancers Revealed by Chromatin Accessibility and Transcriptional Profiles in Pig Liver.

Surgical castration can effectively avoid boar taint and improve pork quality by removing the synthesis of androstenone in the testis, thereby reducing its deposition in adipose tissue. The expression of genes involved in testis-derived hormone metabolism was altered following surgical castration, but the upstream regulatory factors and underlying mechanism remain unclear. In this study, we systematically profiled chromatin accessibility and transcriptional dynamics in liver tissue of castrated and intact full-sibling Yorkshire pigs. First, we identified 897 differentially expressed genes and 6864 differential accessible regions (DARs) using RNA- and ATAC-seq. By integrating the RNA- and ATAC-seq results, 227 genes were identified, and a significant positive correlation was revealed between differential gene expression and the ATAC-seq signal. We constructed a transcription factor regulatory network after motif analysis of DARs and identified a candidate transcription factor (TF) SP1 that targeted the HSD3B1 gene, which was responsible for the metabolism of androstenone. Subsequently, we annotated DARs by incorporating H3K27ac ChIP-seq data, marking 2234 typical enhancers and 245 super enhancers involved in the regulation of all testis-derived hormones. Among these, four typical enhancers associated with HSD3B1 were identified. Furthermore, an in-depth investigation was conducted on the androstenone-related enhancers, and an androstenone-related mutation was identified in a newfound candidatetypical enhancer (andEN) with dual-luciferase assays. These findings provide further insights into how enhancers function as links between phenotypic and non-coding area variations. The discovery of upstream TF and enhancers of HSD3B1 contributes to understanding the regulatory networks of androstenone metabolism and provides an important foundation for improving pork quality.

Physical and functional interaction among Irf8 enhancers during dendritic cell differentiation.

The production of type 1 conventional dendritic cells (cDC1s) requires high expression of the transcription factor IRF8. Three enhancers at the Irf8 3' region function in a differentiation stage-specific manner. However, whether and how these enhancers interact physically and functionally remains unclear. Here, we show that the Irf8 3' enhancers directly interact with each other and contact the Irf8 gene body during cDC1 differentiation. The +56 kb enhancer, which functions from multipotent progenitor stages, activates the other 3' enhancers through an IRF8-dependent transcription factor program, that is, in trans. Then, the +32 kb enhancer, which operates in cDC1-committed cells, reversely acts in cis on the other 3' enhancers to maintain the high expression of Irf8. Indeed, mice with compound heterozygous deletion of the +56 and +32 kb enhancers are unable to generate cDC1s. These results illustrate how multiple enhancers cooperate to induce a lineage-determining transcription factor gene during cell differentiation.

Intergenic sequences harboring potential enhancer elements contribute to Axenfeld-Rieger syndrome by regulating PITX2.

Recent studies have uncovered that noncoding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese family with ARS and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between paired-like homeodomain transcription factor 2 (PITX2) and family with sequence similarity 241 member A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-Seq and real-time quantitative PCR revealed a significant reduction in Pitx2 gene expression in LOH-1-/- mice, while forkhead box C1 expression remained unchanged. ChIP-Seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a substantial downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594-111,399,691) that is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence that is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent.

Spatial enhancer activation influences inhibitory neuron identity during mouse embryonic development.

The mammalian telencephalon contains distinct GABAergic projection neuron and interneuron types, originating in the germinal zone of the embryonic basal ganglia. How genetic information in the germinal zone determines cell types is unclear. Here we use a combination of in vivo CRISPR perturbation, lineage tracing and ChIP-sequencing analyses and show that the transcription factor MEIS2 favors the development of projection neurons by binding enhancer regions in projection-neuron-specific genes during mouse embryonic development. MEIS2 requires the presence of the homeodomain transcription factor DLX5 to direct its functional activity toward the appropriate binding sites. In interneuron precursors, the transcription factor LHX6 represses the MEIS2-DLX5-dependent activation of projection-neuron-specific enhancers. Mutations of Meis2 result in decreased activation of regulatory enhancers, affecting GABAergic differentiation. We propose a differential binding model where the binding of transcription factors at cis-regulatory elements determines differential gene expression programs regulating cell fate specification in the mouse ganglionic eminence.

Identifying promoter and enhancer sequences by graph convolutional networks.

Identification of promoters, enhancers, and their interactions helps understand genetic regulation. This study proposes a graph-based semi-supervised learning model (GCN4EPI) for the enhancer-promoter classification problem. We adopt a graph convolutional network (GCN) architecture to integrate interaction information with sequence features. Nodes of the constructed graph hold word embeddings of DNA sequences while edges hold the Enhancer-Promoter Interaction (EPI) information. By means of semi-supervised learning, much less data (16%) and time are needed in model training. Comparisons on a benchmark dataset of six human cell lines show that the proposed approach outperforms the state-of-the-art methods by a large margin (10% higher F1 score) and has the fastest training time (up to 3 times). Moreover, GCN4EPI's performance on cross-cell line data is also better than the baselines (3% higher F1 score). Our qualitative analyses with graph explainability models prove that GCN4EPI learns from both text and graph structure. The results suggest that integrating interaction information with sequence features improves predictive performance and compensates for the number of training instances.

Osteoarthritis as an Enhanceropathy: Gene Regulation in Complex Musculoskeletal Disease.

PURPOSE OF REVIEW: Osteoarthritis is a complex and highly polygenic disease. Over 100 reported osteoarthritis risk variants fall in non-coding regions of the genome, ostensibly conferring functional effects through the disruption of regulatory elements impacting target gene expression. In this review, we summarise the progress that has advanced our knowledge of gene enhancers both within the field of osteoarthritis and more broadly in complex diseases. RECENT FINDINGS: Advances in technologies such as ATAC-seq have facilitated our understanding of chromatin states in specific cell types, bolstering the interpretation of GWAS and the identification of effector genes. Their application to osteoarthritis research has revealed enhancers as the principal regulatory element driving disease-associated changes in gene expression. However, tissue-specific effects in gene regulatory mechanisms can contribute added complexity to biological interpretation. Understanding gene enhancers and their altered activity in specific cell and tissue types is the key to unlocking the genetic complexity of osteoarthritis. The use of single-cell technologies in osteoarthritis research is still in its infancy. However, such tools offer great promise in improving our functional interpretation of osteoarthritis GWAS and the identification of druggable targets. Large-scale collaborative efforts will be imperative to understand tissue and cell-type specific molecular mechanisms underlying enhancer function in disease.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

Enhancer-promoter interactions become more instructive in the transition from cell-fate specification to tissue differentiation.

To regulate expression, enhancers must come in proximity to their target gene. However, the relationship between the timing of enhancer-promoter (E-P) proximity and activity remains unclear, with examples of uncoupled, anticorrelated and correlated interactions. To assess this, we selected 600 characterized enhancers or promoters with tissue-specific activity in Drosophila embryos and performed Capture-C in FACS-purified myogenic or neurogenic cells during specification and tissue differentiation. This enabled direct comparison between E-P proximity and activity transitioning from OFF-to-ON and ON-to-OFF states across developmental conditions. This showed remarkably similar E-P topologies between specified muscle and neuronal cells, which are uncoupled from activity. During tissue differentiation, many new distal interactions emerge where changes in E-P proximity reflect changes in activity. The mode of E-P regulation therefore appears to change as embryogenesis proceeds, from largely permissive topologies during cell-fate specification to more instructive regulation during terminal tissue differentiation, when E-P proximity is coupled to activation.

Yeast heterochromatin stably silences only weak regulatory elements by altering burst duration.

Transcriptional silencing in Saccharomyces cerevisiae involves the generation of a chromatin state that stably represses transcription. Using multiple reporter assays, a diverse set of upstream activating sequence enhancers and core promoters were investigated for their susceptibility to silencing. We show that heterochromatin stably silences only weak and stress-induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements, and the partial repression of these elements did not result in bistable expression states. Permutation analysis of enhancers and promoters indicates that both elements are targets of repression. Chromatin remodelers help specific regulatory elements to resist repression, most probably by altering nucleosome mobility and changing transcription burst duration. The strong enhancers/promoters can be repressed if silencer-bound Sir1 is increased. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating-type gene regulatory elements but not strong housekeeping gene regulatory sequences.

Increased enhancer-promoter interactions during developmental enhancer activation in mammals.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer-promoter physical proximity for developmental gene activation in mammals.

Super-enhancers: Implications in gastric cancer.

Gastric cancer (GC) is the fifth most prevalent malignancy and the third leading cause of cancer-related mortality globally. Despite intensive efforts to enhance the efficiencies of various therapeutics (chemotherapy, surgical interventions, molecular-targeted therapies, immunotherapies), the prognosis for patients with GC remains poor. This might be predominantly due to the limited understanding of the complicated etiology of GC. Importantly, epigenetic modifications and alterations are crucial during GC development. Super-enhancers (SEs) are a large cluster of adjacent enhancers that greatly activate transcription. SEs sustain cell-specific identity by enhancing the transcription of specific oncogenes. In this review, we systematically summarize how SEs are involved in GC development, including the SE landscape in GC, the SE target genes in GC, and the interventions related to SE functions for treating GC.