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Cellular homeostasis is constantly challenged by a myriad of extrinsic and intrinsic stressors. To mitigate the stress-induced damage, cells activate transient survival programs. The heat shock response (HSR) is an evolutionarily well-conserved survival program that is activated in response to proteotoxic stress. The HSR encompasses a dual regulation of transcription, characterized by rapid activation of genes encoding molecular chaperones and concomitant global attenuation of non-chaperone genes. Recent genome-wide approaches have delineated the molecular depth of stress-induced transcriptional reprogramming. The dramatic rewiring of gene and enhancer networks is driven by key transcription factors, including heat shock factors (HSFs), that together with chromatin-modifying enzymes remodel the 3D chromatin architecture, determining the selection of either gene activation or repression. Here, we highlight the current advancements of molecular mechanisms driving transcriptional reprogramming during acute heat stress. We also discuss the emerging implications of HSF-mediated stress signaling in the context of physiological and pathological conditions. Protein-damaging stress endangers cellular and organismal homeostasis. Cell survival depends on transcriptional rewiring, which is coordinated by chromatin-modifying enzymes and stress-responsive transcription factors. Apart from acute stress, proteostasis is also challenged in a variety of physiological and pathological conditions. Pessa et al. discuss the diversity of mechanisms regulating these processes. [ABSTRACT FROM AUTHOR]

Enhancers are located upstream or downstream of key deoxyribonucleic acid (DNA) sequences in genes and can adjust the transcription activity of neighboring genes. Identifying enhancers and determining their functions are important for understanding gene regulatory networks and expression regulatory mechanisms. However, traditional enhancer recognition relies on manual feature engineering, which is time-consuming and labor-intensive, making it difficult to perform large-scale recognition analysis. In addition, if the original dataset is too small, there is a risk of overfitting. In recent years, emerging methods, such as deep learning, have provided new insights for enhancing identification. However, these methods also present certain challenges. Deep learning models typically require a large amount of high-quality data, and data acquisition demands considerable time and resources. To address these challenges, in this paper, we propose a data-augmentation method based on generative adversarial networks to solve the problem of small datasets. Moreover, we used regularization methods such as weight decay to improve the generalizability of the model and alleviate overfitting. The Transformer encoder was used as the main component to capture the complex relationships and dependencies in enhancer sequences. The encoding layer was designed based on the principle of k-mers to preserve more information from the original DNA sequence. Compared with existing methods, the proposed approach made significant progress in enhancing the accuracy and strength of enhancer identification and prediction, demonstrating the effectiveness of the proposed method. This paper provides valuable insights for enhancer analysis and is of great significance for understanding gene regulatory mechanisms and studying disease correlations. [ABSTRACT FROM AUTHOR]

Background: Despite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed. Results: We examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1. Conclusions: Our study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study. [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]

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]

Nuclear receptor subfamily 5 group A member 1 (NR5A1) is expressed in the pituitary gonadotrope and regulates their differentiation. Although several regulatory regions were implicated in Nr5a1 gene expression in the pituitary gland, none of these regions have been verified using mouse models. Furthermore, the molecular functions of NR5A1 in the pituitary gonadotrope have not been fully elucidated. In the present study, we generated mice lacking the pituitary enhancer located in the 6th intron of the Nr5a1 gene. These mice showed pituitary gland-specific disappearance of NR5A1, confirming the functional importance of the enhancer. Enhancer-deleted male mice demonstrated no defects at fetal stages. Meanwhile, androgen production decreased markedly in adult, and postnatal development of reproductive organs, such as the seminal vesicle, prostate, and penis was severely impaired. We further performed transcriptomic analyses of the whole pituitary gland of the enhancer-deleted mice and controls, as well as gonadotropes isolated from Ad4BP-BAC-EGFP mice. These analyses identified several genes showing gonadotrope-specific, NR5A1-dependent expressions, such as Spp1, Tgfbr3l, Grem1, and Nr0b2. These factors are thought to function downstream of NR5A1 and play important roles in reproductive organ development through regulation of pituitary gonadotrope functions. [ABSTRACT FROM AUTHOR]

Many enhancers exist as clusters in the genome and control cell identity and disease genes; however, the underlying mechanism remains largely unknown. Here, we introduce an algorithm, eNet, to build enhancer networks by integrating single-cell chromatin accessibility and gene expression profiles. The complexity of enhancer networks is assessed by two metrics: the number of enhancers and the frequency of predicted enhancer interactions (PEIs) based on chromatin co-accessibility. We apply eNet algorithm to a human blood dataset and find cell identity and disease genes tend to be regulated by complex enhancer networks. The network hub enhancers (enhancers with frequent PEIs) are the most functionally important. Compared with super-enhancers, enhancer networks show better performance in predicting cell identity and disease genes. eNet is robust and widely applicable in various human or mouse tissues datasets. Thus, we propose a model of enhancer networks containing three modes: Simple, Multiple and Complex, which are distinguished by their complexity in regulating gene expression. Taken together, our work provides an unsupervised approach to simultaneously identify key cell identity and disease genes and explore the underlying regulatory relationships among enhancers in single cells. [ABSTRACT FROM AUTHOR]

Development and regeneration are orchestrated by gene regulatory networks that operate in part through transcriptional enhancers. Although many enhancers are pleiotropic and are active in multiple tissues, little is known about whether enhancer pleiotropy is due to 1) site pleiotropy, in which individual transcription factor binding sites (TFBS) are required for activity in multiple tissues, or 2) multiple distinct sites that regulate expression in different tissues. Here, we investigated the pleiotropy of an intronic enhancer of the stickleback Bone morphogenetic protein 6 (Bmp6) gene. This enhancer was previously shown to regulate evolved changes in tooth number and tooth regeneration, and is highly pleiotropic, with robust activity in both fins and teeth throughout embryonic, larval, and adult life, and in the heart and kidney in adult fish. We tested the hypothesis that the pleiotropy of this enhancer is due to site pleiotropy of an evolutionarily conserved predicted Foxc1 TFBS. Transgenic analysis and site-directed mutagenesis experiments both deleting and scrambling this predicted Foxc1 TFBS revealed that the binding site is required for enhancer activity in both teeth and fins throughout embryonic, larval, and adult development, and in the heart and kidney in adult fish. Collectively these data support a model where the pleiotropy of this Bmp6 enhancer is due to site pleiotropy and this putative binding site is required for enhancer activity in multiple anatomical sites from the embryo to the adult. [Display omitted] • Enhancer in intron 4 of stickleback Bone morphogenetic protein 6 is pleiotropic. • Enhancer is active in multiple sites including teeth, fins, heart, and kidney. • Enhancer contains predicted Foxc1 transcription factor binding site (TFBS). • This TFBS is required for enhancer activity in all domains. • Results suggest further genetic similarities between tooth and hair regeneration. [ABSTRACT FROM AUTHOR]

How cone photoreceptors are formed during retinal development is only partially known. This is in part because we do not fully understand the gene regulatory network responsible for cone genesis. We reasoned that cis-regulatory elements (enhancers) active in nascent cones would be regulated by the same upstream network that controls cone formation. To dissect this network, we searched for enhancers active in developing cones. By electroporating enhancer-driven fluorescent reporter plasmids, we observed that a sequence within an intron of the cone-specific Pde6c gene acted as an enhancer in developing mouse cones. Similar fluorescent reporter plasmids were used to generate stable transgenic human induced pluripotent stem cells that were then grown into three-dimensional human retinal organoids. These organoids contained fluorescently labeled cones, demonstrating that the Pde6c enhancer was also active in human cones. We observed that enhancer activity was transient and labeled a minor population of developing rod photoreceptors in both mouse and human systems. This cone-enriched pattern argues that the Pde6c enhancer is activated in cells poised between rod and cone fates. Additionally, it suggests that the Pde6c enhancer is activated by the same regulatory network that selects or stabilizes cone fate choice. To further understand this regulatory network, we identified essential enhancer sequence regions through a series of mutagenesis experiments. This suggested that the Pde6c enhancer was regulated by transcription factor binding at five or more locations. Binding site predictions implicated transcription factor families known to control photoreceptor formation and families not previously associated with cone development. These results provide a framework for deciphering the gene regulatory network that controls cone genesis in both human and mouse systems. Our new transgenic human stem cell lines provide a tool for determining which cone developmental mechanisms are shared and distinct between mice and humans. [Display omitted] • An intronic enhancer within Pde6c drives expression in developing cones. • Transgenic retinal organoid systems show that the enhancer is active in human cones. • The Pde6c enhancer is activated by cells deciding between rod and cone fates. • The enhancer likely has five or more essential transcription factor binding regions. • Moderate conservation suggests flexibility of enhancer function between species. [ABSTRACT FROM AUTHOR]

Identifying the general principles by which genotypes are converted into phenotypes remains a challenge in the post-genomic era. We still lack a predictive understanding of how genes shape interactions among cells and tissues in response to signalling and environmental cues, and hence how regulatory networks generate the phenotypic variation required for adaptive evolution. Here, we discuss how techniques borrowed from synthetic biology may facilitate a systematic exploration of evolvability across biological scales. Synthetic approaches permit controlled manipulation of both endogenous and fully engineered systems, providing a flexible platform for investigating causal mechanisms in vivo. Combining synthetic approaches with multi-level phenotyping (phenomics) will supply a detailed, quantitative characterization of how internal and external stimuli shape the morphology and behaviour of living organisms. We advocate integrating high-throughput experimental data with mathematical and computational techniques from a variety of disciplines in order to pursue a comprehensive theory of evolution. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'. [ABSTRACT FROM AUTHOR]

Simple Summary: Understanding how genes are regulated is a vital area of current biological research and a crucial adjunct to ongoing efforts to sequence entire genomes. Knowing the DNA sequences responsible for gene regulation—transcriptional cis-regulatory modules (CRMs, e.g., "enhancers") and transcription factor binding sites (TFBSs)—is important for many areas of research including interpretation and validation of data developed by large-scale genomics projects, providing training data for machine-learning CRM-discovery methods, genome annotation, modeling gene-regulatory networks, studying the evolution of gene regulation, and numerous aspects of the basic biology of transcriptional regulation. Knowledge of insect CRMs is also an important step in developing biotechnology methods for control of insect disease vectors and for eliminating pathogen transmission. The REDfly (Regulatory Element Database for Fly) database integrates all of the available insect cis-regulatory information from multiple sources to provide a comprehensive collection of known regulatory elements. In this paper, we describe REDfly's basic contents and data model, emphasizing recently added features, and provide illustrated walk-throughs of some common search scenarios. We provide here an updated description of the REDfly (Regulatory Element Database for Fly) database of transcriptional regulatory elements, a unique resource that provides regulatory annotation for the genome of Drosophila and other insects. The genomic sequences regulating insect gene expression—transcriptional cis-regulatory modules (CRMs, e.g., "enhancers") and transcription factor binding sites (TFBSs)—are not currently curated by any other major database resources. However, knowledge of such sequences is important, as CRMs play critical roles with respect to disease as well as normal development, phenotypic variation, and evolution. Characterized CRMs also provide useful tools for both basic and applied research, including developing methods for insect control. REDfly, which is the most detailed existing platform for metazoan regulatory-element annotation, includes over 40,000 experimentally verified CRMs and TFBSs along with their DNA sequences, their associated genes, and the expression patterns they direct. Here, we briefly describe REDfly's contents and data model, with an emphasis on the new features implemented since 2020. We then provide an illustrated walk-through of several common REDfly search use cases. [ABSTRACT FROM AUTHOR]

• Transcriptional networks in the schizophrenia neural cells were reconstructed. • Regulation via enhancers occupied more than half of the edges of the networks. • Meta-analysis revealed commonly used network modules over multiple datasets. • Enrichment of SNPs suggested associations of schizophrenia with several functions. There have been a number of reports about the transcriptional regulatory networks in schizophrenia. However, most of these studies were based on a specific transcription factor or a single dataset, an approach that is inadequate to understand the diverse etiology and underlying common characteristics of schizophrenia. Here we reconstructed and compared the transcriptional regulatory network for lipid metabolism enzymes using 15 public transcriptome datasets of neural cells from schizophrenia patients. Since many of the well-known schizophrenia-related SNPs are in enhancers, we reconstructed a network including enhancer-dependent regulation and found that 53.3 % of the total number of edges (7,577 pairs) involved regulation via enhancers. By examining multiple datasets, we found common and unique transcriptional modes of regulation. Furthermore, enrichment analysis of SNPs that were connected with genes in the transcriptional regulatory networks by eQTL suggested an association with hematological cell counts and some other traits/diseases, whose relationship to schizophrenia was either not or insufficiently reported in previous studies. Based on these results, we suggest that in future studies on schizophrenia, information on genotype, comorbidities and hematological cell counts should be included, along with the transcriptome, for a more detailed genetic stratification and mechanistic exploration of schizophrenia. [ABSTRACT FROM AUTHOR]

Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. PAX3-FOXO1 degradation impaired RNA polymerase pause release and transcription elongation at most regulated gene targets. Moreover, the activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective, affecting single elements in super-enhancers. This combinatorial analysis indicated that PAX3-FOXO1 was continuously required to maintain chromatin accessibility and enhancer architecture at regulated enhancers. [Display omitted] • PAX3-FOXO1 degradation leads to cell death and myogenic differentiation • PAX3-FOXO1 degradation causes loss of transcription of a small number of target genes • PAX3-FOXO1 regulates RNA polymerase II pause release at regulated genes • PAX3-FOXO1 controls co-regulatory complex assembly at regulated enhancers Zhang et al. use rapid protein degradation to define the mechanism of transcriptional control by, PAX3-FOXO1, a driver of aRMS. PAX3-FOXO1 functions at a limited number of enhancers to maintain chromatin accessibility and assemble critical co-regulatory complexes to trigger RNA polymerase II pause release and drive target gene expression. [ABSTRACT FROM AUTHOR]

Retinal neurogenesis is driven by concerted actions of transcription factors, some of which are expressed in a continuum and across several cell subtypes throughout development. While seemingly redundant, many factors diversify their regulatory outcome on gene expression, by coordinating variations in chromatin landscapes to drive divergent retinal specification programs. Recent studies have furthered the understanding of the epigenetic contribution to the progression of age-related macular degeneration, a leading cause of blindness in the elderly. The knowledge of the epigenomic mechanisms that control the acquisition and stabilization of retinal cell fates and are evoked upon damage, holds the potential for the treatment of retinal degeneration. Herein, this review presents the state-of-the-art approaches to investigate the retinal epigenome during development, disease, and reprogramming. A pipeline is then reviewed to functionally interrogate the epigenetic and transcriptional networks underlying cell fate specification, relying on a truly unbiased screening of open chromatin states. The related work proposes an inferential model to identify gene regulatory networks, features the first footprinting analysis and the first tentative, systematic query of candidate pioneer factors in the retina ever conducted in any model organism, leading to the identification of previously uncharacterized master regulators of retinal cell identity, such as the nuclear factor I, NFI. This pipeline is virtually applicable to the study of genetic programs and candidate pioneer factors in any developmental context. Finally, challenges and limitations intrinsic to the current next-generation sequencing techniques are discussed, as well as recent advances in super-resolution imaging, enabling spatio-temporal resolution of the genome. [ABSTRACT FROM AUTHOR]