Dynamic exchange at regulatory elements during chromatin remodeling underlies assisted loading mechanism.

The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.

Transcription factor AP1 potentiates chromatin accessibility and glucocorticoid receptor binding.

Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome.

Tissue-specific SMARCA4 binding at active and repressed regulatory elements during embryogenesis.

The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4(FLAG) knock-in mouse line. Using ChIP-seq, we identified ∼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development.

Interaction of the glucocorticoid receptor with the chromatin landscape.

The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function are unknown. We have characterized glucocorticoid receptor (GR) binding events and chromatin structural transitions across GR-induced or -repressed genes. This analysis reveals that GR binding invariably occurs at nuclease-accessible sites (DHS). A remarkable diversity of mechanisms, however, render these sites available for GR binding. Accessibility of the GR binding sites is either constitutive or hormone inducible. Within each category, some DHS sites require the Brg1-containing Swi/Snf complex, but others are Brg1 independent, implicating a different remodeling complex. The H2A.Z histone variant is highly enriched at both inducible and constitutive DHS sites and is subject to exchange during hormone activation. The DHS profile is highly cell specific, implicating cell-selective organization of the chromatin landscape as a critical determinant of tissue-selective receptor function. Furthermore, the widespread requirement for chromatin remodeling supports the recent hypothesis that the rapid exchange of receptor proteins occurs during nucleosome reorganization.

DNA methylation status predicts cell type-specific enhancer activity.

Cell-selective glucocorticoid receptor (GR) binding to distal regulatory elements is associated with cell type-specific regions of locally accessible chromatin. These regions can either pre-exist in chromatin (pre-programmed) or be induced by the receptor (de novo). Mechanisms that create and maintain these sites are not well understood. We observe a global enrichment of CpG density for pre-programmed elements, and implicate their demethylated state in the maintenance of open chromatin in a tissue-specific manner. In contrast, sites that are actively opened by GR (de novo) are characterized by low CpG density, and form a unique class of enhancers devoid of suppressive effect of agglomerated methyl-cytosines. Furthermore, treatment with glucocorticoids induces rapid changes in methylation levels at selected CpGs within de novo sites. Finally, we identify GR-binding elements with CpGs at critical positions, and show that methylation can affect GR-DNA interactions in vitro. The findings present a unique link between tissue-specific chromatin accessibility, DNA methylation and transcription factor binding and show that DNA methylation can be an integral component of gene regulation by nuclear receptors.

DNA-binding factor footprints and enhancer RNAs identify functional non-coding genetic variants.

BACKGROUND: Genome-wide association studies (GWAS) have revealed a multitude of candidate genetic variants affecting the risk of developing complex traits and diseases. However, the highlighted regions are typically in the non-coding genome, and uncovering the functional causative single nucleotide variants (SNVs) is challenging. Prioritization of variants is commonly based on genomic annotation with markers of active regulatory elements, but current approaches still poorly predict functional variants. To address this, we systematically analyze six markers of active regulatory elements for their ability to identify functional variants. RESULTS: We benchmark against molecular quantitative trait loci (molQTL) from assays of regulatory element activity that identify allelic effects on DNA-binding factor occupancy, reporter assay expression, and chromatin accessibility. We identify the combination of DNase footprints and divergent enhancer RNA (eRNA) as markers for functional variants. This signature provides high precision, but with a trade-off of low recall, thus substantially reducing candidate variant sets to prioritize variants for functional validation. We present this as a framework called FINDER-Functional SNV IdeNtification using DNase footprints and eRNA. CONCLUSIONS: We demonstrate the utility to prioritize variants using leukocyte count trait and analyze variants in linkage disequilibrium with a lead variant to predict a functional variant in asthma. Our findings have implications for prioritizing variants from GWAS, in development of predictive scoring algorithms, and for functionally informed fine mapping approaches.

Contribution of nucleosome binding preferences and co-occurring DNA sequences to transcription factor binding.

BACKGROUND: Chromatin plays a critical role in regulating transcription factors (TFs) binding to their canonical transcription factor binding sites (TFBS). Recent studies in vertebrates show that many TFs preferentially bind to genomic regions that are well bound by nucleosomes in vitro. Co-occurring secondary motifs sometimes correlated with functional TFBS. RESULTS: We used a logistic regression to evaluate how well the propensity for nucleosome binding and co-occurrence of a secondary motif identify which canonical motifs are bound in vivo. We used ChIP-seq data for three transcription factors binding to their canonical motifs: c-Jun binding the AP-1 motif (TGA(C)/(G)TCA), GR (glucocorticoid receptor) binding the GR motif (G-ACA---(T)/(C)GT-C), and Hoxa2 (homeobox a2) binding the Pbx (Pre-B-cell leukemia homeobox) motif (TGATTGAT). For all canonical TFBS in the mouse genome, we calculated intrinsic nucleosome occupancy scores (INOS) for its surrounding 150-bps DNA and examined the relationship with in vivo TF binding. In mouse mammary 3134 cells, c-Jun and GR proteins preferentially bound regions calculated to be well-bound by nucleosomes in vitro with the canonical AP-1 and GR motifs themselves contributing to the high INOS. Functional GR motifs are enriched for AP-1 motifs if they are within a nucleosome-sized 150-bps region. GR and Hoxa2 also bind motifs with low INOS, perhaps indicating a different mechanism of action. CONCLUSION: Our analysis quantified the contribution of INOS and co-occurring sequence to the identification of functional canonical motifs in the genome. This analysis revealed an inherent competition between some TFs and nucleosomes for binding canonical TFBS. GR and c-Jun cooperate if they are within 150-bps. Binding of Hoxa2 and a fraction of GR to motifs with low INOS values suggesting they are not in competition with nucleosomes and may function using different mechanisms.

Dynamic regulation of glucocorticoid signalling in health and disease.

Activation of the glucocorticoid receptor (GR) by endogenous and synthetic glucocorticoids regulates hundreds of genes to control regulatory networks in development, metabolism, cognition and inflammation. Elucidation of the mechanisms that regulate glucocorticoid action has highlighted the dynamic nature of hormone signalling and provides novel insights into genomic glucocorticoid actions. The major factors that regulate GR function include chromatin structure, epigenetics, genetic variation and the pattern of glucocorticoid hormone secretion. We review our current understanding of the mechanisms that contribute to GR signalling and how these contribute to glucocorticoid sensitivity, resistance and side effects.

MUC4 is not expressed in cell lines used for live cell imaging.

Background: The ability to visualise specific mammalian gene loci in living cells is important for understanding the dynamic processes linked to transcription. However, some of the tools used to target mammalian genes for live cell imaging, such as dCas9, have been reported to themselves impede processes linked to transcription. The MUC4 gene is a popular target for live cell imaging studies due to the repetitive nature of sequences within some exons of this gene. Methods: We set out to compare the impact of dCas9 and TALE-based imaging tools on MUC4 expression, including in human cell lines previously reported as expressing MUC4. Results: We were unable to detect MUC4 mRNA in these cell lines. Moreover, analysis of publicly available data for histone modifications associated with transcription, and data for transcription itself, indicate that neither MUC4, nor any of the mucin gene family are significantly expressed in the cell lines where dCas9 targeting has been reported to repress MUC4 and MUC1 expression, or in the cell lines where dCas13 has been used to report MUC4 RNA detection in live cells. Conclusions: Methods for visualising specific gene loci and gene transcripts in live human cells are very challenging. Our data suggest that care should be given to the choice of the most appropriate cell lines for these analyses and that orthogonal methods of assaying gene expression be carefully compared.

From bedside to bench: regulation of host factors in SARS-CoV-2 infection.

The zoonotic coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), which causes COVID-19 (coronavirus disease-2019), has resulted in a pandemic. This has led to an urgent need to understand the molecular determinants of SARS-CoV-2 infection, factors associated with COVID-19 heterogeneity and severity, and therapeutic options for these patients. In this review, we discuss the role of host factors in SARS-CoV-2 infection and describe variations in host factor expression as mechanisms underlying the symptoms and severity of COVID-19. We focus on two host factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), implicated in SARS-CoV-2 infection. We also discuss genetic variants associated with COVID-19 severity revealed in selected patients and based on genome-wide association studies (GWASs). Furthermore, we highlight important advances in cell and chromatin biology, such as single-cell RNA and chromatin sequencing and chromosomal conformation assays, as methods that may aid in the discovery of viral-host interactions in COVID-19. Understanding how regulation of host factor genes varies in physiological and pathological states might explain the heterogeneity observed in SARS-CoV-2 infection, help identify pathways for therapeutic development, and identify patients most likely to progress to severe COVID-19.

Glucocorticoid receptor dynamics and gene regulation.

The glucocorticoid receptor regulates the expression of a large number of genes in mammalian cells. The interaction of this receptor with regulatory elements has been discovered to be highly dynamic, with occupancy states measured in seconds, rather than minutes or hours. This finding has led to a paradigm shift in our understanding of receptor function throughout the genome. The mechanisms involved in these rapid exchange events, as well as the implications for receptor function, are discussed.

DNA Methylation: Basic Biology and Application to Traumatic Brain Injury.

This article reviews the literature pertinent to epigenetic changes, and in particular, DNA methylation following traumatic brain injury (TBI). TBI is a heterogeneous disease that is a major cause of death and long-term disability. The links between TBI and epigenetics, the process by which environmental factors alter gene expression without changing the underlying DNA sequence, is an expanding area of research that may have profound consequences for understanding the disease, and for clinical care. There are various epigenetic changes that may occur as a direct result of TBI, including DNA methylation, histone modification, and changes in the levels of non-coding RNA. This review focuses on DNA methylation, its potential to alter the degree of injury, and the extent of recovery, including development of post-traumatic neurodegeneration, response to therapies, and the hereditable consequences of injury. The functional consequences of non-coding RNA and histone modifications are well described in the literature; however, the mechanism by which these three mechanisms interact are often overlooked. Here, we briefly describe the interaction of DNA methylation with the two other key epigenetic changes, and highlight key work being performed to understand the functional relevance of those mechanisms. The field of epigenetics is rapidly advancing as a result of the advent of less invasive and more versatile methods for measuring epigenetic proteins and their functional impact on cells; however, the evidence specific to TBI is limited. This review identifies several important outstanding questions that remain from the work already conducted, and highlights directions for the future.

Minireview: Conversing with chromatin: the language of nuclear receptors.

Nuclear receptors are transcription factors that are activated by physiological stimuli to bind DNA in the context of chromatin and regulate complex biological pathways. Major advances in nuclear receptor biology have been aided by genome scale examinations of receptor interactions with chromatin. In this review, we summarize the roles of the chromatin landscape in regulating nuclear receptor function. Chromatin acts as a central integrator in the nuclear receptor-signaling axis, operating in distinct temporal modalities. Chromatin effects nuclear receptor action by specifying its genomic localization and interactions with regulatory elements. On receptor binding, changes in chromatin operate as an effector of receptor signaling to modulate transcriptional events. Chromatin is therefore an integral component of the pathways that guide nuclear receptor action in cell-type-specific and cell state-dependent manners.

Epigenetics: a lasting impression?

Epigenetics is the term that has been classically used to describe inheritable nongenetic factors that regulate genes. Although these factors were originally thought to act in a long time domain only, it is now clear that they can also be highly dynamic, changing over minutes. Transcription factors, including the glucocorticoid, oestrogen and androgen receptors, interact with these epigenetic mechanisms in a very dynamic manner to modify transcription of genes and consequently contribute to physiological processes, health and disease. Modern usage of the term epigenetics encompasses both longer-term and transient changes and is relevant to multiple biological systems.

Transcription factor interactions in genomic nuclear receptor function.

Transcription factors (TF) regulate gene expression acting as DNA sequence-specific binding factors, orchestrating cofactor recruitment and assembly of the transcriptional machinery. Nuclear receptors, a ligand-inducible TF class, regulate a large proportion of the genome, yet achieve highly cell-specific and context-dependent transcription, despite their widespread expression. High-throughput genome-wide profiling of TF binding reveals a startling proportion of colocalized cell- and context-specific TF-binding patterns, implying TF interactions play a critical role in transcription. These interactions depend on the chromatin architecture, that predominantly acts to predetermine accessibility of TF-binding sites at regulatory elements. Here, we summarize recent findings that highlight the importance of combinatorial TF interactions in determining diverse nuclear receptor-mediated transcriptional responses, emphasizing the significance of chromatin structure in directing TF and nuclear receptor recruitment. Interactions between TFs are likely to be a general mechanism of regulatory factors, contributing to transcriptional control in health and disease.

Chromatin accessibility pre-determines glucocorticoid receptor binding patterns.

Development, differentiation and response to environmental stimuli are characterized by sequential changes in cellular state initiated by the de novo binding of regulated transcriptional factors to their cognate genomic sites. The mechanism whereby a given regulatory factor selects a limited number of in vivo targets from a myriad of potential genomic binding sites is undetermined. Here we show that up to 95% of de novo genomic binding by the glucocorticoid receptor, a paradigmatic ligand-activated transcription factor, is targeted to preexisting foci of accessible chromatin. Factor binding invariably potentiates chromatin accessibility. Cell-selective glucocorticoid receptor occupancy patterns appear to be comprehensively predetermined by cell-specific differences in baseline chromatin accessibility patterns, with secondary contributions from local sequence features. The results define a framework for understanding regulatory factor-genome interactions and provide a molecular basis for the tissue selectivity of steroid pharmaceuticals and other agents that intersect the living genome.

Genome-wide mechanisms of nuclear receptor action.

Nuclear receptors are involved in a myriad of physiological processes, responding to ligands and binding to DNA at sequence-specific cis-regulatory elements. This binding occurs in the context of chromatin, a critical factor in regulating eukaryotic transcription. Recent high-throughput assays have examined nuclear receptor action genome-wide, advancing our understanding of receptor binding to regulatory elements. Here, we discuss current knowledge of genome-wide response element occupancy by receptors and the function of transcription factor networks in regulating nuclear receptor action. We highlight emerging roles for the epigenome, chromatin remodeling, histone modification, histone variants and long-range chromosomal interactions in nuclear receptor binding and receptor-dependent gene regulation. These mechanisms contribute importantly to the action of nuclear receptors in health and disease.

Glucocorticoid receptor activation of the Ciz1-Lcn2 locus by long range interactions.

The cellular response to glucocorticoid receptor (GR) activation involves a highly orchestrated series of regulatory actions influenced at multiple levels by a variety of mechanisms including the action of transcription factors and chromatin modifiers. Because the majority of GR binding sites (glucocorticoid-responsive elements (GREs)) are distant from promoters, it is likely that interactions at a distance play an important role in GR action. To determine whether long range chromosomal associations play a role in transcription regulation by GR, we utilized a chromosome conformation capture-based technique (associated chromosome trap) to identify unknown, remote sequences that interact with the GR-induced Lipocalin2 (Lcn2) gene. Our screen revealed that the Lcn2 GRE interacts with the Ciz1 gene, nearly 30 kb upstream. Ciz1 was subsequently found to be a novel GR-responsive gene. The GRE proximal to the Lcn2 promoter apparently functions to regulate both the Lcn2 gene and the distal Ciz1 gene. Using quantitative chromosome conformation capture, we find that a loop structure is organized between these two genes. This structure is hormone-independent and present only in cell types where the genes are active. The strong correlation between gene expression and loop structure in different cell lines suggests that high order interactions play a role in determining tissue-specific gene regulation.

Kinetic complexity of the global response to glucocorticoid receptor action.

We have characterized the kinetic response of gene targets throughout the murine genome to transcriptional modulation by the glucocorticoid receptor (GR). In contrast to a model in which multiple genes are either repressed or activated during the GR response, the vast majority of responsive genes are subject to complex regulation profiles, frequently with alternate activation and repression phases. We also observe that GR binding at response elements does not always correlate with the target gene response profile. Thus, the cellular response to GR stimulation involves a highly orchestrated series of regulatory actions and not simply a binary response to hormone.