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Breast cancers are a diverse group of diseases and are often characterized by their expression of receptors for hormones such as estrogen and progesterone. Recently, another steroid hormone receptor, the glucocorticoid receptor has been shown to be a key player in breast cancer progression, metastasis, and treatment. These receptors bind to chromatin to elicit transcriptional changes within cells, which are often inhibited by the structure of chromatin itself. Chromatin-remodeling proteins, such as Brahma-related gene 1, function to overcome this physical inhibition of transcription factor function and have been linked to many cancers including breast cancer. Recent efforts to understand the interactions of Brahma-related gene 1 and the glucocorticoid receptor, including genomic and single-cell analyses, within breast cancers may give insight into personalized medicine and other potential treatments.

Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation. However, inhibiting proteasome activity can lead to an increase in transcriptional output that is correlated with enriched levels of trimethyl H3K4 and phosphorylated forms of RNA polymerase (Pol) II at the promoter and gene body. Here, we perform gene expression analysis and ChIP followed by sequencing (ChIP-seq) in MCF-7 breast cancer cells treated with the proteasome inhibitor MG132, and we further explore genome-wide effects of proteasome inhibition on the chromatin state and RNA Pol II transcription. Analysis of gene expression programs and chromatin architecture reveals that chemically inhibiting proteasome activity creates a distinct chromatin state, defined by spreading of the H3K4me3 mark into the gene bodies of differentially-expressed genes. The distinct H3K4me3 chromatin profile and hyperacetylated nucleosomes at transcription start sites establish a chromatin landscape that facilitates recruitment of Ser-5- and Ser-2–phosphorylated RNA Pol II. Subsequent transcriptional events result in diverse gene expression changes. Alterations of H3K36me3 levels in the gene body reflect productive RNA Pol II elongation of transcripts of genes that are induced, underscoring the requirement for proteasome activity at multiple phases of the transcriptional cycle. Finally, by integrating genomics data and pathway analysis, we find that the differential effects of proteasome inhibition on the chromatin state modulate genes that are fundamental for cancer cell survival. Together, our results uncover underappreciated downstream effects of proteasome inhibitors that may underlie targeting of distinct chromatin states and key steps of RNA Pol II–mediated transcription in cancer cells.

Synthetic glucocorticoids such as Dexamethasone (Dex) are widely prescribed drugs used to treat a variety of human diseases including auto-immune disorders, asthma, cancer, and COVID-19. The transcriptional response to glucocorticoids is elicited by the Glucocorticoid Receptor (GR), which enters the nucleus upon Dex treatment and interacts with thousands of enhancer elements throughout the genome. We recently demonstrated that the Dex response in human breast cancer cells is highly heterogeneous and that individual cells have unique transcriptional responses to Dex. To examine whether this heterogeneity arises from differential utilization of distinct GR-bound enhancers, we focused on the Dex response at the DNA Damage Inducible Transcript 4 (DDIT4) gene. Using a variety of genomic techniques, we identified four GR binding sites (GBSs) 18-30kb upstream of the DDIT4 TSS with differential patterns of chromatin accessibility, histone acetylation, SWI/SNF recruitment, and enhancer RNA (eRNA) transcription. To determine whether these GBSs had unique requirements for DDIT4 transcription, we used CRISPR-CAS9 to generate homozygous deletions of each site. Using ChIP-seq, 4C-seq, single molecule fluorescent in situ hybridization (smFISH), and RT-PCR, we demonstrated GR binding to these GBSs was independent and each GBS deletion had unique effects on DDIT4 and eRNA transcription, local histone acetylation, and chromatin looping. Deletion of any of the first three GBSs resulted in delayed and/or decreased induction of DDIT4 transcription whereas deletion of the fourth GBS resulted in significant upregulation of both DDIT4 and eRNA transcription. Thus, three of the GBSs acted as enhancers of DDIT4 expression while the fourth functioned as a suppressor. Strikingly, smFISH also revealed that these enhancers contributed to cellular heterogeneity, as deleting the GBSs altered the frequency and amplitude of DDIT4 transcription across cell populations. Taken together, these results demonstrate that individual GBSs uniquely contribute to cell-to-cell heterogeneity within the transcriptional response of DDIT4 to Dex. Furthermore, they underscore the possibility that targeted modification of individual GBSs could be utilized to tailor custom, patient-specific strategies for the treatment of human diseases.

The SWI/SNF complex is a critical regulator of pluripotency in human embryonic stem cells (hESCs), and individual subunits have varied and specific roles during development and in diseases. The core subunit SMARCB1 is required for early embryonic survival, and mutations can give rise to atypical teratoid/rhabdoid tumors (AT/RTs) in the pediatric central nervous system. We report that in contrast to other studied systems, SMARCB1 represses bivalent genes in hESCs and antagonizes chromatin accessibility at super-enhancers. Moreover, and consistent with its established role as a CNS tumor suppressor, we find that SMARCB1 is essential for neural induction but dispensable for mesodermal or endodermal differentiation. Mechanistically, we demonstrate that SMARCB1 is essential for hESC super-enhancer silencing in neural differentiation conditions. This genomic assessment of hESC chromatin regulation by SMARCB1 reveals a novel positive regulatory function at super-enhancers and a unique lineage-specific role in regulating hESC differentiation., eLife digest Our bodies contain trillions of cells that play a wide variety of roles. Despite looking and behaving very differently to one another, all of these ‘mature’ cells somehow descend from a single fertilized egg that contains just one set of genes. This process is partially controlled by how ‘accessible’ genetic material is to the cell machinery that switches genes on or off. For example, in immature brain cells, genes required for memory are accessible, but genes needed to produce bone are not. The developing embryo needs to control gene accessibility carefully to ensure that the right genes become available at the right time, and that crucial genes are not incorrectly ‘hidden’. In humans, the protein SMARCB1 plays an important role in this process: if damaged or deleted, development will be severely disrupted, sometimes causing brain cancer early in life. However, it remains unclear how exactly SMARCB1 regulates the accessibility of its ‘target’ genes. Now, Langer et al. set out to answer this question, and also to determine which parts of the body need SMARCB1 to develop properly. Human stem cells can develop into multiple mature cell types if given the right signals. Langer et al. found reducing levels of SMARCB1 prevented stem cells from maturing into brain cells, but not other kinds of cells. This suggests that SMARCB1 has a specific role in brain development, which is consistent with its devastating effect on brain health when damaged. A detailed analysis of genetic activity and DNA accessibility showed that SMARCB1 was doing this by switching off specific regions of DNA, called stem cell super-enhancers. These regions normally enhance the activity of genes that maintain stem cells in their immature state: when certain super-enhancers are turned off by SMARCB1, this allows stem cells to progress towards a brain cell fate. These results help us understand why damage to SMARCB1 during development causes brain cancer more often than other kinds of cancer. In the future, they could also help explain how certain types of cancer form, which would be the first step towards knowing how to treat them.

Background Transcription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse. Results Here we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET. Conclusions These data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0897-0) contains supplementary material, which is available to authorized users.

The Glucocorticoid Receptor (GR) alters transcriptional activity in response to hormones by interacting with chromatin at GR binding sites (GBSs) throughout the genome. Our work in human breast cancer cells identifies three classes of GBSs with distinct epigenetic characteristics and reveals that BRG1 interacts with GBSs prior to hormone exposure. The GBSs pre-occupied by BRG1 are more accessible and transcriptionally active than other GBSs. BRG1 is required for a proper and robust transcriptional hormone response and knockdown of BRG1 blocks recruitment of the pioneer factors FOXA1 and GATA3 to GBSs. Finally, GR interaction with FOXA1 and GATA3 binding sites was restricted to sites pre-bound by BRG1. These findings demonstrate that BRG1 establishes specialized chromatin environments that define multiple classes of GBS. This in turn predicts that GR and other transcriptional activators function via multiple distinct chromatin-based mechanisms to modulate the transcriptional response., eLife digest Steroid hormones play a number of roles in the body, including controlling the immune system and the body’s response to stress. Artificially produced steroid hormones may also be used as part of treatments for cancer. The hormones affect the behavior of cells by binding to and activating hormone receptor proteins. The receptors can then interact with the cell’s DNA to change the activity of nearby genes. Gaining access to particular sites on a strand of DNA is not always easy. Cells pack DNA into a structure called chromatin. In some regions the DNA is so tightly wrapped in the chromatin that the receptors cannot access it. The structure of the chromatin therefore affects how a cell responds to steroid hormones. Inaccessible regions of chromatin can be ‘opened up’ by two groups of proteins, known as remodeling proteins and pioneer factors. Hormone receptors can work with these proteins to access particular DNA regions, but exactly how all these proteins work together was not fully understood. Hoffman et al. have now used DNA and RNA sequencing technologies to examine the roles of a hormone receptor called the glucocorticoid receptor, a remodeling protein called BRG1, and various pioneer factors in human breast cancer cells. This revealed three ways in which the glucocorticoid receptors worked with the other proteins when binding to chromatin. These could be distinguished by the pattern of BRG1 molecules bound to the DNA. Further investigation showed that BRG1 controls how the glucocorticoid receptor affects the activity of genes. In addition, BRG1 influences how the receptor interacts with pioneer factors when it is bound to DNA. Future research into how these proteins work together could ultimately help us to improve how we use steroid hormones to treat diseases.

Epigenetic enzymes regulate higher-order chromatin architecture and cell-type specific gene expression. The ATPase BRG1 and the SWI/SNF chromatin remodeling complex are epigenetic enzymes that regulate chromatin accessibility during steady and transitional cell states. Experiments in mice show that the loss of BRG1 inhibits cellular reprogramming, while studies using human cells demonstrate that the overexpression of BRG1 enhances reprogramming. We hypothesized that the variation of SWI/SNF subunit expression in the human population would contribute to variability in the efficiency of induced pluripotent stem cells (iPSC) reprogramming. To examine the impact of an individual's sex, ancestry, and age on iPSC reprogramming, we created a novel sex and ancestry balanced cohort of 240 iPSC lines derived from human dermal fibroblasts (DF) from 80 heathy donors. We methodically assessed the reprogramming efficiency of each DF line and then quantified the individual and demographic-specific variations in SWI/SNF chromatin remodeling proteins and mRNA expression. We identified BRG1, BAF155, and BAF60a expression as strongly correlating with iPSC reprogramming efficiency. Additionally, we discovered that high efficiency iPSC reprograming is negatively correlated with donor age, positively correlated with African American descent, and uncorrelated with donor sex. These results show the variations in chromatin remodeling protein expression have a strong impact on iPSC reprogramming. Additionally, our cohort is unique in its large size, diversity, and focus on healthy donors. Consequently, this cohort can be a vital tool for researchers seeking to validate observational results from human population studies and perform detailed mechanistic studies in a controlled cell culture environment.

The regulatory networks of differentiation programs and the molecular mechanisms of lineage-specific gene regulation in mammalian embryos remain only partially defined. We document differential expression and temporal switching of BRG1-associated factor (BAF) subunits, core pluripotency factors and cardiac-specific genes during post-implantation development and subsequent early organogenesis. Using affinity purification of BRG1 ATPase coupled to mass spectrometry, we characterized the cardiac-enriched remodeling complexes present in E8.5 mouse embryos. The relative abundance and combinatorial assembly of the BAF subunits provides functional specificity to Switch/Sucrose NonFermentable (SWI/SNF) complexes resulting in a unique gene expression profile in the developing heart. Remarkably, the specific depletion of the BAF250a subunit demonstrated differential effects on cardiac-specific gene expression and resulted in arrhythmic contracting cardiomyocytes in vitro. Indeed, the BAF250a physically interacts and functionally cooperates with Nucleosome Remodeling and Histone Deacetylase (NURD) complex subunits to repressively regulate chromatin structure of the cardiac genes by switching open and poised chromatin marks associated with active and repressed gene expression. Finally, BAF250a expression modulates BRG1 occupancy at the loci of cardiac genes regulatory regions in P19 cell differentiation. These findings reveal specialized and novel cardiac-enriched SWI/SNF chromatin-remodeling complexes, which are required for heart formation and critical for cardiac gene expression regulation at the early stages of heart development.

Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment., Author Summary Gene transcription is regulated by the coordinated interaction of genetic, epigenetic and trans-acting factors. The chromatin environment at gene promoters, including positioned nucleosomes that may display functional histone modifications, is a key regulator of gene expression, contributing to transcriptional activation and repression. In addition to sense-strand transcription of gene sequences, antisense transcription is prevalent at gene promoters. Often resulting in a short-lived non-coding RNA transcript, the function of antisense transcription is poorly understood. Using next-generation sequencing techniques, we characterized transcription in human breast cancer cells and found extensive correlations between antisense transcription and the chromatin environment at promoters. We found that downstream antisense transcription initiates from between regularly positioned nucleosomes and that those nucleosomes between sense and downstream antisense transcription start sites display histone modifications associated with active gene promoters. Chromatin remodelers and other protein complexes responsible for creation and maintenance of the promoter chromatin environment associate with this same region, suggesting an important role of antisense transcription in the regulation of gene expression.

Steroid hormone receptor (SR) signaling leads to widespread changes in gene expression, and aberrant SR signaling can lead to malignancies including breast, prostate, and lung cancers. Chromatin remodeling is an essential component of SR signaling, and defining the process of chromatin and nucleosome remodeling during signaling is critical to the continued development of related therapies. The glucocorticoid receptor (GR) is a key SR that activates numerous promoters including the well defined MMTV promoter. The activation of MMTV by GR provides an excellent model for teasing apart the sequence of events between hormone treatment and changes in gene expression. Comparing hormone-induced transcription from stably integrated promoters with defined nucleosomal structure to that from transiently expressed, unstructured promoters permits key distinctions between interactions that require remodeling and those that do not. The importance of co-activators and histone modifications prior to remodeling and the formation of the preinitiation complex that follows can also be clarified by defining key transition points in the propagation of hormonal signals. Combined with detailed mapping of proteins along the promoter, a temporal and spatial understanding of the signaling and remodeling processes begins to emerge. In this review, we examine SR signaling with a focus on GR activation of the MMTV promoter. We also discuss the ATP-dependent remodeling complex SWI/SNF, which provides the necessary remodeling activity during GR signaling and interacts with several SRs. BRG1, the central ATPase of SWI/SNF, also interacts with a set of BAF proteins that help determine the specialized function and fine-tuned regulation of BRG1 remodeling activity. BRG1 regulation comes from its own subdomains as well as its interactive partners. In particular, the HSA domain region of BRG1 and unique features of its ATPase homology appear to play key roles in regulating remodeling function. Details of the inter-workings of this chromatin remodeling protein continue to be revealed and promise to improve our understanding of the mechanism of chromatin remodeling during steroid hormone signaling. This article is part of a Special Issue entitled: Chromatin in time and space.

Environmental exposure to low concentration hormones can have permanent epigenetic effects in animals and humans. The consequence of long-term low concentration glucocorticoid exposure was investigated in cell culture using glucocorticoid responsive genes organized in alternative chromatin structures. The MMTV promoter is induced by short-term glucocorticoid exposure on either an integrated (normal chromatin) or transient (unstructured chromatin) promoter. Longer hormone treatment causes a transient refractory repression of only the integrated promoter. Exposure to low concentrations of hormone for several passages persistently represses the integrated MMTV and endogenous glucocorticoid responsive promoters. The glucocorticoid receptor cannot bind to persistently repressed promoters. Induction by androgens is also inhibited on the repressed MMTV promoter. Similarly, osmotic stress induction of the endogenous Sgk gene is repressed. Persistent repression by glucocorticoids targets glucocorticoid responsive genes using a chromatin-dependent mechanism that disrupts binding of both GR-dependent and GR-independent transcription complexes.

The mechanisms and interplay of coactivators that underlie transcription activation is a critical avenue of investigation in biology today. Using nuclear receptor (NR) mediated transcription activation as a model, the nature of coactivator recruitment and chromatin modifications has been found to be highly dynamic. Progress in understanding the kinetics and regulation of coactivator recruitment, and subsequent effects on transcriptional readout, has greatly improved our understanding of nuclear receptor mediated transcription, the subject of discussion in this 'At the Cutting Edge' review.

Eukaryotic genetic information is stored within the association of DNA and histone proteins resulting in a dynamic polymer called chromatin. The fundamental structural unit of chromatin is the nucleosome which consists of approximately 146 bp of DNA wrapped around an octamer of histones containing two copies each of four core histones, H2A, H2B, H3 and H4. It is this DNA/protein fiber that transcription factors and other agents of chromatin metabolism must access and regulate. We have developed model systems to study the mechanisms by which steroid receptors control physiological activities by regulating gene expression within a higher order chromatin organization. Our studies have focused on the glucocorticoid receptor and its ability to remodel chromatin which is mediated by the BRG1 complex. Using novel cell systems, we demonstrate that GR-mediated transactivation from chromatin templates requires BRG1 remodeling activity and that other ATP-dependent remodeling proteins cannot substitute for this activity.

SWI/SNF (switching/sucrose nonfermenting)-dependent chromatin remodeling establishes coordinated gene expression programs during development, yet important functional details remain to be elucidated. We show that the Brg1 (Brahma-related gene 1; Smarca4) ATPase is globally expressed at high levels during postimplantation development and its conditional ablation, beginning at gastrulation, results in increased apoptosis, growth retardation, and, ultimately, embryonic death. Global gene expression analysis revealed that genes upregulated in Rosa26CreERT2; Brg1(flox/flox) embryos (here referred to as Brg1(d/d) embryos to describe embryos with deletion of the Brg1(flox/flox) alleles) negatively regulate cell cycle progression and cell growth. In addition, the p53 (Trp53) protein, which is virtually undetectable in early wild-type embryos, accumulated in the Brg1(d/d) embryos and activated the p53-dependent pathways. Using P19 cells, we show that Brg1 and CHD4 (chromodomain helicase DNA binding protein 4) coordinate to control target gene expression. Both proteins physically interact and show a substantial overlap of binding sites at chromatin-accessible regions adjacent to genes differentially expressed in the Brg1(d/d) embryos. Specifically, Brg1 deficiency results in reduced levels of the repressive histone H3 lysine K27 trimethylation (H3K27me3) histone mark and an increase in the amount of open chromatin at the regulatory region of the p53 and p21 (Cdkn1a) genes. These results provide insights into the mechanisms by which Brg1 functions, which is in part via the p53 program, to constrain gene expression and facilitate rapid embryonic growth.

MicroRNAs and chromatin remodeling complexes represent powerful epigenetic mechanisms that regulate the pluripotent state. miR-302 is a strong inducer of pluripotency, which is characterized by a distinct chromatin architecture. This suggests that miR-302 regulates global chromatin structure; however, a direct relationship between miR-302 and chromatin remodelers has not been established. Here, we provide data to show that miR-302 regulates Brg1 chromatin remodeling complex composition in human embryonic stem cells (hESCs) through direct repression of the BAF53a and BAF170 subunits. With the subsequent overexpression of BAF170 in hESCs, we show that miR-302's inhibition of BAF170 protein levels can affect the expression of genes involved in cell proliferation. Furthermore, miR-302-mediated repression of BAF170 regulates pluripotency by positively influencing mesendodermal differentiation. Overexpression of BAF170 in hESCs led to biased differentiation toward the ectoderm lineage during EB formation and severely hindered directed definitive endoderm differentiation. Taken together, these data uncover a direct regulatory relationship between miR-302 and the Brg1 chromatin remodeling complex that controls gene expression and cell fate decisions in hESCs and suggests that similar mechanisms are at play during early human development. Stem Cells 2015;33:2925—2935

BRG1, the central ATPase of the human SWI/SNF complex, is critical for biological functions, including nuclear receptor (NR)-regulated transcription. Analysis of BRG1 mutants demonstrated that functional motifs outside the ATPase domain are important for transcriptional activity. In the course of experiments examining protein interactions mediated through these domains, Ku70 (XRCC6) was found to associate with a BRG1 fragment encompassing the conserved helicase-SANT-associated (HSA) and BRK domains of BRG1. Subsequent transcriptional activation assays and chromatin immunoprecipitation studies showed that Ku70/86 and components of the topoisomerase IIβ (TOP2β)/poly(ADP ribose) polymerase 1 (PARP1) complex are necessary for NR-mediated SWI/SNF-dependent transcriptional activation from endogenous promoters. In addition to establishing Ku-BRG1 binding and TOP2β/PARP1 recruitment by nuclear receptor transactivation, we demonstrate that the transient appearance of glucocorticoid receptor (GR)/BRG1-dependent, TOP2β-mediated double-strand DNA breaks is required for efficient GR-stimulated transcription. Taken together, these results suggest that a direct interaction between Ku70/86 and BRG1 brings together SWI/SNF remodeling capabilities and TOP2β activity to enhance the transcriptional response to hormone stimulation.

Nuclear receptors (NRs) are a large family of ligand-dependent transcription factors that regulate important physiological processes. To activate or repress genes assembled naturally as chromatin, NRs recruit two distinct enzymatic activities, namely histone-modifying enzymes and ATP-dependent chromatin remodeling complexes, to alter local chromatin structure at target gene promoters. In this review, we examine the functional relationship between ATP-dependent chromatin remodeling complexes and NRs in the context of transcriptional regulation. Using the steroid-responsive mouse mammary tumor virus promoter as a model system, we discuss in detail the molecular mechanisms underlying the recruitment of these complexes and subsequent chromatin structure changes catalyzed by this group of enzymes. In addition, we extend the discussion to other NR-regulated promoters including the pS2 promoter. Finally, we summarize specific principles governing this critical relationship, identify unanswered questions and discuss the potential application of these principles in rational drug design.

SWI/SNF- and ISWI-based complexes have distinct yet overlapping chromatin-remodeling activities in vitro and perform different roles in vivo. This leads to the hypothesis that the distinct remodeling functions of these complexes are specifically required for distinct biological tasks. By creating and characterizing chimeric proteins of BRG1 and SNF2h, the motor proteins of human SWI/SNF- and ISWI-based complexes, respectively, we found that a region that includes the ATPase domain specifies the outcome of the remodeling reaction in vitro. A chimeric protein based on BRG1 but containing the SNF2h ATPase domain formed an intact SWI/SNF complex that remodeled like SNF2h. This altered-function complex was active for remodeling and could stimulate expression from some, but not all, SWI/SNF responsive promoters in vivo. Thus, we were able to separate domains of BRG1 responsible for function from those responsible for SWI/SNF complex formation and demonstrate that remodeling functions are not interchangeable in vivo.

Lipophilic hormones, including steroids, exert their physiological effects through binding to high-affinity superfamily of steroid hormone receptor (SR) proteins that function as ligand-dependent DNA binding transcription factors. To date, SR proteins are among a few transcription factors shown to directly interact with higher order chromatin structures to regulate gene expression. To perturb chromatin, SRs employ enzymatic multicomplexes that can either remodel or modify chromatin. Here we examine the current state of knowledge concerning multicomplex chromatin remodeling/modification machines and SR-dependent transcription. We will focus on the role of these protein–protein and chromatin–protein interactions in vivo with the MMTV promoter as a primary model. In addition, we discuss emerging evidence implicating chaperone proteins and proteasome degradation machinery in SR-mediated gene regulation within chromatin.

Nuclear hormone receptors are ligand-dependent transcriptional regulators that modulate chromatin structure. However, the precise molecular mechanisms by which receptors recruit chromatin-remodeling activity are not fully elucidated. We show that in the absence of its ligand-binding domain, the glucocorticoid receptor (GR) is able to interact with both nuclear receptor coactivators and the BRG1 chromatin-remodeling complex in vivo. Individually, the GR makes direct interactions with BRG1-associated factor 60a (BAF60a) and BAF57, but not with BRG1, BAF155, or BAF170. Further, BAF60a possesses at least two interaction surfaces, one for GR and BRG1 and a second for BAF155 and BAF170. A GR mutant, GR(R488Q), that fails to interact with BAF60a in vitro has reduced chromatin-remodeling activity and reduced transcriptional activity from the promoter assembled as chromatin in vivo. Stable expression of a BAF60a truncation mutant, BAF60a4-140, caused chromatin-specific loss of GR functions in vivo. In the presence of the BAF60a mutant, the GR fails to interact with the BRG1 complex and consequently is also deficient in its ability to activate transcription from chromatin. Thus, in addition to previously identified BAF250, BAF60a may provide another critical and direct link between nuclear receptors and the BRG1 complex that is required for promoter recruitment and subsequent chromatin remodeling.

The mouse mammary tumor virus (MMTV) promoter has been used as a model to study how the glucocorticoid receptor (GR) remodels chromatin to allow other transcription factors to bind and activate transcription. To dissect the precise role of nuclear factor 1 (NF1) in chromatin remodeling and transcriptional activation, we used linker-scanning mutants of transcription factor binding sites on the MMTV promoter. We compared the NF1 mutant MMTV promoter in the context of transiently transfected templates (transient transfection) and templates organized as chromatin (stable transfection) to understand the effect of chromatin on factor binding and transcription. We show that on a transiently transfected template, mutation in the NF1 binding site reduces both basal and hormone-dependent transcription. This suggests that NF1 is required for transcription in the absence of organized chromatin. We also found that binding of NF1 on a transiently transfected template is independent of mutation in hormone response elements or the octamer transcription factor (OTF) binding site. In contrast, the binding of OTF proteins to a transiently transfected template was found to be dependent on the binding of NF1, which may imply that NF1 has a stabilizing effect on OTF binding. On a chromatin template, mutation in the NF1 binding site does not affect the positioning of nucleosomes on the promoter. We also show that in the absence of NF1 binding, GR-mediated chromatin remodeling of nucleosome B is reduced and hormone-dependent activation of transcription is abolished. Further, we demonstrate that NF1 is required for both the association of BRG1 chromatin remodeling complex and the GR on the promoter in vivo. These results suggest the novel possibility that NF1 may participate in chromatin remodeling activities in addition to directly enhancing transcription and that in the absence of its binding site the GR is unable to effectively bind the promoter and recruit the remodeling complex.

Chromatin structure of eukaryotic genes regulates gene expression by controlling the accessibility of regulatory factors. To overcome the inhibitory nature of chromatin, protein complexes that modify higher order chromatin organization and histone–DNA contacts are critical players in regulating transcription. For example, nuclear hormone receptors regulate transcription by interacting with ATP-dependent chromatin-remodeling complexes and coactivators, which include histone acetyltransferases and histone methylases that modify the basic residues of histones. A growing number of tissue-specific nuclear hormone receptor ligands, termed "selective modulators", owe their specificity, at least in part, to the differential recruitment of these chromatin-modifying coactivators. The molecular mechanisms by which these compounds modulate the functions of nuclear hormone receptors are discussed here.Key words: chromatin, BRG1, SWI–SNF, nuclear receptor, glucorticoid receptor, transcription, MMTV.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that induces expression of many genes. The GR has been useful for understanding how chromatin structure regulates steroid-induced transcription in model systems. However, the effect of glucocorticoids on chromatin structure has been examined on few endogenous mammalian promoters. We investigated the effect of glucocorticoids on the in vivo chromatin structure of the glucocorticoid-responsive I kappa B alpha gene promoter, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NF kappa B). Glucocorticoids inhibit NF kappa B activity in some tissues by elevating the levels of I kappa B alpha. We found that glucocorticoids activated the I kappa B alpha promoter in human T47D/A1-2 cells containing the GR. We then investigated the chromatin structure of the I kappa B alpha promoter in the absence and presence of glucocorticoids with the use of micrococcal nuclease, restriction enzyme, and deoxyribonuclease (DNaseI) analyses. In untreated cells, the promoter assembles into regularly positioned nucleosomes, and glucocorticoid treatment did not alter nucleosomal position. Restriction enzyme accessiblity studies indicated that the I kappa B alpha promoter is assembled as phased nucleosomes that adopt an "open" chromatin architecture in the absence of hormone. However, glucocorticoids may be required for transcription factor binding, because DNaseI footprinting studies suggested that regulatory factors bind to the promoter upon glucocorticoid treatment.

We have previously shown a connection between histone H1 phosphorylation and the transcriptional competence of the hormone inducible mouse mammary tumor virus (MMTV) promoter. Prolonged exposure of mouse cells to dexamethasone concurrently dephosphorylated histone H1 and rendered the MMTV promoter refractory to hormonal stimulation and, therefore, transcriptionally unresponsive. Using electrospray mass spectrometry, we demonstrate here that prolonged dexamethasone treatment differentially effects a subset of the six somatic H1 isoforms in mouse cells. H1 isoforms H1.0, H1.1, and H1.2 are non-responsive to hormone whereas prolonged dexamethasone treatment effectively dephosphorylated the H1.3, H1.4, and H1.5 isoforms. The protein kinase inhibitor staurosporine, shown to dephosphorylate histone H1 and down-regulate MMTV in cultured cells, appears only to completely dephosphorylate the H1.3 isoform. These results suggest that dephosphorylation of specific histone H1 isoforms may contribute to the previously observed decrease in transcriptional competence of the MMTV promoter through the modulation of chromatin structure. In a broader sense, this work advances the hypothesis that post-translational modifications of individual histone H1 isoforms directly influence the transcriptional activation/repression of specific genes.

The compaction of DNA into chromatin provides an additional level of gene regulation in eukaryotes that may not be available to prokaryotes. When packaged as chromatin, most promoters are transcriptionally repressed, and transcription factors have reduced access to their binding sites. The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates the activity of genes involved in many physiological processes. To regulate eukaryotic genes, the GR binds to target sites within promoter regions of genes assembled as chromatin. This interaction alters the nucleosomal architecture to allow binding of other transcription factors, and formation of the preinitiation complex. The mouse mammary tumor virus (MMTV) promoter has been used extensively as a model to explore the processes by which the GR remodels chromatin and activates transcription. Significant progress has been made in our understanding of the mechanisms used by the GR to modify chromatin structure, and the limits placed on the GR by post-translational modifications of histones. We will describe recent developments in the processes used by the GR to activate transcription in vivo via chromatin remodeling complexes, histone H1 phosphorylation, and recruitment of diverse coactivators.

The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc from Saccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the beta-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.

We examined the mechanism(s) by which the progesterone receptor (PR) is able to inhibit glucocorticoid receptor (GR) activation from the mouse mammary tumor virus (MMTV) promoter in vivo. Using specific hormone antagonists, we demonstrate that the PR complexed with an type II antiprogestin blocks glucocorticoid-induced activation of the MMTV promoter. However, when complexed with a type I antiprogestin the PR is unable to block glucocorticoid-induced activation. PR repression of GR activity results from the inhibition of the ability of the GR to remodel chromatin such that the antiprogestin-occupied/PR prevents the glucocorticoid induced assembly of a preinitiation complex at MMTV promoter. These experiments suggest that the specific chromatin organization of the MMTV promoter provides a mechanism for regulating cross-talk between the GR and PR in vivo.

A number of important nuclear processes including replication, recombination, repair, and transcription involve the interaction of soluble nuclear proteins with DNA assembled as chromatin. Recent progress in a number of experimental systems has focused attention on the influence chromatin structure may exert on gene regulation in eukaryotes. With the advent of new technologies for the analysis of chromatin structurein vivo,studies evaluating the influence of chromatin structure on gene transcription have become feasible for a number of systems. This article serves as an introduction to the use of restriction endonucleases to define nucleosomal organization and characterize changes in this organization that accompany transcriptional activationin vivo.The procedure includes the isolation of intact transcriptionally competent nuclei, limited digestion with specific restriction endonucleases, and purification of the DNA. This DNA serves as the substrate for a linear amplification using single primers that generate enzyme-specific DNA fragments, which are then resolved by electrophoresis. Specific examples related to our studies of the influence of chromatin structure on steroid hormone regulation of transcription from the mouse mammary tumor virus promoter are provided to illustrate this technique and several novel variations. Alternative methods for analysis of chromatin architecture using DNase I, micrococcal nuclease, permanganate, and methidiumpropyl-EDTA-iron(II) are also described. Through the use of these methodologies one is able to determine both the translational and the rotational positions for a given nucleosome as well as quantify changes at a specific nucleosome in response to regulatory and developmental signals.

There is increasing evidence that specific chromatin structures play an important role in the regulation of transcription in eukaryotes. The mouse mammary tumor virus (MMTV) promoter, which is reproducibly assembled into a phased array of six nucleosomes when introduced into cells, represents a particularly well-studied example. The second or B nucleosome of the phased array is disrupted in response to hormone stimulation, allowing the assembly of a preinitiation complex and the activation of transcription. In vitro experiments demonstrate that the assembly of the proximal promoter into chromatin is sufficient to prevent the binding of transcription factors such as nuclear factor 1. Consequently it is argued that chromatin serves to restrict the access of ubiquitous transcription factors in the absence of hormone stimulation. We have employed an in vivo exonuclease III (ExoIII)/Taq polymerase footprinting assay to study the hormone-dependent loading of transcription factors on the MMTV promoter. This assay makes use of stable mouse and human cell lines harboring bovine papilloma virus chimeras of the MMTV promoter attached to a reporter gene. To ascertain the significance of protein-chromatin interactions vs protein-DNA interactions, we examined transcription factor binding to chromatin and nonchromatin templates of the MMTV promoter within the same cells. By the use of primers specific for each of the two distinct reporter genes, and restriction enzymes that generate entry sites for ExoIII, we can distinguish chromatin and nonnucleosomal templates in vivo. This system has allowed us to visualize the assembly of multicomponent transcription preinitiation complexes and to ascertain the consequences of defined chromatin structures on the binding of individual transcription factors in vivo.

The glucocorticoid receptor (GR) functions to regulate a wide group of physiological processes through hormone inducible interaction with genomic loci and subsequent manipulation of the transcriptional output of target genes. Despite expression in a wide variety of tissues, the GR has diverse roles that are regulated tightly in a cell type specific manner. With the advent of whole genome approaches, the details of that diversity and the mechanisms regulating them are beginning to be elucidated. This review aims describe the recent advances detailing the role chromatin structure plays in dictating GR specificity.

Steroid hormones act via a group of high affinity receptors that regulate transcription by binding to hormone response elements located in the promoters of hormone-inducible genes. Our understanding of these processes has been greatly enhanced by the use of steroid hormone antagonists in both clinical and experimental procedures. However, despite their usefulness in these applications, much about their mechanisms of action remains to be elucidated. Using in vivo analysis techniques, we investigated the influence of type I (ZK98299) and type II (RU486 and ZK112993) steroid hormone antagonists on glucocorticoid-regulated transcription from the mouse mammary tumor virus promoter. Both type I and type II antagonists substantially reduced glucocorticoid-induced expression from the mouse mammary tumor virus promoter stably maintained as chromatin. Concurrent treatment with glucocorticoid and type I or type II antagonists reduced the receptor-dependent chromatin remodeling and loading of transcription factor NF1 that are signature responses of this promoter in the context of chromatin. Treatment with either type I or type II antagonists alone did not induce chromatin remodeling or transcription factor loading. Although type II antagonist-occupied receptor can bind DNA, our results show that this binding is not functionally equivalent to that of agonist-occupied receptor, as it can not interact productively with the cellular apparatus required to open a repressive chromatin structure.

We have investigated whether constitutive binding by the progesterone receptor (PR) to a promoter is required for the maintenance of an open chromatin structure in vivo. For these experiments, we used human T47D breast cancer cells in which the mouse mammary tumor virus (MMTV) promoter, stably assembled as chromatin, is constitutively hypersensitive to endonucleolytic cleavage. In vivo footprinting revealed that transcription factors nuclear factor 1 and the PR were constitutively bound to the MMTV promoter in these cells. Treatment of these cells for 1 hr with the steroid antagonist ZK98299 prevented PR binding to chromatin in vivo and reversed hypersensitivity, leading to the loss of transcription factor binding. The reduction in hypersensitivity induced by ZK98299 was readily reversed by treatment with the progestin R5020. The chromatin organization of the promoter could be cycled between the open and closed states by consecutive treatments with agonist or antagonist. The antagonist RU486 also blocked activation of transcription and the assembly of a transcription preinitiation complex, but in contrast to ZK98299, maintained the hypersensitive chromatin state. Taken together, these results suggest that PR binding to chromatin is sufficient to induce hypersensitivity to endonucleolytic cleavage. Furthermore, they indicate that the PR binding to DNA and the resulting chromatin hypersensitivity is functionally separate from transcriptional activation in vivo.

The ability to respond to small signalling molecules such as steroid hormones is important for many physiological processes. Steroid hormones act through a group of high affinity receptors that regulate transcription by binding to hormone response elements (HREs) located within the promoters of target genes, which themselves are organized with nuclear proteins to form chromatin. To dissect the mechanisms(s) of steroid hormone action we have used the steroid inducible mouse mammary tumor virus (MMTV) promoter as a model system. The MMTV promoter is assembled into a phased array of nucleosomes that are specifically positioned in rodent cells. Induction of transcription by glucocorticoids is accompanied by the appearance of a hypersensitive region in the proximal promoter which allows the hormone dependent assembly of a preinitiation complex including transcription factors such as nuclear factor 1 (NF1) and the octamer transcription factor (OTF). Surprisingly, when introduced by transient transfection, the progesterone receptor (PR) is unable to activate this promoter in vivo, a finding that may result from its inability to alter MMTV promoter chromatin. In an attempt to investigate the failure of the PR to activate the promoter, we have stably introduced the MMTV promoter into human T47D breast cancer cells that express high levels of the PR. In contrast to what has been observed previously in rodent cells, the MMTV templates resident in human breast cancer cells adopt a novel and constitutively open chromatin structure. The constitutively open chromatin structure is accompanied by the hormone independent loading of transcription factors including the PR and NF1. In T47D cells that stably express the glucocorticoid receptor, the MMTV promoter responds to glucocorticoids, but not progestins, and displays glucocorticoid induced restriction enzyme hypersensitivity and transcription factor loading. These findings suggest that the organization of the MMTV chromatin structure is dependent upon the cell type and receptor status of the recipient cell into which the MMTV promoter is stably introduced.

The anticancer drug cis-diamminedichloro-platinum(II) (cisplatin) covalently modifies DNA, and these lesions are thought to lead to cell death by inhibiting DNA and RNA synthesis. By using in vivo analysis techniques, we have investigated the influence of cisplatin on hormone-induced transcription from the mouse mammary tumor virus (MMTV) promoter. Cisplatin substantially reduced glucocorticoid-induced expression from the MMTV promoter stably incorporated into mouse tumor cells. The glucocorticoid-receptor-dependent chromatin remodeling and loading of transcription factors that is a signature response of this promoter in the context of chromatin were significantly reduced by cisplatin but not by the clinically ineffective trans-isomer trans-diamminedichloroplatinum(II) (transplatin). Additional in vivo studies on transiently introduced nonchromatin MMTV templates demonstrated that cisplatin modification of DNA blocked binding of the transcription factor NF1. These results provide strong evidence that cisplatin influences transcription by interfering with the opening of repressive chromatin structures and by blocking transcription factor binding directly, each of which could contribute substantially to its toxicity.

We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (2221 to 275) was found to be constitutively hypersensitive to restrictionenzymecleavageintheabsenceofhormone.Thisregionisnormallyencompassedwithinthesecond nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained inmousecells.Characteristically,intheserodentcells,theidenticalDNAsequencesshowincreasedrestriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR 1 cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sitesfornuclearfactor1andthePRwereobservedbeforeandafterhormonetreatment.Theseresultsindicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin. Mouse mammary tumor virus (MMTV) is a well-developed model system with which to evaluate the regulation of transcription by steroid hormones in the eukaryotic nucleus. It represents one of a handful of promoters for which there is extensive knowledge of the cis control elements and transacting proteins and for which a well-defined chromatin struc

Steroid hormone receptors initiate a genetic program tightly regulated by the chromatin environment of the responsive regions. Using the glucocorticoid receptor (GR) as a model factor for transcriptional initiation, we classified chromatin structure through formaldehyde-assisted isolation of regulatory elements (FAIRE). We looked at dynamic changes in FAIRE signals during GR activation specifically at regions of receptor interaction. We found a distribution of GR-responsive regions with diverse responses to activation and chromatin modulation. The majority of GR binding regions demonstrate increases in FAIRE signal in response to ligand. However, the majority GR-responsive regions shared a similar FAIRE signal in the basal chromatin state, suggesting a common chromatin structure for GR recruitment. Supporting this notion, global FAIRE sequencing (seq) data indicated an enrichment of signal surrounding the GR binding site prior to activation. Brg-1 knockdown showed response element-specific effects of ATPase-dependent chromatin remodeling. FAIRE induction was universally decreased by Brg-1 depletion, but to varying degrees in a target specific manner. Taken together, these data suggest classes of nuclear receptor response regions that react to activation through different chromatin regulatory events and identify a chromatin structure that classifies the majority of response elements tested.

The T47D (A1-2) cell line is a human mammary carcinoma-derived cell line that has been engineered to constitutively express comparable levels of both glucocorticoid and progesterone receptors. In addition, these cells possess a stably integrated mouse mammary tumor virus (MMTV) luciferase reporter gene. Because the MMTV promoter is recognized similarly by both receptors, we have used this cell line to examine the transcriptional regulatory mechanisms employed by the two receptors. The stably integrated MMTV luciferase gene is highly inducible by glucocorticoids, whereas it is almost entirely refractory to induction by progestins. In contrast, a transiently transfected MMTV chloroamphenicol acetyl transferase reporter, while much more inducible by glucocorticoids, can be induced significantly by progestins. The differential inducibility of the stably integrated template is reflected in the superior ability of glucocorticoids to initiate alterations in the chromatin structure of the promoter. Concomitant with the changes in nuclease accessibility, glucocorticoids, unlike progestins, recruit transcription factors to the MMTV promoter. These results emphasize a central role for the modulation of the chromatin environment by steroid receptors in defining their capacity to regulate gene expression in vivo.

The packaging of eukaryotic DNA into nucleosomes, the fundamental unit of chromatin, creates a barrier to nuclear processes, such as transcription, DNA replication, recombination, and repair(1). This obstructive nature of chromatin can be overcome by the enzymatic activity of chromatin remodeling complexes which creates a more favorable environment for the association of essential factors and regulators to sequences within target genes. Here we describe a detailed approach for analyzing chromatin architecture and remodeling by restriction endonuclease hypersensitivity assay. This procedure uses restriction endonucleases to characterize changes in chromatin that accompany nucleosome remodeling. The specific experimental example described in this article is the BRG1 complex dependent chromatin remodeling of the steroid hormone-responsive mouse mammary tumor virus promoter. Through the use of these methodologies one is able to quantify changes at specific nucleosomes in response to regulatory signals.

Nucleosomes can block access to transcription factors. Thus the precise localization of nucleosomes relative to transcription start sites and other factor binding sites is expected to be a critical component of transcriptional regulation. Recently developed microarray approaches have allowed the rapid mapping of nucleosome positions over hundreds of kilobases (kb) of human genomic DNA, although these approaches have not yet been widely used to measure chromatin changes associated with changes in transcription. Here, we use custom tiling microarrays to reveal changes in nucleosome positions and abundance that occur when hormone-bound glucocorticoid receptor (GR) binds to sites near target gene promoters in human osteosarcoma cells. The most striking change is an increase in measured nucleosome occupancy at sites spanning ∼1 kb upstream and downstream of transcription start sites, which occurs one hour after addition of hormone, but is lost at 4 hours. Unexpectedly, this increase was seen both on GR-regulated and GR-non-regulated genes. In addition, the human SWI/SNF chromatin remodeling factor (a GR co-activator) was found to be important for increased occupancy upon hormone treatment and also for low nucleosome occupancy without hormone. Most surprisingly, similar increases in nucleosome occupancy were also seen on both regulated and non-regulated promoters during differentiation of human myeloid leukemia cells and upon activation of human CD4+ T-cells. These results indicate that dramatic changes in chromatin structure over ∼2 kb of human promoters may occur genomewide and in response to a variety of stimuli, and suggest novel models for transcriptional regulation.

The mechanisms by which nuclear hormone receptors (NHRs) regulate transcription are highly dynamic and require interplay between a myriad of regulatory protein complexes including the 26S proteasome. Protein degradation is the most well-established role of the proteasome; however, an increasing body of evidence suggests that the 26S proteasome may regulate transcription in proteolytic and nonproteolytic mechanisms. Here we review how these mechanisms may apply to NHR-mediated transcriptional regulation. This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!

Nuclear receptors are a class of tightly regulated and highly inducible transcription factors and thus represent an excellent model for the study of transcription. The activity of these transcription factors is controlled by their interactions with co-regulatory proteins that act to remodel chromatin, modify histones, and initiate the transcriptional process. This review will focus on the use of nuclear receptors in understanding the role of ATP-dependent chromatin remodeling enzymes in transcription.

A fragment of the mouse mammary tumor virus (MMTV) promoter was reconstituted from pure histones into a dinucleosome with uniquely positioned octamer cores. Core boundaries for the in vitro-assembled dinucleosome corresponded to the observed in vivo phasing pattern for long terminal repeat nucleosomes A and B. Nuclear factor 1 (NF1), a constituent of the MMTV transcription initiation complex, was excluded from the assembled dinucleosome, whereas the glucocorticoid receptor was able to bind. During transcription of MMTV in vivo, displacement of nucleosome B was necessary to permit assembly of the initiation complex. These results indicate that the nucleoprotein structure of the promoter can provide differential access to sequence-specific DNA-binding proteins and that active chromatin remodeling can occur during transcription activation.

Part 1 of this review described the importance of histone acetylases, deacetylases, methylases and demethylases in transcriptional control and their potential as therapeutic targets. However, precise gene regulation requires the involvement of more than just the addition or removal of acetyl and methyl groups on histones. Histone phosphorylation, ubiquitylation, SUMOylation and poly-ADP-ribosylation, as well as ATP-dependent nucleosome remodeling complexes, play equally pivotal roles in the maintenance of transcriptional fidelity. Accordingly, the enzymes responsible for these modifications are also misregulated in various disease states.To review the complex roles of chromatin-modifying enzymes in gene regulation and to highlight their potential as therapeutic targets.This review is based on recent published literature and online resources.In this second and final part of the review, we discuss the importance of these other histone and nucleosome modifying enzymes in gene transcription as well as their therapeutic potential.

Disease pathogenesis may result from genetic alterations and/or a more diverse group of epigenetic changes. While events such as DNA methylation are well established, there is significant interest in nucleosome remodeling, RNA interference and histone modifications, as mechanisms that underlie epigenetic effects. While genetic mutations are permanent, epigenetic changes can be transitory. The potential to reverse epigenetic changes has led to the development of therapeutic strategies targeting chromatin-modifying enzymes.To review the roles of chromatin-modifying enzymes in gene regulation and to highlight their potentials as therapeutic targets.This review is based on recently published literature and online resources.This paper focuses on enzymes responsible for histone acetylation, deacetylation, methylation and demethylation, and their potential as targets for epigenetic therapies. A subsequent paper will do the same for enzymes responsible for histone phosphorylation, ubiquitylation, SUMOylation and poly-ADP-ribosylation as well as ATP-dependent nucleosome remodeling.

Telomeric regions in Drosophila are composed of three subdomains. A chromosome cap distinguishes the chromosome end from a DNA double-strand break; an array of retrotransposons, HeT-A, TART, and TAHRE (HTT), maintains telomere length by targeted transposition to chromosome ends; and telomere-associated sequence (TAS), which consists of a mosaic of complex repeated sequences, has been identified as a source of gene silencing. Heterochromatin protein 1 (HP1) and HP1-ORC-associated protein (HOAP) are major protein components of the telomere cap in Drosophila and are required for telomere stability. Besides the chromosome cap, HP1 is also localized along the HTT array and in TAS. Mutants for Su(var)205, the gene encoding HP1, have decreased the HP1 level in the HTT array and increased transcription of individual HeT-A elements. This suggests that HP1 levels directly affect HeT-A activity along the HTT array, although they have little or no effect on transcription of a white reporter gene in the HTT. Chromatin immunoprecipitation to identify other heterochromatic proteins indicates that TAS and the HTT array may be distinct from either heterochromatin or euchromatin.

Steroid hormone receptors are ligand-activated transcription factors that enhance or repress gene expression. They act by binding to target sites within the promoters of genes assembled as chromatin. Chromatin structure is modified in response to steroid hormones and represents a critical step in steroid receptor signaling. Recent experiments demonstrate that the progesterone and glucocorticoid receptors are differentially influenced by this arrangement of DNA and histones. One of the most important developments in the steroid hormone receptor field has been the identification of coactivators and cointegrators, some of which are histone acetyltransferases. These proteins appear to play an important role in mediating ligand activation of transcription, although their exact role on chromatin templates is undefined.

Steroid hormone receptors, like glucocorticoid (GR) and estrogen receptors (ER), are master regulators of genes that control many biological processes implicated in health and disease. Gene expression is dependent on receptor levels which are tightly regulated by the ubiquitin-proteasome system. Previous studies have shown that proteasome inhibition increases GR, but decreases ER-mediated gene expression. At the gene expression level this divergent role of the proteasome in receptor-dependent transcriptional regulation is not well understood. We have used a genomic approach to examine the impact of proteasome activity on GR- and ER-mediated gene expression in MCF-7 breast cancer cells treated with dexamethasone (DEX) or 17beta-estradiol (E2), the proteasome inhibitor MG132 (MG) or MG132 and either hormone (MD or ME2) for 24 h. Transcript profiling reveals that inhibiting proteasome activity modulates gene expression by GR and ER in a similar manner in that several GR and ER target genes are upregulated and downregulated after proteasome inhibition. In addition, proteasome inhibition modulates receptor-dependent genes involved in the etiology of a number of human pathological states, including multiple myeloma, leukemia, breast/prostate cancer, HIV/AIDS, and neurodegenerative disorders. Importantly, our analysis reveals that a number of transcripts encoding histone and DNA modifying enzymes, prominently histone/DNA methyltransferases and demethylases, are altered after proteasome inhibition. As proteasome inhibitors are currently in clinical trials as therapy for multiple myeloma, HIV/AIDS and leukemia, the possibility that some of the target molecules are hormone regulated and chromatin modifying enzymes is intriguing in this era of epigenetic therapy.