Smarcd3 is an epigenetic modulator of the metabolic landscape in pancreatic ductal adenocarcinoma.

Pancreatic cancer is characterized by extensive resistance to conventional therapies, making clinical management a challenge. Here we map the epigenetic dependencies of cancer stem cells, cells that preferentially evade therapy and drive progression, and identify SWI/SNF complex member SMARCD3 as a regulator of pancreatic cancer cells. Although SWI/SNF subunits often act as tumor suppressors, we show that SMARCD3 is amplified in cancer, enriched in pancreatic cancer stem cells and upregulated in the human disease. Diverse genetic mouse models of pancreatic cancer and stage-specific Smarcd3 deletion reveal that Smarcd3 loss preferentially impacts established tumors, improving survival especially in context of chemotherapy. Mechanistically, SMARCD3 acts with FOXA1 to control lipid and fatty acid metabolism, programs associated with therapy resistance and poor prognosis in cancer. These data identify SMARCD3 as an epigenetic modulator responsible for establishing the metabolic landscape in aggressive pancreatic cancer cells and a potential target for new therapies.

BRD9 regulates interferon-stimulated genes during macrophage activation via cooperation with BET protein BRD4.

Macrophages induce a number of inflammatory response genes in response to stimulation with microbial ligands. In response to endotoxin Lipid A, a gene-activation cascade of primary followed by secondary-response genes is induced. Epigenetic state is an important regulator of the kinetics, specificity, and mechanism of gene activation of these two classes. In particular, SWI/SNF chromatin-remodeling complexes are required for the induction of secondary-response genes, but not primary-response genes, which generally exhibit open chromatin. Here, we show that a recently discovered variant of the SWI/SNF complex, the noncanonical BAF complex (ncBAF), regulates secondary-response genes in the interferon (IFN) response pathway. Inhibition of bromodomain-containing protein 9 (BRD9), a subunit of the ncBAF complex, with BRD9 bromodomain inhibitors (BRD9i) or a degrader (dBRD9) led to reduction in a number of interferon-stimulated genes (ISGs) following stimulation with endotoxin lipid A. BRD9-dependent genes overlapped highly with a subset of genes differentially regulated by BET protein inhibition with JQ1 following endotoxin stimulation. We find that the BET protein BRD4 is cobound with BRD9 in unstimulated macrophages and corecruited upon stimulation to ISG promoters along with STAT1, STAT2, and IRF9, components of the ISGF3 complex activated downstream of IFN-alpha receptor stimulation. In the presence of BRD9i or dBRD9, STAT1-, STAT2-, and IRF9-binding is reduced, in some cases with reduced binding of BRD4. These results demonstrate a specific role for BRD9 and the ncBAF complex in ISG activation and identify an activity for BRD9 inhibitors and degraders in dampening endotoxin- and IFN-dependent gene expression.

Bromodomain containing 9 (BRD9) regulates macrophage inflammatory responses by potentiating glucocorticoid receptor activity.

In macrophages, homeostatic and immune signals induce distinct sets of transcriptional responses, defining cellular identity and functional states. The activity of lineage-specific and signal-induced transcription factors are regulated by chromatin accessibility and other epigenetic modulators. Glucocorticoids are potent antiinflammatory drugs; however, the mechanisms by which they selectively attenuate inflammatory genes are not yet understood. Acting through the glucocorticoid receptor (GR), glucocorticoids directly repress inflammatory responses at transcriptional and epigenetic levels in macrophages. A major unanswered question relates to the sequence of events that result in the formation of repressive regions. In this study, we identify bromodomain containing 9 (BRD9), a component of SWI/SNF chromatin remodeling complex, as a modulator of glucocorticoid responses in macrophages. Inhibition, degradation, or genetic depletion of BRD9 in bone marrow-derived macrophages significantly attenuated their responses to both liposaccharides and interferon inflammatory stimuli. Notably, BRD9-regulated genes extensively overlap with those regulated by the synthetic glucocorticoid dexamethasone. Pharmacologic inhibition of BRD9 potentiated the antiinflammatory responses of dexamethasone, while the genetic deletion of BRD9 in macrophages reduced high-fat diet-induced adipose inflammation. Mechanistically, BRD9 colocalized at a subset of GR genomic binding sites, and depletion of BRD9 enhanced GR occupancy primarily at inflammatory-related genes to potentiate GR-induced repression. Collectively, these findings establish BRD9 as a genomic antagonist of GR at inflammatory-related genes in macrophages, and reveal a potential for BRD9 inhibitors to increase the therapeutic efficacies of glucocorticoids.

A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function.

Regulatory T (Treg) cells play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg cell development and function are dependent on the transcription factor Foxp3. Here, we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome-remodeling and SAGA chromatin-modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc) BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg cell function in vitro. Brd9 ablation compromised Treg cell function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg cell function.

CRISPR screen in regulatory T cells reveals modulators of Foxp3.

Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

Control of Stimulus-Dependent Responses in Macrophages by SWI/SNF Chromatin Remodeling Complexes.

Epigenetic regulation plays an important role in controlling the activation, timing, and resolution of innate immune responses in macrophages. Previously, SWI/SNF chromatin remodeling was found to define the kinetics and selectivity of gene activation in response to microbial ligands; however, these studies do not reflect a comprehensive understanding of SWI/SNF complex regulation. In 2018, a new variant of the SWI/SNF complex was identified with unknown function in inflammatory gene regulation. Here, we summarize the biochemical and genomic properties of SWI/SNF complex variants and the potential for increased regulatory control of innate immune transcriptional programs in light of such biochemical diversity. Finally, we review the development of SWI/SNF complex chemical inhibitors and degraders that could be used to modulate immune responses.

A non-canonical BRD9-containing BAF chromatin remodeling complex regulates naive pluripotency in mouse embryonic stem cells.

The role of individual subunits in the targeting and function of the mammalian BRG1-associated factors (BAF) complex in embryonic stem cell (ESC) pluripotency maintenance has not yet been elucidated. Here we find that the Bromodomain containing protein 9 (BRD9) and Glioma tumor suppressor candidate region gene 1 (GLTSCR1) or its paralog GLTSCR1-like (GLTSCR1L) define a smaller, non-canonical BAF complex (GBAF complex) in mouse ESCs that is distinct from the canonical ESC BAF complex (esBAF). GBAF and esBAF complexes are targeted to different genomic features, with GBAF co-localizing with key regulators of naive pluripotency, which is consistent with its specific function in maintaining naive pluripotency gene expression. BRD9 interacts with BRD4 in a bromodomain-dependent fashion, which leads to the recruitment of GBAF complexes to chromatin, explaining the functional similarity between these epigenetic regulators. Together, our results highlight the biological importance of BAF complex heterogeneity in maintaining the transcriptional network of pluripotency.

Racheting Up Repair.

In this issue of Immunity, Daniel et al. (2018a) demonstrate that the nuclear receptor PPARγ acts in a ligand-insensitive manner to impart transcriptional memory and enhanced functionality to IL-4 polarized macrophages. Their findings shed light on the mechanisms that control priming of the epigenome in response to inflammatory signals.

Small Molecule Targeting of Specific BAF (mSWI/SNF) Complexes for HIV Latency Reversal.

The persistence of a pool of latently HIV-1-infected cells despite combination anti-retroviral therapy treatment is the major roadblock for a cure. The BAF (mammalian SWI/SNF) chromatin remodeling complex is involved in establishing and maintaining viral latency, making it an attractive drug target for HIV-1 latency reversal. Here we report a high-throughput screen for inhibitors of BAF-mediated transcription in cells and the subsequent identification of a 12-membered macrolactam. This compound binds ARID1A-specific BAF complexes, prevents nucleosomal positioning, and relieves transcriptional repression of HIV-1. Through this mechanism, these compounds are able to reverse HIV-1 latency in an in vitro T cell line, an ex vivo primary cell model of HIV-1 latency, and in patient CD4+ T cells without toxicity or T cell activation. These macrolactams represent a class of latency reversal agents with unique mechanism of action, and can be combined with other latency reversal agents to improve reservoir targeting.

Accessibility of the histone H3 tail in the nucleosome for binding of paired readers.

Combinatorial polyvalent contacts of histone-binding domains or readers commonly mediate localization and activities of chromatin-associated proteins. A pair of readers, the PHD fingers of the protein CHD4, has been shown to bivalently recognize histone H3 tails. Here we describe a mechanism by which these linked but independent readers bind to the intact nucleosome core particle (NCP). Comprehensive NMR, chemical reactivity, molecular dynamics, and fluorescence analyses point to the critical roles of intra-nucleosomal histone-DNA interactions that reduce the accessibility of H3 tails in NCP, the nucleosomal DNA, and the linker between readers in modulating nucleosome- and/or histone-binding activities of the readers. We show that the second PHD finger of CHD4 initiates recruitment to the nucleosome, however both PHDs are required to alter the NCP dynamics. Our findings reveal a distinctive regulatory mechanism for the association of paired readers with the nucleosome that provides an intricate balance between cooperative and individual activities of the readers.

Covalent Modifications of Histone H3K9 Promote Binding of CHD3.

Chromatin remodeling is required for genome function and is facilitated by ATP-dependent complexes, such as nucleosome remodeling and deacetylase (NuRD). Among its core components is the chromodomain helicase DNA binding protein 3 (CHD3) whose functional significance is not well established. Here, we show that CHD3 co-localizes with the other NuRD subunits, including HDAC1, near the H3K9ac-enriched promoters of the NuRD target genes. The tandem PHD fingers of CHD3 bind histone H3 tails and posttranslational modifications that increase hydrophobicity of H3K9-methylation or acetylation (H3K9me3 or H3K9ac)-enhance this interaction. Binding of CHD3 PHDs promotes H3K9Cme3-nucleosome unwrapping in vitro and perturbs the pericentric heterochromatin structure in vivo. Methylation or acetylation of H3K9 uniquely alleviates the intra-nucleosomal interaction of histone H3 tails, increasing H3K9 accessibility. Collectively, our data suggest that the targeting of covalently modified H3K9 by CHD3 might be essential in diverse functions of NuRD.

A Unique pH-Dependent Recognition of Methylated Histone H3K4 by PPS and DIDO.

The protein partner of Sans-fille (PPS) and its human homolog DIDO mediate diverse chromatin activities, including the regulation of stemness genes in embryonic stem cells and splicing in Drosophila. Here, we show that the PHD fingers of PPS and DIDO recognize the histone mark H3K4me3 in a pH-dependent manner: the binding is enhanced at high pH values but is decreased at low pH. Structural analysis reveals that the pH dependency is due to the presence of a histidine residue in the K4me3-binding aromatic cage of PPS. The pH-dependent mechanism is conserved in DIDO but is lost in yeast Bye1. Acidification of cells leads to the accelerated efflux of endogenous DIDO, indicating the pH-dependent sensing of H3K4me3 in vivo. This novel mode for the recognition of H3K4me3 establishes the PHD fingers of PPS and DIDO as unique epigenetic readers and high pH sensors and suggests a role for the histidine switch during mitosis.

PHF1 Tudor and N-terminal domains synergistically target partially unwrapped nucleosomes to increase DNA accessibility.

The Tudor domain of human PHF1 recognizes trimethylated lysine 36 on histone H3 (H3K36me3). PHF1 relies on this interaction to regulate PRC2 methyltransferase activity, localize to DNA double strand breaks and mediate nucleosome accessibility. Here, we investigate the impact of the PHF1 N-terminal domain (NTD) on the Tudor domain interaction with the nucleosome. We show that the NTD is partially ordered when it is natively attached to the Tudor domain. Through a combination of FRET and single molecule studies, we find that the increase of DNA accessibility within the H3K36me3-containing nucleosome, instigated by the Tudor binding to H3K36me3, is dramatically enhanced by the NTD. We demonstrate that this nearly order of magnitude increase is due to preferential binding of PHF1 to partially unwrapped nucleosomes, and that PHF1 alters DNA-protein binding within the nucleosome by decreasing dissociation rates. These results highlight the potency of a PTM-binding protein to regulate DNA accessibility and underscores the role of the novel mechanism by which nucleosomes control DNA-protein binding through increasing protein dissociation rates.

Structural Insight into Recognition of Methylated Histone H3K4 by Set3.

The plant homeodomain (PHD) finger of Set3 binds methylated lysine 4 of histone H3 in vitro and in vivo; however, precise selectivity of this domain has not been fully characterized. Here, we explore the determinants of methyllysine recognition by the PHD fingers of Set3 and its orthologs. We use X-ray crystallographic and spectroscopic approaches to show that the Set3 PHD finger binds di- and trimethylated states of H3K4 with comparable affinities and employs similar molecular mechanisms to form complexes with either mark. Composition of the methyllysine-binding pocket plays an essential role in determining the selectivity of the PHD fingers. The finding that the histone-binding activity is not conserved in the PHD finger of Set4 suggests different functions for the Set3 and Set4 paralogs.

Chromatin condensation and recruitment of PHD finger proteins to histone H3K4me3 are mutually exclusive.

Histone post-translational modifications, and specific combinations they create, mediate a wide range of nuclear events. However, the mechanistic bases for recognition of these combinations have not been elucidated. Here, we characterize crosstalk between H3T3 and H3T6 phosphorylation, occurring in mitosis, and H3K4me3, a mark associated with active transcription. We detail the molecular mechanisms by which H3T3ph/K4me3/T6ph switches mediate activities of H3K4me3-binding proteins, including those containing plant homeodomain (PHD) and double Tudor reader domains. Our results derived from nuclear magnetic resonance chemical shift perturbation analysis, orthogonal binding assays and cell fluorescence microscopy studies reveal a strong anti-correlation between histone H3T3/T6 phosphorylation and retention of PHD finger proteins in chromatin during mitosis. Together, our findings uncover the mechanistic rules of chromatin engagement for H3K4me3-specific readers during cell division.

Regulation of Methyllysine Readers through Phosphorylation.

Methyllysine post-translational modifications (PTMs) of histones create binding sites for evolutionarily conserved reader domains that link nuclear host proteins and chromatin-modifying complexes to specific genomic regions. In the context of these events, adjacent histone PTMs are capable of altering the binding activity of readers toward their target marks. This provides a mechanism of "combinatorial readout" of PTMs that can enhance, decrease, or eliminate the association of readers with chromatin. In this Perspective, we focus on recent studies describing the impact of dynamic phospho-serine/threonine/tyrosine marks on the interaction of methyllysine readers with histones, summarize mechanistic aspects of the phospho/methyl readout, and highlight the significance of crosstalk between these PTMs. We also demonstrate that in addition to inhibiting binding and serving as a true switch, promoting dissociation of the methyllysine readers from chromatin, the phospho/methyl combination can act together in a cooperative manner--thus adding a new layer of regulatory information that can be encoded in these dual histone PTMs.

An aromatic cage is required but not sufficient for binding of Tudor domains of the Polycomblike protein family to H3K36me3.

Polycomblike (Pcl) proteins are important transcriptional regulators and components of the Polycomb Repressive Complex 2 (PRC2). The Tudor domains of human homologs PHF1 and PHF19 have been found to recognize trimethylated lysine 36 of histone H3 (H3K36me3); however, the biological role of Tudor domains of other Pcl proteins remains poorly understood. Here, we characterize the molecular basis underlying histone binding activities of the Tudor domains of the Pcl family. In contrast to a predominant view, we found that the methyl lysine-binding aromatic cage is necessary but not sufficient for recognition of H3K36me3 by these Tudor domains and that a hydrophobic patch, adjacent to the aromatic cage, is also required.

Binding of PHF1 Tudor to H3K36me3 enhances nucleosome accessibility.

The Tudor domain of human PHF1 recognizes trimethylated lysine 36 of histone H3 (H3K36me3). This interaction modulates the methyltransferase activity of the PRC2 complex and has a role in retention of PHF1 at DNA damage sites. We have previously determined the structural basis for the association of Tudor with a methylated histone peptide. Here we detail the molecular mechanism of binding of the Tudor domain to the H3KC36me3-nucleosome core particle (H3KC36me3-NCP). Using a combination of TROSY NMR and FRET, we show that Tudor concomitantly interacts with H3K36me3 and DNA. Binding of the PHF1 Tudor domain to the H3KC36me3-NCP stabilizes the nucleosome in a conformation in which the nucleosomal DNA is more accessible to DNA-binding regulatory proteins. Our data provide a mechanistic explanation for the consequence of reading of the active mark H3K36me3 by the PHF1 Tudor domain.

Dido3 PHD modulates cell differentiation and division.

Death Inducer Obliterator 3 (Dido3) is implicated in the maintenance of stem cell genomic stability and tumorigenesis. Here, we show that Dido3 regulates the expression of stemness genes in embryonic stem cells through its plant homeodomain (PHD) finger. Binding of Dido3 PHD to histone H3K4me3 is disrupted by threonine phosphorylation that triggers Dido3 translocation from chromatin to the mitotic spindle. The crystal structure of Dido3 PHD in complex with H3K4me3 reveals an atypical aromatic-cage-like binding site that contains a histidine residue. Biochemical, structural, and mutational analyses of the binding mechanism identified the determinants of specificity and affinity and explained the inability of homologous PHF3 to bind H3K4me3. Together, our findings reveal a link between the transcriptional control in embryonic development and regulation of cell division.

Ligand-dependent perturbation of the conformational ensemble for the GPCR ß2 adrenergic receptor revealed by HDX.

Mechanism of G protein-coupled receptor (GPCR) activation and their modulation by functionally distinct ligands remains elusive. Using the technique of amide hydrogen/deuterium exchange coupled with mass spectrometry, we examined the ligand-induced changes in conformational states and stability within the beta-2-adrenergic receptor (ß(2)AR). Differential HDX reveals ligand-specific alterations in the energy landscape of the receptor's conformational ensemble. The inverse agonists timolol and carazolol were found to be most stabilizing even compared with the antagonist alprenolol, notably in intracellular regions where G proteins are proposed to bind, while the agonist isoproterenol induced the largest degree of conformational mobility. The partial agonist clenbuterol displayed conformational effects found in both the inverse agonists and the agonist. This study highlights the regional plasticity of the receptor and characterizes unique conformations spanning the entire receptor sequence stabilized by functionally selective ligands, all of which differ from the profile for the apo receptor.