Selection of epigenetically privileged HIV-1 proviruses during treatment with panobinostat and interferon-α2a.

CD4+ T cells with latent HIV-1 infection persist despite treatment with antiretroviral agents and represent the main barrier to a cure of HIV-1 infection. Pharmacological disruption of viral latency may expose HIV-1-infected cells to host immune activity, but the clinical efficacy of latency-reversing agents for reducing HIV-1 persistence remains to be proven. Here, we show in a randomized-controlled human clinical trial that the histone deacetylase inhibitor panobinostat, when administered in combination with pegylated interferon-α2a, induces a structural transformation of the HIV-1 reservoir cell pool, characterized by a disproportionate overrepresentation of HIV-1 proviruses integrated in ZNF genes and in chromatin regions with reduced H3K27ac marks, the molecular target sites for panobinostat. By contrast, proviruses near H3K27ac marks were actively selected against, likely due to increased susceptibility to panobinostat. These data suggest that latency-reversing treatment can increase the immunological vulnerability of HIV-1 reservoir cells and accelerate the selection of epigenetically privileged HIV-1 proviruses.

Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.

Condensed Chromatin Behaves like a Solid on the Mesoscale In Vitro and in Living Cells.

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.

The Implication of Early Chromatin Changes in X Chromosome Inactivation.

During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing.

Dosage Compensation of the X Chromosome: A Complex Epigenetic Assignment Involving Chromatin Regulators and Long Noncoding RNAs.

X chromosome regulation represents a prime example of an epigenetic phenomenon where coordinated regulation of a whole chromosome is required. In flies, this is achieved by transcriptional upregulation of X chromosomal genes in males to equalize the gene dosage differences in females. Chromatin-bound proteins and long noncoding RNAs (lncRNAs) constituting a ribonucleoprotein complex known as the male-specific lethal (MSL) complex or the dosage compensation complex mediate this process. MSL complex members decorate the male X chromosome, and their absence leads to male lethality. The male X chromosome is also enriched with histone H4 lysine 16 acetylation (H4K16ac), indicating that the chromatin compaction status of the X chromosome also plays an important role in transcriptional activation. How the X chromosome is specifically targeted and how dosage compensation is mechanistically achieved are central questions for the field. Here, we review recent advances, which reveal a complex interplay among lncRNAs, the chromatin landscape, transcription, and chromosome conformation that fine-tune X chromosome gene expression.

RNA Binding to CBP Stimulates Histone Acetylation and Transcription.

CBP/p300 are transcription co-activators whose binding is a signature of enhancers, cis-regulatory elements that control patterns of gene expression in multicellular organisms. Active enhancers produce bi-directional enhancer RNAs (eRNAs) and display CBP/p300-dependent histone acetylation. Here, we demonstrate that CBP binds directly to RNAs in vivo and in vitro. RNAs bound to CBP in vivo include a large number of eRNAs. Using steady-state histone acetyltransferase (HAT) assays, we show that an RNA binding region in the HAT domain of CBP-a regulatory motif unique to CBP/p300-allows RNA to stimulate CBP's HAT activity. At enhancers where CBP interacts with eRNAs, stimulation manifests in RNA-dependent changes in the histone acetylation mediated by CBP, such as H3K27ac, and by corresponding changes in gene expression. By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which, in turn, is required for regulation of target genes.

Histone Acetylome-wide Association Study of Autism Spectrum Disorder.

The association of histone modification changes with autism spectrum disorder (ASD) has not been systematically examined. We conducted a histone acetylome-wide association study (HAWAS) by performing H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) on 257 postmortem samples from ASD and matched control brains. Despite etiological heterogeneity, ≥68% of syndromic and idiopathic ASD cases shared a common acetylome signature at >5,000 cis-regulatory elements in prefrontal and temporal cortex. Similarly, multiple genes associated with rare genetic mutations in ASD showed common "epimutations." Acetylome aberrations in ASD were not attributable to genetic differentiation at cis-SNPs and highlighted genes involved in synaptic transmission, ion transport, epilepsy, behavioral abnormality, chemokinesis, histone deacetylation, and immunity. By correlating histone acetylation with genotype, we discovered >2,000 histone acetylation quantitative trait loci (haQTLs) in human brain regions, including four candidate causal variants for psychiatric diseases. Due to the relative stability of histone modifications postmortem, we anticipate that the HAWAS approach will be applicable to multiple diseases.

LKB1 controls inflammatory potential through CRTC2-dependent histone acetylation.

Deregulated inflammation is a critical feature driving the progression of tumors harboring mutations in the liver kinase B1 (LKB1), yet the mechanisms linking LKB1 mutations to deregulated inflammation remain undefined. Here, we identify deregulated signaling by CREB-regulated transcription coactivator 2 (CRTC2) as an epigenetic driver of inflammatory potential downstream of LKB1 loss. We demonstrate that LKB1 mutations sensitize both transformed and non-transformed cells to diverse inflammatory stimuli, promoting heightened cytokine and chemokine production. LKB1 loss triggers elevated CRTC2-CREB signaling downstream of the salt-inducible kinases (SIKs), increasing inflammatory gene expression in LKB1-deficient cells. Mechanistically, CRTC2 cooperates with the histone acetyltransferases CBP/p300 to deposit histone acetylation marks associated with active transcription (i.e., H3K27ac) at inflammatory gene loci, promoting cytokine expression. Together, our data reveal a previously undefined anti-inflammatory program, regulated by LKB1 and reinforced through CRTC2-dependent histone modification signaling, that links metabolic and epigenetic states to cell-intrinsic inflammatory potential.

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex.

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

The landscape of pioneer factor activity reveals the mechanisms of chromatin reprogramming and genome activation.

Upon fertilization, embryos undergo chromatin reprogramming and genome activation; however, the mechanisms that regulate these processes are poorly understood. Here, we generated a triple mutant for Nanog, Pou5f3, and Sox19b (NPS) in zebrafish and found that NPS pioneer chromatin opening at >50% of active enhancers. NPS regulate acetylation across core histones at enhancers and promoters, and their function in gene activation can be bypassed by recruiting histone acetyltransferase to individual genes. NPS pioneer chromatin opening individually, redundantly, or additively depending on sequence context, and we show that high nucleosome occupancy facilitates NPS pioneering activity. Nucleosome position varies based on the input of different transcription factors (TFs), providing a flexible platform to modulate pioneering activity. Altogether, our results illuminate the sequence of events during genome activation and offer a conceptual framework to understand how pioneer factors interpret the genome and integrate different TF inputs across cell types and developmental transitions.

Multimodal regulatory elements within a hormone-specific super enhancer control a heterogeneous transcriptional response.

The hormone-stimulated glucocorticoid receptor (GR) modulates transcription by interacting with thousands of enhancers and GR binding sites (GBSs) throughout the genome. Here, we examined the effects of GR binding on enhancer dynamics and investigated the contributions of individual GBSs to the hormone response. Hormone treatment resulted in genome-wide reorganization of the enhancer landscape in breast cancer cells. Upstream of the DDIT4 oncogene, GR bound to four sites constituting a hormone-dependent super enhancer. Three GBSs were required as hormone-dependent enhancers that differentially promoted histone acetylation, transcription frequency, and burst size. Conversely, the fourth site suppressed transcription and hormone treatment alleviated this suppression. GR binding within the super enhancer promoted a loop-switching mechanism that allowed interaction of the DDIT4 TSS with the active GBSs. The unique functions of each GR binding site contribute to hormone-induced transcriptional heterogeneity and demonstrate the potential for targeted modulation of oncogene expression.

Glucose starvation induces a switch in the histone acetylome for activation of gluconeogenic and fat metabolism genes.

Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.

Immune Sensing of Cell Death through Recognition of Histone Sequences by C-Type Lectin-Receptor-2d Causes Inflammation and Tissue Injury.

The immune system monitors the health of cells and is stimulated by necrosis. Here we examined the receptors and ligands driving this response. In a targeted screen of C-type lectin receptors, a Clec2d reporter responded to lysates from necrotic cells. Biochemical purification identified histones, both free and bound to nucleosomes or neutrophil extracellular traps, as Clec2d ligands. Clec2d recognized poly-basic sequences in histone tails and this recognition was sensitive to post-translational modifications of these sequences. As compared with WT mice, Clec2d-/- mice exhibited reduced proinflammatory responses to injected histones, and less tissue damage and improved survival in a hepatotoxic injury model. In macrophages, Clec2d localized to the plasma membrane and endosomes. Histone binding to Clec2d did not stimulate kinase activation or cytokine production. Rather, histone-bound DNA stimulated endosomal Tlr9-dependent responses in a Clec2d-dependent manner. Thus, Clec2d binds to histones released upon necrotic cell death, with functional consequences to inflammation and tissue damage.

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation.

Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. Here we describe a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. We find that the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis.

Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction in vitro. Although extensively studied in Drosophila, the functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.

Exploiting dynamic enhancer landscapes to decode macrophage and microglia phenotypes in health and disease.

The development and functional potential of metazoan cells is dependent on combinatorial roles of transcriptional enhancers and promoters. Macrophages provide exceptionally powerful model systems for investigation of mechanisms underlying the activation of cell-specific enhancers that drive transitions in cell fate and cell state. Here, we review recent advances that have expanded appreciation of the diversity of macrophage phenotypes in health and disease, emphasizing studies of liver, adipose tissue, and brain macrophages as paradigms for other tissue macrophages and cell types. Studies of normal tissue-resident macrophages and macrophages associated with cirrhosis, obese adipose tissue, and neurodegenerative disease illustrate the major roles of tissue environment in remodeling enhancer landscapes to specify the development and functions of distinct macrophage phenotypes. We discuss the utility of quantitative analysis of environment-dependent changes in enhancer activity states as an approach to discovery of regulatory transcription factors and upstream signaling pathways.

Recognition of acetylated histone by Yaf9 regulates metabolic cycling of transcription initiation and chromatin regulatory factors.

How transcription programs rapidly adjust to changing metabolic and cellular cues remains poorly defined. Here, we reveal a function for the Yaf9 component of the SWR1-C and NuA4 chromatin regulatory complexes in maintaining timely transcription of metabolic genes across the yeast metabolic cycle (YMC). By reading histone acetylation during the oxidative and respiratory phase of the YMC, Yaf9 recruits SWR1-C and NuA4 complexes to deposit H2A.Z and acetylate H4, respectively. Increased H2A.Z and H4 acetylation during the oxidative phase promotes transcriptional initiation and chromatin machinery occupancy and is associated with reduced RNA polymerase II levels at genes-a pattern reversed during transition from oxidative to reductive metabolism. Prevention of Yaf9-H3 acetyl reading disrupted this pattern of transcriptional and chromatin regulator recruitment and impaired the timely transcription of metabolic genes. Together, these findings reveal that Yaf9 contributes to a dynamic chromatin and transcription initiation factor signature that is necessary for the proper regulation of metabolic gene transcription during the YMC. They also suggest that unique regulatory mechanisms of transcription exist at distinct metabolic states.

Toll-like Receptor Signaling Rewires Macrophage Metabolism and Promotes Histone Acetylation via ATP-Citrate Lyase.

Toll-like receptor (TLR) activation induces inflammatory responses in macrophages by activating temporally defined transcriptional cascades. Whether concurrent changes in the cellular metabolism that occur upon TLR activation influence the quality of the transcriptional responses remains unknown. Here, we investigated how macrophages adopt their metabolism early after activation to regulate TLR-inducible gene induction. Shortly after TLR4 activation, macrophages increased glycolysis and tricarboxylic acid (TCA) cycle volume. Metabolic tracing studies revealed that TLR signaling redirected metabolic fluxes to generate acetyl-Coenzyme A (CoA) from glucose resulting in augmented histone acetylation. Signaling through the adaptor proteins MyD88 and TRIF resulted in activation of ATP-citrate lyase, which in turn facilitated the induction of distinct LPS-inducible gene sets. We postulate that metabolic licensing of histone acetylation provides another layer of control that serves to fine-tune transcriptional responses downstream of TLR activation. Our work highlights the potential of targeting the metabolic-epigenetic axis in inflammatory settings.

p53 protects against alcoholic fatty liver disease via ALDH2 inhibition.

The tumor suppressor p53 is critical for tumor suppression, but the regulatory role of p53 in alcohol-induced fatty liver remains unclear. Here, we show a role for p53 in regulating ethanol metabolism via acetaldehyde dehydrogenase 2 (ALDH2), a key enzyme responsible for the oxidization of alcohol. By repressing ethanol oxidization, p53 suppresses intracellular levels of acetyl-CoA and histone acetylation, leading to the inhibition of the stearoyl-CoA desaturase-1 (SCD1) gene expression. Mechanistically, p53 directly binds to ALDH2 and prevents the formation of its active tetramer and indirectly limits the production of pyruvate that promotes the activity of ALDH2. Notably, p53-deficient mice exhibit increased lipid accumulation, which can be reversed by ALDH2 depletion. Moreover, liver-specific knockdown of SCD1 alleviates ethanol-induced hepatic steatosis caused by p53 loss. By contrast, overexpression of SCD1 in liver promotes ethanol-induced fatty liver development in wild-type mice, while it has a mild effect on p53-/- or ALDH2-/- mice. Overall, our findings reveal a previously unrecognized function of p53 in alcohol-induced fatty liver and uncover pyruvate as a natural regulator of ALDH2.