Histone Acylation Research Articles

The histone acylation reader ENL/AF9 regulates aging in Drosophila melanogaster

Histone acylation plays a pivotal role in modulating gene expression, ensuring proper neurogenesis and responsiveness to various signals. Recently, the evolutionary conserved YAF9, ENL, AF9, TAF41, SAS5 (YEATS) domain found in four human paralogs, has emerged as a new class of histone acylation reader with a preference for the bulkier crotonyl group lysine over acetylation. Despite advancements, the role of either histone crotonylation or its readers in neurons remains unclear. In this study, we employed Drosophila melanogaster to investigate the role of ENL/AF9 (dENL/AF9) in the nervous system. Pan-neuronal dENL/AF9 knockdown not only extended the lifespan of flies but also enhanced their overall fitness during aging, including improved sleep quality and locomotion. Moreover, a decreased activity of dENL/AF9 in neurons led to an up-regulation of catalase gene expression which combined with reduced levels of malondialdehyde (MDA) and an enhanced tolerance to oxidative stress in aging flies. This study unveiled a novel function of histone crotonylation readers in aging with potential implications for understanding age-related conditions in humans.

Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity.

Engineering histone acylation states can inform mechanistic epigenetics and catalyze therapeutic epigenome editing opportunities. Here, we developed engineered lysine acyltransferases that enable the programmable deposition of acetylation and longer-chain acylations. We show that targeting an engineered lysine crotonyltransferase results in weak levels of endogenous enhancer activation yet retains potency when targeted to promoters. We further identify a single mutation within the catalytic core of human p300 that preserves enzymatic activity while substantially reducing cytotoxicity, enabling improved viral delivery. We leveraged these capabilities to perform single-cell CRISPR activation screening and map enhancers to the genes they regulate in situ. We also discover acylation-specific interactions and find that recruitment of p300, regardless of catalytic activity, to prime editing sites can improve editing efficiency. These new programmable epigenome editing tools and insights expand our ability to understand the mechanistic role of lysine acylation in epigenetic and cellular processes and perform functional genomic screens.

A potent and selective ENL degrader suppresses oncogenic gene expression and leukemia progression.

The histone acylation reader eleven-nineteen leukemia (ENL) plays a pivotal role in sustaining oncogenesis in acute leukemias, particularly in mixed-lineage leukemia-rearranged (MLL-r) leukemia. ENL relies on its reader domain to recognize histone lysine acylation promoting oncogenic gene expression and leukemia progression. Here, we report the development of MS41, a highly potent and selective von Hippel-Lindau-recruiting ENL degrader that effectively inhibits the growth of ENL-dependent leukemia cells. MS41-induced ENL degradation reduces the chromatin occupancy of ENL-associated transcription elongation machinery, resulting in the suppression of key oncogenic gene expression programs and the activation of differentiation genes. MS41 is well-tolerated in vivo and substantially suppresses leukemia progression in a xenograft mouse model of MLL-r leukemia. Notably, MS41 also induces the degradation of mutant ENL proteins identified in Wilms' tumors. Our findings emphasize the therapeutic potential of pharmacological ENL degradation for treating ENL-dependent cancers, making MS41 not only a valuable chemical probe but also potential anticancer therapeutic for further development.

A MOZ-TIF2 leukemia mouse model displays KAT6-dependent H3K23 propionylation and overexpression of a set of active developmental genes

Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alterations of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of acute myeloid leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Furthermore, transcription levels in MOZ-TIF2 cells positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of unique gene sets, and their cellular models exhibit distinct sensitivities to multiple small-molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate that these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation.

Improved Mass Spectrometry–Based Methods Reveal Abundant Propionylation and Tissue-Specific Histone Propionylation Profiles

Histone posttranslational modifications (PTMs) have crucial roles in a multitude of cellular processes, and their aberrant levels have been linked with numerous diseases, including cancer. Although histone PTM investigations have focused so far on methylations and acetylations, alternative long-chain acylations emerged as new dimension, as they are linked to cellular metabolic states and affect gene expression through mechanisms distinct from those regulated by acetylation. Mass spectrometry is the most powerful, comprehensive, and unbiased method to study histone PTMs. However, typical mass spectrometry–based protocols for histone PTM analysis do not allow the identification of naturally occurring propionylation and butyrylation. Here, we present improved state-of-the-art sample preparation and analysis protocols to quantitate these classes of modifications. After testing different derivatization methods coupled to protease digestion, we profiled common histone PTMs and histone acylations in seven mouse tissues and human normal and tumor breast clinical samples, obtaining a map of propionylations and butyrylations found in different tissue contexts. A quantitative histone PTM analysis also revealed a contribution of histone acylations in discriminating different tissues, also upon perturbation with antibiotics, and breast cancer samples from the normal counterpart. Our results show that profiling only classical modifications is limiting and highlight the importance of using sample preparation methods that allow the analysis of the widest possible spectrum of histone modifications, paving the way for deeper insights into their functional significance in cellular processes and disease states.

Histone acylation at a glance.

An important mechanism of gene expression regulation is the epigenetic modification of histones. The cofactors and substrates for these modifications are often intermediary metabolites, and it is becoming increasingly clear that the metabolic and nutritional state of cells can influence these marks. These connections between the balance of metabolites, histone modifications and downstream transcriptional changes comprise a metabolic signaling program that can enable cells to adapt to changes in nutrient availability. Beyond acetylation, there is evidence now that histones can be modified by other acyl groups. In this Cell Science at a Glance article and the accompanying poster, we focus on these histone acylation modifications and provide an overview of the players that govern these acylations and their connections with metabolism.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

Histone acylations as a mechanism for regulation of intestinal epithelial cells.

Histone post-translational modifications are reversible epigenetic mechanisms that regulate chromatin structure and gene transcription. In recent years, in addition to the well-characterized histone acetylation, new acylations such as propionylation, crotonylation, butyrylation and beta-hydroxybutyrylation have been described and explored in different cell types at contexts of health and disease. Understanding how histone acylations contribute to gene expression regulation is especially important in intestinal epithelial cells (IECs) because they receive many different signals from other cells and the external environment and must adapt to maintain essential functions such as nutrient and water absorption, maintenance of tolerance and protection against pathogens. In this review, we describe how cells regulate these modifications, how they are recognized by other proteins and impact gene expression. We summarize recent studies that explored the role of these distinct epigenetic marks in the regulation of IECs and discuss their biological importance for the intestinal epithelium's adaptations to changes in metabolism and to respond to environmental signals provided, for example, by the diet, components of the intestinal microbiota and pathogens. Finally, we discuss how the histone acylations are affected by inflammatory signals and how this knowledge may provide new targets for treatment of pathologies such as the inflammatory bowel diseases.

Histone butyrylation in the mouse intestine is mediated by the microbiota and associated with regulation of gene expression.

Post-translational modifications (PTMs) on histones are a key source of regulation on chromatin through impacting cellular processes, including gene expression1. These PTMs often arise from metabolites and are thus impacted by metabolism and environmental cues2-7. One class of metabolically regulated PTMs are histone acylations, which include histone acetylation, butyrylation, crotonylation and propionylation3,8. As these PTMs can be derived from short-chain fatty acids, which are generated by the commensal microbiota in the intestinal lumen9-11, we aimed to define how microbes impact the host intestinal chromatin landscape, mainly in female mice. Here we show that in addition to acetylation, intestinal epithelial cells from the caecum and distal mouse intestine also harbour high levels of butyrylation and propionylation on lysines 9 and 27 of histone H3. We demonstrate that these acylations are regulated by the microbiota and that histone butyrylation is additionally regulated by the metabolite tributyrin. Tributyrin-regulated gene programmes are correlated with histone butyrylation, which is associated with active gene-regulatory elements and levels of gene expression. Together, our study uncovers a regulatory layer of how the microbiota and metabolites influence the intestinal epithelium through chromatin, demonstrating a physiological setting in which histone acylations are dynamically regulated and associated with gene regulation.

Acetyl-CoA synthetase (ACSS2) does not generate butyryl- and crotonyl-CoA

Acetyl and other acyl groups from different short-chain fatty acids (SCFA) competitively modify histones at various lysine sites. To fully understand the functional significance of such histone acylation, a key epigenetic mechanism, it is crucial to characterize the cellular sources of the corresponding acyl-CoA molecules required for the lysine modification. Like acetate, SCFAs such as propionate, butyrate and crotonate are thought to be the substrates used to generate the corresponding acyl-CoAs by enzymes known as acyl-CoA synthetases. The acetyl-CoA synthetase, ACSS2, which produces acetyl-CoA from acetate in the nucleocytoplasmic compartment, has been proposed to also mediate the synthesis of acyl-CoAs such as butyryl- and crotonyl-CoA from the corresponding SCFAs. This idea is now widely accepted and is sparking new research projects. However, based on our direct in vitro experiments with purified or recombinant enzymes and structural considerations, we demonstrate that ACSS2 is unable to mediate the generation of non-acetyl acyl-CoAs like butyryl- and crotonyl-CoA. It is therefore essential to re-examine published data and corresponding discussions in the light of this new finding.

Histone Acyl Code in Precision Oncology: Mechanistic Insights from Dietary and Metabolic Factors.

Cancer etiology involves complex interactions between genetic and non-genetic factors, with epigenetic mechanisms serving as key regulators at multiple stages of pathogenesis. Poor dietary habits contribute to cancer predisposition by impacting DNA methylation patterns, non-coding RNA expression, and histone epigenetic landscapes. Histone post-translational modifications (PTMs), including acyl marks, act as a molecular code and play a crucial role in translating changes in cellular metabolism into enduring patterns of gene expression. As cancer cells undergo metabolic reprogramming to support rapid growth and proliferation, nuanced roles have emerged for dietary- and metabolism-derived histone acylation changes in cancer progression. Specific types and mechanisms of histone acylation, beyond the standard acetylation marks, shed light on how dietary metabolites reshape the gut microbiome, influencing the dynamics of histone acyl repertoires. Given the reversible nature of histone PTMs, the corresponding acyl readers, writers, and erasers are discussed in this review in the context of cancer prevention and treatment. The evolving 'acyl code' provides for improved biomarker assessment and clinical validation in cancer diagnosis and prognosis.

The short-chain fatty acid crotonate reduces invasive growth and immune escape of Candida albicans by regulating hyphal gene expression.

Macrophages curtail the proliferation of the pathogen Candida albicans within human body niches. Within macrophages, C. albicans adapts its metabolism and switches to invasive hyphal morphology. These adaptations enable fungal growth and immune escape by triggering macrophage lysis. Transcriptional programs regulate these metabolic and morphogenetic adaptations. Here we studied the roles of chromatin in these processes and implicate lysine crotonylation, a histone mark regulated by metabolism, in hyphal morphogenesis and macrophage interactions by C. albicans. We show that the short-chain fatty acid crotonate increases histone crotonylation, reduces hyphal formation within macrophages, and slows macrophage lysis and immune escape of C. albicans. Crotonate represses hyphal gene expression, and we propose that C. albicans uses diverse acylation marks to regulate its cell morphology in host environments. Hyphal formation is a virulence property of C. albicans. Therefore, a further importance of our study stems from identifying crotonate as a hyphal inhibitor.

Role of histone acylation and related technical developments

Role of histone acylation and related technical developments

A chemical catalyst enabling histone acylation with endogenous acyl-CoA

Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst, mBnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables mBnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease.

Molecular Basis of KAT2A Selecting Acyl-CoA Cofactors for Histone Modifications.

Emerging discoveries about undocumented acyltransferase activities of known histone acetyltransferases (HATs) advance our understandings in the regulation of histone modifications. However, the molecular basis of HATs selecting acyl coenzyme A (acyl-CoA) substrates for histone modification is less known. We here report that lysine acetyltransferase 2A (KAT2A) as an illustrative instance of HATs can selectively utilize acetyl-CoA, propionyl-CoA, butyryl-CoA, and succinyl-CoA to directly deposit 18 histone acylation hallmarks in nucleosome. By analyzing the co-crystal structures of the catalytic domain of KAT2A in complex with acetyl-CoA, propionyl-CoA, butyryl-CoA, malonyl-CoA, succinyl-CoA, and glutaryl-CoA, we conclude that the alternative substrate-binding pocket of KAT2A and the length and electrostatic features of the acyl chain cooperatively determine the selection of the acyl-CoA substrates by KAT2A. This study reveals the molecular basis underlying the pluripotency of HATs that selectively install acylation hallmarks in nucleosomes, which might serve as instrumental mechanism to precisely regulate histone acylation profiles in cells.

Biological functions and therapeutic potential of acylation by histone acetyltransferases

Histone lysine acylation is a major class of histone post-translational modifications involved in essential biological activities, such as transcriptional regulation, DNA-damage repair, and cell-cycle progression. Abnormal acylation is strongly associated with various diseases, such as cancer. Thus, histone acetyltransferases (HATs), the “writers” that catalyze histone acylation, are promising targets for cancer treatment. Rapid developments in structural biology and artificial intelligence have facilitated the development of drugs targeting HATs. To provide new ideas for exploring novel HAT modifiers with high efficiency and selectivity, this article reviews the relationships between acylation and diseases, illustrates HAT catalytic mechanisms through structural biology, and summarizes research progress in HAT modifiers.

Nuclear Condensation of CDYL Links Histone Crotonylation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease.

Emerging evidence indicates that epigenetic modulation of gene expression plays a key role in the progression of autosomal dominant polycystic kidney disease (ADPKD). However, the molecular basis for how the altered epigenome modulates transcriptional responses, and thereby disease progression in ADPKD, remains largely unknown. Kidneys from control and ADPKD mice were examined for the expression of CDYL and histone acylations. CDYL expression and its correlation with disease severity were analyzed in a cohort of patients with ADPKD. Cdyl transgenic mice were crossed with Pkd1 knockout mice to explore CDYL's role in ADPKD progression. Integrated cistromic and transcriptomic analyses were performed to identify direct CDYL target genes. High-sensitivity mass spectrometry analyses were undertaken to characterize CDYL-regulated histone lysine crotonylations (Kcr). Biochemical analysis and zebrafish models were used for investigating CDYL phase separation. CDYL was downregulated in ADPKD kidneys, accompanied by an increase of histone Kcr. Genetic overexpression of Cdyl reduced histone Kcr and slowed cyst growth. We identified CDYL-regulated cyst-associated genes, whose downregulation depended on CDYL-mediated suppression of histone Kcr. CDYL assembled nuclear condensates through liquid-liquid phase separation in cultured kidney epithelial cells and in normal kidney tissues. The phase-separating capacity of CDYL was required for efficient suppression of locus-specific histone Kcr, of expression of its target genes, and of cyst growth. These results elucidate a mechanism by which CDYL nuclear condensation links histone Kcr to transcriptional responses and cystogenesis in ADPKD.

Acylations in cardiovascular diseases: advances and perspectives.

Acylations in cardiovascular diseases: advances and perspectives.

Decoding the dynamic H3K9cr landscapes during neural commitment of P19 embryonal carcinoma cells

Histone lysine crotonylation (Kcr) is a novel hydrophobic histone acylation modification, and we recently report its crucial roles in neural differentiation. However, it is still unclear how histone Kcr involve in early neural commitment. Here, we systematically investigate the H3K9cr landscapes during neuroectodermal differentiation of pluripotent P19 embryonal carcinoma cells (ECCs). We reveal that the genome-wide changes in H3K9cr favor neural fate specification, and identify potential co-factors binding H3K9cr. We also uncover that H3K9cr collaborates with H3K9ac to regulate gene expression changes. Our results provide novel insights into the epigenetic mechanisms underlying neural commitment.

Does chromatin function as a metabolite reservoir?

Alternative histone acylations integrate gene expression with cellular metabolic states. Recent measurements of cellular acyl-coenzyme A (acyl-CoA) pools highlight the potential that histone post-translational modifications (PTMs) contribute directly to the regulation of metabolite pools. A metabolite-centric view throws new light onto roles and evolution of histone PTMs.