Histone H3K18 & H3K23 acetylation directs establishment of MLL-mediated H3K4 methylation.

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA, but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin "switch" governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects, and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

H3K18 & H3K23 acetylation directs establishment of MLL-mediated H3K4 methylation.

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA, but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin "switch" governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects, and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression.

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress.

BACKGROUND: Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses of RNA-Seq data uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. RESULTS: Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top-down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression datasets suggest a role for epigenetic modification of DNA in regulation of gene expression in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. CONCLUSIONS: Our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research.

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress.

Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top-down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28.8°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression data suggest a role for epigenetic modification of DNA in gene regulation in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. Taken together, our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research.

An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability.

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g. K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has a broader extension. Here, we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation 'chromatin switch' on the H3 tail that modulates read-write accessibility in nucleosomes and resolves the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.

Histone H4 proteoforms and post-translational modifications in the Mus musculus brain with quantitative comparison of ages and brain regions.

Histone proteins are essential to the regulation of the eukaryotic genome. Histone post-translational modifications (PTMs) and single-molecule combinations of these modifications (proteoforms) allow for the regulation of many DNA-templated processes, most notably transcription. Histone H4 is a part of the core histone octamer, which packages DNA into nucleosomes. Top-down proteomics allows for the inquiry of the epigenetic landscape with proteoform-level specificity. Although these approaches are well-demonstrated ex vivo, our knowledge of in vivo histone proteoform biology remains sparse. Here, we demonstrate the first in vivo quantitative top-down analysis of histone H4 and analyze the forebrains and hindbrains of differently aged mice. This reveals novel differences between the mouse forebrain and hindbrain and region-specific changes during adolescence in histone H4 PTMs and proteoforms. At 25 days of age (P25), histone H4 of the hindbrain is more acetylated than the forebrain. At 47 days of age (P47), there are fewer significant differences in histone H4 PTMs and their combinations between regions. Histone H4 of the forebrain is more acetylated in P47 than in P25 forebrains. Hindbrains exhibit the opposite difference with histone H4 of the P25 hindbrain being more acetylated than that of P47 hindbrains. These differences are mainly driven by less abundant hyperacetylated proteoforms. Transcription of histone acetyltransferases such as p300, CBP, and HAT1 is known to be higher in cortical neurons, consistent with the observed acetylation levels. Lysine 20 methylation (K20me1, K20me2, and K20me3) is notably invariant with brain region and age difference.

Histone variant-specific post-translational modifications.

Post-translational modifications (PTMs) of histones play a key role in DNA-based processes and contribute to cell differentiation and gene function by adding an extra layer of regulation. Variations in histone sequences within each family of histones expands the chromatin repertoire and provide further mechanisms for regulation and signaling. While variants are known to be present in certain genomic loci and carry out important functions, much remains unknown about variant-specific PTMs and their role in regulating chromatin. This ambiguity is in part due to the limited technologies and appropriate reagents to identify and quantitate variant-specific PTMs. Nonetheless, histone variants are an integral portion of the chromatin system and the understanding of their modifications and resolving how PTMs function differently on specific variants is paramount to the advancement of the field. Here we review the current knowledge on post-translational modifications specific to histone variants, with an emphasis on well-characterized PTMs of known function. While not every possible PTM is addressed, we present key variant-specific PTMs and what is known about their function and mechanisms in convenient reference tables.

High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array.

Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. SIGNIFICANCE: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.

The histone H3.3 chaperone HIRA restrains erythroid-biased differentiation of adult hematopoietic stem cells.

Histone variants contribute to the complexity of the chromatin landscape and play an integral role in defining DNA domains and regulating gene expression. The histone H3 variant H3.3 is incorporated into genic elements independent of DNA replication by its chaperone HIRA. Here we demonstrate that Hira is required for the self-renewal of adult hematopoietic stem cells (HSCs) and to restrain erythroid differentiation. Deletion of Hira led to rapid depletion of HSCs while differentiated hematopoietic cells remained largely unaffected. Depletion of HSCs after Hira deletion was accompanied by increased expression of bivalent and erythroid genes, which was exacerbated upon cell division and paralleled increased erythroid differentiation. Assessing H3.3 occupancy identified a subset of polycomb-repressed chromatin in HSCs that depends on HIRA to maintain the inaccessible, H3.3-occupied state for gene repression. HIRA-dependent H3.3 incorporation thus defines distinct repressive chromatin that represses erythroid differentiation of HSCs.

Gene therapy using Aß variants for amyloid reduction.

Numerous aggregation inhibitors have been developed with the goal of blocking or reversing toxic amyloid formation in vivo. Previous studies have used short peptide inhibitors targeting different amyloid ß (Aß) amyloidogenic regions to prevent aggregation. Despite the specificity that can be achieved by peptide inhibitors, translation of these strategies has been thwarted by two key obstacles: rapid proteolytic degradation in the bloodstream and poor transfer across the blood-brain barrier. To circumvent these problems, we have created a minigene to express full-length Aß variants in the mouse brain. We identify two variants, F20P and F19D/L34P, that display four key properties required for therapeutic use: neither peptide aggregates on its own, both inhibit aggregation of wild-type Aß in vitro, promote disassembly of pre-formed fibrils, and diminish toxicity of Aß oligomers. We used intraventricular injection of adeno-associated virus (AAV) to express each variant in APP/PS1 transgenic mice. Lifelong expression of F20P, but not F19D/L34P, diminished Aß levels, plaque burden, and plaque-associated neuroinflammation. Our findings suggest that AAV delivery of Aß variants may offer a novel therapeutic strategy for Alzheimer's disease. More broadly our work offers a framework for identifying and delivering peptide inhibitors tailored to other protein-misfolding diseases.

Expeditious Extraction of Histones from Limited Cells or Tissue Samples and Quantitative Top-Down Proteomic Analysis.

Histones are the primary protein component of chromatin and are involved in virtually all DNA-templated processes. Histones are abundantly post-translationally modified by a variety of chromatin-modifying machinery. These post-translational modifications (PTMs) are recognized by a range of "reader" proteins, which recruit additional proteins to specific locations on chromatin and impart precise and powerful effects on gene regulation. Each PTM typically exerts a positive or negative effect on transcription, and recent studies have shown that histone PTMs function in a combinatorial histone code: that is, histone PTMs function in combination to exert precise DNA-templated regulation. Thus, there is a need to identify and understand proteoforms, or unambiguously defined single protein molecules with all combinations of modifications. Top-down proteomics is currently the only viable approach for identifying and quantitating histone proteoforms, and mass spectrometry instruments have become sufficiently powerful to perform these quantitative analyses in a robust and high-throughput fashion. These recent innovations have enabled new experimental directions in chromatin research but have also introduced temporal and other constraints. This has led us to develop the protocols described here, which increase throughput, reduce sample requirements, and maintain robust quantitation. Although originally designed for high-throughput quantitative top-down proteomics, the protocols described here are useful for a wide range of chromatin biology applications. Starting with small amounts of cells or tissue, we describe two basic protocols for exceptionally rapid and efficient nuclei isolation, acid extraction of histones, and high-performance liquid chromatography fractionation of histones into histone families. We additionally describe the quantitative top-down proteomic analysis of histone H4 proteoforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation and acid extraction of histones from mammalian cells in culture/tissues Basic Protocol 2: HPLC fractionation of histones and histone H4 HPLC-MS/MS Support Protocol: Preparation of intact H3 histone tails by Glu-C digestion.

Combinations of histone post-translational modifications.

Histones are essential proteins that package the eukaryotic genome into its physiological state of nucleosomes, chromatin, and chromosomes. Post-translational modifications (PTMs) of histones are crucial to both the dynamic and persistent regulation of the genome. Histone PTMs store and convey complex signals about the state of the genome. This is often achieved by multiple variable PTM sites, occupied or unoccupied, on the same histone molecule or nucleosome functioning in concert. These mechanisms are supported by the structures of 'readers' that transduce the signal from the presence or absence of PTMs in specific cellular contexts. We provide background on PTMs and their complexes, review the known combinatorial function of PTMs, and assess the value and limitations of common approaches to measure combinatorial PTMs. This review serves as both a reference and a path forward to investigate combinatorial PTM functions, discover new synergies, and gather additional evidence supporting that combinations of histone PTMs are the central currency of chromatin-mediated regulation of the genome.

Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery.

Despite the established roles of the epigenetic factor UHRF1 in oncogenesis, no UHRF1-targeting therapeutics have been reported to date. In this study, we use fragment-based ligand discovery to identify novel scaffolds for targeting the isolated UHRF1 tandem Tudor domain (TTD), which recognizes the heterochromatin-associated histone mark H3K9me3 and supports intramolecular contacts with other regions of UHRF1. Using both binding-based and function-based screens of a ~ 2300-fragment library in parallel, we identified 2,4-lutidine as a hit for follow-up NMR and X-ray crystallography studies. Unlike previous reported ligands, 2,4-lutidine binds to two binding pockets that are in close proximity on TTD and so has the potential to be evolved into more potent inhibitors using a fragment-linking strategy. Our study provides a useful starting point for developing potent chemical probes against UHRF1.

Mechanistic insights into KDM4A driven genomic instability.

Alterations in global epigenetic signatures on chromatin are well established to contribute to tumor initiation and progression. Chromatin methylation status modulates several key cellular processes that maintain the integrity of the genome. KDM4A, a demethylase that belongs to the Fe-II dependent dioxygenase family that uses α-ketoglutarate and molecular oxygen as cofactors, is overexpressed in several cancers and is associated with an overall poor prognosis. KDM4A demethylates lysine 9 (H3K9me2/3) and lysine 36 (H3K36me3) methyl marks on histone H3. Given the complexity that exists with these marks on chromatin and their effects on transcription and proliferation, it naturally follows that demethylation serves an equally important role in these cellular processes. In this review, we highlight the role of KDM4A in transcriptional modulation, either dependent or independent of its enzymatic activity, arising from the amplification of this demethylase in cancer. KDM4A modulates re-replication of distinct genomic loci, activates cell cycle inducers, and represses proteins involved in checkpoint control giving rise to proliferative damage, mitotic disturbances and chromosomal breaks, ultimately resulting in genomic instability. In parallel, emerging evidence of non-nuclear substrates of epigenetic modulators emphasize the need to investigate the role of KDM4A in regulating non-nuclear substrates and evaluate their contribution to genomic instability in this context. The existence of promising KDM-specific inhibitors makes these demethylases an attractive target for therapeutic intervention in cancers.

Orsay Virus CP-δ Adopts a Novel ß-Bracelet Structural Fold and Incorporates into Virions as a Head Fiber.

Fiber proteins are commonly found in eukaryotic and prokaryotic viruses, where they play important roles in mediating viral attachment and host cell entry. They typically form trimeric structures and are incorporated into virions via noncovalent interactions. Orsay virus, a small RNA virus which specifically infects the laboratory model nematode Caenorhabditis elegans, encodes a fibrous protein δ that can be expressed as a free protein and as a capsid protein-δ (CP-δ) fusion protein. Free δ has previously been demonstrated to facilitate viral exit following intracellular expression; however, the biological significance and prevalence of CP-δ remained relatively unknown. Here, we demonstrate that Orsay CP-δ is covalently incorporated into infectious particles, the first example of any attached viral fibers known to date. The crystal structure of δ(1-101) (a deletion mutant containing the first 101 amino acid [aa] residues of δ) reveals a pentameric, 145-Å long fiber with an N-terminal coiled coil followed by multiple ß-bracelet repeats. Electron micrographs of infectious virions depict particle-associated CP-δ fibers with dimensions similar to free δ. The δ proteins from two other nematode viruses, Le Blanc and Santeuil, which both specifically infect Caenorhabditis briggsae, were also found to form fibrous molecules. Recombinant Le Blanc δ was able to block Orsay virus infection in worm culture and vice versa, suggesting these two viruses likely compete for the same cell receptor(s). Thus, we propose that while CP-δ likely mediates host cell attachment for all three nematode viruses, additional downstream factor(s) ultimately determine the host specificity and range of each virus.IMPORTANCE Viruses often have extended fibers to mediate host cell recognition and entry, serving as promising targets for antiviral drug development. Unlike other known viral fibers, the δ proteins from the three recently discovered nematode viruses are incorporated into infectious particles as protruding fibers covalently linked to the capsid. Crystal structures of δ revealed novel pentameric folding repeats, which we term ß-bracelets, in the intermediate shaft region. Based on sequence analysis, the ß-bracelet motif of δ is conserved in all three nematode viruses and could account for ∼60% of the total length of the fiber. Our study indicated that δ plays important roles in cell attachment for this group of nematode viruses. In addition, the tightly knitted ß-bracelet fold, which presumably allows δ to survive harsh environments in the worm gut, could be applicable to bioengineering applications given its potentially high stability.

Characterization of SETD3 methyltransferase-mediated protein methionine methylation.

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain-containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310 Our results suggest that placing methionine properly in the active site-within close proximity to and in line with the incoming methyl group of SAM-would allow some SET domain proteins to selectively methylate methionine in proteins.

High-Throughput Quantitative Top-Down Proteomics: Histone H4.

Proteins physiologically exist as "proteoforms" that arise from one gene and acquire additional function by post-translational modifications (PTM). When multiple PTMs coexist on single protein molecules, top-down proteomics becomes the only feasible method of characterization; however, most top-down methods have limited quantitative capacity and insufficient throughput to truly address proteoform biology. Here we demonstrate that top-down proteomics can be quantitative, reproducible, sensitive, and high throughput. The proteoforms of histone H4 are well studied both as a challenging proteoform identification problem and due to their essential role in the regulation of all eukaryotic DNA-templated processes. Much of histone H4's function is obfuscated from prevailing methods due to combinatorial mechanisms. Starting from cells or tissues, after an optimized protein purification process, the H4 proteoforms are physically separated by on-line C3 chromatography, narrowly isolated in MS1 and sequenced with ETD fragmentation. We achieve more than 30 replicates from a single 35-mm tissue culture dish by loading 55 ng of H4 on column. Parallelization and automation yield a sustained throughput of 12 replicates per day. We achieve reproducible quantitation (average biological Pearson correlations of 0.89) of hundreds of proteoforms (about 200-300) over almost six orders of magnitude and an estimated LLoQ of 0.001% abundance. We demonstrate the capacity of the method to precisely measure well-established changes with sodium butyrate treatment of SUM159 cells. We show that the data produced by a quantitative top-down method can be amenable to parametric statistical comparisons and is capable of delineating relevant biological changes at the full proteoform level.

AMP-activated protein kinase links acetyl-CoA homeostasis to BRD4 recruitment in acute myeloid leukemia.

Altered metabolism fuels 2 hallmark properties of cancer cells: unlimited proliferation and differentiation blockade. Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of bioenergetics crucial for glucose metabolism in acute myeloid leukemia (AML), and its inhibition delays leukemogenesis, but whether the metabolic function of AMPK alters the AML epigenome remains unknown. Here, we demonstrate that AMPK maintains the epigenome of MLL-rearranged AML by linking acetyl-coenzyme A (CoA) homeostasis to Bromodomain and Extra-Terminal domain (BET) protein recruitment to chromatin. AMPK deletion reduced acetyl-CoA and histone acetylation, displacing BET proteins from chromatin in leukemia-initiating cells. In both mouse and patient-derived xenograft AML models, treating with AMPK and BET inhibitors synergistically suppressed AML. Our results provide a therapeutic rationale to target AMPK and BET for AML therapy.