One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry.

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.

Two factor authentication: Asf1 mediates crosstalk between H3 K14 and K56 acetylation.

The ability of histone chaperone Anti-silencing factor 1 (Asf1) to direct acetylation of lysine 56 of histone H3 (H3K56ac) represents an important regulatory step in genome replication and DNA repair. In Saccharomyces cerevisiae, Asf1 interacts functionally with a second chaperone, Vps75, and the lysine acetyltransferase (KAT) Rtt109. Both Asf1 and Vps75 can increase the specificity of histone acetylation by Rtt109, but neither alter selectivity. However, changes in acetylation selectivity have been observed in histones extracted from cells, which contain a plethora of post-translational modifications. In the present study, we use a series of singly acetylated histones to test the hypothesis that histone pre-acetylation and histone chaperones function together to drive preferential acetylation of H3K56. We show that pre-acetylated H3K14ac/H4 functions with Asf1 to drive specific acetylation of H3K56 by Rtt109-Vps75. Additionally, we identified an exosite containing an acidic patch in Asf1 and show that mutations to this region alter Asf1-mediated crosstalk that changes Rtt109-Vps75 selectivity. Our proposed mechanism suggests that Gcn5 acetylates H3K14, recruiting remodeler complexes, allowing for the Asf1-H3K14ac/H4 complex to be acetylated at H3K56 by Rtt109-Vps75. This mechanism explains the conflicting biochemical data and the genetic links between Rtt109, Vps75, Gcn5 and Asf1 in the acetylation of H3K56.

T cell activation triggers reversible inosine-5'-monophosphate dehydrogenase assembly.

T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca2+-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5'-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.

IL-35 (Interleukin-35) Suppresses Endothelial Cell Activation by Inhibiting Mitochondrial Reactive Oxygen Species-Mediated Site-Specific Acetylation of H3K14 (Histone 3 Lysine 14).

OBJECTIVE: IL-35 (interleukin-35) is an anti-inflammatory cytokine, which inhibits immune responses by inducing regulatory T cells and regulatory B cells and suppressing effector T cells and macrophages. It remains unknown whether atherogenic stimuli induce IL-35 and whether IL-35 inhibits atherogenic lipid-induced endothelial cell (EC) activation and atherosclerosis. EC activation induced by hyperlipidemia stimuli, including lysophosphatidylcholine is considered as an initiation step for monocyte recruitment and atherosclerosis. In this study, we examined the expression of IL-35 during early atherosclerosis and the roles and mechanisms of IL-35 in suppressing lysophosphatidylcholine-induced EC activation. APPROACH AND RESULTS: Using microarray and ELISA, we found that IL-35 and its receptor are significantly induced during early atherosclerosis in the aortas and plasma of ApoE (apolipoprotein E) knockout mice-an atherosclerotic mouse model-and in the plasma of hypercholesterolemic patients. In addition, we found that IL-35 suppresses lysophosphatidylcholine-induced monocyte adhesion to human aortic ECs. Furthermore, our RNA-sequencing analysis shows that IL-35 selectively inhibits lysophosphatidylcholine-induced EC activation-related genes, such as ICAM-1 (intercellular adhesion molecule-1). Mechanistically, using flow cytometry, mass spectrometry, electron spin resonance analyses, and chromatin immunoprecipitation-sequencing analyses, we found that IL-35 blocks lysophosphatidylcholine-induced mitochondrial reactive oxygen species, which are required for the induction of site-specific H3K14 (histone 3 lysine 14) acetylation, increased binding of proinflammatory transcription factor AP-1 in the promoter of ICAM-1, and induction of ICAM-1 transcription in human aortic EC. Finally, IL-35 cytokine therapy suppresses atherosclerotic lesion development in ApoE knockout mice. CONCLUSIONS: IL-35 is induced during atherosclerosis development and inhibits mitochondrial reactive oxygen species-H3K14 acetylation-AP-1-mediated EC activation.

HDAC8 substrate selectivity is determined by long- and short-range interactions leading to enhanced reactivity for full-length histone substrates compared with peptides.

Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins. To date, HDAC substrate specificity and selectivity have been largely estimated using peptide substrates. However, it is unclear whether peptide substrates accurately reflect the substrate selectivity of HDAC8 toward full-length proteins. Here, we compare HDAC8 substrate selectivity in the context of peptides, full-length proteins, and protein-nucleic acid complexes. We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40-300-fold higher than those for corresponding peptide substrates. Thus, we conclude that additional contacts with protein substrates enhance catalytic efficiency. However, the catalytic efficiency decreases for larger multiprotein complexes. These differences in HDAC8 substrate selectivity for peptides and full-length proteins suggest that HDAC8 substrate preference is based on a combination of short- and long-range interactions. In summary, this work presents detailed kinetics for HDAC8-catalyzed deacetylation of singly-acetylated, full-length protein substrates, revealing that HDAC8 substrate selectivity is determined by multiple factors. These insights provide a foundation for understanding recognition of full-length proteins by HDACs.

Impact of a High-fat Diet on Tissue Acyl-CoA and Histone Acetylation Levels.

Cellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of Acly in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. Butyryl-CoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity in vitro This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver.

The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions.

The organization of the eukaryotic genome into nucleosomes dramatically affects the regulation of gene expression. The delicate balance between transcription and DNA compaction relies heavily on nucleosome dynamics. Surprisingly, little is known about the free energy required to assemble these large macromolecular complexes and maintain them under physiological conditions. Here, we describe the thermodynamic parameters that drive nucleosome formation in vitro. To demonstrate the versatility of our approach, we test the effect of DNA sequence and H3K56 acetylation on nucleosome thermodynamics. Furthermore, our studies reveal the mechanism of action of the histone chaperone nucleosome assembly protein 1 (Nap1). We present evidence for a paradigm in which nucleosome assembly requires the elimination of competing, nonnucleosomal histone-DNA interactions by Nap1. This observation is confirmed in vivo, wherein deletion of the NAP1 gene in yeast results in a significant increase in atypical histone-DNA complexes, as well as in deregulated transcription activation and repression.

Measuring specificity in multi-substrate/product systems as a tool to investigate selectivity in vivo.

Multiple substrate enzymes present a particular challenge when it comes to understanding their activity in a complex system. Although a single target may be easy to model, it does not always present an accurate representation of what that enzyme will do in the presence of multiple substrates simultaneously. Therefore, there is a need to find better ways to both study these enzymes in complicated systems, as well as accurately describe the interactions through kinetic parameters. This review looks at different methods for studying multiple substrate enzymes, as well as explores options on how to most accurately describe an enzyme's activity within these multi-substrate systems. Identifying and defining this enzymatic activity should help clear the way to using in vitro systems to accurately predicting the behavior of multi-substrate enzymes in vivo. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Overlapping and Divergent Actions of Structurally Distinct Histone Deacetylase Inhibitors in Cardiac Fibroblasts.

Inhibitors of zinc-dependent histone deacetylases (HDACs) profoundly affect cellular function by altering gene expression via changes in nucleosomal histone tail acetylation. Historically, investigators have employed pan-HDAC inhibitors, such as the hydroxamate trichostatin A (TSA), which simultaneously targets members of each of the three zinc-dependent HDAC classes (classes I, II, and IV). More recently, class- and isoform-selective HDAC inhibitors have been developed, providing invaluable chemical biology probes for dissecting the roles of distinct HDACs in the control of various physiologic and pathophysiological processes. For example, the benzamide class I HDAC-selective inhibitor, MGCD0103 [N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide], was shown to block cardiac fibrosis, a process involving excess extracellular matrix deposition, which often results in heart dysfunction. Here, we compare the mechanisms of action of structurally distinct HDAC inhibitors in isolated primary cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. TSA, MGCD0103, and the cyclic peptide class I HDAC inhibitor, apicidin, exhibited a common ability to enhance histone acetylation, and all potently blocked cardiac fibroblast cell cycle progression. In contrast, MGCD0103, but not TSA or apicidin, paradoxically increased expression of a subset of fibrosis-associated genes. Using the cellular thermal shift assay, we provide evidence that the divergent effects of HDAC inhibitors on cardiac fibroblast gene expression relate to differential engagement of HDAC1- and HDAC2-containing complexes. These findings illustrate the importance of employing multiple compounds when pharmacologically assessing HDAC function in a cellular context and during HDAC inhibitor drug development.

Lack of global epigenetic methylation defects in CBS deficient mice.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.

Mitochondrial Reactive Oxygen Species Mediate Lysophosphatidylcholine-Induced Endothelial Cell Activation.

OBJECTIVE: Hyperlipidemia-induced endothelial cell (EC) activation is considered as an initial event responsible for monocyte recruitment in atherogenesis. However, it remains poorly defined what is the mechanism underlying hyperlipidemia-induced EC activation. Here, we tested a novel hypothesis that mitochondrial reactive oxygen species (mtROS) serve as signaling mediators for EC activation in early atherosclerosis. APPROACH AND RESULTS: Metabolomics and transcriptomics analyses revealed that several lysophosphatidylcholine (LPC) species, such as 16:0, 18:0, and 18:1, and their processing enzymes, including Pla2g7 and Pla2g4c, were significantly induced in the aortas of apolipoprotein E knockout mice during early atherosclerosis. Using electron spin resonance and flow cytometry, we found that LPC 16:0, 18:0, and 18:1 induced mtROS in primary human aortic ECs, independently of the activities of nicotinamide adenine dinucleotide phosphate oxidase. Mechanistically, using confocal microscopy and Seahorse XF mitochondrial analyzer, we showed that LPC induced mtROS via unique calcium entry-mediated increase of proton leak and mitochondrial O2 reduction. In addition, we found that mtROS contributed to LPC-induced EC activation by regulating nuclear binding of activator protein-1 and inducing intercellular adhesion molecule-1 gene expression in vitro. Furthermore, we showed that mtROS inhibitor MitoTEMPO suppressed EC activation and aortic monocyte recruitment in apolipoprotein E knockout mice using intravital microscopy and flow cytometry methods. CONCLUSIONS: ATP synthesis-uncoupled, but proton leak-coupled, mtROS increase mediates LPC-induced EC activation during early atherosclerosis. These results indicate that mitochondrial antioxidants are promising therapies for vascular inflammation and cardiovascular diseases.

Modulation of p300/CBP Acetylation of Nucleosomes by Bromodomain Ligand I-CBP112.

The histone acetyltransferase (HAT) enzymes p300 and CBP are closely related paralogs that serve as transcriptional coactivators and have been found to be dysregulated in cancer and other diseases. p300/CBP is a multidomain protein and possesses a highly conserved bromodomain that has been shown to bind acetylated Lys residues in both proteins and various small molecules, including I-CBP112 and CBP30. Here we show that the ligand I-CBP112 can stimulate nucleosome acetylation up to 3-fold while CBP30 does not. Activation of p300/CBP by I-CBP112 is not observed with the isolated histone H3 substrate but requires a nucleosome substrate. I-CBP112 does not impact nucleosome acetylation by the isolated p300 HAT domain, and the effects of I-CBP112 on p300/CBP can be neutralized by CBP30, suggesting that I-CBP112 likely allosterically activates p300/CBP through bromodomain interactions. Using mass spectrometry and Western blots, we have found that I-CBP112 particularly stimulates acetylation of Lys18 of histone H3 (H3K18) in nucleosomes, an established in vivo site of p300/CBP. In addition, we show that I-CBP112 enhances H3K18 acetylation in acute leukemia and prostate cancer cells in a concentration range commensurate with its antiproliferative effects. Our findings extend the known pharmacology of bromodomain ligands in the regulation of p300/CBP and suggest a novel approach to modulating histone acetylation in cancer.

Interaction with the DNA Repair Protein Thymine DNA Glycosylase Regulates Histone Acetylation by p300.

How protein-protein interactions regulate and alter histone modifications is a major unanswered question in epigenetics. The histone acetyltransferase p300 binds thymine DNA glycosylase (TDG); utilizing mass spectrometry to measure site-specific changes in histone acetylation, we found that the absence of TDG in mouse embryonic fibroblasts leads to a reduction in the rate of histone acetylation. We demonstrate that TDG interacts with the CH3 domain of p300 to allosterically promote p300 activity to specific lysines on histone H3 (K18 and K23). However, when TDG concentrations approach those of histones, TDG acts as a competitive inhibitor of p300 histone acetylation. These results suggest a mechanism for how histone acetylation is fine-tuned via interaction with other proteins, while also highlighting a connection between regulators of two important biological processes: histone acetylation and DNA repair/demethylation.

Quantitative Measurement of Histone Tail Acetylation Reveals Stage-Specific Regulation and Response to Environmental Changes during Drosophila Development.

Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as γ-irradiation altered histone acetylation patterns. Strikingly, γ-irradiation dramatically increased the level of acetylation at H3K18, a site linked to DNA repair via nonhomologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in the level of H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation.

Global Profiling of Acetyltransferase Feedback Regulation.

Lysine acetyltransferases (KATs) are key mediators of cell signaling. Methods capable of providing new insights into their regulation thus constitute an important goal. Here we report an optimized platform for profiling KAT-ligand interactions in complex proteomes using inhibitor-functionalized capture resins. This approach greatly expands the scope of KATs, KAT complexes, and CoA-dependent enzymes accessible to chemoproteomic methods. This enhanced profiling platform is then applied in the most comprehensive analysis to date of KAT inhibition by the feedback metabolite CoA. Our studies reveal that members of the KAT superfamily possess a spectrum of sensitivity to CoA and highlight NAT10 as a novel KAT that may be susceptible to metabolic feedback inhibition. This platform provides a powerful tool to define the potency and selectivity of reversible stimuli, such as small molecules and metabolites, that regulate KAT-dependent signaling.

Site specificity analysis of Piccolo NuA4-mediated acetylation for different histone complexes.

We have a limited understanding of the site specificity of multi-subunit lysine acetyltransferase (KAT) complexes for histone-based substrates, especially in regards to the different complexes formed during nucleosome assembly. Histone complexes could be a major factor in determining the acetylation specificity of KATs. In the present study, we utilized a label-free quantitative MS-based method to determine the site specificity of acetylation catalysed by Piccolo NuA4 on (H3/H4)2 tetramer, tetramer bound DNA (tetrasome) and nucleosome core particle (NCP). Our results show that Piccolo NuA4 can acetylate multiple lysine residues on these three histone complexes, of which NCP is the most favourable, (H3/H4)2 tetramer is the second and tetrasome is the least favourable substrate for Piccolo NuA4 acetylation. Although Piccolo NuA4 preferentially acetylates histone H4 (H4K12), the site specificity of the enzyme is altered with different histone complex substrates. Our results show that before nucleosome assembly is complete, H3K14 specificity is almost equal to that of H4K12 and DNA-histone interactions suppress the acetylation ability of Piccolo NuA4. These data suggest that the H2A/H2B dimer could play a critical role in the increase in acetylation specificity of Piccolo NuA4 for NCP. This demonstrates that histone complex formation can alter the acetylation preference of Piccolo NuA4. Such findings provide valuable insight into regulating Piccolo NuA4 specificity by modulating chromatin dynamics and in turn manipulating gene expression.

A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation.

Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell.

Histone acetyltransferase-deficient p300 mutants in diffuse large B cell lymphoma have altered transcriptional regulatory activities and are required for optimal cell growth.

BACKGROUND: Recent genome-wide studies have shown that approximately 30% of diffuse large B-cell lymphoma (DLBCL) cases harbor mutations in the histone acetyltransferase (HAT) coactivators p300 or CBP. The majority of these mutations reduce or eliminate the catalytic HAT activity. We previously demonstrated that the human DLBCL cell line RC-K8 expresses a C-terminally truncated, HAT-defective p300 protein (p300ΔC-1087), whose expression is essential for cell proliferation. METHODS: Using results from large-scale DLBCL studies, we have identified and characterized a second C-terminally truncated, HAT-defective p300 mutant, p300ΔC-820, expressed in the SUDHL2 DLBCL cell line. Properties of p300ΔC-820 were characterized in the SUDHL2 DLBCL cell line by Western blotting, co-immunoprecipitation, and shRNA gene knockdown, as well by using cDNA expression vectors for p300ΔC-820 in pull-down assays, transcriptional reporter assays, and immunofluorescence experiments. A mass spectrometry-based method was used to compare the histone acetylation profile of DLBCL cell lines expressing various levels of wild-type p300. RESULTS: We show that the SUDHL2 cell line expresses a C-terminally truncated, HAT-defective form of p300 (p300ΔC-820), but no wild-type p300. The p300ΔC-820 protein has a wild-type ability to localize to subnuclear "speckles," but has a reduced ability to enhance transactivation by transcription factor REL. Knockdown of p300ΔC-820 in SUDHL2 cells reduced their proliferation and soft agar colony-forming ability. In RC-K8 cells, knockdown of p300ΔC-1087 resulted in increased expression of mRNA and protein for REL target genes A20 and IκBα, two genes that have been shown to limit the growth of RC-K8 cells when overexpressed. Among a panel of B-lymphoma cell lines, low-level expression of full-length p300 protein, which is characteristic of the SUDHL2 and RC-K8 cells, was associated with decreased acetylation of histone H3 at lysines 14 and 18. CONCLUSIONS: The high prevalence of p300 mutations in DLBCL suggests that HAT-deficient p300 activity defines a subtype of DLBCL, which we have investigated using human DLBCL cell lines RC-K8 and SUDHL2. Our results suggest that truncated p300 proteins contribute to DLBCL cell growth by affecting the expression of specific genes, perhaps through a mechanism that involves alterations in global histone acetylation.

Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4.

Although p300 and CBP lysine acetyltransferases are often treated interchangeably, the inability of one enzyme to compensate for the loss of the other suggests unique roles for each. As these deficiencies coincide with aberrant levels of histone acetylation, we hypothesized that the key difference between p300 and CBP activity is differences in their specificity/selectivity for lysines within the histones. Utilizing a label-free, quantitative mass spectrometry based technique, we determined the kinetic parameters of both CBP and p300 at each lysine of H3 and H4, under conditions we would expect to encounter in the cell (either limiting acetyl-CoA or histone). Our results show that while p300 and CBP acetylate many common residues on H3 and H4, they do in fact possess very different specificities, and these specificities are dependent on whether histone or acetyl-CoA is limiting. Steady-state experiments with limiting H3 demonstrate that both CBP and p300 acetylate H3K14, H3K18, H3K23, with p300 having specificities up to 10¹°-fold higher than CBP. Utilizing tetramer as a substrate, both enzymes also acetylate H4K5, H4K8, H4K12, and H4K16. With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300. With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹8-fold higher than CBP. This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.