Acetylation Turnover Research Articles

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.

Mechanisms of stimulation of SAGA-mediated nucleosome acetylation by a transcriptional activator

Eukaryotic gene expression requires the coordination of multiple factors to overcome the repressive nature of chromatin. However, the mechanistic details of this coordination are not well understood. The SAGA family of transcriptional coactivators interacts with DNA-binding activators to establish regions of hyperacetylation. We have previously shown that, contrary to the prevailing model in which activator protein increases SAGA affinity for nucleosome substrate, the Gal4-VP16 activator model system augments the rate of acetylation turnover for the SAGA complex from budding yeast. To better understand how this stimulation occurs, we have identified necessary components using both kinetics assays and binding interactions studies. We find that Gal4-VP16-mediated stimulation requires activator binding to DNA flanking the nucleosome, as it cannot be reproduced in trans by activator protein alone or by exogenous DNA containing the activator binding site in combination with the activator protein. Further, activator-mediated stimulation requires subunits outside of the histone acetylation (HAT) module, with the Tra1 subunit being responsible for the majority of the stimulation. Interestingly, for the HAT module alone, nucleosome acetylation is inhibited by activator proteins due to non-specific binding of the activator to the nucleosomes. This inhibition is not observed for the yeast ADA complex, a small complex comprised mostly of the HAT module, suggesting that subunits outside of the HAT module in both it and SAGA can overcome non-specific activator binding to nucleosomes. However, this activity appears distinct from activator-mediated stimulation, as ADA complex acetylation is not stimulated by Gal4-VP16.

Integrated Analysis of Acetyl-CoA and Histone Modification via Mass Spectrometry to Investigate Metabolically Driven Acetylation.

Acetylation is a highly abundant and dynamic post-translational modification (PTM) on histone proteins which, when present on chromatin-bound histones, facilitates the accessibility of DNA for gene transcription. The central metabolite, acetyl-CoA, is a substrate for acetyltransferases, which catalyze protein acetylation. Acetyl-CoA is an essential intermediate in diverse metabolic pathways, and cellular acetyl-CoA levels fluctuate according to extracellular nutrient availability and the metabolic state of the cell. The Michaelis constant (Km) of most histone acetyltransferases (HATs), which specifically target histone proteins, falls within the range of cellular acetyl-CoA concentrations. As a consequence, global levels of histone acetylation are often restricted by availability of acetyl-CoA. Such metabolic regulation of histone acetylation is important for cell proliferation, differentiation, and a variety of cellular functions. In cancer, numerous oncogenic signaling events hijack cellular metabolism, ultimately inducing an extensive rearrangement of the epigenetic state of the cell. Understanding metabolic control of the epigenome through histone acetylation is essential to illuminate the molecular mechanisms by which cells sense, adapt, and occasionally disengage nutrient fluctuations and environmental cues from gene expression. In particular, targeting metabolic regulators or even dietary interventions to impact acetyl-CoA availability and histone acetylation is a promising target in cancer therapy. Liquid chromatography coupled to mass spectrometry (LC-MS) is the most accurate methodology to quantify protein PTMs and metabolites. In this chapter, we present state-of-the-art protocols to analyze histone acetylation and acetyl-CoA. Histones are extracted and digested into short peptides (4-20 aa) prior to LC-MS. Acetyl-CoA is extracted from cells and analyzed using an analogous mass spectrometry-based procedure. Model systems can be fed with isotopically labeled substrates to investigate the metabolic preference for acetyl-CoA production and the metabolic dependence and turnover of histone acetylation. We also present an example of data integration to correlate changes in acetyl-CoA production with histone acetylation.

Distinct requirements of linker DNA and transcriptional activators in promoting SAGA-mediated nucleosome acetylation

The Spt-Ada-Gcn5 acetyltransferase (SAGA) family of transcriptional coactivators are prototypical nucleosome acetyltransferase complexes that regulate multiple steps in gene transcription. The size and complexity of both the SAGA enzyme and the chromatin substrate provide numerous opportunities for regulating the acetylation process. To better probe this regulation, here we developed a bead-based nucleosome acetylation assay to characterize the binding interactions and kinetics of acetylation with different nucleosomal substrates and the full SAGA complex purified from budding yeast (Saccharomyces cerevisiae). We found that SAGA-mediated nucleosome acetylation is stimulated up to 9-fold by DNA flanking the nucleosome, both by facilitating the binding of SAGA and by accelerating acetylation turnover. This stimulation required that flanking DNA is present on both sides of the nucleosome and that one side is >15 bp long. The Gal4-VP16 transcriptional activator fusion protein could also augment nucleosome acetylation up to 5-fold. However, contrary to our expectations, this stimulation did not appear to occur by stabilizing the binding of SAGA toward nucleosomes containing an activator-binding site. Instead, increased acetylation turnover by SAGA stimulated nucleosome acetylation. These results suggest that the Gal4-VP16 transcriptional activator directly stimulates acetylation via a dual interaction with both flanking DNA and SAGA. Altogether, these findings uncover several critical mechanisms of SAGA regulation by chromatin substrates.

Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.

Cellular substrate limitations of lysine acetylation turnover by sirtuins investigated with engineered futile cycle enzymes

Metabolic activity and epigenetic regulation of gene expression are intimately coupled. The mechanisms linking the two are incompletely understood. Sirtuins catalyse the removal of acetyl groups from lysine side chains of proteins using NAD+ as a stoichiometric cofactor, thereby connecting the acetylation state of histones to energy supply of the cell. Here, we investigate the impact of lysine acetylation turnover by sirtuins on cell physiology by engineering Sirtase, an enzyme that self-acetylates and deacetylates in futile cycles. Expression of Sirtase in E. coli leads to the consumption of the majority of the cellular NAD+ supply, indicating that there is little negative feedback from reaction products, O-acetyl-ADP-ribose and nicotinamde, on sirtuin activity. Targeting Sirtase to a partially defective E silencer of the budding yeast mating type locus restores silencing, indicating that lysine acetylation turnover stabilizes heterochromatin in yeast. We speculate that this could be the consequence of local acetyl-CoA depletion because the effect is equally pronounced if the sirtuin moiety of Sirtase is exchanged with Hos3, a NAD+-independent deacetylase. Our findings support the concept that metabolism and epigenetic regulation are linked via modulation of heterochromatin stability by lysine acetylation turnover.

Nucleus-Translocated ACSS2 Promotes Gene Transcription for Lysosomal Biogenesis and Autophagy

Overcoming metabolic stress is a critical step in tumor growth. Acetyl coenzyme A (acetyl-CoA) generated from glucose and acetate uptake is important for histone acetylation and gene expression. However, how acetyl-CoA is produced under nutritional stress is unclear. We demonstrate here that glucose deprivation results in AMP-activated protein kinase (AMPK)-mediated acetyl-CoA synthetase 2 (ACSS2) phosphorylation at S659, which exposed the nuclear localization signal of ACSS2 for importin α5 binding and nuclear translocation. In the nucleus, ACSS2 binds to transcription factor EB and translocates to lysosomal and autophagy gene promoter regions, where ACSS2 incorporates acetate generated from histone acetylation turnover to locally produce acetyl-CoA for histone H3 acetylation in these regions and promote lysosomal biogenesis, autophagy, cell survival, and brain tumorigenesis. In addition, ACSS2 S659 phosphorylation positively correlates with AMPK activity in glioma specimens and grades of glioma malignancy. These results underscore the significance of nuclear ACSS2-mediated histone acetylation in maintaining cell homeostasis and tumor development.

The Roles of Flanking DNA and Transcriptional Activators in Regulating SAGA‐Mediated Nucleosome Acetylation

The SAGA family of transcriptional coactivators are prototypical nucleosome acetyltransferase complexes that regulate multiple steps of gene transcription. The size and complexity of both the SAGA enzyme and the chromatin substrate provide opportunities to regulate the acetylation process. To better probe this regulation, we have characterized the binding interactions and kinetics of acetylation using different nucleosomal substrates and the full SAGA complex purified from budding yeast. We find that SAGA‐mediated nucleosome acetylation proceeds through a multiple‐turnover burst phase, suggesting rapid nucleosome acetylation at multiple sites prior to nucleosome release. The rate of nucleosome acetylation is affected by DNA flanking the nucleosome, both by facilitating the binding of SAGA and by accelerating acetylation turnover. This stimulation requires flanking DNA on both sides of the nucleosome, where one side needs to be longer than 15 base pairs. Gal4‐VP16 activator can also augment nucleosome acetylation. However, contrary to our expectations, this stimulation does not appear to occur by stabilizing the binding of SAGA toward nucleosomes containing an activator binding site. Instead, stimulation of nucleosome acetylation occurs by increasing acetylation turnover by SAGA. Altogether, these studies uncover several novel mechanisms of SAGA regulation by chromatin substrates.Support or Funding InformationThis work was supported by American Cancer Society Research Scholar Grant 1206501 to M. A. S.‐K. Work by S. J. O. was supported in part by a Gary Roewe Faculty Research Award.

Panobinostat: The Small Molecule Metalloenzyme Inhibitor with Marvelous Anticancer Activity

Histone acetyl transferases (HATs) and histone deacetylases (HDACs) are antagonistic enzymes regulating the turnover of histone acetylation thereby governing gene expression in a precise manner. Histone acetylation deregulation caused by aberrant expression of classical HDACs plays a crucial role in tumour onset and progression making these enzymes as striking targets for anticancer drugs and therapy. Small molecule inhibitors targetting HDACs (HDACi) have shown multiple biological effects including cell cycle arrest, differentiation and apoptosis in cancer cell models. The current review deals with the recently approved pan-HDAC inhibitor panobinostat (LBH589) and its antiproliferative activity against distinct cancers (breast, ovarian, lung and multiple myeloma). The intricate details about the different mechanisms triggered by panobinostat to exert cytotoxic effect in these cancers have also been provided. The article also highlights the different combination strategies of panobinostat which can be utilized for overcoming conventional therapy resistant cases and for achieving the enhanced therapeutic benefit from this marvelous inhibitor in the upcoming future.

Measurement of acetylation turnover at distinct lysines in human histones identifies long-lived acetylation sites

Histone acetylation has long been determined as a highly dynamic modification associated with open chromatin and transcriptional activation. Here we develop a metabolic labeling scheme using stable isotopes to study the kinetics of acetylation turnover at 19 distinct lysines on histones H3, H4, and H2A. Using human HeLa S3 cells, the analysis reveals 12 sites of histone acetylation with fast turnover and 7 sites stable over a 30 hour experiment. The sites showing fast turnover (anticipated from classical radioactive measurements of whole histones) have half-lives between ~1–2 hours. To support this finding, we use a broad-spectrum deacetylase inhibitor to verify that only fast turnover sites display 2–10 fold increases in acetylation whereas long-lived sites clearly do not. Most of these stable sites lack extensive functional studies or localization within global chromatin, and their role in non-genetic mechanisms of inheritance is as yet unknown.

Regulation of Primary Response Genes in B Cells

Deregulated gene expression in B cells often results in various lymphoid malignancies and immune deficiencies. Therefore, understanding signal-induced gene regulatory pathways involved during B cell activation is important to tackle pathologies associated with altered B cell function. Primary response genes (PRGs) are rapidly induced upon signaling in B cells and other cell types and often encode oncogenic transcription factors, which are associated with various malignancies. However, an important issue that remains unclear is whether the fundamental mechanism of activation of these genes is essentially the same under such diverse conditions. c-fos is a PRG that is induced rapidly upon activation of B cells in response to a wide variety of stimuli. Using the c-fos gene as a candidate PRG, we addressed here how it is regulated in response to tumor-promoting and antigen-mimicking signals. Our results show that although the mRNA was induced and extinguished within minutes in response to both signals, surprisingly, apparently full-length unspliced pre-mRNA persisted for several hours in both cases. However, although the mitogenic signal resulted in a more sustained mRNA response that persisted for 4 h, antigenic signaling resulted in a more robust but very transient response that lasted for <1 h. Moreover, the pre-mRNA profile exhibited significant differences between the two signals. Additionally, the splicing regulation was also observed with egr-2, but not with c-myc. Together, these results suggest a previously underappreciated regulatory step in PRG expression in B cells.

Quantitative Dynamics of the Link between Cellular Metabolism and Histone Acetylation

Acetylation on the tails of histones plays an important role in controlling transcription initiation. Although the steady-state abundances of histone acetyl groups have been reported, the rate at which histones are acetylated and deacetylated on a residue-specific basis has not been quantitatively established. We added [(13)C]glucose to human cells and monitored the dynamic incorporation of (13)C-labeled acetyl groups onto specific histone lysines with quantitative mass spectrometry. We determined the turnover of acetylation to be generally slower than phosphorylation, but fast relative to methylation, and that the rate varied depending on the histone, the residue modified, and also the neighboring modifications. Cells were also treated with a deacetylase inhibitor to determine the rate due to histone acetyltransferase activity alone and in the absence of deacetylase activity. Introduction of (13)C-labeled glucose also resulted in the incorporation of (13)C into alanine, which allowed us to partition histones into existing and newly synthesized protein categories. Newly synthesized histones were slower to accumulate histone modifications, especially modifications associated with silent chromatin. Finally, we applied our new approaches to find that quiescent fibroblasts exhibited lower levels of labeled acetyl accumulation compared with proliferating fibroblasts. This suggests that acetylation rates can be modulated in cells in different biological states and that these changes can be detected with the approach presented here. The methods we describe can be broadly applied to defining the turnover of histone acetylation in other cell states such as during cellular reprogramming and to quantify non-histone protein acetylation dynamics.

Histone deacetylase inhibitors in the generation of the anti‐tumour immune response

Acetylation of lysine residues acts to modify the function of a wide range of proteins. In histones, it affects chromatin structure, which can impact gene transcription, whereas acetylation of transcription factors and heat-shock proteins affect their activity. Deacetylase inhibitors block the dynamic turnover of acetylation resulting in hyperacetylation of target proteins. This can affect a wide range of cellular functions, and in a wide range of tumour cell types promote cytostatic and cytotoxic effects, but has little effect on normal cells. The inhibitors are being used clinically as anti-cancer agents. Although direct effects of the histone deacetylase (HDAC) inhibitors on cancers are beginning to be elucidated, the prospect of concurrent stimulation of the immune response raises hopes for immune attack of the tumour as part of the initial anti-cancer therapy and long-term immune-surveillance of residual or recurrent tumour. This review will examine the evidence for the generation of anti-tumour immunity after treatment of cancers with HDAC inhibitors.

Dynamic acetylation of all lysine-4 trimethylated histone H3 is evolutionarily conserved and mediated by p300/CBP

Histone modifications are reported to show different behaviors, associations, and functions in different genomic niches and organisms. We show here that rapid, continuous turnover of acetylation specifically targeted to all K4-trimethylated H3 tails (H3K4me3), but not to bulk histone H3 or H3 carrying other methylated lysines, is a common uniform characteristic of chromatin biology in higher eukaryotes, being precisely conserved in human, mouse, and Drosophila. Furthermore, dynamic acetylation targeted to H3K4me3 is mediated by the same lysine acetyltransferase, p300/cAMP response element binding (CREB)-binding protein (CBP), in both mouse and fly cells. RNA interference or chemical inhibition of p300/CBP using a newly discovered small molecule inhibitor, C646, blocks dynamic acetylation of H3K4me3 globally in mouse and fly cells, and locally across the promoter and start-site of inducible genes in the mouse, thereby disrupting RNA polymerase II association and the activation of these genes. Thus, rapid dynamic acetylation of all H3K4me3 mediated by p300/CBP is a general, evolutionarily conserved phenomenon playing an essential role in the induction of immediate-early (IE) genes. These studies indicate a more global function of p300/CBP in mediating rapid turnover of acetylation of all H3K4me3 across the nuclei of higher eukaryotes, rather than a tight promoter-restricted function targeted by complex formation with specific transcription factors.

Undifferentiated hematopoietic cells are characterized by a genome-wide undermethylation dip around the transcription start site and a hierarchical epigenetic plasticity

AbstractEvidence for the epigenetic regulation of hematopoietic stem cells (HSCs) is growing, but the genome-wide epigenetic signature of HSCs and its functional significance remain unclear. In this study, from a genome-wide comparison of CpG methylation in human CD34+ and CD34− cells, we identified a characteristic undermethylation dip around the transcription start site of promoters and an overmethylation of flanking regions in undifferentiated CD34+ cells. This “bivalent-like” CpG methylation pattern around the transcription start site was more prominent in genes not associated with CpG islands (CGI−) than CGI+ genes. Undifferentiated hematopoietic cells also exhibited dynamic chromatin associated with active transcription and a higher turnover of histone acetylation than terminally differentiated cells. Interestingly, inhibition of chromatin condensation by chemical treatment (5-azacytidine, trichostatin A) enhanced the self-renewal of “stimulated” HSCs in reconstituting bone marrows but not “steady-state” HSCs in stationary phase bone marrows. In contrast, similar treatments on more mature cells caused partial phenotypic dedifferentiation and apoptosis at levels correlated with their hematopoietic differentiation. Taken together, our study reveals that the undifferentiated state of hematopoietic cells is characterized by a unique epigenetic signature, which includes dynamic chromatin structures and an epigenetic plasticity that correlates to level of undifferentiation.

The Gcn5 Bromodomain of the SAGA Complex Facilitates Cooperative and Cross-tail Acetylation of Nucleosomes

Bromodomains are acetyl lysine binding modules found in many complexes that regulate gene transcription. In budding yeast, the coactivator complex SAGA (Spt-Ada-Gcn5-acetyl-transferase) predominantly facilitates transcription of stress-activated genes and requires the bromodomain of the Gcn5 subunit for full activation of a number of these genes. This bromodomain has previously been shown to promote retention of the complex to H3 and H4 acetylated nucleosomes. Because the SAGA complex mediates histone H3 acetylation, we sought to determine to what extent the Gcn5 bromodomain directly modulates histone acetylation activity. Kinetic analysis of SAGA-mediated acetylation of nucleosomal substrates reveals that this bromodomain: 1) is required for the cooperative acetylation of nucleosomes, 2) enhances acetylation of an H3 histone tail when the other H3 tail within a nucleosome is already acetylated, and 3) augments the acetylation turnover of nucleosomes previously acetylated at lysine 16 of the histone H4 tails. These results indicate that the Gcn5 bromodomain promotes the establishment of nucleosome acetylation through multiple mechanisms and more generally show how chromatin recognition domains can modulate the enzymatic activity of chromatin modifying complexes.

Developmental and Environmental Signals Induce Distinct Histone Acetylation Profiles on Distal and Proximal Promoter Elements of theC4-PepcGene in Maize

The maize C(4)-Pepc gene is expressed in an organ- and cell-type-specific manner, inducible by light and modulated by nutrient availability and the metabolic state of the cell. We studied the contribution of histone acetylation at five lysine residues to the integration of these signals into a graduated promoter response. In roots and coleoptiles, where the gene is constitutively inactive, three of the five lysines were acetylated and the modifications showed unique patterns with respect to their distribution on the gene. A similar pattern was observed in etiolated leaves, where the gene is poised for activation by light. Here, illumination selectively induced the acetylation of histone H4 lysine 5 and histone H3 lysine 9 in both the promoter and the transcribed region, again with unique distribution patterns. Induction was independent of transcription and fully reversible in the dark. Nitrate and hexose availability modulated acetylation of all five lysines restricted to a distal promoter region, whereas proximal promoter acetylation was highly resistant to these stimuli. Our data suggest that light induction of acetylation is controlled by regulating HDAC activity, whereas metabolic signals regulate HAT activity. Acetylation turnover rates were high in the distal promoter and the transcribed regions, but low on the proximal promoter. On the basis of these results, we propose a model with three levels of stimulus-induced histone modifications that collectively adjust promoter activity. The results support a charge neutralization model for the distal promoter and a stimulus-mediated, but transcription-independent, histone acetylation pattern on the core promoter, which might be part of a more complex histone code.

Dynamic histone H3 methylation during gene induction: HYPB/Setd2 mediates all H3K36 trimethylation

Understanding the function of histone modifications across inducible genes in mammalian cells requires quantitative, comparative analysis of their fate during gene activation and identification of enzymes responsible. We produced high-resolution comparative maps of the distribution and dynamics of H3K4me3, H3K36me3, H3K79me2 and H3K9ac across c-fos and c-jun upon gene induction in murine fibroblasts. In unstimulated cells, continuous turnover of H3K9 acetylation occurs on all K4-trimethylated histone H3 tails; distribution of both modifications coincides across promoter and 5′ part of the coding region. In contrast, K36- and K79-methylated H3 tails, which are not dynamically acetylated, are restricted to the coding regions of these genes. Upon stimulation, transcription-dependent increases in H3K4 and H3K36 trimethylation are seen across coding regions, peaking at 5′ and 3′ ends, respectively. Addressing molecular mechanisms involved, we find that Huntingtin-interacting protein HYPB/Setd2 is responsible for virtually all global and transcription-dependent H3K36 trimethylation, but not H3K36-mono- or dimethylation, in these cells. These studies reveal four distinct layers of histone modification across inducible mammalian genes and show that HYPB/Setd2 is responsible for H3K36 trimethylation throughout the mouse nucleus.

Chromatin at the crossroads. Meeting on Signalling to Chromatin Epigenetics

![][1] In June 2006, about 90 scientists came together at the Wellcome Trust Conference Centre in Hinxton (near Cambridge, UK), to attend the first European Science Foundation (ESF)–Wellcome Trust Conference on Signalling to Chromatin Epigenetics. This meeting addressed how cellular processes both signal and respond to chromatin. Chromatin is composed of nucleosomes that each comprise 147 base pairs of DNA wrapped around an octamer of two copies of each histone H2A, H2B, H3 and H4. Nucleosomes are folded into higher‐order structures that are stabilized by linker histones. Chromatin structure can be altered by enzymes that post‐translationally modify histones (for example, through phosphorylation, acetylation, methylation or ubiquitination) or by ATP‐driven chromatin‐remodelling complexes that alter nucleosome position and/or composition. The role of chromatin in cellular processes, such as transcription, DNA‐damage repair and cell differentiation, was discussed at the meeting. In this report, we summarize a few of the highlights from the broad range of topics discussed. Histone modifications have an important role in the regulation of gene transcription. L. Mahadevan (Oxford, UK) and co‐workers have studied how a particular set of histone modifications controls the rapid induction of the immediate‐early genes c‐jun and c‐fos . Mahadevan showed that on induction by extracellular signals, the extracellular‐signal‐regulated kinase (ERK) and the mitogen‐ and stress‐activated protein kinase 1/2 (MSK1/2) pathways direct a dynamic turnover of acetylation and phosphorylation marks on histone H3 at these genes (Hazzalin & Mahadevan, 2005). Sequential chromatin immunoprecipitation (ChIP) assays showed that acetylated K9‐phosphorylated S10 (H3K9acS10ph) and trimethylated K4 (H3K4me3) forms of histone H3 co‐exist on the same nucleosomes at the promoter and at the 5′‐coding sequences of these genes when they are transcriptionally active. Intriguingly, H3K9acS10ph is only transiently observed on gene induction, whereas H3K4me3 is present before induction. Mahadevan suggested that H3K4me3 might assist in creating a poised state by … [1]: /embed/graphic-1.gif

Dynamic Acetylation of All Lysine 4–Methylated Histone H3 in the Mouse Nucleus: Analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.