Forms Of H4 Research Articles

The influence of different types of hypobaric hypoxia on histone H3 methylation in rat neocortical and hippocampal neurons

Using the quantitative immunohistochemical method, we analyzed changes in the number of cells that were immunopositive for methylated forms of histone H3 at lysine 4 (H3K4me2,3) and lysine 9 (H3K9me2) in the parietal neocortex and hippocampus (the СА1 and СА3 areas) in rats under the conditions of damaging severe hypobaric hypoxia (SH) and mild adaptogenic hypobaric hypoxia (MH). We found that 3 or 24 hours after SH exposure the quantity of H3K9me2-positive cells (IPC) strongly increased, causing heterochromatin formation and global repression of gene transcription. After three (but not single) MH sessions, as well as after the following SH, the quantity of H3K9me2-positive cells decreased and H3K4me2,3- positive cells increased, causing the formation of euchromatin and an increase in the expression of genes–targets of transcriptional factors and other proadaptive proteins, thus increasing the neuroplasticity and neuroprotection of brain neurons after damage. Thus, we showed that different types of hypobaric hypoxia induce ambiguous changes in immunostaining of various forms of H3 histone methylation in mammalian forebrain structures when applying severe damaging and adaptogenic neuroprotective hypobaric hypoxia.

Abstract SY10-04: Histone tail alterations in cellular senescence

Abstract The process of cellular senescence generates a repressive chromatin environment, however, the role of histone tail modifications, histone variants and histone proteolytic cleavage in senescence remains unclear. Using well-characterized senescence models in human primary lung fibroblasts and melanocytes, we are investigating how chromatin alterations contribute to the senescence phenotype. Here we report novel histone H3 tail cleavage events in oncogene-induced and replicative senescence models mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and that H3.3 is the preferred cleaved form of H3 - an H3 histone variant that is deposited independent of DNA replication. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Furthermore, we find that chromatin incorporation of H3.3cs1 is mediated by the HUCA (HIRA/UBN1/CABIN1/ASF1a) histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our studies have identified histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. Citation Format: Luis F. Duarte, Andrew R. J. Young, Zichen Wang, Hsan-Au Wu, Taniya Panda, Yan Kou, Avnish Kapoor, Dan Hasson, Nicholas R. Mills, Avi Ma'ayan, Masashi Narita, Emily Bernstein. Histone tail alterations in cellular senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY10-04. doi:10.1158/1538-7445.AM2015-SY10-04

Abstract 2247: Histone H3 cleavage via Cathepsin L drives a cellular senescence program

Abstract Oncogene-induced senescence (OIS) is characterized by irreversible loss of proliferative capacity. It has been proposed that OIS functions as a tumor suppressor mechanism, and several studies have demonstrated induction of senescence markers in pre-malignant lesions. In addition to profound morphological changes, significant alterations in chromatin structure occur during senescence including formation of senescence-associated heterochromatin foci (SAHF). We have used well-characterized senescence models in human primary lung fibroblasts and melanocytes to study how chromatin alterations contribute to the senescence phenotype. We discovered that the N'-terminal tail of histone H3 is proteolytically cleaved by the lysosomal protease Cathepsin L1 (CTSL1). We show that CTSL1 is induced during senescence both at the mRNA and protein levels, and that it is present in the chromatin fraction of senescent cells. Chemical and shRNA-mediated inhibition of CTSL1 activity effectively block H3 tail cleavage and leads to a SAHF formation defect. We identified two distinct cleaved forms of H3 in senescent cells, which are associated with chromatin and incorporated into nucleosomes. Using antibodies directed against specific post-translational modifications (PTMs) of the histone H3 tail, we have mapped the approximate locations of the cleavage sites. Furthermore, overexpression of flag-tagged cleaved versions of H3, but not the full-length histone, trigger robust senescence in IMR90 cells accompanied by a modest induction of SAHFs. Interestingly, we also detect cleaved H3 in chromatin extracted from benign nevi tissue samples, providing evidence of H3 tail cleavage in senescence models in vivo. Collectively, our results suggest that H3 tail cleavage may contribute significantly to the senescence phenotype, potentially through transcriptional regulation of key senescence target genes. This area of investigation is currently ongoing. Citation Format: Luis F. Duarte, Andrew R. Young, Hsan-Au Wu, Taniya Panda, Zichen Wang, Alexandre G. Maia, Dan Hasson, Avnish Kapoor, Masashi Narita, Emily Bernstein. Histone H3 cleavage via Cathepsin L drives a cellular senescence program. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2247. doi:10.1158/1538-7445.AM2014-2247

Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules

The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

Synthesis of Metallic Nanoparticles Using Electrogenerated Reduced Forms of [α-SiW12O40]4– as Both Reductants and Stabilizing Agents

In this paper, controlled potential bulk electrolysis at a glassy carbon working electrode was conveniently employed to produce the reduced forms of H4[α-SiW12O40] with little or no supporting electrolyte. Consequently, without the need for separation and purification, these reduced forms of [α-SiW12O40]4– were directly used as both the reductants and the stabilizing agents for the size-controlled synthesis of various types of metallic and alloy nanoparticles, including Au, Ag, Pt, Pd and Ag–Au. Ni nanoparticles were also successfully synthesized using highly reduced [α-SiW12O40]4– under neutral pH condition. The Pt nanoparticles thus obtained displayed attractive electrocatalytic properties in methanol oxidation.

Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism

Histone H4 undergoes extensive post-translational modifications (PTMs) at its N-terminal tail. Many of these PTMs profoundly affect the on and off status of gene transcription. The molecular mechanism by which histone PTMs modulate genetic and epigenetic processes is not fully understood. In particular, how a PTM mark affects the presence and level of other histone modification marks needs to be addressed and is essential for better understanding the molecular basis of histone code hypothesis. To dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. Although certain acetylations present a repressive impact on PRMT1-mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases the activity of PRMT1 but increases that of PRMT5. Furthermore, circular dichroism study and computer simulation demonstrate that hyperacetylation increases the content of ordered secondary structures at the H4 tail region. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation and unravel the complex intercommunications that exist between different the PTM marks in cis. The divergent activities of PRMT1 and PRMT5 with respect to different acetyl-H4 substrates suggest that type I and type II protein-arginine methyltransferases use distinct molecular determinants for substrate recognition and catalysis.

Myc-dependent Mitochondrial Generation of Acetyl-CoA Contributes to Fatty Acid Biosynthesis and Histone Acetylation during Cell Cycle Entry

Cell reprogramming from a quiescent to proliferative state requires coordinate activation of multiple -omic networks. These networks activate histones, increase cellular bioenergetics and the synthesis of macromolecules required for cell proliferation. However, mechanisms that coordinate the regulation of these interconnected networks are not fully understood. The oncogene c-Myc (Myc) activates cellular metabolism and global chromatin remodeling. Here we tested for an interconnection between Myc regulation of metabolism and acetylation of histones. Using [(13)C(6)]glucose and a combination of GC/MS and LC/ESI tandem mass spectrometry, we determined the fractional incorporation of (13)C-labeled 2-carbon fragments into the fatty acid palmitate, and acetyl-lysines at the N-terminal tail of histone H4 in myc(-/-) and myc(+/+) Rat1A fibroblasts. Our data demonstrate that Myc increases mitochondrial synthesis of acetyl-CoA, as the de novo synthesis of (13)C-labeled palmitate was increased 2-fold in Myc-expressing cells. Additionally, Myc induced a forty percent increase in (13)C-labeled acetyl-CoA on H4-K16. This is linked to the capacity of Myc to increase mitochondrial production of acetyl-CoA, as we show that mitochondria provide 50% of the acetyl groups on H4-K16. These data point to a key role for Myc in directing the interconnection of -omic networks, and in particular, epigenetic modification of proteins in response to proliferative signals.

High Throughput Characterization of Combinatorial Histone Codes

We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resolution mass spectrometer, displays an improvement over existing methods with an approximately 100-fold reduction in sample requirements and analysis time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h analysis. The large N-terminal histone peptides are eluted by the pH and organic solvent weak cation exchange-hydrophilic interaction liquid chromatography gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric trimethyl and acetyl modifications are resolved chromatographically and confidently distinguished by the synthesis of mass spectrometric and chromatographic information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells, but it is generally applicable to the analysis of highly modified peptides. We find this methodology very useful for chromatographic separation of isomeric species that cannot be separated well by any other chromatographic means, leading to less complicated tandem mass spectra. The improved separation and increased sensitivity generated novel information about much less abundant forms. In this method demonstration we report over 200 H3.2 forms and 70 H4 forms, including forms not yet detected in human cells, such as the remarkably highly modified histone H3.2 K4me3K9acK14acK18acK23acK27acK36me3. Such detail provided by our proteomics platform will be essential for determining how histone modifications occur and act in combination to propagate the histone code during transcriptional events and could greatly enable sequencing of the histone component of human epigenomes.

Combinatorial Modification of Human Histone H4 Quantitated by Two-dimensional Liquid Chromatography Coupled with Top Down Mass Spectrometry

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

Mild Performic Acid Oxidation Enhances Chromatographic and Top Down Mass Spectrometric Analyses of Histones

Recent developments in top down mass spectrometry have enabled closely related histone variants and their modified forms to be identified and quantitated with unprecedented precision, facilitating efforts to better understand how histones contribute to the epigenetic regulation of gene transcription and other nuclear processes. It is therefore crucial that intact MS profiles accurately reflect the levels of variants and modified forms present in a given cell type or cell state for the full benefit of such efforts to be realized. Here we show that partial oxidation of Met and Cys residues in histone samples prepared by conventional methods, together with oxidation that can accrue during storage or during chip-based automated nanoflow electrospray ionization, confounds MS analysis by altering the intact MS profile as well as hindering posttranslational modification localization after MS/MS. We also describe an optimized performic acid oxidation procedure that circumvents these problems without catalyzing additional oxidations or altering the levels of posttranslational modifications common in histones. MS and MS/MS of HeLa cell core histones confirmed that Met and Cys were the only residues oxidized and that complete oxidation restored true intact abundance ratios and significantly enhanced MS/MS data quality. This allowed for the unequivocal detection, at the intact molecule level, of novel combinatorially modified forms of H4 that would have been missed otherwise. Oxidation also enhanced the separation of human core histones by reverse phase chromatography and decreased the levels of salt-adducted forms observed in ESI-FTMS. This method represents a simple and easily automated means for enhancing the accuracy and sensitivity of top down analyses of combinatorially modified forms of histones that may also be of benefit for top down or bottom up analyses of other proteins.

Observations and models of deuterated in proto-planetary disks

Young, gas-rich proto-planetary disks orbiting around solar-type young stars represent a crucial phase in disk evolution and planetary formation. Of particular relevance is to observationally track the evolution of the gas, which governs the overall evolution of the disk and is eventually dispersed. However, the bulk of the mass resides in the plane, which is so cold and dense that virtually all heavy-element-bearing molecules freeze out onto the dust grains and disappear from the gas phase. In this paper, we show that the ground-state ortho-H2D+ transition is the best, if not the only, tracer of the disk-plane gas. We report the theoretical models of the chemical structure of the plane of the disk, where the deuterated forms of H3+, including H2D+, play a major role. We also compare the theoretical predictions with the observations obtained towards the disk of the young star DM Tau and show that the ionization rate is probably enhanced there, perhaps owing to the penetration of X-rays from the central object through the disk plane. We conclude by remarking that the ground-state ortho-H2D+ transition is such a powerful diagnostic that it may also reveal the matter in the dark halos of external galaxies, if it is hidden in cold, dense and small clouds, as several theories predict.

Histone proteins determined in a human colon cancer by high-performance liquid chromatography and mass spectrometry

The application of reversed-phase high-pressure liquid chromatography under gradient conditions and electrospray ion trap mass spectrometry (LC–ESI–MS) to the analysis of global modification levels of core histones is described. The optimised LC–ESI–MS method was applied for the first time to the characterisation of histones extracted from HT29, a human colon cancer cell line. Eight histones (H1-1, H1-2, H2A-1, H2A-2, H2B, H3-1, H3-2, H4) were separated on a C4 stationary phase with complete resolution, never reached in previous HPLC–MS methods, by using a gradient elution with the combined presence of heptafluorobutyric acid and formic acid as acidic modifiers in the mobile phase. Heptafluorobutyric acid was found to improve selectivity, whereas the presence of formic acid decreased ion suppression. Histones eluted from the column were detected with an ion trap mass spectrometer with an electrospray source. The peak averaged mass spectra were reconstructed by Mag Tran 1.0 software and the mass of the various isoforms of histones were derived. Method validation was conducted by performing the same sample analysis by coupling LC–ESI to a quadrupole-time-of-flight mass spectrometer (Q-TOF). The number of histone forms and their mass were found to differ not significantly from those obtained by ion trap mass spectrometer. Also the relative modifications abundance within the same histone type was found following the same trend as the two mass analysers. This method was then applied to the characterisation of changes in histone modification in HT29, never analysed by LC–MS before, treated with histone deacetylase inhibitors such as valproate and sodium butyrate, also used in preclinical trials as anticancer drugs. In particular, both the inhibitors produced a significant increase in H4 histone acetylated forms: 89% increase of the diacetyl dimethyl H4 form was observed with 1 mM valproate supplementation, whereas 5 mM butyrate led to a 68% increase of the same form. Triacetyl monomethyl H4 (11 377 Da) and triacetyl dimethyl H4 (11 390 Da) were found only in cells treated with butyrate. Selective changes of H3 histone were detected with butyrate, in agreement with recently reported western blotting studies. Modifications in the H2A histone degree of acetylation were revealed by treatment of the cells with butyrate (H2A-1, H2A-2) and valproate (H2A-2). The results of the proposed methodology confirmed that inhibition of histone deacetylases caused histone hyperacetylation, responsible for decondensation and reorganization of interphase dynamic chromatin. This method resulted in selective and sensitive method to monitor variations in the acetylation and methylation state of histones after treatment of HT29 with inhibitors, and is therefore suitable for further application in new drug discovery for tumour therapy.

Release of Hypoacetylated and Trimethylated Histone H4 Is an Epigenetic Marker of Early Apoptosis

Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers.

Systematic strong-coupling expansion of theT ⊗ t Jahn–Teller system

An analytic approach to the strongly coupled problem is described, which allows a systematic expansion ofthe Hamiltonian in inverse powers of the coupling parameterK. The approach rests on the construction of a novel vibronic basis causing the Hilbert spaceto decay into two orthogonal subspaces and such that, for strong coupling, the low-lying states in are separated from all states in by a large energy gap. This constitutes the basis for the construction of an effective HamiltonianH1 defined on and generated by means of projection operators. The form ofH1 is that of an infinite series in increasing powers ofK−1, which is terminatedafter the terms of O (K−2).The eigenvalues of H1 incorporate those already found by Moffitt and Thorson, including the correct splitting of thet2g vibrations.

The Histone Deacetylase Inhibitor Depsipeptide Mediates Distinct Patterns of Histone Acetylation in Cells Overexpressing Bcl-2.

Post-translational modifications of histone proteins including acetylation and methylation are known to be important in the regulation of gene expression, and histone modification patterns are found to be altered in many tumor types. We have established a liquid chromatography/mass spectrometry (LC/MS) analysis technique that allows for detailed interrogation of histone protein post-translational patterns. Application of this technique to histone proteins isolated from Chronic Lymphocytic Leukemia (CLL) patient cells and CD19-selected normal B cells has demonstrated that primary CLL cells have a pattern of H2A modification distinct from normal B cells, and that changes in each of the histone proteins H2A, H2B, H3 and H4 occur following treatment with HDAC inhibitors. We have used this and other techniques to understand how factors that influence apoptotic threshold, such as Bcl-2, can also cause changes in histone post-translational modifications induced by depsipeptide and other HDAC inhibitors. To initially explore this, we utilized the 697 lymphoblastoid cell line transfected either with an empty vector (697-neo) or a vector driving a ten-fold overexpression of Bcl-2 protein (697-Bcl-2). Cells were exposed to varying concentrations of depsipeptide for 4 or 24 hours and analyzed for growth inhibition, cell death, HDAC inhibition, and p21 induction. The results of these studies are summarized in the following table:Effects of Depsipeptide Treatment on 697 Cells at 24 HoursRelative % Cell GrowthApoptotic Cells (%)Relative HDAC Inhibition (%)p21, fold increase697-neo untreated1008.201.0697-neo 3.8 nM DDP66.39.074.01.5697-neo 38 nM DDP41.057.597.02.3697-Bcl-2 untreated1007.601.0697-Bcl-2 3.8 nM DDP90.47.577.31.9697-Bcl-2 38 nM DDP80.610.494.63.9Over-expression of Bcl-2 completely blocked apoptosis induced by depsipeptide, as compared to the empty vector cell line. Despite this, growth inhibition, HDAC inhibition and p21 protein induction was observed in the Bcl-2 over-expressing cell line. Specific acetylation of H4 was analyzed by LC/MS and demonstrated that baseline histone acetylation patterns were identical between the 697-neo and 697-Bcl-2 cell lines. With depsipeptide treatment, however, distinct differences were noted in the pattern of acetylation between the two cell lines at 24 hours. Specifically, 697-neo showed increases in the di-, tri-, and tetra-acetylated H4 forms, with the tri-acetylated form predominating. In the 697-Bcl-2 cells the di-acetylated form was the most abundant followed closely by tri- and tetra- acetylated forms, and in contrast to the 697-neo cells, a penta-acetylated form is strongly detected. Our data suggest that molecular characteristics of tumor cells that alter apoptotic thresholds may influence changes in histone acetylation following HDAC inhibitor treatment. Exploration of the relationship between histone acetylation and Bcl-2 over-expression is currently under study.

Structural determinants for generating centromeric chromatin.

Mammalian centromeres are not defined by a consensus DNA sequence. In all eukaryotes a hallmark of functional centromeres--both normal ones and those formed aberrantly at atypical loci--is the accumulation of centromere protein A (CENP-A), a histone variant that replaces H3 in centromeric nucleosomes. Here we show using deuterium exchange/mass spectrometry coupled with hydrodynamic measures that CENP-A and histone H4 form sub-nucleosomal tetramers that are more compact and conformationally more rigid than the corresponding tetramers of histones H3 and H4. Substitution into histone H3 of the domain of CENP-A responsible for compaction is sufficient to direct it to centromeres. Thus, the centromere-targeting domain of CENP-A confers a unique structural rigidity to the nucleosomes into which it assembles, and is likely to have a role in maintaining centromere identity.

Shotgun annotation of histone modifications: a new approach for streamlined characterization of proteins by top down mass spectrometry.

Eukaryotic histones serve as prototypical examples of posttranslational complexity with diverse modifications (PTMs) on many different residues that comprise a "Histone Code". To help crack this code more efficiently, we demonstrate a new strategy for protein characterization wherein complete PTM descriptions are obtained by database retrieval instead of manual interpretation of information-rich data from high-resolution tandem mass spectrometry (MS/MS). A database of nearly 50 000 modified histone H4 sequences was created and queried with 91 fragment ions from electron capture dissociation of a histone form +112 Da (versus unmodified mass) selectively accumulated in a quadrupole Fourier transform hybrid mass spectrometer. The correct form atop the retrieval list indicated dimethylation at Lys20, acetylation at the N terminus, and acetylation at Lys16 (resolved from trimethylation, Deltam = 0.036 Da). A statistical evaluation reveals the critical role of mass accuracy and that PTM "isomers" are retrieved as next-best matches. The applicability of shotgun annotation to forms of H4 with up to six PTMs is demonstrated, with extensibility to other histones (e.g., H2A, H2B, H3) and other protein classes projected.

Age-dependent response of lymphocytes in the induction of the linker histone variant, H1° and histone H4 acetylation after treatment with the histone deacetylase inhibitor, trichostatin A

In the present study we investigated the age-related response of Phytohemaglutinin (PHA)-activated S phase human lymphocytes isolated from peripheral blood from donors of four different age groups, namely young (25–30 years), mid-aged (40–45 years), senior (60–65 years) and elderly (80–95 years) on the induction of the linker histone variant, H1° and histone H4 acetylation after treatment with the very specific histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). The cell system of peripheral blood lymphocytes is ideal for the study of H1° induction since they do not synthesize this particular linker histone variant. Lymphocytes isolated from peripheral blood were activated with PHA (5 μg/10 6 cells/ml medium) and placed in culture for a duration of 72 h at which time cells are in the S phase. Forty-eight hours after inoculation, TSA (250 ng/10 6 cells/ml medium) was added to the cell cultures for a period of 24 h. Assays were performed 72 h after initiation of cultures. The results showed that the induction of H1° after TSA treatment increased to a statistically significant degree in the elderly age group with respect to both the young and the mid-aged age groups. Moreover histone H4 acetylation was found to increase as a function of increasing donor age. A hyperacetylation pattern was observed even in the youngest age group analyzed. Specifically, the tetra-acetylated (H4.4) H4 form increased to a statistically significant degree with the concomitant decrease in the non-acetylated H4 for (H4.0) as a function of donor age. The other acetylated H4 forms (H4.1, H4.2, and H4.3) remained more or less constant, irrespective of donor age. These results show that the sensitivity of lymphocytes to TSA is enhanced with increasing donor age. Since to date, 11 class I and II HDACs have been isolated that have been found by other investigators to have differential responses to HDAC inhibitors, these findings may indicate that there is also a differential age-related response of certain HDACs or perhaps a senescent-specific HDAC. This line of research warrants further study.

Global Regulation of Post-translational Modifications on Core Histones

Full-length masses of histones were analyzed by mass spectrometry to characterize post-translational modifications of bulk histones and their changes induced by cell stimulation. By matching masses of unique peptides with full-length masses, H4 and the variants H2A.1, H2B.1, and H3.1 were identified as the main histone forms in K562 cells. Mass changes caused by covalent modifications were measured in a dose- and time-dependent manner following inhibition of phosphatases by okadaic acid. Histones H2A, H3, and H4 underwent changes in mass consistent with altered acetylation and phosphorylation, whereas H2B mass was largely unchanged. Unexpectedly, histone H4 became almost completely deacetylated in a dose-dependent manner that occurred independently of phosphorylation. Okadaic acid also partially blocked H4 hyperacetylation induced by trichostatin-A, suggesting that the mechanism of deacetylation involves inhibition of H4 acetyltransferase activity, following perturbation of cellular phosphatases. In addition, mass changes in H3 in response to okadaic acid were consistent with phosphorylation of methylated, acetylated, and phosphorylated forms. Finally, kinetic differences were observed with respect to the rate of phosphorylation of H2A versus H4, suggesting differential regulation of phosphorylation at sites on these proteins, which are highly related by sequence. These results provide novel evidence that global covalent modifications of chromatin-bound histones are regulated through phosphorylation-dependent mechanisms.

A novel chromatin protein, distantly related to histone H2A, is largely excluded from the inactive X chromosome.

Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease-digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.