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PubMed2005

Expression patterns and post-translational modifications associated with mammalian histone H3 variants.

Covalent histone modifications and the incorporation of histone variants bring about changes in chromatin structure that in turn alter gene expression. Interest in non-allelic histone variants has been renewed, in part because of recent work on H3 (and other) histone variants. However, only in mammals do three non-centromeric H3 variants (H3.1, H3.2, and H3.3) exist. Here, we show that mammalian cell lines can be separated into two different groups based on their expression of H3.1, H3.2, and H3.3 at both mRNA and protein levels. Additionally, the ratio of these variants changes slightly during neuronal differentiation of murine ES cells. This difference in H3 variant expression between cell lines could not be explained by changes in growth rate, cell cycle stages, or chromosomal ploidy, but rather suggests other possibilities, such as changes in H3 variant incorporation during differentiation and tissue- or species-specific H3 variant expression. Moreover, quantitative mass spectrometry analysis of human H3.1, H3.2, and H3.3 showed modification differences between these three H3 variants, suggesting that they may have different biological functions. Specifically, H3.3 contains marks associated with transcriptionally active chromatin, whereas H3.2, in contrast, contains mostly silencing modifications that have been associated with facultative heterochromatin. Interestingly, H3.1 is enriched in both active and repressive marks, although the latter marks are different from those observed in H3.2. Although the biological significance as to why mammalian cells differentially employ three highly similar H3 variants remains unclear, our results underscore potential functional differences between them and reinforce the general view that H3.1 and H3.2 in mammalian cells should not be treated as equivalent proteins.

Animals↗
PubMed1999

Role of covalent modifications of histones in regulating gene expression.

DNA is organized into a hierarchy of structures, resulting in the level of compaction required to pack 2m of DNA into a nucleus with a diameter of 10 micrometer. The orderly packaging of DNA in the nucleus plays an important role in the functional aspects of gene regulation. A small percentage of chromatin is made available to transcription factors and the transcription machinery, while the remainder of the genome is in a state that is essentially invisible to the RNA polymerases. Modification of histones has a key role in altering chromatin higher order structure and function. In this review, we will present the latest developments in the study of histone modifications (ubiquitination, acetylation, methylation, and phosphorylation) and the enzymes involved in these processes.

Acetylation↗
PubMed2005

A conserved, extended chromatin opening within alpha-globin locus during development.

Histone modifications play an important role in eukaryotic gene regulation. However, the dynamic alteration of histone modification during development is poorly understood. In addition, the relationship between histone modification and globin gene switching remains unclear. Here, we assessed the dynamic pattern of histone modification (H3 acetylation, H4 acetylation, H3 K4 methylation, and H3 K79 methylation) along the murine alpha-globin locus, as well as along the human alpha-globin locus in transgenic mice, during globin gene switching in vivo. During the switching, histone modification at embryonic zeta-gene and fetal/adult alpha-genes displayed different developmental patterns. The level of histone modification at zeta-gene was developmentally regulated, in accordance with the level of zeta-gene expression, whereas the alpha-genes kept high level of histone modification at both developmental stages, regardless of their expression levels. Histone deacetylase inhibition selectively increased acetylation at the inactive zeta-gene in fetal livers, although it did not reactivate the gene expression. More importantly, an obvious increasing of histone modification level at major regulatory elements and fetal/adult alpha-genes was observed during the switching, suggesting that a conserved, extended chromatin opening within the locus occurs during globin gene switching.

Acetylation↗
PubMed2005

Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases.

The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

Apoptosis↗
PubMed2003

Chromatin remodeling and human disease.

In the past few years, there has been a nascent convergence of scientific understanding of inherited human diseases with epigenetics. Identified epigenetic processes involved in human disease include covalent DNA modifications, covalent histone modifications, and histone relocation. Each of these processes influences chromatin structure and thereby regulates gene expression and DNA methylation, replication, recombination, and repair. The importance of these processes for nearly all aspects of normal growth and development is illustrated by the array of multi-system disorders and neoplasias caused by their dysregulation.

Acetylation↗
PubMed2006

[The relationship between histone acetylation modification and IFN-gamma responsive gene regulation].

OBJECTIVES: To study the role of histone modification in the regulation of IFN-gamma-activated gene using chromatin immunoprecipitation technique. METHODS: Real-time PCR was used to measure the mRNA level of interferon-gamma-inducible protein 10 (IP-10) in Hela cells. Chromatin immunoprecipitation combined with Real-time PCR was used to check the histone H4 acetylation level at IFN-stimulated response element (ISRE) locus of IP-10 gene. RESULTS: IP-10 was strongly activated by IFN-gamma. The histone H4 deacetylation happened at the ISRE locus when IP-10 was induced by IFN-gamma. The activation of IP-10 and the deacetylation of histone H4 at the ISRE site induced by IFN-gamma were inhibited or blocked by histone deacetylase (HDAC) inhibitor trichostatin A (TSA). CONCLUSION: The histone H4 deacetylation at the ISRE site is related with the activation of IP-10 by IFN-gamma.

Acetylation↗
PubMedDate not supplied

Resetting the epigenetic histone code in the MRL-lpr/lpr mouse model of lupus by histone deacetylase inhibition.

The baseline level of gene expression varies between healthy controls and systemic lupus erythematosus (SLE) patients, and among SLE patients themselves. These variations may explain the different clinical manifestations and severity of disease observed in SLE. Epigenetic mechanisms, which involve DNA and histone modifications, are predictably associated with distinct transcriptional states. To understand the interplay between various histone modifications, including acetylation and methylation, and lupus disease, we performed differential expression histone modification analysis in splenocytes from the MRL-lpr/lpr mouse model of lupus. Using stable isotope labeling in combination with mass spectrometry, we found global site-specific hypermethylation (except H3 K4 methylation) and hypoacetylation in histone H3 and H4 MRL-lpr/lpr mice compared to control MRL/MPJ mice. Moreover, we have identified novel histone modifications such as H3 K18 methylation, H4 K31 methylation, and H4 K31 acetylation that are differentially expressed in MRL-lpr/lpr mice compared to controls. Finally, in vivo administration of the histone deacetylase inhibitor trichostatin A (TSA) corrected the site-specific hypoacetylation states on H3 and H4 in MRL-lpr/lpr mice with improvement of disease phenotype. Thus, this study is the first to establish the association between aberrant histone codes and pathogenesis of autoimmune disease SLE. These aberrant post-translational histone modifications can therefore be reset with histone deacetylase inhibition in vivo.

Amino Acid Sequence↗
PubMed2002

In vitro assays to study protein ubiquitination in transcription.

Polyubiquitination is a death signal for proteins and condemns proteins to subsequent degradation by the 26S proteasome. However, recent studies imply that monoubiquitination and polyubiquitination of proteins do not necessarily result in protein degradation but play an important role in the execution of various biological events such as signal transduction and transcription. Ubiquitin was originally identified as a moiety attached to histones, and this as well as other histone modifications may play an important role for transcription and various other DNA-dependent processes. Considerable progress has been made in linking several histone modifications with chromatin dynamics in transcription. Acetylation of histones has been intimately linked to activation of transcription, while deacetylation is concomitant with repression of transcription. Although other histone modifications such as methylation, phosphorylation, and ubiquitination have been correlated with transcriptionally competent or inactive chromatin, the enzymes that mediate these modifications are only now being discovered. The identification of these histone-modifying enzymes may provide valuable insights into the role and function of histone modifications such as ubiquitination in transcription as well as other DNA-dependent processes. Recently, we have used various in vitro assays to show that the coactivator TAF(II)250 possesses both ubiquitin-activating and ubiquitin-conjugating activities, which monoubiquitinate histone H1. Here, we describe the methods used to identify this bifunctional enzyme: (1) in-gel activity assay; (2) protein-transfer membrane activity assay; and (3) in-solution activity assay. These methods have been successfully used to identify various histone-modifying enzymes and protein kinases. In this article we contribute a short review of the history of the methods used to study ubiquitination of proteins and histone modification. We provide protocols for in-gel, protein-transfer membrane, and in-solution ubiquitination assays. A discussion of the general use of the provided protocols, their limitations, and future perspectives are presented. The described methods provide useful tools for the identification of not only novel histone-modifying enzymes but also other protein-modifying enzymes that act in a variety of biological events.

Animals↗
PubMed2006

Organismal differences in post-translational modifications in histones H3 and H4.

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.

Acetylation↗
PubMed2003

Genomewide histone acetylation microarrays.

Histone acetylation and methylation are important regulators of gene activity. Chromatin immunoprecipitation (ChIP or ChrIP) has made it possible to examine not only the state of histone acetylation at a gene but also that of histone methylation and may soon be extended to other histone modifications such as phosphorylation and ubiquitination. In principle such studies are possible as long as an antibody is available to the particular histone modification. Once a target gene is identified it is instructive to see the effect of mutating putative enzymes responsible for the modification to determine how a particular enzyme is responsible for altering chromatin of that gene. Although specific target genes have been studied that contain such modifications recent technical advances have made it possible to study histone modifications genomewide. This not only allows for alternate views of particular paradigms to be investigated, but also uncovers chromosomal patterns of histone modification that would be missed in analyzing individual genes. We describe here an approach to rapidly study histone modifications genomewide by combining chromatin immunoprecipitation and DNA microarrays.

Acetylation↗
PubMed2006

Diverse roles for histone H2A modifications in DNA damage response pathways in yeast.

There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone modifications are involved in many repair processes. We have analyzed the roles of H2A and its modifications in repair by mutagenizing modifiable residues in the N- and C-terminal tails of yeast H2A and by testing strains containing these mutations in multiple DNA repair assays. We show that residues in both tails are important for homologous recombination and nonhomologous end-joining pathways of double-strand break repair, as well as for survival of UV irradiation and oxidative damage. We show that H2A serine 122 is important for repair and/or survival in each of these assays. We also observe a complex pattern of H2A phosphorylation at residues S122, T126, and S129 in response to different damage conditions. We find that overlapping but nonidentical groups of H2A residues in both tails are involved in different pathways of repair. These data suggest the presence of a set of H2A "damage codes" in which distinct patterns of modifications on both tails of H2A may be used to identify specific types of damage or to promote specific repair pathways.

Amino Acid Sequence↗
PubMed2006

Concise review: epigenetic mechanisms contribute to pluripotency and cell lineage determination of embryonic stem cells.

Epigenetic mechanisms, such as histone modifications and DNA methylation, have been shown to play a key role in the regulation of gene transcription. Results of recent studies indicate that a novel "bivalent" chromatin structure marks key developmental genes in embryonic stem cells (ESCs), wherein a number of untranscribed lineage-control genes, such as Sox1, Nkx2-2, Msx1, Irx3, and Pax3, are epigenetically modified with a unique combination of activating and repressive histone modifications that prime them for potential activation (or repression) upon cell lineage induction and differentiation. However, results of these studies also showed that a subset of lineage-control genes, such as Myf5 and Mash1, were not marked by these histone modifications, suggesting that distinct epigenetic mechanisms might exist for lineage-control genes in ESCs. In this review article, we summarize evidence regarding possible mechanisms that control these unique histone modifications at lineage-control gene loci in ESCs and consider their possible contribution to ESC pluripotency. In addition, we propose a novel "histone modification pulsing" model wherein individual pluripotent stem cells within the inner cell mass of blastocysts undergo transient asynchronous histone modifications at these developmental gene loci, thereby conferring differential responsiveness to environmental cues and morphogenic gradients important for cell lineage determination. Finally, we consider how these rapid histone modification exchanges become progressively more stable as ESCs undergo differentiation and maturation into specialized cell lineages.

Animals↗
PubMed2001

ATP-dependent chromatin remodeling factors: nucleosome shufflers with many missions.

This review addresses recent developments in the field of ATP-dependent chromatin remodeling factors. These factors use the energy of ATP hydrolysis to introduce superhelical torsion into DNA, which suggests a common mechanistic basis of action. Chromatin remodeling factors function both in transcriptional activation and repression, but they may have roles outside of transcriptional regulation such as DNA repair. A study of the nucleosome dependent ATPase ISWI in yeast illustrates the involvement of ATP-dependent chromatin remodeling in transcriptional repression by setting up inaccessible chromatin structures at promoters. However, factors such as ISWI are also involved in the restructuring of large chromatin domains and even whole chromosomes. Transcriptional regulation by ATP-dependent chromatin remodeling factors occurs in concert with histone modifying enzymes such as histone acetyltransferases and histone deacetylases: In yeast, SWI/SNF targeting is a requirement for histone acetyltransferases activity at promoters that are active at late stages of mitosis, when the chromatin is still condensed. This demonstrates that ATP-dependent remodeling factors facilitate covalent histone modifications. However, they are also regulated by histone modifications and in some circumstances they function in parallel with histone modifications towards the same goal.

Adenosine Triphosphatases↗
PubMed2005

Inducible covalent posttranslational modification of histone H3.

The physiological state of a eukaryotic cell is determined by endogenous and exogenous signals, and often the endpoint of the pathways that transmit these signals is DNA. DNA is organized into chromatin, a nucleoprotein complex, which not only facilitates the packaging of DNA within the nucleus but also serves as an important factor in the regulation of gene function. The nucleosome is the basic unit of chromatin and generally consists of approximately two turns of DNA wrapped around an octamer of core histone proteins. Each histone also contains an accessible N-terminal tail that extends outside the chromatin complex and is subject to posttranslational modifications that are crucial in the regulation of gene expression. Two distinct categories of histone posttranslational modification have been observed: (i) inducible or stimulation-dependent and (ii) mitosis-dependent. Stimulation by mitogens or stress leads to rapid transient posttranslational modifications of histones, in particular histone H3, which are mechanistically and temporarily distinct from modifications associated with mitosis. This Review focuses mainly on the inducible phosphorylation of histone H3 brought about by different stimuli, such as epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, arsenite, or ultraviolet radiation. We examine the most recent, and at times controversial, research data concerning the identity of the histone H3 kinases responsible for this phosphorylation. In addition, the interdependence of phosphorylation and acetylation will be discussed in light of data showing patterns of inducible modification at specific genes.

Acetylation↗
PubMed2004

Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression.

During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency. To address this point, we employed established models to predict genomic regions with the highest probability of being methylated and, using bisulfite sequencing, analyzed the methylation profiles of these regions. We found no significant methylation of latent DNA isolated from mouse dorsal root ganglia in any of the regions examined, including the ICP4 and LAT promoters. This analysis indicates that methylation is unlikely to play a major role in regulating HSV-1 latent gene expression. Subsequently we focused on differential histone modification as another epigenetic mechanism that could regulate latent transcription. Chromatin immunoprecipitation analysis of the latent HSV-1 DNA repeat regions demonstrated that a portion of the LAT region is associated with histone H3 acetylated at lysines 9 and 14, consistent with a euchromatic and nonrepressed structure. In contrast, the chromatin associated with the HSV-1 DNA polymerase gene located in the unique long segment was not enriched in H3 acetylated at lysines 9 and 14, suggesting a transcriptionally inactive structure. These data suggest that histone composition may be a major regulatory determinant of HSV latency.

Acetylation↗
PubMed2006

Histone H3 acetylation and H3 K4 methylation define distinct chromatin regions permissive for transgene expression.

Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing.

Acetylation↗
PubMed2003

In vivo assays to study histone ubiquitylation.

The importance of histone acetylation, phosphorylation, and methylation in transcription and other DNA-mediated processes is now well established. Histones are also ubiquitylated, but in contrast to the majority of ubiquitylated proteins, ubiquitylated histones are not generally targeted for degradation and may play roles similar to those of other histone modifications. Antibodies against acetylated histones have provided unique insights into the regulation, distribution, and cellular roles of these modified histones. In this report, we describe methods to identify ubiquitylated histones in budding yeast and HeLa cells. We provide protocols to detect ubiquitylated histones that are based on a combination of in vivo genetic and immunological assays. These methods should provide relatively simple and useful tools to study the global regulation of this important but poorly understood histone modification.

Acetylation↗