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PubMed2026

Improved spike-in normalization clarifies the relationship between active histone modifications and transcription.

Spike-in normalization enables quantitative analysis of chromatin immunoprecipitation sequencing (ChIP-seq) signal. Here we introduce a robust dual spike-in normalization approach for ChIP-seq (ChIP-wrangler), optimize parameters and verify its accuracy in quantifying changes in ChIP-seq signal and detecting technical artifacts. We use ChIP-wrangler to revisit recent claims that active histone marks depend on transcription. We show that acute depletion of RNA polymerase II (RNAPII) has a modest impact on H3K27ac levels, with only 6% of peaks significantly changing after RNAPII depletion, indicating that histone acetylation maintenance is not entirely dependent on ongoing transcription. Promoters and enhancers are differentially affected, with 82% of decreasing acetylation peaks located at promoter-distal elements with enhancer-related motifs. ChIP-wrangler provides increased rigor and 'guardrails' for successful spike-in normalization and, as applied here, refines the understanding of crosstalk between RNAPII activity and transcription-associated histone marks.

Histones↗
PubMed2003

DNA hypermethylation in Drosophila melanogaster causes irregular chromosome condensation and dysregulation of epigenetic histone modifications.

The level of genomic DNA methylation plays an important role in development and disease. In order to establish an experimental system for the functional analysis of genome-wide hypermethylation, we overexpressed the mouse de novo methyltransferase Dnmt3a in Drosophila melanogaster. These flies showed severe developmental defects that could be linked to reduced rates of cell cycle progression and irregular chromosome condensation. In addition, hypermethylated chromosomes revealed elevated rates of histone H3-K9 methylation and a more restricted pattern of H3-S10 phosphorylation. The developmental and chromosomal defects induced by DNA hypermethylation could be rescued by mutant alleles of the histone H3-K9 methyltransferase gene Su(var)3-9. This mutation also resulted in a significantly decreased level of genomic DNA methylation. Our results thus uncover the molecular consequences of genomic hypermethylation and demonstrate a mutual interaction between DNA methylation and histone methylation.

Animals↗
PubMed2003

Glucocorticoid suppression of nuclear factor-kappa B: a role for histone modifications.

Corticosteroids are by far the most effective treatment for chronic inflammatory diseases such as asthma. Inflammation in asthma is characterized by the increased expression of multiple inflammatory genes, including those that encode cytokines, chemokines, adhesion molecules, and inflammatory enzymes and receptors. Increased expression of inflammatory genes is regulated by pro-inflammatory transcription factors, such as nuclear factor kappa B (NF-kappa B). These bind to, and activate, co-activator molecules that then acetylate core histones resulting in elevated gene transcription. Corticosteroids reverse histone acetylation at the site of inflammatory gene transcription, either by direct binding of the activated glucocorticoid receptor to NF-kappa B-associated co-activators or by recruitment of histone deacetylases to the activated transcription complex. Understanding how corticosteroids work in asthma may help in designing novel corticosteroids with fewer systemic effects, as well as novel anti-inflammatory approaches.

Acetylation↗
PubMed2002

Alternate activation of two divergently transcribed mouse genes from a bidirectional promoter is linked to changes in histone modification.

Thymidine kinase (TK) is a growth factor-inducible enzyme that is highly expressed in proliferating mammalian cells. Expression of mouse TK mRNA is controlled by transcriptional and posttranscriptional mechanisms including antisense transcription. Here we report the identification of a novel gene that is divergently transcribed from the bidirectional TK promoter. This gene encodes kynurenine formamidase (KF), an enzyme of the tryptophan metabolism. Whereas the TK gene is induced upon interleukin-2-mediated activation of resting T cells, the KF gene becomes simultaneously repressed. The TK promoter is regulated by E2F, SP1, histone acetyltransferases, and deacetylases. The binding site for the growth-regulated transcription factor E2F is beneficial for TK promoter activity but not required for KF expression. In contrast, the SP1 binding site is crucial for transcription in both directions. Inhibition of histone deacetylases by trichostatin A leads to increased histone acetylation at the TK/KF promoter and thereby to selective activation of the TK promoter and simultaneous shut-off of KF expression. Similarly, TK gene activation by interleukin-2 is linked to histone hyperacetylation, whereas KF expression correlates with reduced histone acetylation. The KF gene is the rare example of a mammalian gene whose expression is linked to histone hypoacetylation at its promoter.

Acetylation↗
PubMed2025

Integrative analysis of gene expression and histone modifications for DES, DSP, GJA1 and SMOC2 in adipose tissue reveals potential relationship to cardiometabolic health.

BACKGROUND: Adipose tissue influences cardiometabolic health through its endocrine activity and its role in regulating inflammation, lipid metabolism, and cardiovascular function. The expression of cardiac-associated genes within adipose tissue may reflect or contribute to cardiometabolic risk, yet this relationship remains poorly understood. This study investigates the expression profiles of the cardiac function associated genes GJA1, DES, DSP and SMOC2 in human adipose tissue, and analyses their associations with cardiometabolic traits. Additionally, we explore epigenomic mechanisms that may underlie their differential gene expression. METHODS: Expression profiling and functional enrichment analyses were conducted to identify depot-specific cardiac gene expression patterns. Quantitative PCR validated gene expression in paired subcutaneous (SAT) and omental visceral adipose tissue (OVAT) samples from 78 individuals with obesity. Gene expression was further validated in three independent cohorts (N = 1,548 total). Associations with clinical traits were assessed using Spearman correlations and multivariate linear regression, adjusted for age, sex, and BMI. Integration with transcriptomic and proteomic datasets publicly available from the Adipose Tissue Knowledge Portal was performed to strengthen clinical relevance. Epigenomic profiling using genome-wide ChIP-seq for histone marks (H3K4me3, H3K4me1, H3K27ac, H3K27me3) was conducted in paired SAT and OVAT samples from five individuals. RESULTS: DES, DSP, GJA1, and SMOC2 were significantly upregulated in OVAT compared to SAT. DES, DSP, and SMOC2 showed consistent expression patterns across all cohorts, while GJA1 exhibited context-dependent regulation. Gene expression in SAT was negatively correlated with cardiometabolic traits, including blood pressure, insulin resistance, and liver function markers. These associations were confirmed by regression analysis and supported by publicly available multi-omics data. Epigenetic analyses revealed OVAT-specific enrichment of active histone marks and reduced repressive marks, supporting higher differential transcriptional activity in OVAT. CONCLUSIONS: Depot-specific gene expression of DES, DSP, and SMOC2 in adipose tissue is robustly linked to cardiometabolic traits and supported by distinct epigenetic landscapes in OVAT vs SAT, highlighting their potential as novel biomarkers for cardiometabolic health.

Humans↗
PubMed2002

Centromere domain organization and histone modifications.

Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles. Centromere structure appears to be organized in different, separable domains in order to accomplish these functions. Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences. Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms. The recent analysis of the centromere structure in the yeast S. pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere. The structural organization of the centromere is generally multilayered with a heterochromatin domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore. It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histones and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core. Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes.

Animals↗
PubMed1986

Histones and their modifications.

Histones constitute the protein core around which DNA is coiled to form the basic structural unit of the chromosome known as the nucleosome. Because of the large amount of new histone needed during chromosome replication, the synthesis of histone and DNA is regulated in a complex manner. During RNA transcription and DNA replication, the basic nucleosomal structure as well as interactions between nucleosomes must be greatly altered to allow access to the appropriate enzymes and factors. The presence of extensive and varied post-translational modifications to the otherwise highly conserved histone primary sequences provides obvious opportunities for such structural alterations, but despite concentrated and sustained effort, causal connections between histone modifications and nucleosomal functions are not yet elucidated.

Amino Acid Sequence↗
PubMed2003

Methods and tips for the purification of human histone methyltransferases.

Recently developed biochemical techniques have enabled researchers to study histone modifications more easily and accurately. One of these modifications, histone lysine methylation, has been shown to be highly stable and to represent an epigenetic alteration. Extensive biochemical analyses have led to discoveries about the nature and functions of this modification, thus accelerating our understanding of this crucial epigenetic event. Here we describe basic methods for purification and biochemical analysis of lysine-directed, histone methyltransferases from HeLa cell-derived extracts. In the section on substrate preparation, we describe a simple method for the preparation of recombinant substrates, although we recommend using native substrates for initial detection of the activities. The purification protocols for several histone methyltransferases have been streamlined so that those researchers with a basic understanding of biochemistry can perform them. We also describe many tips and provide suggestions to avoid common pitfalls in the biochemical analysis of histone methyltransferases.

Amino Acid Sequence↗
PubMed1983

Fine resolution of the poly ADP-ribosylated domains of polynucleosomal chromatin: DNA gene and integrity analysis; mechanism of histone H1 modification.

The focus of our laboratory has been to ascertain how the poly(ADP-ribosyl)ation reaction influences the structure and biological function of nucleosomal chromatin. Antibody to poly(ADP-ribose (ADP-Rib] was coupled to Sepharose to prepare an immunoaffinity column. The following new information concerning poly(ADP-Rib) and chromatin was obtained with this column: 1) those limiting domains of nucleosomal chromatin undergoing the modification (circa 10%) could be isolated in the "bound" fraction, 2) antibody-bound nucleosomes contained all the poly ADP-ribosylated acceptors and polymerase activity of the bulk chromatin, 3) bound nucleosomes contain significant numbers of internal DNA strand breaks, and [3H]-thymidine (TdR) repair incorporation from in vivo "DNA repair label," compared to the unbound, bulk of the chromatin, 4) the presence of actively transcribed genes in "bound" nucleosomes is being investigated, 5) the acetylation modification of histones occurs in the same domains of chromatin as does poly(ADP-ribosyl)ation, and 6) poly ADP-ribosylated histone H1 can be selectively purified by the immunoaffinity method. These same histone H1 molecules appear to be equally accessible to the histone kinase, phosphorylation modification. In addition, new information has been obtained concerning histone H1 cross-linking by poly(ADP-ribosyl)ation, and on polymerase binding sites to chromatin. We have reconstituted histone H1-depleted chromatin with intact H1 and peptide domains of H1, and subsequently studied H1-poly(ADP-Rib)-complex synthesis. The data indicate that elongation of poly(ADP-Rib) proceeds on the amino terminal region of this histone. By utilizing the new techniques of DNA technology, huge advances in our understanding of the programmed structure of the eukaryotic genome have been accomplished in a relatively short time. To complement this explosion of information, it is important to ascertain how these recently appreciated properties of eukaryotic DNA are packaged within extended and condensed domains of chromatin. The research to be discussed is directed, in part, at this latter topic. We have initiated a program aimed at furthering our comprehension of chromatin structure by studying one specific, enzymatically active chromosomal protein.

Chemical Phenomena↗
PubMed2002

Fission yeast CENP-B homologs nucleate centromeric heterochromatin by promoting heterochromatin-specific histone tail modifications.

Heterochromatin is a functionally important chromosomal component, especially at centromeres. In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequent methylation of Lys 9, promote the recruitment of a heterochromatin protein, Swi6, a homolog of the Drosophila heterochromatin protein 1. However, the primary determinants of the positioning of heterochromatin are still unclear. The fission yeast proteins Abp1, Cbh1, and Cbh2 are homologs of the human protein CENP-B that bind to centromeric alpha-satellite DNA and associate with centromeric heterochromatin. We show that the CENP-B homologs are functionally redundant at centromeres, and that Abp1 binds specifically to centromeric heterochromatin. In the absence of Abp1 or Cbh1, the centromeric association of Swi6 is diminished, resulting in a decrease in silencing of the region. CENP-B-homolog double disruptants show a synergistic reduction of Swi6 at centromeric heterochromatin, indicating that the three proteins are functionally redundant in the recruitment of Swi6. Furthermore, using chromatin immunoprecipitation assays, we show that disruption of CENP-B homologs causes a decrease in heterochromatin-specific modifications of histone H3. These results indicate that the CENP-B homologs act as site-specific nucleation factors for the formation of centromeric heterochromatin by heterochromatin-specific modifications of histone tails.

Autoantigens↗
PubMed1980

Effect of histone H3 sulfhydryl modifications on histone-histone interactions and nucleosome formation and structure.

The effect of histone H3 sulfhydryl mnodification and disulfide bridge formation on histone-histone interactions, nucleosome reconstitution and structure has been examined for calf and chicken mononucleosomes. For intramolecular disulfide bridge formation histone H3-H4 complexation is disrupted and no nucleosome-like particle containing all four of the histones could be prepared. Intermolecular disulfide bridge formation between H3 residues 110 and 110 as well as chemical modification of this site with small and with bulky groups allowed histone H3-H4 complexation and the reconstituatioin of a nucleosome-like particle. However, the yield of such particles is decreased and their thermal denaturation properties indicate a reduced stability. These results suggest that the histone core is destabilized or even structurally altered by even a minor modification at H3 position 110, such as carboxymethylation, and therefore this site must be used with caution for the attachment of reporter groups.

Animals↗
PubMed2027

Genomic Profiling of Chromatin State Using CUT&Tag.

Alterations in chromatin state, mediated through histone modifications and the incorporation of histone variants, are fundamental to establishing transcriptional networks and cell identity. Recent advances in low-input epigenome profiling methods, such as CUT&Tag and CUT&RUN, have enabled the study of chromatin states from very limited starting materials. In this chapter, we describe procedures for generating CUT&Tag libraries to profile histone modifications and histone variants in early-developing zebrafish embryos.

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↗
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↗
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↗