Specific Histone Variants Research Articles

RNA Polymerase II coordinates histone deacetylation at active promoters.

Nucleosomes at actively transcribed promoters have specific histone post-transcriptional modifications and histone variants. These features are thought to contribute to the formation and maintenance of a permissive chromatin environment. Recent reports have drawn conflicting conclusions about whether these histone modifications depend on transcription. We used triptolide to inhibit transcription initiation and degrade RNA Polymerase II and interrogated the effect on histone modifications. Transcription initiation was dispensable for de novo and steady-state histone acetylation at transcription start sites (TSSs) and enhancers. However, at steady state, blocking transcription initiation increased the levels of histone acetylation and H2AZ incorporation at active TSSs. These results demonstrate that deposition of specific histone modifications at TSSs is not dependent on transcription and that transcription limits the maintenance of these marks.

GCNA is a histone binding protein required for spermatogonial stem cell maintenance.

Recycling and de-novo deposition of histones during DNA replication is a critical challenge faced by eukaryotic cells and is coordinated by histone chaperones. Spermatogenesis is highly regulated sophisticated process necessitating not only histone modification but loading of testis specific histone variants. Here, we show that Germ Cell Nuclear Acidic protein (GCNA), a germ cell specific protein in adult mice, can bind histones and purified GCNA exhibits histone chaperone activity. GCNA associates with the DNA replication machinery and supports progression through S-phase in murine undifferentiated spermatogonia (USGs). Whilst GCNA is dispensable for embryonic germ cell development, it is required for the maintenance of the USG pool and for long-term production of sperm. Our work describes the role of a germ cell specific histone chaperone in USGs maintenance in mice. These findings provide a mechanistic basis for the male infertility observed in patients carrying GCNA mutations.

Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice.

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.

Centromere Identity and the Regulation of Chromosome Segregation.

Eukaryotes segregate their chromosomes during mitosis and meiosis by attaching chromosomes to the microtubules of the spindle so that they can be distributed into daughter cells. The complexity of centromeres ranges from the point centromeres of yeast that attach to a single microtubule to the more complex regional centromeres found in many metazoans or holocentric centromeres of some nematodes, arthropods and plants, that bind to dozens of microtubules per kinetochore. In vertebrates, the centromere is defined by a centromere specific histone variant termed Centromere Protein A (CENP-A) that replaces histone H3 in a subset of centromeric nucleosomes. These CENP-A nucleosomes are distributed on long stretches of highly repetitive DNA and interspersed with histone H3 containing nucleosomes. The mechanisms by which cells control the number and position of CENP-A nucleosomes is unknown but likely important for the organization of centromeric chromatin in mitosis so that the kinetochore is properly oriented for microtubule capture. CENP-A chromatin is epigenetically determined thus cells must correct errors in CENP-A organization to prevent centromere dysfunction and chromosome loss. Recent improvements in sequencing complex centromeres have paved the way for defining the organization of CENP-A nucleosomes in centromeres. Here we discuss the importance and challenges in understanding CENP-A organization and highlight new discoveries and advances enabled by recent improvements in the human genome assembly.

Chaperoning histones at the DNA repair dance

Unlike all other biological molecules that are degraded and replaced if damaged, DNA must be repaired as chromosomes cannot be replaced. Indeed, DNA endures a wide variety of structural damage that need to be repaired accurately to maintain genomic stability and proper functioning of cells and to prevent mutation leading to disease. Given that the genome is packaged into chromatin within eukaryotic cells, it has become increasingly evident that the chromatin context of DNA both facilitates and regulates DNA repair processes. In this review, we discuss mechanisms involved in removal of histones (chromatin disassembly) from around DNA lesions, by histone chaperones and chromatin remodelers, that promotes accessibility of the DNA repair machinery. We also elaborate on how the deposition of core histones and specific histone variants onto DNA (chromatin assembly) during DNA repair promotes repair processes, the role of histone post translational modifications in these processes and how chromatin structure is reestablished after DNA repair is complete.

Bromodomain‐Containing Protein 4 (BRD4) Inhibitors as Emerging Therapeutics for Opioid Use Disorder

Aims Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain-containing (BET) family of chromatin “reader” proteins. Small molecule BET inhibitors have been developed within the context of cancer and inflammation pathologies, and more recently are of interest for central nervous system (CNS) disorders, including opioid use disorder (OUD). BRD4 has two bromodomains (BDs) that recognize specific post-translational histone variants to control downstream transcriptional outcomes. The protein-protein interaction between BRD4 and lysine residues of histones is driven through the two highly conserved N-terminal bromodomains BD1 and BD2. The aim of the present study is to pharmacologically characterize BRD4-BD1-specific inhibitors to facilitate investigations into the role of BRD4 mechanisms in preclinical models of opioid use disorder. Methods Selected potent BRD4 inhibitors were synthesized and scaled up for analyses of in vitro and in vivo drug metabolism and pharmacokinetics (DMPK), blood-brain barrier (BBB) penetration, and pharmacological profiles against a panel of CNS receptors, channels and transporters via the Psychoactive Drug Screening Program (PDSP). Based on initial profiles, we further evaluated one compound for in vivo efficacy in male Sprague-Dawley rats trained to stability on intravenous oxycodone self-administration (0.1 mg/kg/infusion). Results ZL0513, ZL0516, ZL0742, and ZL0969 exhibit selectivity for BRD4-BD1 over other BD proteins and are the subject of ongoing profiling. To date, ZL0513, ZL0516, and ZL0969 exhibit excellent in vitro DMPK profiles, including bioavailability greater than 35%, aqueous solubility, metabolic stability, and weak inhibition of CYP450 enzymes. ZL0987 (HCl salt form of ZL0516) exhibited a brain/plasma ratio (intravenous, 1 hr) of 0.08 which was improved to 0.62 for ZL0969. However, ZL0516 exhibited a cleaner PDSP profile than did ZL0969. ZL0742 exhibited a good in vivo DMPK, however its penetration of the BBB was suboptimal. Additional backup molecules ZL0513 and ZL0742 are undergoing further DMPK, BBB, and PDSP screening. Based upon our initial profiling of ZL0987, we assessed its efficacy in vivo and found that ZL0987 at the dose of 10 mg/kg suppressed oxycodone intake (p<0.05) which was sustained across the entire 180-minute self-administration session. Conclusion We have characterized several BRD4-BD1 selective inhibitors with compelling profiles of drug-like properties. Our initial in vivo data revealed that ZL0987 elicited sustained suppression of oxycodone self-administration. The development and characterization of specific BRD4 inhibitors will allow exploration of BRD4 mechanisms in preclinical models of opioid use and validate the BRD4 as a novel drug target for the development of epigenetic therapeutics for OUD.

Establishment and function of chromatin modification at enhancers.

How a single genome can give rise to distinct cell types remains a fundamental question in biology. Mammals are able to specify and maintain hundreds of cell fates by selectively activating unique subsets of their genome. This is achieved, in part, by enhancers—genetic elements that can increase transcription of both nearby and distal genes. Enhancers can be identified by their unique chromatin signature, including transcription factor binding and the enrichment of specific histone post-translational modifications, histone variants, and chromatin-associated cofactors. How each of these chromatin features contributes to enhancer function remains an area of intense study. In this review, we provide an overview of enhancer-associated chromatin states, and the proteins and enzymes involved in their establishment. We discuss recent insights into the effects of the enhancer chromatin state on ongoing transcription versus their role in the establishment of new transcription programmes, such as those that occur developmentally. Finally, we highlight the role of enhancer chromatin in new conceptual advances in gene regulation such as condensate formation.

Similar yet critically different: the distribution, dynamics and function of histone variants.

Organization of the genetic information into chromatin plays an important role in the regulation of all DNA template-based reactions. The incorporation of different variant versions of the core histones H3, H2A, and H2B, or the linker histone H1 results in nucleosomes with unique properties. Histone variants can differ by only a few amino acids or larger protein domains and their incorporation may directly affect nucleosome stability and higher order chromatin organization or indirectly influence chromatin function through histone variant-specific binding partners. Histone variants employ dedicated histone deposition machinery for their timely and locus-specific incorporation into chromatin. Plants have evolved specific histone variants with unique expression patterns and features. In this review, we discuss our current knowledge on histone variants in Arabidopsis, their mode of deposition, variant-specific post-translational modifications, and genome-wide distribution, as well as their role in defining different chromatin states.

Proteomic Analysis of Histones H2A/H2B and Variant Hv1 in Tetrahymena thermophila Reveals an Ancient Network of Chaperones.

Epigenetic information, which can be passed on independently of the DNA sequence, is stored in part in the form of histone posttranslational modifications and specific histone variants. Although complexes necessary for deposition have been identified for canonical and variant histones, information regarding the chromatin assembly pathways outside of the Opisthokonts remains limited. Tetrahymena thermophila, a ciliated protozoan, is particularly suitable to study and unravel the chromatin regulatory layers due to its unique physical separation of chromatin states in the form of two distinct nuclei present within the same cell. Using a functional proteomics pipeline, we carried out affinity purification followed by mass spectrometry of endogenously tagged T. thermophila histones H2A, H2B and variant Hv1.We identified a set of interacting proteins shared among the three analyzed histones that includes the FACT-complex, as well as H2A- or Hv1-specific chaperones. We find that putative subunits of T. thermophila versions of SWR- and INO80-complexes, as well as transcription-related histone chaperone Spt6Tt specifically copurify with Hv1. We also identified importin β6 and the T. thermophila ortholog of nucleoplasmin 1 (cNpl1Tt) as H2A–H2B interacting partners. Our results further implicate Poly [ADP-ribose] polymerases in histone metabolism. Molecular evolutionary analysis, reciprocal affinity purification coupled to mass spectrometry experiments, and indirect immunofluorescence studies using endogenously tagged Spt16Tt (FACT-complex subunit), cNpl1Tt, and PARP6Tt underscore the validity of our approach and offer mechanistic insights. Our results reveal a highly conserved regulatory network for H2A (Hv1)–H2B concerning their nuclear import and assembly into chromatin.

Post-Translational Modifications of H2A Histone Variants and Their Role in Cancer.

Histone variants are chromatin components that replace replication-coupled histones in a fraction of nucleosomes and confer particular characteristics to chromatin. H2A variants represent the most numerous and diverse group among histone protein families. In the nucleosomal structure, H2A-H2B dimers can be removed and exchanged more easily than the stable H3-H4 core. The unstructured N-terminal histone tails of all histones, but also the C-terminal tails of H2A histones protrude out of the compact structure of the nucleosome core. These accessible tails are the preferential target sites for a large number of post-translational modifications (PTMs). While some PTMs are shared between replication-coupled H2A and H2A variants, many modifications are limited to a specific histone variant. The present review focuses on the H2A variants H2A.Z, H2A.X, and macroH2A, and summarizes their functions in chromatin and how these are linked to cancer development and progression. H2A.Z primarily acts as an oncogene and macroH2A and H2A.X as tumour suppressors. We further focus on the regulation by PTMs, which helps to understand a degree of context dependency.

Characterization of Posttranslational Modifications on Histone Variants.

The study of histone variants and histone posttranslational modifications (PTMs) is a trending topic in different fields including developmental biology, neurobiology, and immunology; as well as in the understanding of molecular mechanisms leading to diverse diseases, such as cancer. Since the establishment of histone PTMs starts immediately after their synthesis and it continues once they are assembled into chromatin, here we describe a classic protocol of subcellular fractionation aiming to study histones at different stages of maturation. This includes newly synthesized histones enriched in cytosolic fractions; a pool of newly synthesized, evicted, and stored histones enriched in nuclear soluble fractions; and chromatin-associated histones enriched in chromatin pellet. To study specific histone variants and the establishment of their PTMs, we describe a protocol for obtaining histone variants expressed in bacteria. In addition, we describe a Triton-Acetic acid-Urea (TAU) gel electrophoresis protocol adapted to work on mini-gels, which can be coupled to Western blot to analyze PTMs on histone variants. Finally, we describe a Chromatin immunoprecipitation (ChIP) assay for studying histone PTMs, or tagged histone variants, on specific DNA sequences.

Histone profiling reveals the H1.3 histone variant as a prognostic biomarker for pancreatic ductal adenocarcinoma

BackgroundEpigenetic alterations have been recognized as important contributors to the pathogenesis of PDAC. However, the role of histone variants in pancreatic tumor progression is still not completely understood. The aim of this study was to explore the expression and prognostic significance of histone protein variants in PDAC patients.MethodsLiquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for qualitative analysis of histone variants and histone related post-translational modifications (PTMs) in PDAC and normal pancreatic tissues. Survival analysis was conducted using the Kaplan-Meier method and Cox proportional hazards regression.ResultsHistone variant H1.3 was found to be differentially expressed (p = 0.005) and was selected as a PDAC specific histone variant candidate. The prognostic role of H1.3 was evaluated in an external cohort of patients with resected PDAC using immunohistochemistry. Intratumor expression of H1.3 was found to be an important risk factor for overall survival in PDAC, with an adjusted HR value of 2.6 (95% CI 1.1–6.1), p = 0.029.ConclusionWe suggest that the intratumor histone H1.3 expression as reported herein, may serve as a new epigenetic biomarker for PDAC.

Histonvarianten - Gleiche Gene bedeuten nicht gleiches Schicksal

Chromatin is one of the most significant structures to regulate gene expression. Active deposition and incorporation of specialized histone variants can lead to severe alterations in gene expression, cell cycle progression and embryonic development. Further, deregulation or mutations of histone variants are often implicated in diseases, most notably cancer development. Our work focuses on the discovery of novel human histone variants and variant-interactors to better understand fundamental epigenetic processes.

A Rapid and Efficient ChIP Protocol to Profile Chromatin Binding Proteins and Epigenetic Modifications in Arabidopsis.

Chromatin immunoprecipitation (ChIP) is a widely used and very powerful procedure to identify the proteins that are associated with the DNA to regulate developmental processes. These proteins can be transcription factors, or specific histone variants and modified histones, which are all crucial for gene regulation. In order to obtain reliable results, ChIP must be carried out under highly reproducible conditions. Here, we describe a simple and fast ChIP protocol adapted for Arabidopsis seedlings, which can serve as a basis for other species, organs or more sophisticated procedures, such as the sequential ChIP. We also provide user-oriented troubleshooting to increase the chances of successful applications.

Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation.

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines.

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

H3.Y discriminates between HIRA and DAXX chaperone complexes and reveals unexpected insights into human DAXX-H3.3-H4 binding and deposition requirements.

Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive post-translational histone modifications. H3.Y mutational gain-of-function screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, chromatin immunoprecipitation sequencing experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin.

Recent Advances in Understanding Histone Modification Events

This review will discuss histone modification events that have been recently discovered or significant technical advancements made in the last year. The physiological state of the eukaryotic genome is chromatin. Chromatin is a complex of DNA and primarily histone proteins. The post-translational modification of histones and the occupancy of specific histone variants in a given nucleosome play a central role in the regulation of the genome. Histone variants and modifications exist in complex and unique combinations. The resulting diversity allows for histones to play key roles in almost all cellular processes and their dysregulation is often the root cause of many diseases. In particular, epigenetic alterations have been noted in cancer, are associated with its progression, aggressiveness, and metastasis, and are useful in its prognosis. Thus, a deep understanding of histone variants and modifications is needed to comprehend both normal cellular function and disease. Technological advancements have dramatically improved our quantitative understanding of histone modification events, decreased sample requirements, and revealed nuances about how these events work together. Specifically, the miniaturization of ChIP-seq and the continuing use and development of mass spectrometric approaches for histone analysis have revealed interesting and diverse new aspects of chromatin biology. This review will explore the causes, mechanisms, and downstream functions of these newly discovered events. We will conclude with an outlook on the future of histone modification analysis and its biological impact.

Effects of H2A Histone Variants on DNA Sequence and Nucleosome Structure using Coarse Grain Simulations

Coarse grain molecular dynamics (CG-MD) simulations are seeing a rising tendency in a wide range of applications because their potential to enhance the sampling of all-atomistic simulations (AA-MD) and the capacity to make near-atomistic millisecond-timescale simulations practical, putting the second threshold on the horizon. In this field, the coarse-grained Martini force field has a prominent position and has recently extended its already range of applications to DNA-containing biomolecules [1].Eukaryotic genomic DNA exists as highly compacted nucleosome arrays called chromatin. Each nucleosome contains a 147-base-pair (bp) stretch of DNA, which is sharply bent and tightly wrapped around a histone protein octamer [2]. Several mechanisms regulate DNA accessibility, including replacement of canonical histones with specialized histone variants. Among the core histone variants, the H2A family is the biggest one -to date 5 histones variants are documented- showing also the highest sequence divergence among histone families. H2A variants show striking differences in three sites that are critical in intra- and inter-nucleosome interactions: the docking domain, which is close to the DNA entry/exit region, the L1 loop and the acidic patch, which is involved in nucleosome-nucleosome interactions. We have studied the structural and dynamic differences in those regions among H2A histone variants-containing nucleosome structures.Furthermore, it has been shown that DNA sequence effects vary between nucleosomes resulting in a significant factor in their stability. For this reason, we have also analyzed the differences in the associated DNA flexibility along the wrapped DNA sequence derived from MD simulations.