Linker Histone H1 Variants Research Articles

Label-Free Mass Spectrometry-Based Quantification of Linker Histone H1 Variants in Clinical Samples

Epigenetic aberrations have been recognized as important contributors to cancer onset and development, and increasing evidence suggests that linker histone H1 variants may serve as biomarkers useful for patient stratification, as well as play an important role as drivers in cancer. Although traditionally histone H1 levels have been studied using antibody-based methods and RNA expression, these approaches suffer from limitations. Mass spectrometry (MS)-based proteomics represents the ideal tool to accurately quantify relative changes in protein abundance within complex samples. In this study, we used a label-free quantification approach to simultaneously analyze all somatic histone H1 variants in clinical samples and verified its applicability to laser micro-dissected tissue areas containing as low as 1000 cells. We then applied it to breast cancer patient samples, identifying differences in linker histone variants patters in primary triple-negative breast tumors with and without relapse after chemotherapy. This study highlights how label-free quantitation by MS is a valuable option to accurately quantitate histone H1 levels in different types of clinical samples, including very low-abundance patient tissues.

Joint Effect of Histone H1 Amino Acid Sequence and DNA Nucleotide Sequence on the Structure of Chromatosomes: Analysis by Molecular Modeling Methods

A chromatosome consisting of a nucleosome core, linker DNA, and linker histone (LH) is an important structural element of chromatin and plays a role in replication and transcription regulation. There are two experimentally confirmed modes of LH binding to the nucleosome and linker DNA that differ in their geometry: on-dyad and off-dyad binding. It was shown that the LH amino acid sequence influences the type of histone binding and the conformation of the chromatosome. However, the geometry of linker DNA bound with LH also changes. Thus, the mutual influence of these factors and the molecular basis determining the type of LH binding to nucleosomes remain unclear. In this study, molecular modeling methods, including homology modeling, atom–atom interaction analysis, and DNA deformation energy analysis, were applied to study the joint effect of the LH amino acid sequence and the DNA nucleotide sequence on the configuration of the chromatosome. The known crystal and NMR structures of the chromatosome for atom–atom interactions of LH and DNA, as well as the energy of DNA deformation in these structures for various DNA sequences, were analyzed. For various LH H1 variants, the analysis was carried out using homology modeling methods. Sequence-dependent differences in the bending energy of the linker DNA for two different conformations of the chromatosome were found, and nucleotide sequences preferred for these structures were proposed. As a result of the analysis, it was shown that the DNA nucleotide sequence, along with the LH amino acid sequence, influences the type of binding to the nucleosome. Hypotheses for experimental verification have been formulated, according to which the type of LH binding can change with different DNA nucleotide sequences.

Post-translational modifications of linker histone H1 variants in mammals

The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2–H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

Modulation of chromatin function through linker histone H1 variants.

In this review, the structural aspects of linker H1 histones are presented as a background for characterization of the factors influencing their function in animal and human chromatin. The action of H1 histone variants is largely determined by dynamic alterations of their intrinsically disordered tail domains, posttranslational modifications and allelic diversification. The interdependent effects of these factors can establish dynamic histone H1 states that may affect the organization and function of chromatin regions.

In situ detection of histone variants and modifications in mouse brain using imaging mass spectrometry.

Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity, and availability of antihistone antibodies. Here, we describe a novel MALDI imaging MS based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available.

HP1BP3 is a novel histone H1 related protein with essential roles in viability and growth.

The dynamic architecture of chromatin is vital for proper cellular function, and is maintained by the concerted action of numerous nuclear proteins, including that of the linker histone H1 variants, the most abundant family of nucleosome-binding proteins. Here we show that the nuclear protein HP1BP3 is widely expressed in most vertebrate tissues and is evolutionarily and structurally related to the H1 family. HP1BP3 contains three globular domains and a highly positively charged C-terminal domain, resembling similar domains in H1. Fluorescence recovery after photobleaching (FRAP) studies indicate that like H1, binding of HP1BP3 to chromatin depends on both its C and N terminal regions and is affected by the cell cycle and post translational modifications. HP1BP3 contains functional motifs not found in H1 histones, including an acidic stretch and a consensus HP1-binding motif. Transcriptional profiling of HeLa cells lacking HP1BP3 showed altered expression of 383 genes, suggesting a role for HP1BP3 in modulation of gene expression. Significantly, Hp1bp3−/− mice present a dramatic phenotype with 60% of pups dying within 24 h of birth and the surviving animals exhibiting a lifelong 20% growth retardation. We suggest that HP1BP3 is a ubiquitous histone H1 like nuclear protein with distinct and non-redundant functions necessary for survival and growth.

Histone variants: key players of chromatin.

Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.

Polymorphic linker histone H1 variants in breeding and conservative duck populations

Abstract A purpose of this study was to evaluate genetic diversity in duck populations based on polymorphic variants (H1.a, H1.b and H1.z) of linker histone H1. The study was performed using conservative brown-feathered Khaki Campbell (Kh1) and Orpington (Or) populations and white-feathered Pekin (P77) duck breeding line. While no significant distortion between both brown-feathered duck populations was noted (P>0.05), the allele frequencies at histone H1 polymorphic loci were found to differ significantly between brown-feathered and white-feathered duck flocks (P<0.001). While the alleles a1, b1 and z1 were detected in all three duck lines, the alleles a2 and b2 missed in the line P77 were found in the Kh1 and Or populations. A rare allele z2 not detected in a homozygous state during screening our duck populations was found to occur only in heterozygous P77 duck individuals (z1z2). After a purpose mating of these heterozygous P77 birds, the homozygous individuals (z2) were obtained in their offspring. The uneven distribution of the alleles for polymorphic histone H1 variants among duck populations seems to suggest that they are not functionally equivalent and, therefore, might have a differential influence on chromatin structure and/or function leading to line-specific phenotypic effects in duck.

Mapping of six somatic linker histone H1 variants in human breast cancer cells uncovers specific features of H1.2

Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagged recombinant H1 variants expressed in breast cancer cells, we have investigated the distribution of six H1 variants in promoters and genome-wide. H1 is depleted at promoters depending on its transcriptional status and differs between variants. Notably, H1.2 is less abundant than other variants at the transcription start sites of inactive genes, and promoters enriched in H1.2 are different from those enriched in other variants and tend to be repressed. Additionally, H1.2 is enriched at chromosomal domains characterized by low guanine–cytosine (GC) content and is associated with lamina-associated domains. Meanwhile, other variants are associated with higher GC content, CpG islands and gene-rich domains. For instance, H1.0 and H1X are enriched at gene-rich chromosomes, whereas H1.2 is depleted. In short, histone H1 is not uniformly distributed along the genome and there are differences between variants, H1.2 being the one showing the most specific pattern and strongest correlation with low gene expression.

Citrullination regulates pluripotency and histone H1 binding to chromatin

Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

Abstract 199: Distinctive Roles of Linker Histone H1 Variants in the Hypertrophic Response of Cardiomyocytes

Heart failure results when cardiac output is insufficient to meet physiological requirements and is often preceded by development of cardiomyocyte hypertrophy. As cardiac myocytes respond to hypertrophic stresses they re-express developmentally important genes, normally senescent in the adult. The chromatin structural events underlying this “fetal gene program” are unknown. We previously showed by proteomics that histones, components of the chromatin protein functional unit, the nucleosome, are altered during hypertrophic and failing phases of pressure overload in mouse: linker histone variants H1.2 and H1.5 decreased in hypertrophied myocardium while H1.0 increased during the transition to failure. The linker histone H1 family influences higher order chromatin structure and gene expression, although the role of this family in the heart is unknown. To assess the role of linker histones in hypertrophy, neonatal rat ventricular cardiomyocytes (NRVMs) were transfected with siRNAs individually targeting six H1 variants. Loss of H1.3 and H1.4 respectively induced a significant 26.1% (76 of 90) and 13.5% (80 of 94) increase in cell size area (µm2). A role of H1 in the hypertrophic response is evidenced by its influence on myosin heavy chain (MHC) mRNA expression. Knock-down of individual H1 variants significantly altered the MHC isoform ratio: loss of H1.3 increased α-MHC levels 1.5 fold and decreased β-MHC 1.6 fold while H1.5 depletion decreased α-MHC 2.5 fold. Both H1.3 and H1.4 knock-down increased atrial natriuretic factor (ANF) 1.3 fold while H1.5 loss decreased ANF 6.2 fold shown by qRT-PCR. Treatment with hypertrophy-inducing agents Isoproterenol (1μM), Endothelin (2nM) or Phenylephrine (10μM), reduced H1 mRNA levels however with subtle effects on protein abundance. To evaluate whether H1 loss shifted NRVM nuclei from a predominantly heterochromatic toward euchromatic state favoring gene accessibility we examined distinct histone markers of chromatin states. Histone H1.5 knock-down significantly decreased H3K9Me3 levels, a silencing mark associated with heterochromatin, 1.7 fold. Therefore we conclude that variants package distinctive regions of the genome and that H1.3 and H1.4 controls genes involved in the hypertrophic response.

Evidence for Insertional RNA Editing in Humans

Large-scale analysis directly at the protein level holds the promise of uncovering features not apparent or present at the gene level [1-3]. Although mass spectrometry (MS)-based proteomics can now identify and quantify thousands of cellular proteins in large-scale proteomics experiments, much of the peptide information contained in these experiments remains unassigned [4]. Here, we use such information to discover a previously unreported mechanism creating altered protein forms. Linker histones H1 and high-mobility group (HMG) proteins are abundant nuclear proteins that regulate gene expression through modulation of chromatin structure [5-8]. In the high-resolution MS analysis of histone H1 and HMG protein fractions isolated from human cells, we discovered peptides that mapped upstream of the known translation start sites of these genes. No alternative upstream start site exists in the genome, but analysis of Expressed Sequence Tag (EST) databases revealed that these N-terminally extended (ET) proteins are due to in-frame translation of the 5' untranslated region (5'UTR) sequences of the transcripts. The new translation start sites are created by a single uridine insertion between AG, reflecting a previously unreported RNA-editing mechanism. To our knowledge, this is the first report of RNA-insertion editing in humans and may be an example of the type of discoveries possible with modern proteomics methods.

Mass Spectrometric Mapping of Linker Histone H1 Variants Reveals Multiple Acetylations, Methylations, and Phosphorylation as Well as Differences between Cell Culture and Tissue

Posttranslational modifications of histones are involved in regulation of chromatin structure and gene activity. Whereas the modifications of the core histones H2A, H2B, H3, and H4 have been extensively studied, our knowledge of H1 modifications remained mainly limited to its phosphorylation. Here we analyzed the composition of histone H1 variants and their modifications in two human cell lines and nine mouse tissues. Use of a hybrid linear ion trap-orbitrap mass spectrometer facilitated assignment of modifications by high resolution and low ppm mass accuracy for both the precursor and product mass spectra. Across different tissues we identified a range of phosphorylation, acetylation, and methylation sites. We also mapped sites of ubiquitination and report identification of formylated lysine residues. Interestingly many of the mapped modifications are located within the globular domain of the histones at sites that are thought to be involved in binding to nucleosomal DNA. Investigation of mouse tissue in addition to cell lines uncovered a number of interesting differences. For example, whereas methylation sites are frequent in tissues, this type of modification was much less abundant in cultured cells and escaped detection. Our study significantly extends the known spectrum of linker histone variability.