Characterisation of human histone H1x

The members of the H1 histone family can be classified into three groups, which are the main class subtypes expressed in somatic cells, the developmental- and tissue-specific subtypes, and the replacement subtype H1(o). Until now, the subtype H1x was not classified, since it has not yet been thoroughly examined. The results of this study show that H1x shares similarities but also exhibits slight differences in its biochemical behaviour in comparison to the main class H1 histones. In HeLa cells it is located in the nucleus and partially associated with nucleosomes. Nevertheless, it is, like H1(o), mainly located in chromatin regions that are not affected by micrococcal nuclease digestion. Further common features of H1x and the replacement histone H1(o) are that the genes of both subtypes are solitarily located and give rise to polyadenylated mRNA. However, comparison of the inducibility of their expression revealed that their genes are regulated differentially.     

Reduction of Hox gene expression by histone H1 depletion

The evolutionarily conserved homeotic (Hox) genes are organized in clusters and expressed collinearly to specify body patterning during embryonic development. Chromatin reorganization and decompaction are intimately connected with Hox gene activation. Linker histone H1 plays a key role in facilitating folding of higher order chromatin structure. Previous studies have shown that deletion of three somatic H1 subtypes together leads to embryonic lethality and that H1c/H1d/H1e triple knockout (TKO) embryonic stem cells (ESCs) display bulk chromatin decompaction. To investigate the potential role of H1 and higher order chromatin folding in the regulation of Hox gene expression, we systematically analyzed the expression of all 39 Hox genes in triple H1 null mouse embryos and ESCs by quantitative RT-PCR. Surprisingly, we find that H1 depletion causes significant reduction in the expression of a broad range of Hox genes in embryos and ESCs. To examine if any of the three H1 subtypes (H1c, H1d and H1e) is responsible for decreased expression of Hox gene in triple-H1 null ESCs, we derived and characterized H1c(-/-), H1d(-/-), and H1e(-/-) single-H1 null ESCs. We show that deletion of individual H1 subtypes results in down-regulation of specific Hox genes in ESCs. Finally we demonstrate that, in triple-H1- and single-H1-null ESCs, the levels of H3K4 trimethylation (H3K4me3) and H3K27 trimethylation (H3K27me3) were affected at specific Hox genes with decreased expression. Our data demonstrate that marked reduction in total H1 levels causes significant reduction in both expression and the level of active histone mark H3K4me3 at many Hox genes and that individual H1 subtypes may also contribute to the regulation of specific Hox gene expression. We suggest possible mechanisms for such an unexpected role of histone H1 in Hox gene regulation.     

H1 subtype expression during cell proliferation and growth arrest

H1 histone subtype genes differ in their expression patterns during the different stages of the cell cycle interphase. While the group of replication-dependent H1 histone subtypes is synthesized during S phase, the replacement histone subtype H1.0 is also expressed replication-independently in non-proliferating cells. The present study is the first report about the analysis of the cell cycle-dependent expression of all five replication-dependent H1 subtypes, the replacement histone H1.0 and the ubiquitously expressed subtype H1x. The expression of these H1 histone subtypes in HeLa cells was analysed on mRNA level by quantitative real-time RT-PCR as well as on protein level by immunoblotting. We found that after arrest of HeLa cells in G1 phase by treatment with sodium butyrate, the mRNA levels of all replication-dependently expressed H1 subtypes decreased, but to very different extent. During S phase the individual replication-dependently expressed H1 subtypes show similar kinetics regarding their mRNA levels. However, the variations in their protein amounts partially differ from the respective RNA levels which especially applies to histone H1.3. In contrast, the mRNA as well as the protein level of H1x remained nearly unchanged in G1 as well as during S phase progression. The results of the present study demonstrate that the cell cycle-dependent mRNA and protein expression of various H1 subtypes is differentially regulated, supporting the hypothesis of a functional heterogeneity.     

Linker histone subtypes and their allelic variants

Members of histone H1 family bind to nucleosomal and linker DNA to assist in stabilization of higher‐order chromatin structures. Moreover, histone H1 is involved in regulation of a variety of cellular processes by interactions with cytosolic and nuclear proteins. Histone H1, composed of a series of subtypes encoded by distinct genes, is usually differentially expressed in specialized cells and frequently non‐randomly distributed in different chromatin regions. Moreover, a role of specific histone H1 subtype might be also modulated by post‐translational modifications and/or presence of polymorphic isoforms. While the significance of covalently modified histone H1 subtypes has been partially recognized, much less is known about the importance of histone H1 polymorphic variants identified in various plant and animal species, and human cells as well. Recent progress in elucidating amino acid composition‐dependent functioning and interactions of the histone H1 with a variety of molecular partners indicates a potential role of histone H1 polymorphic variation in adopting specific protein conformations essential for chromatin function. The histone H1 allelic variants might affect chromatin in order to modulate gene expression underlying some physiological traits and, therefore could modify the course of diverse histone H1‐dependent biological processes. This review focuses on the histone H1 allelic variability, and biochemical and genetic aspects of linker histone allelic isoforms to emphasize their likely biological relevance.     

The alterations in H1 histone complement during mouse spermatogenesis and their significance for H1 subtype function

The principal histone H1 subtypes of mouse prepachytene spermatocytes were identified as H1a and H1c, proteins that are metabolically unstable and have been postulated to promote extended or flexible chromatin conformations. We suggest that the unusual H1 subtype composition in these cells favors chromatin conformations compatible with the precise chromosomal pairing necessary for genetic recombination and also that it may expedite histone replacement during spermiogenesis.     

G1 phase-dependent nucleolar accumulation of human histone H1x.

BACKGROUND INFORMATION: H1 histones are a protein family comprising several subtypes. Although specific functions of the individual subtypes could not be determined so far, differential roles are indicated by varied nuclear distributions as well as differential expression patterns of the H1 subtypes. Although the group of replication-dependent H1 subtypes is synthesized during S phase, the replacement H1 subtype, H1 degrees , is also expressed in a replication-independent manner in non-proliferating cells. Recently we showed, by protein biochemical analysis, that the ubiquitously expressed subtype H1x is enriched in the micrococcal nuclease-resistant part of chromatin and that, although it shares common features with H1 degrees , its expression is differentially regulated, since, in contrast to H1 degrees , growth arrest or induction of differentiation did not induce an accumulation of H1x. RESULTS: In the present study, we show that H1x exhibits a cell-cycle-dependent change of its nuclear distribution. This H1 subtype showed a nucleolar accumulation during the G(1) phase, and it was evenly distributed in the nucleus during S phase and G(2). Immunocytochemical analysis of the intranucleolar distribution of H1x indicated that it is located mainly in the condensed nucleolar chromatin. In addition, we demonstrate that the amount of H1x protein remained nearly unchanged during S phase progression, which is in contrast to the replication-dependent subtypes. CONCLUSION: These results suggest that the differential localization of H1x provides a mechanism for a control of H1x activity by means of shuttling between nuclear subcompartments instead of a controlled turnover of the protein.     

Genome Distribution of Replication-independent Histone H1 Variants Shows H1.0 Associated with Nucleolar Domains and H1X Associated with RNA Polymerase II-enriched Regions

Unlike core histones, the linker histone H1 family is more evolutionarily diverse, and many organisms have multiple H1 variants or subtypes. In mammals, the H1 family includes seven somatic H1 variants; H1.1 to H1.5 are expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. Using ChIP-sequencing data and cell fractionation, we have compared the genomic distribution of H1.0 and H1X in human breast cancer cells, in which we previously observed differential distribution of H1.2 compared with the other subtypes. We have found H1.0 to be enriched at nucleolus-associated DNA repeats and chromatin domains, whereas H1X is associated with coding regions, RNA polymerase II-enriched regions, and hypomethylated CpG islands. Further, H1X accumulates within constitutive or included exons and retained introns and toward the 3′ end of expressed genes. Inducible H1X knockdown does not affect cell proliferation but dysregulates a subset of genes related to cell movement and transport. In H1X-depleted cells, the promoters of up-regulated genes are not occupied specifically by this variant, have a lower than average H1 content, and, unexpectedly, do not form an H1 valley upon induction. We conclude that H1 variants are not distributed evenly across the genome and may participate with some specificity in chromatin domain organization or gene regulation.     

Regulation of specific genes during the cell cycle. Utilization of homologous cDNAs and cloned sequences for studying histone gene expression in human cells

Evidence for differential gene expression during the cell cycle and approaches for studying cell-cycle-stage specific gene expression are summarized. Attention is focused on regulation of histone gene expression during the cell cycle of continuously dividing cells and after stimulation of nondividing cells to proliferate. The level(s) at which control of histone gene expression occurs and the possible involvement of chromosomal proteins in the regulation of histone gene expression are discussed. The preparation of cloned human histone sequences and their use in studying the structural and functional properties of human histone genes are presented. Index Entries: Cell cycle, gene regulation during; gene regulation, during the cell cycle; regulation of specific genes, during the cell cycle; DNAs, homologous, and histone gene expression; cloned DNAs, and histone gene expression; histone gene expression; gene expression, histone; cloned human histone sequences.     

G1 phase-dependent nucleolar accumulation of human histone H1x

Background information: H1 histones are a protein family comprising several subtypes. Although specific functions of the individual subtypes could not be determined so far, differential roles are indicated by varied nuclear distributions as well as differential expression patterns of the H1 subtypes. Although the group of replication‐dependent H1 subtypes is synthesized during S phase, the replacement H1 subtype, H1°, is also expressed in a replication‐independent manner in non‐proliferating cells. Recently we showed, by protein biochemical analysis, that the ubiquitously expressed subtype H1x is enriched in the micrococcal nuclease‐resistant part of chromatin and that, although it shares common features with H1°, its expression is differentially regulated, since, in contrast to H1°, growth arrest or induction of differentiation did not induce an accumulation of H1x. Results: In the present study, we show that H1x exhibits a cell‐cycle‐dependent change of its nuclear distribution. This H1 subtype showed a nucleolar accumulation during the G₁ phase, and it was evenly distributed in the nucleus during S phase and G₂. Immunocytochemical analysis of the intranucleolar distribution of H1x indicated that it is located mainly in the condensed nucleolar chromatin. In addition, we demonstrate that the amount of H1x protein remained nearly unchanged during S phase progression, which is in contrast to the replication‐dependent subtypes. Conclusion: These results suggest that the differential localization of H1x provides a mechanism for a control of H1x activity by means of shuttling between nuclear subcompartments instead of a controlled turnover of the protein.     

A series of repetitive DNA sequences are associated with human core and H1 histone genes

Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following³²P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)₁₆ simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.     

Molecular and evolutionary analysis of mussel histone genes (Mytilus spp.): possible evidence of an "orphon origin" for H1 histone genes

Linker histones are a divergent group of histone proteins with an independent evolutionary history in which, besides somatic subtypes, tissue- and differentiation-specific subtypes are included. In the present work H1 histone coding and noncoding segments from five Mytilus mussel species (Mollusca: Bivalvia) widely distributed throughout the world have been determined and characterized. Analysis of promoter regions shows clear homologies among Mytilus H1 genes, sea urchin H1 genes, and vertebrate differentiation-specific H1 subtypes (H5 and H10), all having an H4 box motif in common. The amino acid sequence of the H1 protein central conserved domain is also closely related to that previously defined for the vertebrate divergent subtypes. A phylogenetic tree reconstructed from different H1 genes from several species strengthens the hypothesis of an "orphon" origin for the Mytilus H1 genes, as well as for the H10/H5 genes from vertebrates and the H1D gene from the sea urchin Strongylocentrotus purpuratus, is suggested. As additional data, the average copy number of the H1 genes in the species analyzed was estimated as being 100 to 110 copies per haploid genome, where FISH revealed telomeric chromosomal location for several H1 copies in M. galloprovincialis. The contribution of such proximity to heterochromatic regions over the amount of codon bias detected for H1 genes is discussed.     

N- and C-terminal domains determine differential nucleosomal binding geometry and affinity of linker histone isotypes H1(0) and H1c

Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin binding interactions. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H1(0) and H1c. Exchanging the N-terminal domains between H1(0) and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.     

A solitary human H3 histone gene on chromosome 1

A solitary histone H3 gene encoding a novel H3 protein sequence has been isolated. This H3 gene maps to chromosome 1 (1g42), whereas we have shown previously that the majority of the human histone genes form a large cluster on chromosome 6 (6p21.3). In addition, a small cluster has been described at 1q21. The clustered histone genes are expressed during the S-phase of the cell cycle, hence their definition as replication-dependent histone genes. In contrast, expression of replacement histone genes is essentially cell-cycle independent; they are solitary genes and map outside the major clusters. The newly described H3 gene maps outside all known histone gene clusters and varies by four amino acid residues from the consensus mammalian H3 structure. In contrast to other solitary histone genes, this human H3 gene shows the consensus promoter and 3 flanking portions that are typical for replication-dependent genes.