The histone H1(0)-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H1(0) promoter, a specific regulatory element, named the H4 box, exhibits features that implicate a role in mediating H1(0) gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H1(0) H4 box were therefore expected to link differentiation-dependent expression of H1(0) to the cell cycle control machinery. The aim of this work has been to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, the cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacts specifically with the H1(0) H4 box and moreover is expressed in a differentiation-dependent manner. HBP1-encoding gene is able to produce different forms of HBP1. Both HBP1 and RB are involved in the activation of H1(0) gene expression. It is therefore proposed that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H1(0), HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells (Lemercier, 2000).

Two Tetrahymena strains were created by gene replacement. One contained H1 with all phosphorylation sites mutated to alanine, preventing phosphorylation. The other had these sites changed to glutamic acid, mimicking the fully phosphorylated state. Global gene expression was not detectably changed in either strain. Instead, H1 phosphorylation activates or represses specific genes in a manner that is remarkably similar to the effects of knocking out the gene encoding H1. These studies demonstrate a role for H1 phosphorylation in the regulation of transcription in vivo and suggest that it acts by mimicking the partial removal of H1 (Dou, 1999).

Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. The major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila are described. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine is detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kinase consensus sequences. Phosphoserine is detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it is observed only when a nearby p34(cdc2) site is also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites are viable even though macronuclear H1 phosphorylation is abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena (Mizen, 1999).

Histone H1, thought previously to have global effects on gene regulation, regulates specific gene expression but not global transcription in Tetrahymena. In an H1 knockout strain, the number of mature RNAs produced by genes transcribed by RNA polymerase I and pol III and for most genes transcribed by pol II remains unchanged. However, H1 is required for the normal basal repression of a gene (ngoA) in growing cells but is not required for its activated expression in starved cells. Surprising, H1 is required for the activated expression of another gene (CyP) in starved cells but not for its repression in growing cells. Thus, H1 does not have a major effect on global transcription but can act as either a positive or negative gene-specific regulator of transcription in vivo (Shen, 1996)

The existence of histone H1 in the yeast, Saccharomyces cerevisiae, has long been debated. In this report the presence of histone H1 in yeast is described. YPL127c, a gene encoding a protein with a high degree of similarity to histone H1 from other species was sequenced as part of the contribution of the Montreal Yeast Genome Sequencing Group to chromosome XVI. To reflect this similarity, the gene designation has been changed to HHO1 (Histone H One). The HHO1 gene is highly expressed as poly A+ RNA in yeast. Although deletion of this gene had no detectable effect on cell growth, viability or mating, it significantly alters the expression of beta-galactosidase from a CYC1-lacZ reporter. Fluorescence observed in cells expressing a histone H1-GFP protein fusion indicates that histone H1 is localized to the nucleus (Ushinsky, 1997).

There is currently no published report on the isolation and definitive identification of histone H1 in Saccharomyces cerevisiae. It was, however, recently shown that the yeast HHO1 gene codes for a predicted protein homologous to H1 of higher eukaryotes, although there is no biochemical evidence that shows that Hho1p is, indeed, yeast histone H1. Purified recombinant Hho1p (rHho1p) has electrophoretic and chromatographic properties similar to linker histones. The protein forms a stable ternary complex with a reconstituted core di-nucleosome in vitro at molar rHho1p:core ratios up to 1. Reconstitution of rHho1p with H1-stripped chromatin confers a kinetic pause at approximately 168 base pairs in the micrococcal nuclease digestion pattern of the chromatin. These results strongly suggest that Hho1p is a bona fide linker histone. The HHO1 gene was deleted and the strain was shown to be viable and has no growth or mating defects. Hho1p is not required for telomeric silencing, basal transcriptional repression, or efficient sporulation. Unlike core histone mutations, a hho1Delta strain does not exhibit a Sin or Spt phenotype. The absence of Hho1p does not lead to a change in the nucleosome repeat length of bulk chromatin nor to differences in the in vivo micrococcal nuclease cleavage sites in individual genes as detected by primer extension mapping (Patterton, 1998).

In Tetrahymena, histone H1 phosphorylation can regulate transcription and mimics loss of H1 from chromatin. The mechanism by which H1 phosphorylation affects transcription was investigated. Tetrahymena strains were created containing mutations in H1 that mimic the charge of the phosphorylated region without mimicking the structure or increased hydrophilicity of the phosphorylated residues. Whenever the charge resembles that of the phosphorylated state, the induced expression of the CyP1 gene is greatly inhibited. Whenever the charge is similar to that of the dephosphorylated state, the CyP1 gene is induced normally. These results argue strongly that phosphorylation of H1 acts by changing the overall charge of a small domain, not by phosphate recognition or by creating a site-specific charge (Dou, 2000).

How is the phosphorylation-induced change in the charge of a small domain of H1 transduced into a change in transcriptional activity? Several mechanisms are possible. One is that the negative charge can cause a structural change in H1, which affects its interaction with a transcription activator or repressor or with DNA. However, computer searches of the Tetrahymena H1 primary sequence for structural motifs or secondary structure suggest that the charge patch region is unstructured, making this mechanism unlikely. Another possibility is that introduction of negative charges in the N-terminal domain of H1 could affect transcription by reducing the electrostatic binding of H1 to DNA in chromatin. In theory, this electrostatic effect could completely dissociate the H1 molecule from chromatin at physiological ionic strength. However, complete dissociation of H1 is unlikely as both the A5 and the E5 mutants contain indistinguishable amounts of H1 that dissociate from chromatin at similar, but not identical, salt concentrations. This result argues that phosphorylation affects the affinity of H1 binding without complete dissociation of the whole molecule. H1 has been shown to dissociate slowly from chromatin in vitro at physiological ionic strength and can exchange between the nuclei of mammalian tissue culture cell heterokaryons. At equilibrium, H1 binding to chromatin is strongly favored. Any change that alters this equilibrium might significantly increase the accessibility of a particular H1 binding site on DNA to trans-acting protein complexes without greatly changing the steady-state amount of total H1 bound to chromatin. These factors could be (co-) activators or (co-) repressors that compete with H1 for chromatin binding sites. A phosphorylation-mediated shift of the equilibrium in favor of unbound H1 might allow more of these complexes to bind stably. Consistent with this hypothesis, H1 readily dissociates from isolated fragments of sea urchin chromatin only when phosphorylated. In addition, the A5 and E5 H1s show a small difference in the salt concentration at which they dissociate, with the E5 version dissociating at a slightly lower salt concentration. This difference could reflect either weakened binding due to localized dissociation of the charge patch region or an increased dissociation rate of the entire molecule at physiological ionic strengths. A final possibility is that phosphorylation could cause localized dissociation of the charge patch region itself, increasing the access of (activating or repressing) transcription factors to DNA. Clearly, understanding the detailed mechanism(s) by which the phosphorylation-induced creation of a charge patch affects the H1 molecule and chromatin structure warrants further study (Dou, 2000).

A gene encoding a protein that shows sequence similarity with the histone H1 family was cloned in the filamentous fungus Ascobolus immersus. The deduced peptide sequence presents the characteristic three-domain structure of metazoan linker histones, with a central globular region, an N-terminal tail, and a long positively charged C-terminal tail. By constructing an artificial duplication of this gene, named H1, it was possible to methylate and silence it by the MIP (methylation induced premeiotically) process. This results in the complete loss of the Ascobolus H1 histone. Mutant strains lacking H1 display normal methylation-associated gene silencing, undergo MIP, and show the same methylation-associated chromatin modifications as do wild-type strains. However, they display an increased accessibility of micrococcal nuclease to chromatin, whether DNA is methylated or not, and exhibit a hypermethylation of the methylated genome compartment. These features are taken to imply that Ascobolus H1 histone is a ubiquitous component of chromatin which plays no role in methylation-associated gene silencing. Mutant strains lacking histone H1 reproduce normally through sexual crosses and display normal early vegetative growth. However, between 6 and 13 days after germination, they abruptly and consistently stop growing, indicating that Ascobolus H1 histone is necessary for long life span. This constitutes the first observation of a physiologically important phenotype associated with the loss of H1 (Barra, 1999).

Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopus sperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development (Lu, 1998).

In Xenopus, cells from the animal hemisphere are competent to form mesodermal tissues from the morula through to the blastula stage. Loss of mesodermal competence at early gastrula is programmed cell-autonomously, and occurs even in single cells at the appropriate stage. To determine the mechanism by which this occurs, a concomitant, global change in expression of H1 linker histone subtypes has been investigated. H1 histones are usually considered to be general repressors of transcription, but in Xenopus they are increasingly thought to have selective functions in transcriptional regulation. Xenopus eggs and embryos at stages before the midblastula transition are deficient in histone H1 protein, but contain an oocyte-specific variant called histone B4 or H1M. After the midblastula transition, histone B4 is progressively substituted by three somatic histone H1 variants, and replacement is complete by early neurula. Accumulation of somatic H1 protein is rate limiting for the loss of mesodermal competence. This involves selective transcriptional silencing of regulatory genes required for mesodermal differentiation pathways (for example, muscle development) by somatic (but not maternal) H1 protein (Steinbach, 1997).

There are major transitions in the type and modification of chromatin-associated proteins during the early development of Xenopus laevis. Histone H4 is stored in the diacetylated form in the egg and is progressively deacetylated during normal development. If histone deacetylases are inhibited with sodium butyrate, only hyperacetylated histone H4 accumulates after the mid-blastula transition. The type of linker histone in chromatin also changes during embryogenesis, from predominantly the B4 protein at the mid-blastula transition to predominantly histone H1 at the end of gastrulation. These transitions in chromatin composition correlate with major changes in the replicative and transcriptional activity of embryonic nuclei (Dimitrov, 1993).

The molecular mechanisms responsible for the remodeling of entire somatic erythrocyte nuclei in Xenopus laevis egg cytoplasm have been examined. These transitions in chromosomal composition are associated with the capacity to activate new patterns of gene expression and the re-acquisition of replication competence. Somatic linker histone variants H1 and H1 (0) are released from chromatin in egg cytoplasm, whereas the oocyte-specific linker histone B4 and HMG1 are efficiently incorporated into remodeled chromatin. Histone H1 (0) is released from chromatin preferentially in comparison with histone H1. Core histones H2A and H4 in the somatic nucleus are phosphorylated during this remodeling process. These transitions recapitulate the chromosomal environment found within the nuclei of the early Xenopus embryo. Phosphorylation of somatic linker histone variants is demonstrated not to direct their release from chromatin, nor does direct competition with cytoplasmic stores of linker histone B4 determine their release. However, the molecular chaperone nucleoplasmin does have an important role in the selective removal of linker histones from somatic nuclei. For Xenopus erythrocyte nuclei, this disruption of chromatin structure leads to activation of the 5S rRNA genes. These results provide a molecular explanation for the remodeling of chromatin in Xenopus egg cytoplasm and indicate the capacity of molecular chaperones to disrupt a natural chromosomal environment, thereby facilitating transcription (Dimitrov, 1996).

Xenopus oocyte 5S RNA genes are normally activated at the mid-blastula transition and are subsequently repressed as gastrulation proceeds. The regulated expression of histone H1 during Xenopus development has a specific and dominant role in mediating the differential expression of the oocyte and somatic 5S rRNA genes. The incorporation of histone H1 into chromatin during embryogenesis directs the specific repression of the Xenopus oocyte 5S rRNA genes before gastrulation is complete. The only Xenopus genes known to be influenced by H1 protein are the oocyte 5s rRNA genes. An increase in histone H1 content specifically restricts transcription factor TFIIIA-activated transcription, and a decrease in histone H1 within chromatin facilitates the activation of the oocyte 5S rRNA genes by TFIIIA. Variation in the amount of histone H1 in chromatin does not significantly influence somatic 5S rRNA gene transcription. This example demonstrates that histones can exert dominant repressive effects on the transcription of a gene in vivo in spite of an abundance of transcription factors for that gene (Bouvet, 1994).

The potential role of histone hyperacetylation in gene activation during Xenopus development was examined using Trichostatin A, (TSA), a specific inhibitor of histone deacetylase. TSA is very effective in inducing both core histone hyperacetylation and histone H1 (0) gene expression in a Xenopus somatic cell line. In contrast, TSA does not induce histone hyperacetylation or histone H1 (0) transcription in Xenopus oocytes. Histone hyperacetylation is developmentally regulated during Xenopus embryogenesis; hyperacetylated histones first accumulate early in gastrulation. The capacity of TSA to induce histone H1 (0) gene expression correlates with the induction of histone hyperacetylation. Concentrations of TSA sufficient to induce histone hyperacetylation in Xenopus embryos delay gastrulation and cause diminished midtrunk and posterior formation, suggesting defects in mesoderm formation. Although the constitutive hyperacetylation of the histones does not prevent either the cell division or differentiation sufficient for early morphogenesis it has a role in establishing stable states of differential gene activity during gastrulation (Almouzni, 1994).

There exists a close relationship between core histone acetylation and the induced expression of the histone H1 (0) gene. The influence of chromatin hyperacetylation was examined on the developmentally regulated expression of Xenopus histone H1 (0). Two stages of development were examined: gastrula stage, when H1 (0) is not expressed and not inducible by butyrate treatment, and stage 27, when H1 (0) is not expressed but is inducible by butyrate. At stage 27 of development the early induced accumulation of histone H1 (0) under butyrate treatment occurs mainly in tissues that normally express the protein during later development. These experiments suggest that histone acetylation may be part of a pathway that in a specific set of cells keeps H1 (0) (and probably a series of specific genes) competent for transcription, but cell-specific factors are involved in the induced expression of these genes (Seigneurin, 1995).

One molecule of a linker histone such as histone H1 is incorporated into every metazoan nucleosome. Histone H1 has three distinct structural domains: the positively charged amino-terminal and carboxy-terminal tails are separated by a globular domain that is similar to the winged-helix motif found in sequence-specific DNA-binding proteins. The globular domain interacts with DNA immediately contiguous to that wrapped around the core histones, whereas the tail domains are important for the compaction of nucleosomal arrays. Experiments in vivo indicate that histone H1 does not function as a global transcriptional repressor, but instead has more specific regulatory roles. In Xenopus, maternal stores of the B4 linker histone that are assembled into chromatin during the early cleavage divisions are replaced by somatic histone H1 during gastrulation. This transition in chromatin composition causes the repression of genes encoding oocyte-type 5S rRNAs, and restricts the competence of ectodermal cells to differentiate into mesoderm. It is demonstrated that the globular domain of histone H1 is sufficient for directing gene-specific transcriptional repression and for restricting the mesodermal competence of embryonic ectoderm. These results are discussed in the context of specific structural roles for this domain in the nucleosome (Vermaak, 1998).