Histone H1


EVOLUTIONARY HOMOLOGS (part 1/2)

Regulation of Histone H1 expression

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).

Phosphorylation of Histone H1

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).

PTEN interacts with histone H1 and controls chromatin condensation

Chromatin organization and dynamics are integral to global gene transcription. Histone modification influences chromatin status and gene expression. PTEN plays multiple roles in tumor suppression, development, and metabolism. This study, performed with HeLa cells, reports on the interplay of PTEN, histone H1, and chromatin. Loss of PTEN leads to dissociation of histone H1 from chromatin and decondensation of chromatin. PTEN deletion also results in elevation of histone H4 acetylation at lysine 16, an epigenetic marker for chromatin activation. PTEN and histone H1 physically interact through their C-terminal domains. Disruption of the PTEN C terminus promotes the chromatin association of MOF acetyltransferase and induces H4K16 acetylation. Hyperacetylation of H4K16 impairs the association of PTEN with histone H1, which constitutes regulatory feedback that may reduce chromatin stability. These results demonstrate that PTEN controls chromatin condensation, thus influencing gene expression. It is proposed that PTEN regulates global gene transcription profiling through histones and chromatin remodeling (Chen, 2014: PubMed).

Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin

Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. This study describes a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulates the dephosphorylation of H1 in vitro and modulates the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilizes H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation are associated with transcriptional activation independent of RNA polymerase II. This study thus has identified a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin (Raghuram, 2013).

Yeast and Protozoan Histone H1

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).

Xenopus Histone H1

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).

Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts

Maresca, T. J., Freedman, B. S. and Heald, R. (2005). Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts. J. Cell Biol. 169: 859-869. PubMed citation: 15967810

During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. This study has investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Thus, these results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis (Maresca, 2005).

Chicken histone H1

Histone H5 is a variant of histone H1 and is found in chicken erythrocytes. In transcriptionally repressed nuclei, such as those from chicken erythrocytes and sea urchin sperm, distinctive H1 variants (H5 and spH1 respectively) replace H1 and contribute significantly to the enhanced stability of chromatin higher order structures from these sources. Globular domains of H1 and H5 have two basic clusters on opposite sides of the domains. These proteins bind two DNA duplexes forming structures called "tramline" complexes, continuous arrays of globular domains bridging two strands of DNA. The ability to form tramlines is abolished or impaired by removal of charges by mutagenesis at either location. The mutant forms of H5 globular domains also fail to protect the additional 20 bp of nucleosomal DNA characteristically protected by H1 and H5. These mutant H5 globular domains still bind to H1/H5 depleted chromatin, but it seems that both binding sites are required to position the globular domain correctly on the nucleosome (Goytisolo, 1996).

Globular domains of histones H1 and H5 bind cooperatively to DNA. Isolated globular domains of H5 show little if any tendency to self-associate in dilute solution, and H1 shows none. However they both bind in a highly cooperative fashion to DNA. The resulting complexes contain tramline structures of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with globular H5, probably resulting from association of the primary complexes, possibly with binding of additional H5. The highly cooperative nature of the binding, in close apposition, of H1 and H5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding (Thomas, 1992).

Mammalian Histone H1: Early development

The transition from a late 1-cell mouse embryo to a 4-cell embryo, the period when zygotic gene expression begins, is accompanied by an increasing ability to repress the activities of promoters and replication origins. Since this repression can be relieved by either butyrate or enhancers, it appears to be mediated through chromatin structure. Oocytes, which can repress promoter activity, synthesize a full complement of histones, and histone synthesis up to the early 2-cell stage originate from mRNA inherited from the oocyte. However, while histones H3 and H4 continue to be synthesized in early 1-cell embryos, synthesis of histones H2A, H2B and H1 (proteins required for chromatin condensation) is delayed until the late 1-cell stage, reaching amaximum rate in early 2-cell embryos. Histone H4 in both 1-cell and 2-cell embryos is predominantly diacetylated (a modification that facilitates transcription). Deacetylation towards the unacetylated and monoacetylated H4 population in fibroblasts begin at the late 2-cell to 4-cell stage. Arresting development at the beginning of S-phase in 1-cell embryos prevents both the appearance of chromatin-mediated repression of transcription in paternal pronuclei and synthesis of new histones. These changes correlate with the establishment of chromatin-mediated repression during formation of a 2-cell embryo, and the increase in repression from the 2-cell to 4-cell stage as linker histone H1 accumulates and core histones are deacetylated (Wiekowski, 1997).

The distribution of histone H1 has been examined in oocytes and preimplantation embryos of the mouse. No somatic histone H1 is found in germinal vesicle (GV)-stage oocytes. 1- and 2-cell embryos examined do not contain detectable somatic histone H1. At the early 4-cell stage (54-56 hours), 5 of 52 embryos contained somatic histone H1 in one or more nuclei. However, by the late 4-cell stage (66-68 hours post-hCG), 58 of 62 embryos contained somatic histone H1. In 8-cell embryos all nuclei contained somatic histone H1. The transcriptional inhibitor alpha-amanitin inhibited appearance of histone H1. The somatic subtypes first appear at the 4-cell stage, through a process requiring embryonic transcription and DNA replication during the third cell cycle. These results suggest that the deposition of somatic histone H1 on chromatin is developmentally regulated during mouse embryogenesis (Clarke, 1992).

The distribution of somatic histone H1 in bovine oocytes and preimplantation embryos was examined, using an antibody that recognizes histone H1 subtypes present in somatic cells. Immunoreactive H1 was not detectable on the chromosomes of metaphase II of meiosis nor in the nuclei of early cleavage-stage embryos. In most embryos, immunoreactive H1 was assembled onto embryonic chromatin during the fourth to sixth cell cycle after fertilization. No immunoreactive somatic histone H1 was detected, however, when embryos were incubated in the presence of alpha-amanitin beginning early during the fourth cell cycle. These results indicate that somatic subtypes of histone H1 are assembled onto embryonic chromatin in a developmentally regulated manner that requires embryonic transcription. Aphidicolin, an inhibitor of DNA replication, also inhibited the assembly of somatic histone H1 onto chromatin when present at early stages of the 4th cell cycle. It has been suggested that because the bulk of histone gene expression in proliferating cells occurs during DNA replication, expression of genes encoding immunoreactive H1 is inhibited in embryos blocked before or soon after entering the S-phase. Findings in cattle on the control of somatic histone H1 assembly onto chromatin show a remarkable similarity to those found in the mouse. Such evolutionary conservation suggests that the somatic histone H1 complement of chromatin may regulate critical aspects of chromatin activity during mammalian oogenesis or early embryogenesis (Smith, 1995).

A striking feature of early embryogenesis in a number of organisms is the use of embryonic linker histones or high mobility group proteins in place of somatic histone H1. The transition in chromatin composition towards somatic H1 appears to be correlated with a major increase in transcription at the activation of the zygotic genome. Previous studies have supported the idea that the mouse embryo essentially follows this pattern, with the significant difference that the substitute linker histone might be the differentiation variant H1°, rather than an embryonic variant. Histone H1° is shown not to be a major linker histone during early mouse development. Instead, somatic H1 is present throughout this period. Though present in mature oocytes, somatic H1 is not found on maternal metaphase II chromatin. Upon formation of pronuclear envelopes, somatic H1 is rapidly incorporated onto maternal and paternal chromatin, and the amount of somatic H1 steadily increases on embryonic chromatin through to the 8-cell stage. Microinjection of somatic H1 into oocytes, and nuclear transfer experiments, demonstrate that factors in the oocyte cytoplasm and the nuclear envelope, play central roles in regulating the loading of H1 onto chromatin. Exchange of H1 from transferred nuclei onto maternal chromatin requires breakdown of the nuclear envelope and the extent of exchange is inversely correlated with the developmental advancement of the donor nucleus (Adenot, 2000).

Four enzymes cooperate to displace histone H1 during the first minute of hormonal gene activation

Gene regulation by external signals requires access of transcription factors to DNA sequences of target genes, which is limited by the compaction of DNA in chromatin. Althought insight has been gained into how core histones and their modifications influence this process, the role of linker histones remains unclear. This study show that, within the first minute of progesterone action, a complex cooperation between different enzymes acting on chromatin mediates histone H1 displacement as a requisite for gene induction and cell proliferation. First, activated progesterone receptor (PR) recruits the chromatin remodeling complexes NURF and ASCOM (ASC-2 [activating signal cointegrator-2] complex) to hormone target genes. The trimethylation of histone H3 at Lys 4 by the MLL2/MLL3 subunits of ASCOM, enhanced by the hormone-induced displacement of the H3K4 demethylase KDM5B, stabilizes NURF binding. NURF facilitates the PR-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement. Cooperation of ATP-dependent remodeling, histone methylation, and kinase activation, followed by H1 displacement, is a prerequisite for the subsequent displacement of histone H2A/H2B catalyzed by PCAF and BAF. Chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) and expression arrays show that H1 displacement is required for hormone induction of most hormone target genes, some of which are involved in cell proliferation (Vicent, 2011).

These results contribute to a better comprehension of the molecular mechanisms of gene induction by describing the very initial steps of hormonal promoter activation. The data reveal an unexpected complexity in the interactions between enzymatic activities implicated in preparing the chromatin for full access of transcription factors. Apart from previously described enzymatic activities, at least four complexes act 1 min after hormone addition. An ATP-dependent chromatin remodeling complex (NURF), a protein kinase complex (Cdk2/CyclinA), a histone lysine demethylase (JARID1B/KDM5B), and a histone lysine methylase (MLL2 or MLL3)-containing complex cooperate in the displacement of histone H1 from the promoter, an important early step in gene induction by progestins (Vicent, 2011).

It has been shown, in T47D-MTVL cells treated with hormone for 5 min, PR interacts with an exposed HRE on the surface of a nucleosome positioned over the MMTV promoter and recruits Brg1/Brm-containing BAF complexes. This study demonstrates that NURF interacts with PR, and that recruitment of the NURF complex in the first minute following hormone addition is a requisite for subsequent binding of BAF and activation of mammary tumor virus (MMTV) and other progesterone target genes. NURF is anchored at the promoter of progesterone target genes by an interaction with H3K4me3, likely generated by the MLL2/3 histone lysine methylases of the ASCOM complex. This is reminiscent of the role of hormone-induced acetylation of H3K14 in anchoring the BAF complex. At both phases in activation of the promoter, a histone tail modification stabilizes the binding of an ATP-dependent chromatin remodeling complex to the target promoters (Vicent, 2011).

Another similarity between the two subsequent cycles of promoter chromatin remodeling relates to the role of protein kinases. It was found previously that hormone-induced activation of the Src/Ras/Erk cascade leads to phosphorylation of PR at S294 and activation of Msk1, which is targeted to the promoter by PR and phosphorylates H3 at S10, contributing to the displacement of a repressive complex containing HP1γ. This study shows that, prior to this event, 1 min after hormone, PR interacts with a complex of Cdk2 and CyclinA that phosphorylates PR at S400, is recruited to the promoter, and phosphorylates histone H1, leading to its displacement. Thus, there are two similar and consecutive cycles essential for transcriptional activation of hormone-dependent genes, both involving the collaboration between protein kinases, histone-modifying enzymes, and ATP-dependent chromatin remodelers. Each of the remodeling complexes is anchored at the promoter by different epigenetic marks: H3K4me3 established by MLL2/3 anchors NURF, and H3K14ac established by PCAF anchors BAF. The final output of the first cycle is to decompact the chromatin fiber by displacing histone H1, and the outcome of the second cycle is to open the nucleosome by displacing H2A/H2B dimers (Vicent, 2011).

The chromatin remodeling complex NURF has been shown to be necessary for both transcription activation and repression in vivo. Most reports on the role of NURF in gene regulation come from studies in Drosophila, where NURF is involved in the activation of several genes, including the homeotic selector gene engrailed, ultrabithorax, ecdysone-responsive genes, and the roX noncoding RNA. These studies were complemented with mechanistic studies using recombinant Drosophila NURF complex. In contrast, little is known regarding the mechanism of action of NURF in human cells, except for reports on a role in neuronal physiology. It was found that, in T47D-MTVL human breast cancer cells, NURF is essential for efficient hormone-dependent activation of several PR target genes, and is recruited to the target promoters via an interaction with PR. The BAF complex is also recruited to the MMTV promoter within minutes after progestin treatment, but the kinetics of loading of both chromatin remodelers are different. NURF is recruited after 1 min of hormone treatment, while BAF is loaded only after 5 min and its recruitment depends on NURF action. These findings highlight the notion of transcription initiation as a process involving consecutive cycles of enzymatic chromatin remodeling, where each enzyme complex is necessary at a given time point and catalyzes a particular remodeling step. These results support the existence in T47D-MTVL cells of several pools of PR, associated with the different chromatin remodelers. How the coordinated action of each PR population on target promoters is orchestrated is not well understood, but phosphorylation of the receptor by different kinases and post-translational modifications of nucleosomal histones could provide possible mechanisms (Vicent, 2011).

Although H3K4me3 marks transcription start sites (TSSs) of virtually all active genes the role of this modification during MMTV activation has been questioned. This study shows that, in T47D-MTVL cells, the MLL2/3-containing complex ASCOM is recruited to a target promoter after 1 min of hormone and increases H3K4me3. Experiments with siRNA knockdown, ChIP, and peptide pull-down assays showed that H3K4me3 is critical for NURF anchoring at the promoter. The very early and transient appearance of the H3K4me3 mark could explain the apparent controversy with previously published studies. It was found that the H3K4me3 signal observed at the MMTV promoter is due to the concerted recruitment of the ASCOM complex and the localized displacement of the H3K4me3/2/1 demethylase KDM5B. Knockdown of KDM5B increased the basal and hormone-dependent activity of PR target genes and caused an increase in H3K4me3 levels at the promoters in the absence of hormone (Vicent, 2011).

The molecular mechanism underlying hormone-induced displacement of KDM5B is unclear. It has been reported that KDM5B forms a complex with histone deacetylases (HDACs). Ir was shown previously that an HP1γ-containing complex is bound to the MMTV promoter prior to induction, and is displaced by phosphorylation of H3S10 catalyzed by hormone-activated Msk1. However, in coimmunoprecipitation experiments, no interaction between KDM5B and HP1γ was detected. Recently, it has been reported that PARP1 can parylate and inactivate KDM5B catalytic activity. Since nuclear receptors are known to activate PARP1, it is possible that this pathway participates in the inactivation and displacement of KDM5B following progestin treatment (Vicent, 2011).

The PHD finger present in the BPTF subunit of NURF acts as a highly specialized methyl lysine-binding domain critical for NURF loading. H3S10ph and H3K14ac, two other post-translational modifications present in the MMTV promoter chromatin after hormone addition, increase the binding of the PHD domain to H3K4me3. Binding of BPTF to acetylated lysines could be expected, as the protein contains a bromodomain in its C terminus, but the interaction with H3 phosphopeptides was not predicted, as BPTF does not encompass a consensus 14-3-3-like domain. Regarding the role of the H3K9me3 signal in NURF recruitment, peptide pull-down experiments showed no interaction of NURF components with the H3K9me3 mark. Moreover, either knockdown or inhibition of the methyltransferase G9a increased the basal level of transcription in several target genes without affecting the fold induction after hormone. The same effect was observed when cells were depleted of HP1γ, indicating that the H3K9me3 signal anchors HP1γ at the target chromatin (Vicent, 2011).

The NURF complex is recruited after 1 min of hormone, decreased after 2 min, and is almost undetectable after 5 min. How NURF is released from target chromatin is still unknown. Binding of NURF correlates closely with H3K4me3, and therefore a decrease in the trimethylation of H3K4 would explain NURF displacement. It has been proposed that methylation of histone H3R2 by PRMT6 and methylation of H3K4 by MLLs are mutually exclusive. Moreover, H3R2 methylation has been reported to block the binding of effectors that harbor methyl-specific binding domains, including PHD domains, chromodomains, and Tudor domains. Thus, the presence of the H3R2me2 mark could cooperate in erasing the H3K4me3 signal from the promoters and in competing for NURF binding, thus triggering NURF displacement (Vicent, 2011).

MMTV minichromosomes reconstituted with Drosophila embryo extracts were used previously to address the role of histone H1. Histone H1 increases nucleosome spacing and compacts the chromatin, hinders access of general transcription factors to the MMTV promoter, and thus inhibits basal transcription. In the presence of bound PR, H1 is phosphorylated by Cdk2 and subsequently is removed from the promoter upon transcription initiation. The kinase Cdk2 is known to phosphorylate histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions. Histone H1 phosphorylation by Cdk2 has been associated with hormone-dependent transcriptional activation. This study found that NURF facilitates the access of Cdk2/CyclinA to target promoter chromatin, and this could explain its role in H1 displacement from the MMTV promoter and from 15 other PR-binding sites that also contain NURF and recruit Cdk2 after hormone treatment. Along with the general effect of Cdk2 inhibition on gene regulation by progestins, these results support a very general role of Cdk2/CyclinA in histone H1 eviction during the initial steps of hormonal chromatin remodeling (Vicent, 2011).

There is evidence for a direct interaction between PR and Cdk2, CyclinA, or cyclinE that could explain how Cdk2/CyclinA is recruited to the target promoters. In the T47D-MTVL breast cancer cell line, a hormone-independent association of PR with Cdk2 was found and recruitment of CyclinA to this complex upon hormone addition. Therefore, PR could recruit Cdk2/CyclinA to the target promoter upon hormone addition. It is not known whether NURF and Cdk2/CyclinA form a single ternary complex with PR, or rather are in two different PR-associated complexes. Although by coimmunoprecipitation interaction of PR with both complexes was detected after 1 min of hormone addition, a more in-depth analysis performed at 1-min intervals at 30°C revealed that NURF is recruited before Cdk2/CyclinA. These results suggest that NURF recruitment is required for Cdk2/CyclinA loading at target promoters, and support the existence of two independent complexes (Vicent, 2011).

Although H1 displacement takes place locally, it could have a long-range effect on chromatin decompaction, as demonstrated with in vitro assembled condensed chromatin. Displacement of histone H1 could be a prerequisite for all subsequent steps in remodeling, as SWI/SNF remodeling has been reported to be inhibited by the presence of histone H1. A connection between ISWI-containing remodeling machineries and histone H1 dynamics has been reported previously in Drosophila. In this system, ISWI promotes the association of the linker histone H1 with chromatin. Along these lines, it is still possible that NURF is also involved in later steps during hormone induction by helping histone H1 deposition back at the promoter (Vicent, 2011).

How H1 binding is regulated and leads to a more open chromatin structure remains unclear. Some models proposed that Cdk2-dependent H1 phosphorylation leads to the decondensation of chromatin during interphase by disrupting the association of HP1γ with the chromatin fiber. A hormone-dependent displacement of HP1γ from the MMTV promoter was observed without changes in H3K9me3 levels. Whether H1 and Hp1γ are interacting as part of a common repressive complex requires further studies but constitutes an attracting hypothesis. In contrast, PARP-1 possesses the ability to disrupt chromatin structure by PARylating histones (e.g., H1 and H2B) and a variety of nuclear proteins involved in gene regulation. Both PARP-1 and H1 compete for binding to nucleosomes and exhibit a reciprocal pattern of binding at actively transcribed promoters: H1 is depleted and PARP-1 is enriched. Other post-translational modifications of H1 have been proposed to influence its binding and function. Histone H1 is acetylated at Lys 26 in vivo and can be deacetylated by the NAD+-dependent HDAC SirT1, promoting the formation of repressive heterochromatin. This effect was accompanied by an enrichment of H1 at the promoter, and the spreading of heterochromatin marks like H3K9me3 and H4K20me1 throughout the coding region (Vicent, 2011).

Regarding the NURF-mediated changes in chromatin structure, analysis of nucleosome profiles obtained by MNase digestion before hormone treatment showed a preferential location of nucleosomes overlapping with NURF and PR sites that is less pronounced after hormone activation, indicating that chromatin remodeling is involved (Vicent, 2011).

Analysis of the hormone-regulated genes that are affected by depletion of NURF reveals many genes involved in cell cycle and cell proliferation, which could mediate the proliferative response of breast cancer cells to progestins. This may explain the inhibition of cell proliferation in response to progestins that was observed in T47D cells depleted of NURF. A similar inhibition of the proliferative response has been observed in cells depleted of Cdk2. These results indicate that histone H1 displacement may be a prerequisite for the effects of progestins on cell proliferation, and therefore the enzymes involved in this process would be novel targets for the pharmacological control of breast cancer cell proliferation (Vicent, 2011).

A model of the current view of the initial steps in progesterone activation of the MMTV promoter is presented. Although the different steps of remodeling are depicted as a linear time sequence, it cannot be excluded that some of these process occur in parallel and in different time sequences in different target promoters. The model reflects the average time sequence in the cell population. Briefly, after hormone induction, activated PR carrying Erk and Msk1 binds first to the exposed HRE1 on the surface of the MMTV promoter nucleosome in a process that does not require chromatin remodeling. Along with the activated PR kinases, the NURF and ASCOM complexes are recruited to the promoter chromatin in one or several complexes. The combined action of ASCOM recruitment and KDM5B displacement enhances H3K4me3 and stabilizes NURF at the promoter. Other modifications, such as H3S10phos and H3K14ac produced by Msk1 and PCAF, could also contribute to NURF anchoring. Once at the promoter, NURF remodels the nucleosome and facilitates the access of PR and the associated Cdk2/CyclinA kinase, which phosphorylates histone H1 and promotes its displacement, contributing to unfolding of the chromatin fiber. Although it was observed that H3S10 phosphorylation by Msk1 plays a role in HP1γ displacement, it is possible that phosphorylation of histone H1 also contributes to this process. H1-depleted nucleosomes constitute a suitable substrate for recruitment of PR-BAF complexes and further remodeling events catalyzed by BAF and PCAF. H3K14 acetylation by PCAF promotes BAF anchoring. BAF mediates ATP-dependent displacement of histones H2A/H2B, and thus facilitates binding of NF1. Bound NF1 stabilizes the open conformation of the H3/H4 tetramer particle that exposes the previously hidden HREs, allowing synergistic binding of further PR-BAF-kinase complexes and PCAF (Vicent, 2011).

Finally, given that NURF is also recruited to the promoter 30 min after hormone addition, when no H1 is present, it cannot be excluded that NURF catalyzes later steps in chromatin remodeling involving histones or nonhistone chromatin proteins. Indeed, the current results indicate that NURF can act on MMTV minichromosomes lacking histone H1. In this respect, it remains to be established whether NURF and BAF fulfill partly redundant functions, cooperate on the same promoter, or, rather, are mutually exclusive (Vicent, 2011).

Mammalian Histone H1 and transcriptional regulation

Continued: see Histone H1 Evolutionary Homologs part 2/2


Histone H1: Biological Overview | Regulation | Developmental Biology | References

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