The N-terminally acetylated form of mammalian histone H1(o), but not that of avian histone H5, increases with age

We report here on the HPCE separation of two chicken H5 histones, which do not show the heterogeneity (Gln/Arg) at residue 15 first found by Greenaway and Murray [Greenaway and Murray (1971) Nat. New Biol. 229, 233-238]. The two subfractions obtained were identified using reversed-phase HPLC, hydrophilic interaction HPLC, Edman degradation, and MALDI-MS analysis. We found that the two H5 subcomponents differ only by an acetylated (designated H5a) and an unacetylated N-terminus (H5b). In contrast to the N-terminally acetylated form of rat kidney histone H1(o), which increased by about 40% with aging of the animal, the corresponding form of chicken H5 did not: the ratio N-terminally acetylated: unacetylated remained constant (30:70) when histone H5 was extracted from erythrocytes of newly hatched chickens and from adult chickens, respectively. The HPCE technique used in this investigation represents a quick and convenient method for analyzing N-terminally acetylated proteins in the presence of unacetylated forms.     

Autoantibodies to nucleolin cross-react with histone H1 in systemic Lupus erythematosus

IgM autoantibodies to nucleolin and histone H1 are strongly associated in the serum of patients with systemic lupus erythematosus. IgM eluted from immobilized nucleolin specifically stained histone H1 blotted to nitrocellulose; conversely, IgM eluates prepared from immobilized histone H1 stained nucleolin blots. We conclude that the linkage of anti-nucleolin and anti-histone H1 autoantibodies in SLE is due, at least in part, to immunologic cross-reactivity between these two autoantigens, which share certain similar structural features.Abbreviation SLE Systemic Lupus erythematosus     

DNA and nucleosomes direct distinct folding of a linker histone H1 C-terminal domain

We previously documented condensation of the H1 CTD consistent with adoption of a defined structure upon nucleosome binding using a bulk FRET assay, supporting proposals that the CTD behaves as an intrinsically disordered domain. In the present study, by determining the distances between two different pairs of sites in the C-terminal domain of full length H1 by FRET, we confirm that nucleosome binding directs folding of the disordered H1 C-terminal domain and provide additional distance constraints for the condensed state. In contrast to nucleosomes, FRET observed upon H1 binding to naked DNA fragments includes both intra- and inter-molecular resonance energy transfer. By eliminating inter-molecular transfer, we find that CTD condensation induced upon H1-binding naked DNA is distinct from that induced by nucleosomes. Moreover, analysis of fluorescence quenching indicates that H1 residues at either end of the CTD experience distinct environments when bound to nucleosomes, and suggest that the penultimate residue in the CTD (K195) is juxtaposed between the two linker DNA helices, proposed to form a stem structure in the H1-bound nucleosome.     

Crystallization of the globular domain of histone H5

The globular domain of histone H1/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure. We have expressed in Escherichia coli a gene that codes for the globular domain of H5. The protein produced in E. coli is functional in nucleosome binding assays. We have obtained crystals of the protein that diffract to beyond 2.5 A (1 A = 0.1 nm) resolution. The crystals are orthorhombic with unit cell dimensions of a = 80.1 A, b = 67.5 A and c = 38.0 A.     

Relationship of H1(0) histone to differentiation and cancer

The H1(0) histone was first described by Panyim and Chalkley in 1969 as a new electrophoretic band found with histones of non-replicating tissues. Tissues which are active in DNA replication such as ascites tumor cells or thymus cells were reported to lack this band. In this respect the H1(0) histone differs from the bulk of histones which are generally maintained in a constant ratio with respect to each other and to DNA. An inverse relationship between H1(0) histone levels and growth rate was suggested by the decrease in H1(0) histone concentration during regeneration of the pancreas and liver. The synthesis of H1(0) is unusual but not unique in that, unlike the major histone species, it is not restricted to the S phase of the cell cycle. Although there is a general trend for the levels of H1(0) histone to be lower in neoplastic than normal tissues, exceptions have been observed. Compounds such as sodium butyrate and dimethylsulfoxide, which can induce differentiated properties in neoplastic cells, can bring about the accumulation of increased amounts of H1(0) histones. The relative magnitude of these effects exhibits cell-type specificity. There are two H1(0) histone subtypes (a and b) with ratios which differ according to the tissue examined and whose relative importance is not known. The levels of H1(0) histone appear to be more closely related to the degree of differentiation than to the proliferative activity of cells.     

Changes in the synthesis of histone H1(0) and H1 in rat FRTL-5 thyroid cells exposed to thyrotropin

In absence of thyrotropin (TSH), FRTL-5 rat thyroid cells stop proliferating and lose the functional characteristics of thyroid tissue. FRTL-5 cells regain their differentiated state and their proliferation activity upon addition of TSH. In this study we investigated the synthesis of histone H1 variants and H19(0) in FRTL-5 cells exposed to 10(-8) M TSH, two days after TSH withdrawal. TSH induced the synthesis of some H1 variants and H1. This effect was already evident six hours after TSH addition, thus well before proliferation, DNA or thyroglobulin synthesis was induced. These data indicate that the induction of H1(0) and some H1 variants is an early event after TSH stimulation and may thus be related to the functional differentiation of FRTL-5 cells.     

The carboxyl-terminal domain of murine H1(0). Immunochemical and partial amino acid sequence comparisons with other H1(0)/H1/H5 histones

The carboxyl-terminal domain of murine H1(0) histone was compared with that of human H1(0), bovine H1(0) and other H1 and H5 histones. Two sets of antibodies were induced by murine H1(0). One set reacted with only the carboxyl-terminal domain of murine H1(0) and preferred the murine over the bovine and human proteins. The second set of antibodies reacted with the globular domain of murine H1(0) and did not distinguish among murine, bovine and human H1(0) species. There were five positions in the first 60 residues of the carboxyl-terminal domain in which the murine H1(0) differed from the human H1(0). In this region, the murine H1(0) had no more than 49% overall homology with other H1 and H5 histones; however, short sequences in the domain were very similar to short sequences that occur in rabbit H1.3, trout H1 and goose or chicken H5. In comparisons based on these and other published data, the carboxyl-terminal domain of H1(0) is found to be more variable among species than is the globular domain; the first two-thirds of the H1(0) carboxyl-terminal domain is largely unique and does not show great overall homology with H1 or H5, whereas the last third is again more conserved. As the first two-thirds of the domain is the only portion where the homology with H5 is less than 50%, it may be responsible for functional differences between H1(0) and H5.     

Induction of histone H1(0) differs with different treatments among different cell lines

Histone H1(0) is an H1 subfraction whose level on the chromatin is inversely correlated with the mitotic index of the cell. We investigated the increase in H1(0) in 6 cell lines when cell division was blocked by 4 methods: 5 mM butyrate, 2% DMSO, serum withdrawal or density inhibition. The 6 cell lines (HeLa S1 and S3, CHO, 3T6, NIE-115 and Vero) responded differently to the treatments as regards the amount of increase in H1(0) and comparisons among the lines reveal no obvious similarities between the lines in the differential effects of the various treatments on H1(0) levels.     

Immunohistochemical distribution of the histone H1(0)/H5 variant in various tissues of adult Xenopus laevis

The cellular distribution of the histone H1(0)/H5 variant has been examined immunohistochemically in various tissues of adult Xenopus laevis, using monoclonal antibodies against this variant that was isolated from erythrocyte nuclei. The H1(0)/H5 variant appears not to be erythrocyte-specific and appears to be present in all cell types of liver, stomach, and skin. In contrast, in oocyte nuclei the H1(0)/H5 variant cannot be detected, whereas they do contain H1; the nuclei of spermatogenic cells contain the H1(0)/H5 variant, but probably less than the somatic cells. In Xenopus no H1(0) variant distinct from H5 seems to occur and the H1(0)/H5 variant apparently may perform a functional role related to mammalian H1(0).     

Cooperative interaction of the C-terminal domain of histone H1 with DNA

We have studied the interaction of the isolated C-terminal domain of histone H1 with linear DNA using precipitation curves and electron microscopy. The C-terminal domain shows a salt-dependent transition towards cooperative binding, which reaches completion at 60 mM NaCl. At this salt concentration, the C-terminal domain binds to some of the DNA molecules, leaving the rest free. A binding site of 22 base-pairs can be calculated from the stoichiometry of the precipitated fractions. The C-terminal domain condenses the DNA in toroidal particles. The average inner radius of the particles is of the order of 195 A. Consideration of the value of the inner radius of the toroids in the light of counterion condensation theory suggests that in these complexes the isolated C-terminal domain is capable of nearly full electrostatic neutralization of the DNA phosphate charge.     

Accessibility of the globular domain of histones H1 and H5 to antibodies upon folding of chromatin

Antibodies to the globular domain of histones H1 and H5 were purified by affinity chromatography and used to study the accessibility of this region of H1 and H5 in folded and unfolded rat liver and hen erythrocyte chromatin respectively. The different conformations of the chromatin filament were induced by varying the ionic strength from 1 mM to 80 mM NaCl and maintained by fixation with glutaraldehyde. Treatment with glutaraldehyde at a given salt concentration affected neither the orientation of nucleosomes relative to the fiber axis nor the compactness of chromatin. Solid-phase immunoassay and inhibition experiments showed no binding of the antibody against the globular domain of H1 to chromatin at the entire range of salt concentrations, while the antibody to the whole H1 molecule reacted with chromatin at low salt. On the other hand, the antibody to the globular region of H5 reacted with hen erythrocyte chromatin independently of the extent of chromatin condensation. These results indicate that the antigenic determinants of the globular domain of H5 are accessible to the antibody both in folded and unfolded chromatin, while those of the same region of H1 are masked, probably by interaction with DNA or proteins.     

Human histone acetyltransferase 1 (Hat1) acetylates lysine 5 of histone H2A in vivo

The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74 % decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50 %, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions.     

Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis

In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged expression up to elongating spermatids (Sd1) at stage V. A fragment of the equine H1t gene was cloned (GenBank Accession No. AJ865320). The mRNA expression of H1t was found at the level in spermatogonia and in primary spermatocytes up to mid-pachytene at stage VIII/I, whereas H1t protein was found to be expressed up to round spermatides (Sa/Sb1) at stage VIII/I. In peripubertal animals, the H1t protein expression was detected up to elongating spermatids (Sb2) at stage II. Analysis of testes of different ages (< or =2 years) and (> or =3 years) by real-time RT-PCR revealed an increase of H1t mRNA expression, with a wide range of individual variety between 2 and 4 years old animals indicating a stable expression in animals older than 4 years old. This is the first study to show the testis-specific H1t in the stallion and gives evidence that the well-known peripubertal infertility in the stallion may be related to an insufficient histone to protamine exchange. The pattern of protamine gene expression, however, has still to be elucidated.     

Replacement of histone H1 by H5 in vivo does not change the nucleosome repeat length of chromatin but increases its stability.

In vivo competition between histones H1 and H5 for chromatin has been studied in rat sarcoma XC10 cells transfected with a glucocorticoid responsive MMTV-H5 gene. Activation of H5 expression results in accumulation of H5 in the nuclei where it partially replaces H1. H5 displaces H1 from its primary binding sites presumably during chromatin replication and also binds with high affinity to secondary chromatin sites normally not occupied by H1. Replacement of H1 by H5 to levels similar to those of mature chicken erythrocytes does not alter the nucleosome repeat length of chromatin. This indicates that H5 is not solely responsible for the increase in nucleosome spacing of maturing erythroid cells. Exchange of H1 by H5 in vivo or in vitro results in a higher compaction/stability of chromatin.     

Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.     

Ultraviolet laser footprinting of histone H1(0)-four-way junction DNA complexes.

We have used a new light footprinting technique to study the interaction of histone H1(0) and a deletion mutant delta CH1(0) (lacking H1(0) COOH-terminal domain) with a synthetic four-way junction DNA. This technique is based on a single 5-ns UV laser pulse and has the ability to map protein-DNA interactions within unperturbed complexes at time scales far faster than molecular rearrangements. We found both H1(0) and delta CH1(0) to affect the photoreactivity of specific guanine residues located on the central part of four-way junction DNA. These observations demonstrate specific recognition of H1(0) for the central domain of four-way junction DNA. In addition, histone H1(0) decreases the photoreactivity of selected guanines located some distance from the crossover, indicating specific involvement of the H1(0) COOH-terminal tail with this region. Immunofractionation of delta CH1(0)-four-way DNA junction complexes with monoclonal anti-H1 antibody combined with the UV laser footprinting method demonstrated the existence of two types of delta CH1(0)-four-way DNA junction complexes.     

Histones H1(0) and H5 share common epitopes with RNA polymerase II

We report here the cross-reaction of RNA polymerase II antiserum with histones H1(0) and H5 and the complementary cross-reactions of antisera to the globular domain of histone H1(0) (GH1(0)) and histone H5 (GH5) with RNA polymerase II. Immunoblotting of RNA polymerase II antiserum with fragments of histone H1(0) localized the cross-reaction at the junction of the globular and C-terminal domains of histone H1(0). The structural homology implied by these cross-reactions is interesting in light of reports that suggest H1(0) may play a role in differentiation and development.