Human spleen histone H1. Isolation and amino acid sequences of three minor variants, H1a, H1c, and H1d

Following the previous determination of the main variant H1b of human spleen histone H1, we have determined the complete amino acid sequence of another variant, H1d. Limited chymotryptic digestion of H1d produced four fragments, I to IV, and one partial fragment I-II, as in the case of H1b. These fragments were aligned with two overlapping peptides, produced by another enzyme from the intact H1d. We also confirmed the C-terminal sequence of H1d by carboxypeptidase digestion. This H1d has an acetylated N-terminal serine, equimolar alanine or valine residue at 17, and is composed of 212 residues. The molecular weight was 21,233 for the alanine variant and 21,261 for the valine variant in the unmodified form. We also deduced the total sequences of H1a and H1c in a similar way, considering the maximum homology with H1b and H1d. Each N-terminal serine residue is acetylated, too. H1a consists of 222 amino acid residues and has a molecular weight of 22,178 in its unmodified form; the H1c consists of 220 residues and has a molecular weight of 22,218 in that form. The human spleen H1 sequences varied to about the same extent in the N-terminal 40 and C-terminal 110 residues. However, the sequences of the about 70 internal residues are well conserved between the variants. The extent of differences among the human H1 variants is similar to, or rather smaller than, those among the mammalian somatic H1 species. The implications of these differences in the sequence for H1 function are discussed from the evolutionary viewpoint.     

Isolation and characterization of two replication-dependent mouse H1 histone genes.

Mice contain at least seven nonallelic forms of the H1 histones, including the somatic variants H1a-e and less closely related variants H1 degrees and H1t. The mouse H1 degrees and H1c (H1var.1) genes were isolated and characterized previously. We have now isolated, sequenced and studied the expression properties of two additional mouse H1 genes, termed H1var.2 and H1var.3. Extensive amino acid and nucleotide sequence comparisons were made between the two genes and other mammalian H1 histone genes. A high degree of nucleotide sequence identity was seen between the H1var.2, rat H1d and human H1b genes, even well beyond the coding region, indicating that these genes are likely homologues. Unlike the previously characterized mouse H1var.1 gene which produces both nonpolyadenylated and polyadenylated mRNAs, the H1var.2 and H1var.3 genes produce only typical, replication dependent, nonpolyadenylated mRNAs.     

Histone H1 heterogeneity in the midge, Chironomus thummi. Structural comparison of the H1 variants in an organism where their intrachromosomal localization is possible

1. Seven subfractions of histone H1 have been isolated and purified from larvae of Chironomus thummi (Diptera). They have been denominated I-1, II-1, II-2, II-3, III-1, III-2, and III-3, according to the order of migration in two steps of preparative electrophoresis. 2. The amino acid compositions are similar to those of other H1 histones. Subfractions I-1 and II-1 were found to contain one methionine and two tyrosine residues, II-2 contained two methionine and three tyrosine residues, and III-1 one methionine and three tyrosine residues. The other subfractions contained one or two methionine and two or three tyrosine residues. For subfractions I-1 and II-1 a chain length of about 252 amino acids was estimated. 3. Peptide pattern analyses after chemical cleavage at the methionine and tyrosine residues, and enzymatic cleavage with thrombin and chymotrypsin, respectively, showed that all subfractions have different individual primary structures. A comparison of peptide sizes and of the positions in the peptide patterns of epitopes recognized by monoclonal antibodies was made to check whether some of the subfractions could arise by proteolytic degradation of others. This possibility can be excluded for five of the subfractions and is very improbable for the two others. Treatment of C. thummi H1 with alkaline phosphatase did not change the pattern of subfractions, while the phosphorylated subfraction of histone H2A disappeared after this treatment. Most and very probably all subfractions are thus H1 sequence variants. 4. Inbred strains and individual larvae of C. thummi were found to comprise all seven variants. The H1 heterogeneity can therefore not be due to allelic polymorphism. Salivary gland nuclei were found to contain variant I-1 and at least some of the other variants. 5. H1 from Drosophila melanogaster and from calf thymus were used as reference molecules in all cleavage experiments and yielded the peptide patterns expected from the sequence. The comparison discriminates the group of C. thummi H1 histones clearly from Drosophila and calf thymus H1. Limited trypsin digestion yielded a protected peptide of uniform size in six of the seven variants which was considerably smaller than the protected central domain of calf thymus H1. 6. Two other species of Chironomidae, C. pallidivittatus and Glyptotendipes barbipes were found to contain five and three H1 subfractions, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subtype-specific cyclic AMP-dependent histone H1 phosphorylation at the differentiation of mouse neuroblastoma cells.

At the initial phase of cell differentiation in mouse neuroblastoma (N18) induced by dibutyrylcyclic AMP (dbcAMP), an additional site of histone H1 was extensively phosphorylated. Forskolin and various phosphodiesterase inhibitors also induced both cell differentiation and H1 phosphorylation at the identical site. The phosphorylation preferentially occurred in a single H1 subtype (H1c) among the five (H1a-e) fractionated by high performance liquid chromatography. The three H1 subtypes of N18 (H1c, H1d, and H1e) were phosphorylated in vitro, and their amino acid sequences of the phosphopeptides were identical to the known sequence of rabbit H1 peptides containing a serine 37 residue. However, the amount of H1a and H1b phosphorylations was negligible. The serine residue was replaced by threonine residue in H1a, and H1b did not have a homologous peptide. The tryptic phosphopeptides of H1 in N18 were identical to that in rat liver H1 induced by glucagon (Langan, T.A. (1969) Proc. Natl. Acad. Sci. USA 64, 1276-1283). The results indicate that 1) the response of H1 subtypes to cAMP-dependent protein kinase in vivo and in vitro is H1 subtype-specific, and 2) the H1c phosphorylation may play an important role in the restrictive area of chromatin in both cell differentiation and hormonal stimulation mediated by cAMP.     

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.     

Preparation and characterization of bovine growth hormones produced in recombinant Escherichia coli.

Two analogues of bovine growth hormone (BGH) have been produced in Escherichia coli by recombinant DNA techniques. In analogue Delta-1, the N-terminal alanine residue of the full-length bovine sequence is replaced by methionine. In analogue Delta-9, which is expressed at much higher levels than is Delta-1, the full-length bovine sequence is truncated at the N-terminus by eight residues and there is a serine-for-glycine substitution in the first position of the truncated protein. Both analogues, which were characterized by isoelectric focusing (i.e.f.), polyacrylamide-gel electrophoresis in the presence of SDS (SDS/PAGE), amino acid analysis and N-terminal amino acid sequence determination using combined g.l.c.-m.s., are compared with BGH isolated from pituitaries. In contrast with pituitary-derived BGH, the recombinant-derived proteins are homogeneous on SDS/PAGE and on i.e.f. In a radioimmunoassay, a radioreceptor assay and a bioassay in vivo (rat tibia), Delta-9 BGH showed very similar characteristics to the pituitary-derived hormone. Similar results have also been obtained with the Delta-1 analogue.     

Individual Somatic H1 Subtypes Are Dispensable for Mouse Development Even in Mice Lacking the H10 Replacement Subtype

H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H1(0), the most divergent subtype, develop normally. Examination of chromatin in H1(0-/-) mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H1(0) to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H1(0) (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434-6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H1(0-/-) mice, each of the three H1 knockout mouse lines was bred with H1(0) knockout mice to generate H1c/H1(0), H1d/H1(0), or H1e/H1(0) double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.     

A new h.p.l.c. isolation procedure for chicken and goose erythrocyte histones.

Total chicken erythrocyte histones were separated by reversed-phase h.p.l.c. using a multi-step acetonitrile gradient in a very short time (35 min). The proteins were eluted in the following order: H1, H5, H2B, H2A.2, H4, H2A.1 and H3.2. Applying a special gradient system adapted for the separation of very-lysine-rich histones, chicken erythrocyte H5 was resolved into two subfractions. Their electrophoretic mobilities were identical in both SDS and acetic acid/urea/Triton polyacrylamide-gel electrophoresis, but different in free-flow electrophoresis. Amino-acid-sequence analyses revealed that the two components only differ with respect to position 15, one having glutamine in that position and the other arginine. A separation of histones prepared from goose erythrocytes disclosed no H5 subfractionation. Furthermore, histones obtained from anaemic-chicken blood were analysed by the above-mentioned h.p.l.c. conditions. An alteration in the relation of H1 to H5 was detected, but no further differences in the number and quantity of the histones and histone variants were observed as compared with the corresponding proteins processed from normal-chicken blood.     

Site-specificity of histone H1 methylation by two H1-specific protein-lysine N-methyltransferases from Euglena gracilis

1. The histone H1 fractions from rat spleen and liver were used as substrates for two H1-specific protein-lysine N-methyltransferases, V-A and V-B (protein methylase III) from Euglena gracilis. 2. When the enzymatically [methyl-3H]labeled H1 fractions were resolved by two-dimensional gel electrophoresis, four subtypes were found to be methylated (H1b, H1c, H1d and H1e). Both enzymes methylated H1c and H1b to approximately the same extent; H1d and H1e were methylated preferentially by enzyme V-B and V-A, respectively. 3. Histone H1c, [methyl-3H]labeled by the methyltransferase V-A, which had been digested by arginine-specific protease (Arg C protease), showed a single radioactive peptide on HPLC, indicating methylation site specificity of the enzyme. 4. Arg C protease-digestion of [methyl-3H]labeled H1c labeled by methyltransferase V-B indicated that this enzyme methylated two sites on the histone molecule. 5. The histone H1c methylation sites of these two enzymes did not overlap, indicating the two enzymes have different site specificity. 6. In combination with the other results, this suggests that the two enzymes serve discrete purposes, possibly involving the presumed different actions of histone H1 subtypes.     

Three novel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 3 isoforms. Expression, regulation, and function of the membranes of the SERCA3 family

Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a-c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a-e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated.     

H 1 histone and histone variants in Trypanosoma cruzi

Trypanosoma cruzi chromatin is not condensed in chromosomes during mitosis. In previous studies a characteristic H 1 was not found in SDS or in acid-urea-PAGE. Consequently, it was proposed that the particular behavior of T. cruzi chromatin in dividing cells was due to the absence of an H 1 histone. In the present work, histones from this parasite were systematically characterized by spectrofluorometric analysis, amino acid composition, PAGE in one and in two dimensions, differential extraction with PCA and TCA, immunological cross-reactivity with antisera, and immunoblotting. We conclude that T. cruzi contains all five histones, H 1 presenting solubility and immunological properties similar to those in other species, but with a particular electrophoretic mobility in Triton-PAGE. Thus an explanation other than the absence of H 1 should be offered in order to understand the behavior of T. cruzi chromatin during mitosis. Moreover, histone variants were described by two-dimensional PAGE. The presence of histone variants suggests that they may participate in the regulation of cell proliferation and differentiation of this parasite, as it has been postulated for higher eukaryotes.     

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 purification and complete amino acid sequence of the 9000-Mr Ca2+-binding protein from rat placenta. Identity with the vitamin D-dependent intestinal Ca2+-binding protein.

A 9000-Mr Ca2+-binding protein was isolated from rat placenta and purified to homogeneity by h.p.l.c. procedures. The complete amino acid sequence was established for the 78-residue placental protein. A sequence analysis of a minor component of the rat intestinal Ca2+-binding protein (residues 4-78) and a tryptic peptide (residues 55-74), both purified by h.p.l.c., showed both proteins to be identical. Thus this placental 9000-Mr Ca2+-binding protein is the same gene product as the intestinal Ca2+-binding protein whose synthesis is dependent on vitamin D.     

Human spleen histone H1. Isolation and amino acid sequence of a main variant, H1b

The complete amino acid sequence of a main variant, H1b, of human spleen histone H1 was determined, following previous determinations of human spleen histones H2B, H2A, H3, and H4. High-performance liquid chromatography on C8 silica of the H1 fraction yielded the homogeneous H1b subfraction; this variant was estimated to account for 60% of the total of the four H1 variants. The sequence determination was performed with four main fragments, I to IV, obtained by limited chymotryptic digestion of H1b. Together with direct sequencing by automated Edman degradation of fragments II, III, and IV, fragment I, blocked at the N-terminal, and fragment IV, the C-terminal half the H1b molecule, were sequenced after further digestion with staphylococcal protease and others. The four fragments were aligned with three overlapping peptides each derived from chymotryptic partial fragments, I-II and I-II-III, and intact H1b. Carboxypeptidase digestion of intact H1b confirmed the C-terminal sequence of the molecule. Thus, the total sequence of H1b was completely determined; it consists of a total of 218 amino acid residues, has a molecular weight of 21,734 in the unmodified form, and is completely acetylated at the N-terminal serine residue and partially methylated at the lysine residue 25. This sequence is compared with two mammalian somatic H1 sequences.     

Hemagglutinin 222D/G Polymorphism Facilitates Fast Intra-Host Evolution of Pandemic (H1N1) 2009 Influenza A Viruses

The amino acid substitution of aspartic acid to glycine in hemagglutinin (HA) in position 222 (HA-D222G) as well as HA-222D/G polymorphism of pandemic (H1N1) 2009 influenza viruses (A(H1N1)pdm09) were frequently reported in severe influenza in humans and mice. Their impact on viral pathogenicity and the course of influenza has been discussed controversially and the underlying mechanism remained unclarified. In the present study, BALB/c mice, infected with the once mouse lung- and cell-passaged A(H1N1)pdm09 isolate A/Jena/5258/09 (mpJena/5258), developed severe pneumonia. From day 2 to 3 or 4 post infection (p.i.) symptoms (body weight loss and clinical score) continuously worsened. After a short disease stagnation or even recovery phase in most mice, severity of disease further increased on days 6 and 7 p.i. Thereafter, surviving mice recovered. A 45 times higher virus titer maximum in the lung than in the trachea on day 2 p.i. and significantly higher tracheal virus titers compared to lung on day 6 p.i. indicated changes in the organ tropism during infection. Sequence analysis revealed an HA-222D/G polymorphism. HA-D222 and HA-G222 variants co-circulated in lung and trachea. Whereas, HA-D222 variant predominated in the lung, HA-G222 became the major variant in the trachea after day 4 p.i. This was accompanied by lower neutralizing antibody titers and broader receptor recognition including terminal sialic acid α-2,3-linked galactose, which is abundant on mouse trachea epithelial cells. Plaque-purified HA-G222-mpJena/5258 virus induced severe influenza with maximum symptom on day 6 p.i. These results demonstrated for the first time that HA-222D/G quasispecies of A(H1N1)pdm09 caused severe biphasic influenza because of fast viral intra-host evolution, which enabled partial antibody escape and minor changes in receptor binding.     

Primary structure of the two variants of a sperm-specific histone H1 from the annelid Platynereis dumerilii

The amino acid sequences of the two variants (H1a 121 residues and H1b 119 residues) of the sperm-specific histone H1 from the polychaete annelid Platynereis dumerilii have been completely established. Comparison of the sequences of these two variants shows one deletion of two residues in histone H1b and 22 substitents, of which most occur in the globular domain. The two variants differ highly in a sequence of nine residues adjacent to the conservative phenylalanine residue of histone H1 (64-72 in H1a, 62-70 in H1b) which makes H1a less hydrophobic than H1b. The small molecular size of Platynereis H1a and H1b is a unique feature among the histones H1 of which the size ranges between 189 residues (chicken erythrocyte H5) and 248 residues (sea urchin sperm H1). H1a and H1b have short N- and C-terminal basic domains but the size of the globular domain (approximately equal to 80 residues) is similar to that of other H1s. In the globular region the variant H1a exhibits a close relationship with somatic or sperm H1s whereas the variant H1b is more related to H5 histones.     

High-Resolution Mapping of H1 Linker Histone Variants in Embryonic Stem Cells

H1 linker histones facilitate higher-order chromatin folding and are essential for mammalian development. To achieve high-resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H1⁰ displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d, and H1e causes pericentric chromocenter clustering and de-repression of major satellites. These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.     

Species- and cell-specific expression of H1 histones in tissue culture cells

The H1 histones of 25 tissue culture cell types of rat, mouse, human, and Chinese hamster origin were studied by ion-exchange chromatography. Fractionation of H1 histones revealed that each cell type expressed multiple H1 histone subfractions. The average number of four subfractions was the same as seen in tissues. The individual H1 histones exhibited species-specific chromatographic properties, and different cell lines from a given animal were capable of expressing cell-specific quantities of the H1 histone subtypes. Neither the species- nor cell-specific properties of the H1 histones could be abolished by alkaline phosphatase treatment, but such treatment did reduce the complexity of H1 histone subfractions in SV40-transformed cells. The direct comparison of rat liver and rat hepatoma cell (H-35) H1 histones by chromatographic and electrophoretic methods demonstrated that a cell line was capable of expressing the same variety of H1 histones as the tissue of origin. Variations in the content of H1 histone subtypes were seen in intraspecies comparisons in all four species. The variations were not reduced by recloning selected cells. The variations among subfractions were dramatic for Chinese hamster cell lines CHO, V79, and DON. Smaller variations could be seen in mouse cell lines and numerous genetic mutant human fibroblast cells and HeLa cells, but the latter did not appear to be specifically related to disease state. Since H1 histones link nucleosomes, the packing of nucleosomes in the chromatin of tissue culture cells has the same potential variability as in tissues.     

Natural allelic variation of duck erythrocyte histone H1b

In our previous work (J. Palyga, Genetic polymorphisms of histone H1. b in duck erythrocytes. Hereditas 114, 85-89, 1991) we reported a genetic polymorphism of duck erythrocyte histone H1.b. Here, we screened H1 preparations in a two-dimensional polyacrylamide gel to refine the distribution of allelic forms of H1.b in fifteen duck populations. We have revealed that the frequency of H1.b allelic variants was significantly different among many conservative and breeding duck groups. While b(1) and b(3) were common in all populations screened, the allele b(2), with a slightly lower apparent molecular weight, was confined mainly to brown-feathered ducks (Khaki Campbell and Orpington) and descendent lines.The C- and N-terminal peptides released upon cleavage with N-bromosuccinimide and Staphylococcus aureus protease V8 from duck allelic histones H1. b2 and H1.b3, respectively, migrated differently in the gel, probably as a result of potential amino acid variation in a C-terminal domain.