Histone H1d Research Articles

Involvement of the linker histone H1Foo in the regulation of oogenesis.

In oocytes, chromatin structure is loosened during their growth, which seems to be essential for the establishment of competence to accomplish the maturation and further development after fertilization. This paper shows that a linker histone variant, H1foo, is involved in the formation of loosened chromatin structure in growing oocytes. During oogenesis, oocytes show a unique mode of division and gene expression patterns. Chromatin structure is thought to be involved in the regulation of these processes. In this study, we investigated the functions of linker histones, which modulate higher-order chromatin structure during oogenesis. Because H1foo is highly expressed in oocytes, we knocked down H1foo using siRNA and observed oocyte growth, maturation, and fertilization. However, H1foo knockdown had no effect on any of these processes. Overexpression of H1b or H1d, which has a high ability to condense chromatin and is expressed at a low level in oocytes, resulting in tightened chromatin and a decreased success rate of oocyte maturation. By contrast, overexpression of H1a, which is expressed at a high level in oocytes and has a low ability to compact chromatin, did not affect growth or maturation. Therefore, H1a, but not other variants, might compensate for the function of H1foo in H1foo-knockdown oocytes. These results implicate H1foo in the formation of loose chromatin structure, which is necessary for oocyte maturation. In addition, the low expression of somatic linker histone variants, for example, H1b and H1d, is important for loosened chromatin and meiotic progression.

Spermatid-specific linker histone HILS1 is a poor condenser of DNA and chromatin and preferentially associates with LINE-1 elements

BackgroundLinker histones establish and maintain higher-order chromatin structure. Eleven linker histone subtypes have been reported in mammals. HILS1 is a spermatid-specific linker histone, and its expression overlaps with the histone–protamine exchange process during mammalian spermiogenesis. However, the role of HILS1 in spermatid chromatin remodeling is largely unknown.ResultsIn this study, we demonstrate using circular dichroism spectroscopy that HILS1 is a poor condenser of DNA and chromatin compared to somatic linker histone H1d. Genome-wide occupancy study in elongating/condensing spermatids revealed the preferential binding of HILS1 to the LINE-1 (L1) elements within the intergenic and intronic regions of rat spermatid genome. We observed specific enrichment of the histone PTMs like H3K9me3, H4K20me3 and H4 acetylation marks (H4K5ac and H4K12ac) in the HILS1-bound chromatin complex, whereas H3K4me3 and H3K27me3 marks were absent.ConclusionsHILS1 possesses significantly lower α-helicity compared to other linker histones such as H1t and H1d. Interestingly, in contrast to the somatic histone variant H1d, HILS1 is a poor condenser of chromatin which demonstrate the idea that this particular linker histone variant may have distinct role in histone to protamine replacement. Based on HILS1 ChIP-seq analysis of elongating/condensing spermatids, we speculate that HILS1 may provide a platform for the structural transitions and forms the higher-order chromatin structures encompassing LINE-1 elements during spermiogenesis.

160 A dynamic model for the linker histone

Linker histones play an important role in the packing of chromatin. This family of proteins generally consists of a short, unstructured N-terminal domain, a central globular domain, and a C-terminal domain (CTD). The CTD, which makes up roughly half of the protein, is intrinsically disordered in solution but adopts a specific fold upon interaction with DNA (Fang et al., 2012). While the globular domain structure is well characterized, the structure of the CTD remains unknown. Sequence alignment alone does not reveal any significant homologs for this region of the protein. Construction of a model thus requires additional information. For example, the atomic model for the rat histone H1d CTD, proposed over a decade ago, used novel bioinformatics tools and biochemical data (Bharath et al., 2002). New fluorescence resonance energy transfer (FRET) studies of the folding of the CTD in the presence of linear DNA, single nucleosomes, and oligonucleosomal arrays (Caterino et al., 2011; Fang et al., 2012) have stimulated our interest in constructing a dynamic model of the protein. We have obtained preliminary information about the structure and dynamics of the linker histone CTD through ab initio folding simulations using the Rosetta modeling package (Rohl et al., 2004). By analyzing a large number of conformations sampled through a Monte Carlo procedure, we get a clearer picture of the preferred states of the protein and its dynamics. Our results show that the CTD may frequently adopt a structure with 3–5 helices and helix-turn-helix motifs in specific regions. Some of the best scoring structures show high similarity with the HMG-box-containing proteins previously used as templates by Bharath et al. Further clustering analysis of our results hints of a preferred set of conformations for the CTD of the linker histone. Comparison of these models with distances measured by FRET may help account for the distinct structures of the CTD observed upon binding to different macromolecular partners.

Sodium tungstate (Na2WO4) exposure increases apoptosis in human peripheral blood lymphocytes

The potential for adverse health effects of using tungsten and its alloys in military munitions are an important concern to both civilians and the US military. The toxicological implications of exposure to tungsten, its alloys, and the soluble tungstate (Na2WO4) are currently under investigation. To examine tungstate toxicity, a series of experiments to determine its in vitro effects on cells of the immune system were performed. We identified alterations in isolated human peripheral blood lymphocytes (PBL) treated in vitro with sodium tungstate (0.01, 0.1, 1.0, and 10 mM). Analyses of apoptosis with annexin V and propidium iodide revealed a dose- and time-dependent increase in the quantity of cells in early apoptosis after tungstate exposure. Reductions in the number of cells entering into the cell cycle were also noted. Exposure of PBL to tungstate (1 mM) and Concanavalin A (ConA) for 72 h reduced the number of cells in S and G2/M phases of the cell cycle. There were alterations in the numbers of cells in G0/G1, S, and G2/M phases of the cell cycle in long-term THP-1 (acute leukemic monocytes) cultures treated with tungstate (0.01, 0.1, 1.0, and 10 mM). Gel electrophoresis, silver staining, and LC-MS/MS showed the cytoplasmic presence of histone H1b and H1d after 72 h of tungstate exposure. The addition of tungstate to cultures resulted in significant reductions in the quantity of interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and IL-6 produced by stimulated [CD3/CD28, ConA, or lipopolysaccharide (LPS)] and tungstate-treated lymphocytes. Taken together, these data indicate that tungstate increases apoptosis of PBL, alters cell cycle progression, reduces cytokine production, and therefore warrants further investigation.

A K52Q substitution in the globular domain of histone H1t modulates its nucleosome binding properties

A comparison of the globular domain sequences of the somatic H1d and testis-specific H1t revealed a single substitution of lysine 52 in H1d to glutamine 54 in H1t, which is one of the three crucial residues within the second DNA binding site. The globular domains of both histones were modeled using the crystal structure of chicken GH5 as a template and was also docked onto the nucleosome structure. The glutamine residue in histone H1t forms a hydrogen bond with main chain carbonyl of methionine-52 (in H1t) and is spatially oriented away from the nucleosome dyad axis. A consequence of this change was a lower affinity of recombinant histone H1t towards Four-way junction DNA and reconstituted 5S mononucleosomes. When Gln-54 in Histone H1t was mutated to lysine, its binding affinity towards DNA substrates was comparable to that of histone H1d. The differential binding of histones H1d and H1t towards reconstituted mononucleosomes was also reflected in the chromatosome-stop assay.

Molecular modeling of the chromatosome particle.

In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by molecular modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homology modeling methods, revealed a three-helical bundle fold that could be described as a helix-turn-helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that observed by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was observed that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.

The rat histone H1d gene has intragenic activating sequences that are absent from the testis-specific variant H1t

In some cases core histone genes in the mouse depend on intragenic sequence elements for high level expression [Gene 176 (1996) 1]. Here we report that the highly expressed gene for rat linker histone H1d also contains an intragenic activating region (IAR). Using transient transfection assays in mouse fibroblast NIH3T3 cells, we showed that rat H1d contains a downstream region (+21 to +116) that imparts a two- to threefold up-regulation of fused reporters. This region also activated expression when moved to the promoter region, though the effect was dependent on its distance from other promoter elements. The IAR contains sequence homologies to the core α and Ω elements identified as functional protein binding sites within the mouse H3.2 coding region activating sequence (CRAS). A pair of Ω elements (+32 and +66) accounts for the activating effect of the H1d intragenic region as shown by targeted mutations as well as stepwise deletions. The H1d and H3.2 Ω sequences bound similar and perhaps identical proteins by gel shift analysis. The H1d α-like sequence at +56 overlaps the translational start codon and was therefore not mutated. Like the mouse H3.2 α element, it bound transcription factor YY1 in gel shift assays. H1t, the gene for the testis-specific linker histone, did not demonstrate an IAR. While H1t has a similar α sequence and did bind YY1, it lacks the Ω homologies of H1d. Sequence comparison shows that the YY1/α site as well as the adjacent Ω site are likely present in genes for other standard H1 variants, but that the +32 Ω site in the 5′ untranslated region (UTR) of H1d is unique. We conclude that the +32 and +66 Ω sequences of the rat H1d gene contribute significantly to its high-level expression.

Identification of a 34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing domain by site-directed mutagenesis.

The C-terminus of histone H1 is necessary for the folding of polynucleosomal arrays into higher-order structure(s) and contains octapeptide repeats each having DNA binding S/TPKK motifs. These repeat motifs were earlier shown to mimic the DNA/chromatin-condensing properties of the C-terminus of histone H1 (Khadake, J. R., and Rao, M. R. S. (1995) Biochemistry 36, 1041-1051). In the present study, we have generated a series of C-terminal mutants of rat histone H1d and studied their DNA-condensation properties. The single proline to alanine mutation in the S/TPKK motifs either singly or in combination resulted in only a 20% decrease in the DNA-condensation property of histone H1. Deletion of all the three S/TPKK motifs resulted in a 45% decrease in DNA condensation. When the three octapeptide repeats encompassing the S/TPKK motifs were deleted, there was again a 45% decrease in DNA condensation. On the other hand, when the entire 34 amino acid stretch (residue 145-178) was deleted, there was nearly a 90% decrease in DNA condensation brought about by histone H1d. Interestingly, deletion of the 10 amino acid spacer between the octapeptide repeats (residues 161-170) also reduced the DNA condensation by 70%. Deletion of the region (residues 115-141) immediately before the 34 amino acid stretch and after the globular domain and the region (residues 184-218) immediately after the 34 amino acid stretch had only a marginal effect on DNA condensation. The importance of the 34 amino acid stretch, including the 10 amino acid spacer, was also demonstrated with the recombinant histone H1d C-terminus. We have also determined the induced alpha-helicity of histone H1 and its various mutants in the presence of 60% trifluoroethanol, and the experimentally determined induced helical contents agree with the theoretical predictions of secondary structural elements in the C-terminus of histone H1d. Thus, we have identified a 34 amino acid stretch in the C-terminus of histone H1d as the DNA-condensing domain.

Sp1 and Sp3 activate the testis-specific histone H1t promoter through the H1t/GC-box.

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in large part through elements located within the proximal promoter. Previous work in transgenic animals showed that a unique 40 bp promoter element designated H1t/TE is essential for spermatocyte-specific expression. The H1t/TE element contains a GC-box, which is a perfect consensus binding site for members of the Sp family of transcription factors. We have shown that Sp1 and Sp3 are present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids and that they can bind to the H1t/GC-box. Mutagenesis of the GC-box reduced H1t promoter activity. Furthermore, a CpG dinucleotide within the GC-box was totally unmethylated in rat testis primary spermatocytes where the gene is transcribed but it was methylated in liver where the gene is silenced. These previous studies supported the importance of the GC-box and suggested that Sp transcription factors contribute to expression of the H1t gene. In this study, we show that co-transfection of Sp1 and Sp3 expression vectors leads to an upregulation of histone H1t promoter activity in several cell lines including testis GC-2spd cells. However, very low H1t promoter activity is seen in GC-2spd cells grown at 39 degrees C, which correlates with lower levels of Sp1 and Sp3 in these cells grown at this elevated temperature. Upregulation of the H1t promoter by Sp1 and Sp3 was also seen in cotransfected NIH3T3 and C127I cell lines. On the other hand, co-transfection of the Sp1 and Sp3 expression vectors does not lead to upregulation of activity of the cell-cycle dependent histone H1d promoter.

Antibacterial peptides in stimulated human granulocytes: characterization of ubiquitinated histone H1A.

Antibacterial peptides were isolated from human peripheral granulocytes of a healthy donor who had been treated with granulocyte-colony stimulating factor (G-CSF) and cortisol. Peptides were solubilized in acidified chloroform/methanol, and partitioned in chloroform/methanol/water. Water- soluble polypeptides were separated by cation-exchange and reversed-phase chromatography. Several previously characterized antibacterial polypeptides were identified; defensins 1-3, defensin 4, lysozyme, eosinophil cationic protein, and calgranulin A. In addition, several histone fragments were isolated and exhibited activity against the Gram- positive bacterium Bacillus megaterium strain Bm11. These fragments included two C-terminal fragments of histone H1A, three C-terminal fragments of histone H1D, one fragment of histone H1B, and two fragments of histone H4. The molecular masses of both histone H1A fragments, as determined by electrospray (ES) MS, were 270 Da higher than those calculated from their amino acid sequences. The two histone H1A fragments corresponded to Lys152-Lys222 (7527 +/- 1 Da) and Lys167-Lys222 (6023 +/- 1 Da). Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa fragments indicated that the post-translational modification is on Lys222, the epsilon-amino group of which was conjugated with the alpha-carboxyl group of the tripeptide Arg-Gly-Gly. This finding was substantiated by digestion of the 7.5-kDa polypeptide with trypsin and analysis of the resulting peptides by ES MS and MS/MS. The tripeptide Arg-Gly-Gly corresponded uniquely to the three C-terminal residues of ubiquitin, demonstrating the presence of ubiquitinated histone H1A.

Localization of upstream elements involved in transcriptional regulation of the rat testis-specific histone H1t gene in somatic cells.

The testis-specific histone H1t is synthesized exclusively in late pachytene primary spermatocytes during spermatogenesis. The mechanisms involved in transcriptional repression of the H1t gene during development before the spermatocyte stage and in later stages of germinal cell maturation and in nonexpressing somatic tissues are unknown. To assess the contribution of the upstream DNA sequence to H1t transcriptional silencing in nonexpressing cells, a set of histone H1t-promoted reporter vectors was constructed. Transient transfection of mouse C127I cells with these reporter vectors allowed us to identify a transcriptional silencer located between 948 base pairs (bp) and 780 bp upstream from the H1t transcriptional initiation site. Histone H1t-promoted luciferase activity increased 4-fold when the region between 948 bp and 875 bp upstream from the transcriptional initiation site was eliminated. Addition of a 73-bp rat H1t promoter fragment (-948 to -875, containing the 5' portion of the silencer region) to a site immediately upstream from the histone H1d proximal promoter led to significantly reduced luciferase expression upon transient transfection (56% in C127I cells and 44% in HeLa cells). Nuclear proteins were found to bind to DNA within the H1t silencer region when assayed by in vitro deoxyribonuclease (DNase) I footprinting. Thus, our data suggest that an active transcriptional silencer mechanism involving a specific and autonomous H1t promoter element (nucleotides -948/-875) may be operative to minimize expression of the H1t gene in nontesticular cells.

Expression of rat histone H1d in Escherichia coli and its purification.

Histone H1 is involved in the folding of linear polynucleosomal filament into a 30-nm fiber. In an effort to understand the role of different domains of histone H1 in chromatin folding, we have now expressed rat histone H1d in Escherichia coli using pTrc99A expression vector by providing a 6-His tag at the C-terminus to facilitate its purification. The expressed protein histone H1d was purified from the soluble extract of E. coli by employing Ni2+ NTA-agarose and heparin-agarose chromatography. The recombinant histone H1d was shown to be authentic by its N-terminal amino acid analysis, its secondary structural characteristics, and its ability to (a) condense DNA and (b) bind specifically to synthetic four-way junction DNA.

Histone H1 isoforms purified from rat liver bind nonspecifically to the nuclear factor 1 recognition sequence and serve as generalized transcriptional repressors.

Two polypeptides with molecular masses of 34 and 30 kDa were copurified from rat liver during DNA affinity purification of a sequence-specific transcription factor binding to the footprint II sequence within the P2 promoter of the rat alpha1B adrenergic receptor (alpha1B AR) gene, and were identified by microsequencing their endoproteinase Lys-C-derived peptides as histone H1d and histone H1c, respectively. Histone H1 was previously reported to bind to the nuclear factor 1 (NF1) recognition sequence, although the specificity of this binding has been controversial. Here, DNA mobility shift and supershift assays, DNase I footprinting and mutational analyses indicated that the binding of histone H1 to the NF1 sites located within footprint II of the alpha1B AR gene P2 promoter is nonspecific. Transient cotransfections into Hep3B cells of histone H1d cDNA with CAT constructs containing promoter regions of different genes resulted in generalized and non-specific suppression of CAT activity. The histone H1d-mediated repression of the activities of the alpha1B AR gene P2/CAT or beta2 AR gene P(-186/1307)/CAT constructs was reversed by the cotransfection of a cDNA encoding the sequence-specific transcription factor NF1/X, and the fold increase in CAT activities was similar to that obtained in the absence of histone H1d. These results suggest that sequence-specific transcription factors counteract the histone H1-mediated transcriptional repression in vivo by a true activation, which is different from the in vitro antirepression in histone H1-repressed chromatin templates (Laybourn and Kadonaga, (1991) Science 254: 238-245).

Condensation of DNA and chromatin by an SPKK-containing octapeptide repeat motif present in the C-terminus of histone H1.

Several DNA binding motifs have been described in the C-terminus of histone H1 (Churchill & Travers, 1991), of these the S/TPKK repeat (Suzuki, 1989) often occurs as a part of an octapeptide repeat of the type XTPKKXKK. We have studied in detail the DNA and chromatin condensing properties of a consensus octapeptide KSPKKAKK (8 mer) present in many histone H1 subtypes and its imperfect repeat ATPKKSTKKTPKKAKK (16 mer TPKK) as it occurs in the C-terminus of rat histone H1d. The 16 mer TPKK peptide containing two S/TPKK motifs was able to condense both rat oligonucleosomal (2-5 kbp) DNA and histone H1-depleted chromatin as revealed by circular dichroism spectroscopy. The 8 mer peptide, however, was unable to condense either the DNA or the histone H1-depleted chromatin. Both the 8 mer peptide and the 16 mer TPKK peptide displaced distamycin A from the drug-DNA complex, although with different efficiency, indicating that while these two peptides could bind DNA, only the 16 mer (TPKK) peptide could bring about condensation of DNA and histone H1-depleted chromatin. A mutant 16 mer (TAKK) peptide wherein two proline residues are replaced by alanine, was ineffective in bringing about condensation of both DNA and histone H1-depleted chromatin. These results suggest that the two beta-turn structures present in the 16 mer (TPKK) peptide could be important in facilitating binding to different regions of duplex DNA thereby bringing about close packing and condensation. The condensation property of the 16 mer (TPKK) peptide was very similar to that of histone H1 in terms of (a) its preference for AT rich DNA, (b) cooperativity of condensation, and (c) salt dependence of condensation. The 16 mer (TPKK) peptide, but not the 8 mer peptide or the 16 mer (TAKK) peptide, could form complexes with a polynucleosomal 5S DNA core resulting in retarded mobility similar to the complexes formed with histone H1 on agarose gel electrophoresis.

Preferential condensation of SAR-DNA by histone H1 and its SPKK containing octapeptide repeat motif

Linker histone H1 binds preferentially the scaffold associated region (SAR) DNA elements that contain characteristic oligo dA·dT tracts. In the present study, we have compared the condensation brought about by histone H1 of a SAR DNA fragment in the histone spacer region of Drosophila melanogaster with that of a random DNA (pBR322 EcoRI- SalI) fragment by circular dichroism spectroscopy. The condensation of the SAR DNA fragment by histone H1 is 3–4-fold higher than that of the random DNA fragment. A 16-mer peptide, ATPKKSTKKTPKKAKK, the sequence that is present in the C-terminus of histone H1d, which has recently been shown to possess DNA and chromatin condensing properties, also condenses the SAR DNA fragment preferentially in a highly cooperative manner. We have proposed a model for the dynamics of chromatin structure involving histone H1-SAR DNA interaction through SPKK containing peptide motifs and its competition by AT-hook peptides present in the nonhistone chromosomal proteins like HMG-I and HMG-Y.

Chromosome mapping of rat histone genes H1fv, H1d, H1t, Th2a and Th2b.

Chromosome assignment of the rat histone genes H1t, H1d (H1.4), H1fv (H10), Th2a and Th2b is described. The testicularly expressed histone genes H1t, Th2a and Th2b could be assigned to rat chromosome (RNO) 17 by PCR analysis of somatic cell hybrid DNAs. The H1d gene was mapped to RNO17p12-->p11 by FISH. These genes might form a histone gene cluster homologous to that found on HSA6p21.3 in humans and MMU13A2-3 in mice. The rat histone H1fv gene was assigned to RNO7 by PCR. This result allows the inclusion of rat H1fv to an established conserved group of syntenic genes in rat, mouse and human on chromosomes RNO7, MMU15 and HSA22, respectively.

DNA- and chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec; H1t is a poor condenser of chromatin.

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using both acid and salt extraction procedures. Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA. Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

A rat histone H2B pseudogene is closely associated with the histone H1d gene

A 9 kb EcoRI restriction fragment was isolated from a recombinant phage out of a rat genomic library. This DNA fragment contains a rat H1d histone gene, its flanking sequences and a H2B histone pseudogene closely associated with the H1d gene. A comparison of the H2B pseudogene with human H2B genes flanking regions reveals sequence homologies to a human H2B histone gene (Albig, W. et al. (1991) Genomics 10, 940–948).

Alteration in proportions of histone H1 variants during the differentiation of murine erythroleukaemic cells.

We have investigated the changes in the relative amounts of histone H1 zero and all five H1 variants during the differentiation in vitro of Friend erythroleukaemic cells. Three different agents were used as inducers of differentiation: dimethyl sulphoxide, hexamethylenebisacetamide and sodium butyrate. By applying a combination of reverse-phase h.p.l.c. and one-dimensional gel electrophoresis we observed that, during differentiation in vitro, (1) the relative amount of each subtype changes upon induction and that (2) dimethyl sulphoxide and hexamethylenebisacetamide produce a similar histone H1 pattern with a strong increase in histones H1 zero and H1c, a modest increase in histone H1e and a decrease in the relative amounts of histone H1a, H1b and H1d, whereas butyrate induces a different pattern, particularly with respect to both histones H1c and H1e: H1c increased slightly, and H1e strongly, during differentiation. These results are compared with changes in the histone H1 pattern during differentiation in vivo in the mouse [Lennox & Cohen (1983) J. Biol. Chem. 258, 262-268] and in the rat [Pina, Martinez & Suau (1987) Eur. J. Biochem. 164, 71-76], and similarities and deviations are discussed.

Purification and partial sequencing of inhibitory factor on renal membrane adenylate cyclase in pancreatic cancer extract: Identity with histones H1B or H1D

Inhibitory activity on renal membrane adenylate cyclase (AC) has previously been found in the extract of a pancreatic cancer associated with humoral hypercalcemia of malignancy (HHM). AC inhibitor was purified employing inhibition of AC activity of renal membrane stimulated by forskolin as its index. N-terminal 9 residues and a digested fragment of purified protein (14 residues) were completely consistent with that of histones H1b and H1d. Not only histone H1 but also histones H2A, H2B and H3 from calf thymus inhibited AC activity. These results indicate that the AC inhibitor in the pancreatic cancer extract is histone H1b or H1d and histones H2A, H2B and H3 also have an AC inhibitory activity.