H1 Content Research Articles

Extent of histone modifications and H1 0 content during cell cycle progression in the presence of butyrate

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1 0 during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain >98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1 0, H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1 0 phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1 0 in chromatin during the S or G2 phases of the cell cycle.

Nucleotide-stimulated proteolysis of histone H1.

We have identified a proteolytic system that selectively degrades histone H1 in normal human lymphocytes. Treatment of permeabilized human lymphocytes with a series of nucleotides produced a marked decrease in their histone H1 content compared to untreated cells. The nucleotide-stimulated process was selective for histone H1 because gel electrophoresis showed that almost all other lymphocyte protein bands remained constant while histone H1 disappeared. The elimination of histone H1 appears to be the result of proteolysis by a trypsin-like enzyme because it was inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor, and diisopropyl fluorophosphate. Proteolysis was stimulated by P1,P4-di(adenosine-5') tetraphosphate, P1,P3-di(adenosine-5') triphosphate, P1,P5-di(adenosine-5') pentaphosphate, adenosine 5'-tetraphosphate, ATP, adenosine 5'-[alpha, beta-methylene]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, ADP, CTP, GTP, UTP, dATP, or pyrophosphate, whereas AMP, adenosine, adenosine diphosphoribose, NAD+, cAMP, or sodium phosphate did not show this stimulation of proteolysis. ATP, [alpha, beta-methylene]ATP, [beta, gamma-methylene]ATP, and pyrophosphate all stimulated proteolysis, suggesting that a pyrophosphate linkage was necessary for this process. Thus, resting human lymphocytes contain a trypsin-like protease that is stimulated by nucleotides or pyrophosphate to selectively degrade histone H1.

Histone H1 depletion with human cellular aging: Biochemical and cytochemical evidences.

Biochemical and immunocytochemical examinations of histone H1 in G1 arrested and senescent human fibroblasts were performed to pursuit the mechanisms of human cell aging in vitro. Examinations of histone synthesis in serum depleted or contact inhibited cell cultures indicated that the ratio of histone synthesis over DNA synthesis and molar synthesis of histone H1 to nucleosomal histones did not decline with accumulation of resting cell populations, while senescent cells had a lower ratio of histone H1 synthesis than young cells. Histone H1 contents plotted as a function of DNA synthesis activity revealed that senescent cultures had a lower content of histone H1 than young cultures at any stage of cell proliferation.Immunocytological study using antiserum against histone H1 on cocultivated young and senescent cells showed that immunofluorescence intensity was very low in senescent nuclei in comparison with young cell nuclei. Thus, we conclude that a decline in content and synthesis of histone H1 relative to nucleosomal histones with passage number was not simply due to the passage-related accumulation of resting cells, but actually reflects age-specific changes in cycling cells or whole cell populations in the senescent cultures. The depletion of histone H1 in chromatin was discussed as a possible cause of DNA strand break and relaxation of gene repression.

Chemical alterations in cell envelopes of polymyxin-resistant mutants of Pseudomonas aeruginosa grown in the absence or presence of polymyxin.

The polymyxin-resistant mutant strains H181 and H185 of Pseudomonas aeruginosa, after growth in the absence or presence of polymyxin, were compared with the polymyxin-sensitive H103 strain as to their cell envelope protein composition (determined by slab polyacrylamide gel electrophoresis) and their lipid composition. When grown in the absence of polymyxin, the H181 and H185 strains had an increased content of the outer membrane protein H1 with a decreased content of the outer membrane proteins D2 and F. After growth in the presence of polymyxin, the content of H1, D2, and F were all decreased. Significant alterations in the lipid composition of the H181 and H185 strains were found after growth in the absence of polymyxin. These lipid alterations were enhanced upon growth in the presence of polymyxin, with both strains having a reduced content of phosphatidylethanolamine and phosphatidylglycerol and an increased content of diphosphatidylglycerol and an unidentified lipid thought to be a neutral lipid. Other workers have proposed that an increased content of H1 protein is the molecular basis for polymyxin resistance in the H181 and H185 strains. Our observations make this hypothesis appear unlikely.

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1 0 protein was predominant in G 0 cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.

Effect of growth stage on histone H1 metabolism in human diploid fibroblasts.

Metabolism of histone H1 at different stages of cell growth was investigated in order to get a better understanding of the role of histone H1 in the cell growth of human diploid fibroblasts. Histone H1 content exhibited some fluctuation during the culture stage of cell growth. When cells entered confluent phase, the ratio of histone H1 to total histones decreased significantly. Histone H1 had a turnover half-life of 80 hours whereas nucleosomal histones did not significantly turn-over regardless of the growth stage. DNA synthesis was drastically diminished with increased cell density whereas histone synthesis was less sensitive to contact-inhibition. The gradual decline of histone H1 content with increased cell density suggests that its degradation is slightly superior to its residual synthesis. When the confluent cells were seeded at low density, cell proliferation resumed and histone H1 was synthesized and deposited to chromatin in a greater amount than nucleosomal histones, thus resulting in an abrupt increase of histone H1 content. The possible role of histone H1 metabolism in normal cells is discussed.

Structure of transcriptionally active and inactive nucleosomes from butyrate-treated and control HeLa cells.

Nuclei from butyrate-treated or control HeLa cells were separated into micrococcal nuclease sensitive and resistant chromatin. Those regions most sensitive to the nuclease, amounting to some 10% of the chromatin, consisted mainly of mononucleosomes with equimolar amounts of the inner histones H2A, H2B, H3, and H4, very little H1, and equimolar amounts of the two small high-mobility group (HMG) proteins, HMG-14 and -17. Both in butyrate-treated and in control cells, these nuclease sensitive monomers were some 5--7-fold enriched in DNA sequences which are transcribed into cytoplasmic polyadenylated RNA, while resistant monomers are depleted in the same sequences. Electrophoretic analyses of the transcriptionally active mononucleosomes revealed heterogeneity. Several subcomponents were resolved when monomers of butyrate-treated or control cells were electrophoresed at low ionic strength. Active monomer subcomponents differ in their molar content of HMG-14 and -17, in their content of H1 and A24, and in the length of their DNA. Some minor differences between nucleosomes of butyrate-treated and control cells were observed.

Accumulation of histone H1(0) during chemically induced differentiation of murine neuroblastoma cells.

A basic nuclear protein with properties similar to lysine-rich histones accumulates during differentiation in vitro of C1300 murine neuroblastoma cells (clone Neuro-2a) induced by either n-butyrate, dimethyl sulfoxide, hexamethylene bisacetamide or 1,6-dibutyryl-adenosine 3',5'-monophosphate. A protein with the same electrophoretic properties, present in adult mouse brain cell nuclei in amounts similar to those in the induced neuroblastoma cells, has been characterized as histone H1(0). Digestion of the two proteins with Staphylococcus aureus V8 protease gives identical peptide maps (sharing no common peptides with those of histones H1A or H1B), which indicates that the induced protein qualifies as H1. Accumulation of histone H1(0) in neuroblastoma cell nuclei accompanies shutoff of DNA synthesis and transgression of the cells into a G1-phase resting state. Upon removal of n-butyrate the cells resume proliferation and their H1 content decreases, indicating an association of H1 with the resting state during which differentiated cellular functions are expressed.

Exchange of histone H1 between segments of chromatin.

Abstract We have asked whether histone H1 is tightly bound in chromatin at relatively low ionic strength (below physiological) or whether it can exchange between binding sites. We have studied this question in chromatin fragments generated by digestion with micrococcal nuclease, by mixing two fragments of known H1 content and different length (either a fragment radioactively labelled in all its histones with an unlabelled fragment, or two labelled fragments, only one of which contains H1) and then separating them again by centrifugation in sucrose gradients in order tom reexamine their H1 contents. At very low ionic strength (5 m m -Tris · HCl (pH 7.1), 1 m m -Na2EDTA, 0.5 m m -phenylmethylsulphonyl fluoride) there was very little (less than 5 to 10%) exchange of H1. In contrast, the presence of 70 m m -NaCl in the buffer caused rapid and complete equilibration of H1 between sites in less (possibly much less) than about one hour. At 30 m m added NaCl, the result was intermediate between those at 0 m m and 70 m m added NaCl, partial exchange occurring with a half-time of one to two hours. The results were essentially the same whether or not both fragments contained H1. We infer from these results that at physiological ionic strength (~150 m m ) there will be rapid and complete equilibration of H1 between sites in chromatin. We do not know whether the exchange of particular H1 subtypes is restricted to a particular class of binding site.

Comparative studies on pancreas chromatin proteins: Species specificity and behaviour during rat pancreas regeneration

1. 1. Chromatin proteins of pancreas were separated into UP, HP and NP fractions and compared, in two aspects, following their electrophoresis on SDS-polyacrylamide gels: species specificity and behaviour during pancreas regeneration. 2. 2. Some species specificity was stated only for UP proteins of rat and calf pancreas. 3. 3. Rat pancreas acinar cell regeneration was accompanied by the following changes in chromatin protein composition: the decrease of 20,000, 62,000, 79,000 and the increase of 14,000, 49,000 components in UP proteins, the lower amount of 18,000 component in NP proteins and the decrease of H1 content in HP fraction.

Histone H1 from prophase aggregates DNA better than histone H1 from S phase

Published data from Physarum polycephalum plasmodia show a correlation between chromosome condensation before metaphase in the cell cycle and a six-fold increase in phosphate content of histone H1. Histone H1 was isolated from naturally synchronous plasmodia of Physarum either in S phase or in early prophase. The interaction of H1 with DNA was studied using a turbidity assay and the results showed that H1 from early prophase was more effective at causing turbidity with DNA than was H1 from S phase, consistent with the proposed role of H1 phosphorylation in chromosome condensation.

Dependence of mononucleosome deoxyribonucleic acid conformation on the deoxyribonucleic acid length and H1/H5 content. Circular dichroism and thermal denaturation studies.

Four mononucleosome preparations were isolated from micrococcal nuclease digests of chicken erythrocyte nuclei which differed in average deoxyribonucleic acid (DNA) length and in H1 and H5 content. The circular dichroism properties of the unperturbed mononucleosome preparations and the corresponding H1- and H5-depleted species demonstrate that the nucleoprotein spectra above 250 nm are all altered relative to protein-free DNA by the addition of a single negative band at 275 nm, similar to the band observed for psi-DNA. The quantitative analysis of the psi-type band intensity for any of the higher molecular weight unperturbed samples relative to core particle mononucleosomes yielded a constant number of DNA base pairs (approximately 140) contributing to this new band. Upon removal of H1 and H5 from the mononucleosome preparations which have sufficiently long linker DNA, the psi-type band intensity indicates an approximately 30 base pair reduction in the number of core DNA base pairs contributing to the altered circular dichroism properties. The psi-type band is proposed to be due to the compact DNA tertiary structure, i.e., the manner in which the DNA is wound around the histone core allowing interactions between adjacent turns of the superhelix. This interpretation implies that approximately 30 base pairs of core DNA are removed from the unique core tertiary structure when the linker DNA is not bound by H1 or H5. The circular dichroism analysis correlates well with the thermal denaturation properties of mononucleosomes. Removal of H1 and H5 causes an overall reduction in the thermal stability of both core and linker DNA. The degree of destabilization is greatest when the average DNA length is maximum. Some core DNA is lost from the highest temperature melting bands when histone-free DNA is present. These results indicate two regions of different conformational and thermodynamic stability in core DNA. The length of attached linker DNA and its histone content influence the two regions of the core to differing extents.

Rearrangements and reconstitution of mononucleosomes as monitored by thermal denaturation measurements.

Derivative melting profiles of calf thymus mononucleosomes have been examined for changes resulting from variations in solvent pH and ionic strength, histone H1 content, and DNA size. Samples of mononucleosomes were found to rearrange during freeze-drying to form an altered monomer and a series of noncovalent multimers. The derivative melting profiles of these particles differ significantly from those for the untreated monomer and dimer. The noncovalent dimer exhibited a new melting transition at 66 degrees C involving approximately 18 base pairs of DNA normally associated with the highest melting transition. Mononucleosomes were reconstituted from 6 M guanidine hydrochloride to give particles with physical properties including melting profile which were virtually indistinguishable from those of the starting material. This result confirms the notion that no structural domains exist in the histone core that can be irreversible denatured by noncovalent perturbations.

Circular dichroism analysis of mononucleosome DNA conformation.

Mononucleosomes were isolated from micrococcal nuclease digests of chicken erythrocyte nuclei. The circular dichroism properties of mononucleosome preparations, differing in average DNA length and in H1 and H5 content, demonstrate that the spectrum of chromatin is due only to the complete structure of its repeating subunits. The nucleoprotein spectra are all altered relative to protein-free DNA by the emergence of a single negative band at 275 nm, similar to the band observed for psi DNA. The intensity of the psi-type band depends on the proportion of DNA condensed in a specific manner. The psi-type band is proposed to be due to the compact DNA tertiary structure; i.e., the manner in which the DNA is wound around the histone core allowing interactions between adjacent turns of the superhelix. This interpretation attributes changes and variability in nucleoprotein circular dichroism spectra under different experimental conditions to alterations in DNA tertiary structure rather than secondary structure.

Nucleosomes and subnucleosomes: heterogeneity and composition

Previous studies ( Varshavsky, Bakayev and Georgiev, 1976a) have shown that chromatin subunits (mononucleosomes) and their oligomers in a mild staphylococcal nuclease digest of chromatin display a heterogeneous content of histone H1. We now report that a mild staphylococcal nuclease digest of either chromatin or nuclei from mouse Ehrlich tumor cells contains mononucleosomes of three discrete kinds. The smallest mononucleosome (MN 1) contains all histones except H1 and a DNA fragment 140 base pairs (bp) long. The intermediate mononucleosome (MN 2) contains all five histones and a DNA fragment 170 bp long. The third mononucleosome (MN 3) also contains all five histones, but its DNA fragment is longer and more heterogeneous in size (180–200 bp). Most of the MN 3 particles are rapidly converted by nuclease into mononucleosomes MN 1 and MN 2 There exists, however, a relatively nuclease-resistant subpopulation of the MN 3 mononucleosomes. These 200 bp MN 1 particles contain not only histones but also nonhistone proteins, and are significantly more resistant to nuclease than the bulk of MN 3 particles and the smaller mononucleosomes MN 1 and MN 2. There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (SN). The SN 1 is a set of naked double-stranded DNA fragments ∼20 bp long. The SN 2 is a complex of a specific basic nonhistone protein (molecular weight ∼16,000 daltons) and a DNA fragment ∼27 bp long. The SN 3 contains histone H4, the above-mentioned specific nonhistone protein and a DNA fragment ∼27 bp long. The SN 4 contains histones H2a, H2b, H4 and a DNA fragment ∼45 bp long. The SN 5 contains histones H2a, H2b, H3 and a DNA fragment ∼55 bp long. The SN 6 is a complex of histone H1 and a DNA fragment ∼35 bp long. Subnucleosomes SN 7 and SN 8 each contain all the histones except H1, and DNA fragments ∼100 and ∼120 bp long, respectively. Nuclease digestion of isolated mono- or dinucleosomes does not produce some of the subnucleosomes. These and related findings indicate that the cleavage required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- and dinucleosomes.

Studies on chromatin. II. Isolation and characterization of chromatin subunits.

Earlier findings /1-10/ bearing on a subunit organization of chromatin were confirmed and in some points detailed. Besides this, a large-scale isolation of chromatin subunits; their protein composition, electron microscopic appearance and CsCl banding pattern are described. Although the purified chromatin subunit contains all five histones, the relative content of histone H1 i in it is two times lower than that in the original chromatin. tit is shown that a mild digestion of chromatin with staphylococcal nuclease produced not only separate chromatin subunits and their "oligomers' but also deoxyribonucleoprotein particles which sediment more slowly than subunits. It appears that these particles and subunits are produced from different initial structures in the chromatin. Finally, a crystallization of the purified chromatin subunit as a cetyltrimethyl ammonium salt is described.