Hyper(ADP-ribosyl)ation of histone H1

Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P]ADP-ribosylated histones on first-dimension acid-urea or acid-urea-Triton gels and on second-dimension acid--urea--cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H1(0). Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.     

Qualitative differences in nuclear proteins correlate with neuronal terminal differentiation

1. Protein composition of neuronal nuclei was studied at two stages of brain maturation, i.e., before (embryonic day 16; E16) and after (postnatal day 10; P10) shortening of the nucleosomal repeat length. Glial nuclei were analyzed in parallel as a control. 2. Total nuclear or HCl- and 5% perchloric acid (PCA)-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1 zero with differentiation, although the H1 class undergoes an overall decrease. 4. The chromatin of mature neurons is also enriched in the ubiquinated form of histone H2A (A24), while the high-mobility group (HMG) proteins 1 and 2 seem to decrease slightly relative to core histones. 5. Both quantitative and qualitative differences in the abundance of nonhistone proteins relative to histones accompany neuronal terminal differentiation.     

Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions.

BACKGROUND: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. METHODS: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. RESULTS: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. CONCLUSIONS: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.     

The sites of deposition of newly synthesized histone.

The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomalous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new H1, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.     

Analysis of putative high-mobility-group (HMG) proteins in neuronal and glial nuclei from rabbit brain

Putative high-mobility-group (HMG) proteins 1, 2, and 17 were detected in neuronal and glial nuclei isolated from the cerebral hemisphere of rabbit brain. Although divergent chromatin structures are present in these two populations of brain nuclei (i.e., neuronal nuclei exhibit a short DNA repeat length), no differences were apparent in the electrophoretic mobilities of putative HMG proteins 1, 2, and 17 on SDS gels.     

THE HISTONES ASSOCIATED WITH CONDENSED AND EXTENDED CHROMATIN OF MOUSE LIVER

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin. Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels. Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin. However, minor differences were found. A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2A1 and F2A2 differed in the two types of chromatin. The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples. Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions.     

Structural organization of the meiotic prophase chromatin in the rat testis

Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA.     

Methylation of Chromosomal Proteins in Neuronal and Glial Nuclei Purified from Cerebral Hemispheres of Rat During Postnatal Development

The process of methylation of chromosomal proteins [histones and nonhistone proteins (NHP)] in neuronal and glial cell nuclei obtained from cerebral hemispheres of rats at 1, 10, and 30 days of age was investigated. Purified neuronal and glial nuclei were incubated in the presence of S-adenosyl[methyl-3H]methionine. Histone and NHPs were extracted and fractionated by polyacrylamide gel electrophoresis. The results obtained indicate remarkable differences in the process of methylation of histones and NHPs between neuronal and glial nuclei, especially during the first period of postnatal development. In both nuclear populations the histone fraction H3 was labeled to a greater degree than the other fractions and showed the major changes during postnatal development. The densitometric and radioactive patterns of NHPs show considerable changes in the two nuclear populations at the various ages examined. The main difference between neuronal and glial nuclei consists in the intense methylation of proteins with a molecular weight of approximately 100,000, which are present in neuronal nuclei and virtually absent in glial ones. The results obtained may be correlated with the different chromatin structures of neuronal and glial nuclei and with the patterns of maturation and differentiation of neuronal and glial cells during postnatal development.     

Histone gene expression and chromatin structure in mammalian cell hybrids

DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1 histone subfractions of the B82 mouse cells. Because these hybrids exhibit the nucleosome repeat length only of the PG19 cells, it appears that if histone H1 plays a role in determining the repeat length it does so in consort with other nonhistone chromosomal proteins.     

Chromatin fine structure of the histone gene complex of Drosophila melanogaster.

We have used salt extractions of nuclei and long agarose gels to dissect the chromatin fine structure of the histone gene repeat of Drosophila melanogaster. Extraction of nuclei with 0.35 M KCl removes many non-histone chromosomal proteins but does not significantly disturb the overall nucleosome arrangement of the repeat unit. After extraction of nuclei with 0.55 M KCl, which also removes histone Hl, the basic arrangement of nucleosome core particles in the repeat unit is not greatly disturbed and the exposed DNA segments near the 5' ends of the histone genes are also retained. Extraction of nuclei with 0.75 M or higher KCl concentrations causes extensive nucleosome sliding and rearrangement with accompanying changes in the nucleoprotein organization of the histone gene complex and loss of the 5' hypersensitive sites. Our results indicate that the histone gene repeat displays a highly organized chromatin structure in vivo.     

Binding form of pollen mother cell protein in the nucleosomes of lily

We have previously reported the existence of pollen mother cell nuclear protein (PMCP) which appears during microsporogenesis in lily (Lilium spesiosum). It is very similar to mammalian testis specific H1 histone, H1t. In this paper, we describe the PMCP distribution in lily nucleosomes. Isolated nuclei were treated with micrococcal nuclease, and template active and inactive chromatin fractions were prepared. The nucleosome repeat length of pollen mother cells was determined to be 210 base pairs. The majority of the PMCP was found in the template inactive chromatin fraction, similar to other histones. PMCP was contained in the nucleosome monomer, but not in the core particle. However, PMCP was mainly found in the nucleosome dimer when slightly digested. Salt extraction from isolated nuclei indicated that PMCP and H1 histone share similar binding affinities to DNA. Judging from our results, it seems probable that PMCP links two core particles more strongly than H1 histone does. Since it is known that meiotic chromatin includes nick transferase and nuclease activity, one possible role of PMCP is the protection of its own chromatin. Other possible functions of PMCP are also discussed.     

Structure of chromatin from the pigeon brain. I. Composition, electrophoretic motility and circular dichroism spectra of nucleosome particles

Composition and structural properties of pigeon brain hemisphere neuron chromatin have been studied. In these cells nucleosomal DNA repeat length is about 165 nucleotide pairs. The content of H1 and H2A histones was found to decrease by 18 and 30% respectively in comparison with the chromatin possessing the normal quantity of those histones. At the same time the content of protein uH2A (A-24), being the conjugate of H2A histone and ubiquitine, is increased. Mononucleosomes isolated from neuron chromatin was found to have relatively low electrophoretic mobility in polyacrylamide gel taking into account the size of their DNA fragment. Circular dichroism spectra of nucleosome particles show that the neuron mononucleosomes are more unfolded than the rat thymus ones. Data obtained allow to suggest that the short DNA repeat and accumulation of protein uH2A in neurons are the factors influencing the compactization of neuron chromatin.     

Histones from Saccharomyces cerevisiae

Total histones and histone fractions isolated from Saccharomyces cerevisiae chromatin were analysed by polyacrylamide gel electrophoresis. The presence of the four histone fractions H2a, H2b, H3 and H4 was demonstrated. In addition, yeast chromatin contained a protein similar to histone H1 from mammals in molecular weight, charge and association properties with Triton X-100. However, it had a much lower lysine to arginine ratio, equal to about 3, as compared with H1 histones from higher eukaryotes. The order of electrophoretic mobilities of yeast histone fractions in acidic urea-polyacrylamide gels was similar to that observed for histones from plant sources, i.e. H4>H3>H2a>H2b>H1. Previously undetected protein (protein X) was extracted from yeast chromatin with 5 % HClO₄. The properties of this protein are under investigation.     

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.     

Differential association of linker histones H1 and H5 with telomeric nucleosomes in chicken erythrocytes.

Rat liver telomeric DNA is organised into nucleosomes characterised by a shorter and more homogeneous average nucleosomal repeat than bulk chromatin as shown by Makarov et al. (1). The latter authors were unable to detect the association of any linker histone with the telomeric DNA. We have confirmed these observations but show that in sharp contrast chicken erythrocyte telomeric DNA is organised into nucleosomes whose spacing length and heterogeneity are indistinguishable from those of bulk chromatin. We further show that chicken erythrocyte telomeric chromatin contains chromatosomes which are preferentially associated with histone H1 relative to histone H5. This contrasts with bulk chromatin where histone H5 is the more abundant species. This observation strongly suggests that telomeric DNA condensed into nucleosome core particles has a higher affinity for H1 than H5. We discuss the origin of the discrimination of the lysine rich histones in terms of DNA sequence preferences, telomere nucleosome preferences and particular constraints of the higher order chromatin structure of telomeres.     

Chromatin structure from the marine sponge Geodia cydonium

The histones isolated from the siliceous sponge Geodia cydonium have been separated using two electrophoretic techniques. A comparison of their mobilities with those of calf thymus and rat liver show that some Geodia histone species (H3, H1 and H1(0) exhibit electrophoretic variance. The results show, that as in other eukaryotic systems the sponge chromatin contains the core histones (H2A, H2B, H3 and H4) and the linker histone (H1). ADP-ribosylation of Geodia histones and separation of the individual histones by electrophoresis resulted in four histones being radiolabeled. Digestion of Geodia chromatin with endogenous endonuclease is shown to result in the formation of nucleosome particles containing approximately 200 base pairs of DNA. A major product of endogenous endonuclease digestion is a relatively stable 110 base pair intermediate. Incubation of chromatin with DNase II and separation of the products under denaturing conditions reveals 20 bands migrating at 10 base intervals.