Repeating oligonucleosomal units. A new element of chromatin structure.

Supranucleosomal chromatin structure has been analysed by the use of histone H1 polymers crosslinked in nuclei and extended chromatin with bifunctional reagents methyl-4-mercaptobutyrimidate (MMB) and dimethyl suberimidate dihydrochloride. Almost pure H1 homopolymers were obtained in milligram amounts and examined for the distribution in molecular weights. The H1 homopolymer molecules both from nuclei and chromatin have been found to be integer multiples of an elementary structure (called "clisone") consisting of 12 histone H1 molecules. This finding strongly suggests that nucleosomal chains of chromatin are not uniform but rather organized as repeating oligonucleosomal units each consisting of 12 nucleosomes. Correlation between oligonucleosomal structures in nuclei and chromatin implies that a linearized nucleosomal chain retains the information on chromatin superstructure. The relation of the disclosed 12-nucleosome units to superbeads (nucleomeres) and other structures is discussed.     

Mutual arrangement of histone H1 molecules in extended chromatin. Chymotryptic digestion of cross-linked H1 histone dimers

Mutual arrangement of histone H1 molecules in chromatin extended in low salt-EDTA buffer and additionally in the presence of urea was studied by means of reversible cross-linking combined with chymotryptic digestion. In the chromatins tested, the chymotryptic halves of H1 were cross-linked in all possible combinations; i.e., C-C, C-N and N-N. The results imply that the mutual arrangement of H1 histones is determined by the structure of extended nucleosomal chain, rather than chromatin superstructure.     

Proximity and accessibility studies of histones in nuclei and free nucleosomes.

Histone proximity in chromatin was studied with the cleavable crosslinking reagent, dithiobissuccinimidyl propionate. Crosslinks between H4 and H2a, H4 and H2b, H4 and H3, H2a and H2b, H2b and H3 were found. H1 is also crosslinked to the nucleosomal histones. In nuclei, unsheared chromatin, and H1 depleted chromatin, the four nucleosomal histones are crosslinked at similar relative rates both in 5 mM salt and 100 mM salt. After micrococcal nuclease treatment to generate nucleosomes, H2a and H2b are crosslinked faster than H4 and H3. C14-NEM titration of thiopropionate residues bound to each histone shows that H2a and H2b are more accessible to this reagent after nuclease treatment but that the increased binding was not sufficient by itself to explain the increase in crosslinking. Bolton Hunter reagent was used to further study the accessibility of the four nucleosomal histones in whole chromatin and nuclease digested chromatin. These studies showed that salt increases the accessibility of all four histones while nuclease treatment decreases H4 accessibility.     

Interaction of sperm histone variants and linker DNA during spermiogenesis in the sea urchin

Several physical properties of sea urchin spermatid chromatin, which contains phosphorylated Sp H1 and Sp H2B histone variants, and mature sperm chromatin, in which these histones are dephosphorylated, were compared. Density, thermal stability, average nucleosomal repeat length, and resistance to micrococcal nuclease digestion are all increased in mature sperm relative to spermatid chromatin. Since the chromatins are identical in histone variant subtypes, the altered physical properties are not a consequence of changes in histone primary structure during spermiogenesis. The data are interpreted to mean that dephosphorylation of the N-terminal regions of Sp H1 and Sp H2B in late spermatid nuclei permits strong ionic binding of these highly basic regions to the extended linker, stabilizing the highly condensed structure of sperm chromatin.     

Internucleosome interactions: detecting the dinucleosome fragmentation of chromatin using micrococcal nuclease. Heterogeneity of nucleosomes and localization of histone H1

The content of histone H1 (H1/H4 ratio) in dinucleosomes with the DNA of various length liberated from L-cell nuclear chromatin by micrococcal nuclease was analyzed. It was found that the histone H1 content in the dichromatosome is two times as low as that in the largest dinucleosome and in the complete mononucleosome. The set of chromatin fragments liberated from the Triton X-100 pretreated nuclei differs considerably from that of chromatin sites devoid of histone H1 (the de novo replicating chromatin and the chromatin formed on the undermethylated DNA). A scheme for asymmetric distribution of histone H1 with molecules oriented along the nucleosomal fibril, which reflects the peculiarities of chromatin fragmentation by micrococcal nuclease with predominant liberation of the dichromatosome, is proposed.     

Structure, chemical modification and interaction of histone H1 with chromatin components

Structure, chemical modification, and interaction of histone H1 and its individual fragments with DNA and structural elements of chromatin are considered. Special attention is paid to phosphorylation of histone H1 molecules. Recent data concerning localization and mobility of histone H1 in chromatin as well as mechanisms of nucleosomal chain condensation are reviewed.     

Sperm histones and chromatin structure of the "primitive" sea urchin Eucidaris tribuloides

The "primitive" sea urchin Eucidaris tribuloides resembles the advanced sea urchins (euechinoids) in many respects, yet some features of its biochemistry and morphogenesis are more similar to other echinoderms such as starfish or sea cucumbers. Two unique characteristics of the sperm chromatin of all known euechinoids are an extremely long average nucleosomal repeat length and the presence of two male germ-line-specific histone variants, Sp H1 and Sp H2B. Histone composition and nucleosomal repeat length of the sperm chromatin of Eucidaris were compared to those of several euechinoids and a starfish. Eucidaris sperm chromatin contained large H1 and H2B histone variants typical of euechinoids. The H1 was about nine amino acids smaller than Sp H1 of the advanced urchin Strongylocentrotus purpuratus. Its Sp H2B molecules were the same size as in the euechinoids. Peptide maps showed that N-terminal regions of Sp H1 and Sp H2B contained repeating basic amino acid motifs characteristic of euechinoids. The smaller size of Eucidaris H1 is accounted for by a smaller C-terminal region. The repeat length of Eucidaris sperm chromatin was slightly shorter than that of two euechinoids, but significantly larger than starfish, which lacks a large H2B. The Sp H2B gene of Eucidaris was expressed during spermatogenesis in the same cell types as for S. purpuratus. Thus Sp histone subtype expression and chromatin structure in this distantly related echinoid closely resemble the euechinoids. The presence of an Sp H2B and a very long repeat length appear to be characteristic of the echinoids only.     

Developmentally Regulated Linker Histone H1c Promotes Heterochromatin Condensation and Mediates Structural Integrity of Rod Photoreceptors in Mouse Retina

Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H1⁰ triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.     

Identification of specific functional subdomains within the linker histone H10 C-terminal domain

Linker histone binding to nucleosomal arrays in vitro causes linker DNA to form an apposed stem motif, stabilizes extensively folded secondary chromatin structures, and promotes self-association of individual nucleosomal arrays into oligomeric tertiary chromatin structures. To determine the involvement of the linker histone C-terminal domain (CTD) in each of these functions, and to test the hypothesis that the functions of this highly basic domain are mediated by neutralization of linker DNA negative charge, four truncation mutants were created that incrementally removed stretches of 24 amino acids beginning at the extreme C terminus of the mouse H1(0) linker histone. Native and truncated H1(0) proteins were assembled onto biochemically defined nucleosomal arrays and characterized in the absence and presence of salts to probe primary, secondary, and tertiary chromatin structure. Results indicate that the ability of H1(0) to alter linker DNA conformation and stabilize condensed chromatin structures is localized to specific C-terminal subdomains, rather than being equally distributed throughout the entire CTD. We propose that the functions of the linker histone CTD in chromatin are linked to the characteristic intrinsic disorder of this domain.     

Characterization and nucleosomal core localization of Achlya histones involved in stress-induced chromatin condensation

To better understand the basis for heat shock-induced chromatin condensation in Achlya, a further characterization of the histones of this organism was carried out. The nucleosomal location (i.e., core vs linker), partial peptide map, and electrophoretic behavior of each Achlya histone was determined and compared to the well-characterized histones of rabbit kidney. The results of this and previous studies suggest that in Achlya, no nucleosome linker-associated histone analogous to histone H1 of higher eucaryotes is observed and that the Achlya histone designated alpha is a novel nucleosomal core histone. These observations may reflect the existence of a mechanism of stress-induced chromatin condensation which does not involve histone H1.     

Features of the chromatin structure of erythrocytes depending on the properties of lysine-rich histones

A comparative analysis of chromatin from erythrocytes of frog, trout and hen has been performed in correlation with properties of the nucleosomal linker histones of H1 family. In the nucleosomes from frog erythrocytes the linker histone is represented by H1(0)-like variant with amino acid sequence highly homologous to that of the hen histone H5, however the arginine content in the proteins differs (3 mol% in the frog erythrocyte H1 and 12 mol% in the hen erythrocyte H5). On the other hand histone H5 from trout being significantly different in the primary structure from the hen histone H5 is at the same time rich in arginine (9 mol%). The nucleosomal repeat length, estimated by using agarose gel electrophoresis is 201, 213 and 213 b.p. in erythrocyte chromatin from frog, trout and hen, correspondingly. Chromatin packing density in fixed nuclei from erythrocytes of frog, trout and hen as determined using cytophotometric measurements is 0.144, 0.444 and 530 pg/mu 3, correspondingly. The data support the previously made suggestion that the increase in arginine content in nucleosomal linker proteins is connected with the increase of chromatin compaction in the nuclei and elongation of the linker in the nucleosome.     

Chemical crosslinking of histone H1° to histone neighbours in nuclei and chromatin

Crosslinking of histones in mouse liver nuclei and extended chromatin with a bifunctional reagent leads to the formation of H1H1° heterodimers as well as H1°H1° homodimers. H1° can be also crosslinked to the core histones. Thus, the location of histone H1° within the basic repeating chromatin structure seems to be analogous to that of H1 histone.     

Principles of nucleosome recognition by chromatin factors and enzymes

The recent torrent of structures of chromatin complexes determined by cryoelectron microscopy provides an opportunity to discern general principles for how chromatin factors and enzymes interact with their nucleosome substrate. We find that many chromatin proteins use a strikingly similar arginine anchor and variant arginine interactions to bind to the nucleosome acidic patch. We also observe that many chromatin proteins target the H3 and H2B histone fold α1-loop1 elbows and the H2B C-terminal helix on the nucleosomal histone face. These interactions with the histones can be complemented with interactions with and distortions of nucleosomal DNA.     

Characteristics of limited trypsinolysis of histone H5 in a solution and as a component of chromatin with different degrees of compactness

Trypsinolysis of histone H5 in solution and as a component of chromatin with different level of compactization was studied. It was demonstrated that the existence of supernucleosomal organization leads to a significant decrease of the degradation rate of histones H1 and H5 in comparison with histones H2A, H2B, H3 and H4. Analysis of trypsinolysis electrophoretic spectra of histone H5 revealed the existence of protease-resistant fragments in chromatin, but not in solution. These fragments contain not only the globular domain of histone H5 but also small-sized unstructured N- and/or C-terminal regions. The peptides were identified with the help of an immune serum specific for the globular region of histone H5. The possible role of resistant fragments in the nucleosomal organization of chromatin is discussed.     

Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase

Yeast disruptor of telomeric silencing-1 (DOT1) is involved in gene silencing and in the pachytene checkpoint during meiotic cell cycle. Here we show that the Dot1 protein possesses intrinsic histone methyltransferase (HMT) activity. When compared with Rmt1, another putative yeast HMT, Dot1 shows very distinct substrate specificity. While Rmt1 methylates histone H4, Dot1 targets histone H3. In contrast to Rmt1, which can only modify free histones, Dot1 activity is specific to nucleosomal substrates. This was also confirmed using native chromatin purified from yeast cells. We also demonstrate that, like its mammalian homolog PRMT1, Rmt1 specifically dimethylates an arginine residue at position 3 of histone H4 N-terminal tail. In surprising contrast, methylation by Dot1 occurs in the globular domain of nucleosomal histone H3. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis suggests that H3 lysine 79 is trimethylated by Dot1. The intrinsic nucleosomal histone H3 methyltransferase activity of Dot1 is certainly a key aspect of its function in gene silencing at telomeres, most likely by directly modulating chromatin structure and Sir protein localization. In agreement with a role in regulating localization of histone deacetylase complexes like SIR, an increase of bulk histone acetylation is detected in dot1- cells.     

Histone H2A C-terminus regulates chromatin dynamics, remodeling, and histone H1 binding

The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A C-terminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformation.     

HMG-D and histone H1 alter the local accessibility of nucleosomal DNA

There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.     

Quantitative analysis of HP1alpha binding to nucleosomal arrays

Elucidating how the metazoan genome is organised into distinct functional domains is fundamental to understanding all aspects of normal cellular growth and development. The "histone code" hypothesis predicts that post-translational modifications of specific histone residues regulate genomic function by selectively recruiting nuclear factors that modify chromatin structure. A paradigm supporting this hypothesis is the preferential binding of the silencing protein heterochromatin protein 1 (HP1) to histone H3 trimethylated at K9. However, a caveat to several in vitro studies is that they employed histone N-terminal tail peptides to determine dissociation constants, thus ignoring any potential role of DNA and/or the underlying chromatin structure in the recruitment of HP1. Using a well-defined in vitro chromatin assembly system (employing a 12-208 DNA template), we describe here, the use of a fluorescence spectroscopic method that enabled us to measure and quantify the relative binding affinities of HP1alpha to unmodified and variant nucleosomal arrays. Using this approach, we previously demonstrated that mouse HP1alpha (i) binds with high affinity to naked DNA, (ii) has an intrinsic affinity for highly folded chromatin, (iii) has a 2-fold higher affinity for nucleosomal arrays when H2A is replaced with H2A.Z, and (iv) binds to DNA or chromatin in a non-cooperative manner.