Characterization of post-translational modifications of the linker histones H1 and H5 from chicken erythrocytes using mass spectrometry

Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified all six major isoforms of the chicken histone H1 (H101, H102, H103, H110, H11R and H11L) and, in addition, the specialist avian isoform H5. In all the histone variants, both the acetylated and nonacetylated N (alpha)-terminal peptides were identified. Mass spectrometry analysis also enabled the identification of a wide range of post-translational modifications including acetylation, methylation, phosphorylation and deamidation. Furthermore, a number of amino acids were identified that were modified with both acetylation and methylation. These results highlight the extensive modifications that are present on the linker histone proteins, indicating that, similar to the core histones, post-translational modifications of the linker histones may play a role in chromatin remodelling and gene regulation.     

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.     

Ethylene-Mediated Programmed Cell Death during Maize Endosperm Development of Wild-Type and shrunken2 Genotypes

We characterized the progression of programmed cell death during maize (Zea mays L.) endosperm development of starchy (Su; wild-type) and shrunken2 (sh2) genotypes and tested the involve ment of ethylene in mediating this process. Histological and viability staining demonstrated that endosperm cell death was initiated earlier and progressed more rapidly in sh2 endosperm compared with Su endosperm. Internucleosomal DNA fragmentation accompanied endosperm cell death and occurred more extensively in sh2 endosperm. 1-Aminocyclopropane-1-carboxylic acid levels peaked approximately 16 d after pollination (dap) in Su endosperm and gradually decreased during subsequent development, whereas two large 1-aminocyclopropane-1-carboxylic acid peaks were observed in sh2 endosperm, the first between 16 and 20 dap and the second at 36 dap. Ethylene levels were elevated in sh2 kernels compared with Su kernels, with an initial peak 20 dap approximately 3-fold higher than in Su kernels and a second peak 36 dap approximately 5-fold higher than that in Su kernels. Ethylene treatment of Su kernels resulted in earlier and more extensive endosperm cell death and DNA fragmentation. Aminoethoxyvinylglycine treatment of sh2 kernels reduced the extent of DNA fragmentation. We conclude that ethylene is involved in triggering programmed cell death in developing maize endosperm and is responsible for the aberrant phenotype of sh2 kernels.     

Interaction of maize chromatin-associated HMG proteins with mononucleosomes: role of core and linker histones

Two groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified. We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini. In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Analyses of linker histone--chromatin interactions in situ.

Recent studies, using cytometric techniques based on fluorescence microscopy, have provided new information on how linker histones interact with chromatin in vivo or in situ. In particular, the use of green fluorescent proteins (GFPs) has enabled detailed studies of how individual H1 subtypes, and specific motifs in them, interact with chromatin in vivo. Furthermore, the development of cytochemical methods to study the interaction between linker histones and chromatin using DNA-binding fluorochromes as indirect probes for linker histone affinity in situ, in combination with highly sensitive and specific analytical methods, has provided additional information on the interactions between linker histones and chromatin in several cell systems. Such results verified that linker histones have a substantially higher affinity for chromatin in mature chicken erythrocytes than in frog erythrocytes, and they also indicated that the affinity decreased during differentiation of the frog erythrocytes. Furthermore, in cultured human fibroblasts, the linker histones showed a relatively high affinity for chromatin in interphase, whereas it showed a significantly lower affinity in highly condensed metaphase chromosomes. This method also enables the analysis of linker histone affinity for chromatin in H1-depleted fibroblasts reconstituted with purified linker histones. No consistent correlation between linker histone affinity and chromatin condensation has so far been detected.     

Histone gene expression during sea urchin embryogenesis: isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity

We have examined the molecular mechanisms responsible for the shifts in histone protein phenotype during embryogenesis in the sea urchinStrongylocentrotus purpuratus. The H1, H2A, and H2B classes of histone synthesized at the earliest stages of cleavage are heterogeneous: These proteins are replaced at late embryogenesis by a different set of histone-like polypeptides, some of which are also heterogeneous. The H3 and H4 histones appear to be homogeneous classes and remain constant. We have isolated from both early and late embryos the individual messenger RNAs coding for each of the multiple protein subtypes. The RNAs were isolated by hybridization to cloned DNA segments coding for a single histone protein or by elution from polyacrylamide gels. Each RNA was then analyzed and identified by its mobility on polyacrylamide gels and by its template activity in the wheat germ cell-free protein synthesizing system. The mRNAs for each of the five early histone protein classes are heterogeneous in size and differ from the late stage templates. The late mRNAs consist of at least 11 separable types coding for the 5 classes of histones. Each of the 11 has been separated and identified. The late stage proteins were shown to be authentic histones since many of their templates hybridize with histone coding DNA. The early and late stage mRNAs are transcribed from different sets of histone genes since (1) late stage H1 and H2A mRNAs fail to hybridize to cloned early stage histone genes under ideal conditions for detecting homologous early stage hybrids, (2) late stage H2B, H3, and H4 RNA/DNA hybrids melt at 14, 11, and 11°C lower, respectively, than do homologous RNA/DNA hybrids, and (3) purified late stage mRNAs direct the synthesis of the variant histone proteins which are synthesized only during later stages. The time course of synthesis of the late stage mRNAs suggests that they appear many hours before the late histone proteins can be detected, possibly as early as fertilization. In addition, early mRNAs are synthesized in small quantities as late as 40 hr after fertilization, during gastrulation. Thus, the major modulations of histone gene expression are neither abrupt nor an absolute on-off switch, and may represent only a gradual and relative repression of early gene expression. Two histones are detected only transiently during early cleavage. The mRNA for one of them, a subtype of H2A, can be detected in the cytoplasm for as long as 40 hr after fertilization. However, template activity for the other, a subtype of H2B, can be detected only at the blastula stage. Thus, the histone genes represent a complex multigene family that is developmentally modulated.     

<Emphasis Type="Italic">Mytilus edulis</Emphasis> Core Histone Genes Are Organized in Two Clusters Devoid of Linker Histone Genes

Abstract Comparison of histone gene cluster arrangements in several species has revealed a broad spectrum of histone gene patterns. To elucidate the core histone gene organization in a mollusk, we have analyzed a Mytilus edulis genomic library and have isolated eight phage clones containing core histone genes. Analysis of insert DNA revealed that the core histone genes are arranged as regular gene repeats of all four core histones. The repeats do not contain linker histone genes. The clones are distributed into two groups of dissimilar repeated units with a common size of about 5.6 kb. The genes of each core histone class in the distinct repeats encode identical histone proteins and have comparable gene arrangements in the two repeat units. However, the intergenic sequences differ significantly. The core histone genes are organized as large clusters of about 100 repeats each. Previously, we have shown that the linker histone genes in M. edulis are also organized in a cluster of repeats of solitary H1 genes. Hence, this is the first case of a separate, clustered organization of both core and linker histone genes, repectively.     

C-terminal phosphorylation of murine testis-specific histone H1t in elongating spermatids

Previous studies gave differing results as to whether the testis-specific histone H1t was phosphorylated during rodent spermatogenesis. We show here that histones extracted from germ cell populations enriched with spermatids at different stages of development in rat testes reveal an electrophoretic shift in the position of H1t to slower mobilities in elongating spermatids as compared to that from preceding stages. Alkaline phosphatase treatment and radioactive labeling with (32)P demonstrated that the electrophoretic shift is due to phosphorylation. Mass spectrometric analysis of histone H1t purified from sexually mature mice and rat testes confirmed the occurrence of singly, doubly, and triply phosphorylated species, with phosphorylation sites predominantly found at the C-terminal end of the molecule. Furthermore, using collision-activated dissociation (CAD) and electron transfer dissociation (ETD), we have been able to identify the major phosphorylation sites. These include a new, previously unidentified putative H1t-specific cdc2 phosphorylation site in linker histones. The presence of phosphorylation at the C-terminal end of H1t and the timing of its appearance suggest that this post-translational modification is involved in the reduction of H1t binding strength to DNA. It is proposed that this could participate in the opening of the chromatin fiber in preparation for histone displacement by transition proteins in the next phase of spermiogenesis.     

Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

The histone database: a comprehensive WWW resource for histones and histone fold-containing proteins

The Histone Database (HDB) is an annotated and searchable collection of all full-length sequences and structures of histone and non-histone proteins containing the histone fold motif. These sequences are both eukaryotic and archaeal in origin. Several new histone fold-containing proteins have been identified, including Spt7p, and a few false positives have been removed from the earlier version of HDB. Database contents include compilations of post-translational modifications for each of the core and linker histones, as well as genomic information in the form of map loci for the human histone gene complement, with the genetic loci linked to Online Mendelian Inheritance in Man (OMIM). Conflicts between similar sequence entries from a number of source databases are also documented. Newly added to the HDB are multiple sequence alignments in which predicted functions of histone fold amino acid residues are annotated. The database is freely accessible through the WWW at http://genome.nhgri.nih.gov/histones/     

Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin

An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5' and 3' nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour.     

Characterization of post-translational modifications of histone H2B-variants isolated from Arabidopsis thaliana

Eukaryotic DNA is structurally packed into chromatin by the basic histone proteins H2A, H2B, H3, and H4. There is increasing evidence that incorporation and post-translational modifications of histone variants have a fundamental role in gene regulation. While modifications of H3 and H4 histones are now well-established, considerably less is known about H2B modifications. Here, we present the first detailed characterization of H2B-variants isolated from the model plant Arabidopsis thaliana. We combined reversed-phase chromatography with tandem mass spectrometry to identify post-translational modifications of the H2B-variants HTB1, HTB2, HTB4, HTB9, and HTB11, isolated from total chromatin and euchromatin-enriched fractions. The HTB9-variant has acetylation sites at lysines 6, 11, 27, 32, 38, and 39, while Lys-145 can be ubiquitinated. Analogous modifications and an additional methylation of Lys-3 were identified for HTB11. HTB2 shows similar acetylation and ubiquitination sites and an additional methylation at Lys-11. Furthermore, the N-terminal alanine residues of HTB9 and HTB11 were found to be mono-, di-, or trimethylated or unmodified. No methylation of arginine residues was detected. The data suggest that most of these modification sites are only partially occupied. Our study significantly expands the map of covalent Arabidopsis histone modifications and is the first step to unraveling the histone code in higher plants.