Recent and rapid amplification of the sperm basic nuclear protein genes in winter flounder

The high molecular weight basic nuclear proteins (HMrBNPs), which are tightly bound to sperm chromatin in winter flounder, are made up of imperfect reiterations of simple peptide sequences that contain phosphorylatable DNA-binding motifs. Genomic Southern blots hybridized with probes to the coding and non-coding regions of HMrBNP mRNA showed that HMrBNP sequences form a complex multi-gene family. Previously, one gene (2B) was used to establish an evolutionary link between histone H1 and the HMrBNPs. Further examination of this complex, multi-gene family has now revealed that the majority of the HMrBNP genes are linked as 4.5 kb direct tandem repeats that each contain a 2.8 kb coding region and a 1.7 kb intergenic region (IR). These findings, combined with the cloning of the IR, established that the tandemly repeated genes lack introns and code for the abundant 3 kb HMrBNP mRNAs that produce the prominent 110 kDa HMrBNP. Southern blotting of DNAs from other righteye flounder species showed that HMrBNP multi-gene families were present in closely related species, though with substantial differences in restriction patterns and band intensities, but were not detected in more distantly related flounders. These observations are consistent with recent and rapid elaboration of the HMrBNP gene family.     

Saccharomyces cerevisiae RAP1 binds to telomeric sequences with spatial flexibility

A wide divergence has been detected in the telomeric sequences among budding yeast species. Despite their length and homogeneity differences, all these yeast telomeric sequences show a conserved core which closely matches the consensus RAP1-binding sequence. We demonstrate that the RAP1 protein binds this sequence core, without involving the diverged sequences outside the core. In Saccharomyces castellii and Saccharomyces dairensis specific classes of interspersed variant repeats are present. We show here that a RAP1-binding site is formed in these species by connecting two consecutive 8 bp telomeric repeats. DNase I footprint analyses specify the binding site as the 13 bp sequence CTGGGTGTCTGGG. The RAP1 protein also binds the variant repeats, although with a lowered affinity. However, a split footprint is produced when RAP1 binds a variant repeat where the two half-sites of the binding site are separated by an additional 6 nt. This is probably caused by the intervening sequence looping out of the RAP1-DNA complex. We suggest that the bipartite subdomain structure of the RAP1 protein allows it to remodel telomeric chromatin, a feature which may be of great relevance for telomeric chromatin assembly and structure in vivo.     

The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain (ΔN-hH1.4). The ΔN-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that ΔN-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.     

Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis

We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).     

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.     

Identification of an ubinuclein 1 region required for stability and function of the human HIRA/UBN1/CABIN1/ASF1a histone H3.3 chaperone complex

The mammalian HIRA/UBN1/CABIN1/ASF1a (HUCA) histone chaperone complex deposits the histone H3 variant H3.3 into chromatin and is linked to gene activation, repression, and chromatin assembly in diverse cell contexts. We recently reported that a short N-terminal fragment of UBN1 containing amino acids 1-175 is necessary and sufficient for interaction with the WD repeats of HIRA and attributed this interaction to a region from residues 120-175 that is highly conserved with the yeast ortholog Hpc2 and so termed the HRD for Hpc2-related domain. In this report, through a more comprehensive and refined biochemical and mutational analysis, we identify a smaller and more moderately conserved region within residues 41-77 of UBN1, which we term the NHRD, that is essential for interaction with the HIRA WD repeats; we further demonstrate that the HRD is dispensable for this interaction. We employ analytical ultracentrifugation studies to demonstrate that the NHRD of UBN1 and the WD repeats of HIRA form a tight 1:1 complex with a dissociation constant in the nanomolar range. Mutagenesis experiments identify several key residues in the NHRD that are required for interaction with the HIRA WD repeat domain, stability of the HUCA complex in vitro and in vivo, and changes in chromatin organization in primary human cells. Together, these studies implicate the NHRD domain of UBN1 as being an essential region for HIRA interaction and chromatin organization by the HUCA complex.     

Effects of H1 histone variant overexpression on chromatin structure

The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic. Here we report a detailed characterization of the chromatin structure of cells overexpressing either H1(0) or H1c. Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H1(0) variant. Overexpression of H1(0) results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H1(0) protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H1(0) and H1c in regulating chromatin structure and function.     

Epigenetic modification of centromeric chromatin: hypomethylation of DNA sequences in the CENH3-associated chromatin in Arabidopsis thaliana and maize

The centromere in eukaryotes is defined by the presence of a special histone H3 variant, CENH3. Centromeric chromatin consists of blocks of CENH3-containing nucleosomes interspersed with blocks of canonical H3-containing nucleosomes. However, it is not known how CENH3 is precisely deposited in the centromeres. It has been suggested that epigenetic modifications of the centromeric chromatin may play a role in centromere identity. The centromeres of Arabidopsis thaliana are composed of megabase-sized arrays of a 178-bp satellite repeat. Here, we report that the 178-bp repeats associated with the CENH3-containing chromatin (CEN chromatin) are hypomethylated compared with the same repeats located in the flanking pericentromeric regions. A similar hypomethylation of DNA in CEN chromatin was also revealed in maize (Zea mays). Hypomethylation of the DNA in CEN chromatin is correlated with a significantly reduced level of H3K9me2 in Arabidopsis. We demonstrate that the 178-bp repeats from CEN chromatin display a distinct distribution pattern of the CG and CNG sites, which may provide a foundation for the differential methylation of these repeats. Our results suggest that DNA methylation plays an important role in epigenetic demarcation of the CEN chromatin.     

H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases

Transcribed inverted repeats are potent triggers for RNA interference and RNA-directed DNA methylation in plants through the production of double-stranded RNA (dsRNA). For example, a transcribed inverted repeat of endogenous genes in Arabidopsis thaliana, PAI1-PAI4, guides methylation of itself as well as two unlinked duplicated PAI genes, PAI2 and PAI3. In previous work, we found that mutations in the SUVH4/KYP histone H3 lysine 9 (H3 K9) methyltransferase cause a loss of DNA methylation on PAI2 and PAI3, but not on the inverted repeat. Here we use chromatin immunoprecipitation analysis to show that the transcribed inverted repeat carries H3 K9 methylation, which is maintained even in an suvh4 mutant. PAI1-PAI4 H3 K9 methylation and DNA methylation are also maintained in an suvh6 mutant, which is defective for a gene closely related to SUVH4. However, both epigenetic modifications are reduced at this locus in an suvh4 suvh6 double mutant. In contrast, SUVH6 does not play a significant role in maintenance of H3 K9 or DNA methylation on PAI2, transposon sequences, or centromere repeat sequences. Thus, SUVH6 is preferentially active at a dsRNA source locus versus targets for RNA-directed chromatin modifications.     

Mice with a targeted disruption of the H1t gene are fertile and undergo normal changes in structural chromosomal proteins during spermiogenesis

H1t is an H1 histone variant unique to late spermatocytes and early spermatids. Using gene targeting and embryonic stem cell technologies, we have produced mice with a disrupted H1t gene. Homozygous H1t-null mice have normal fertility and show no obvious phenotypic consequence due to the lack of this histone. Biochemical and immunohistochemical approaches were used to show that normal changes in chromosomal proteins occurred during spermatid development, including the appearance and disappearance of transition proteins 1 and 2. Both protamines 1 and 2 are present in normal amounts in sonication-resistant spermatid nuclei from H1t-null mice. Analysis of H1 histones by quantitative gel electrophoresis in enriched populations of pachytene spermatocytes and round spermatids showed that the lack of H1t is only partially compensated for by somatic H1s, so that the chromatin of these cells is H1 deficient. Because H1t is thought to create a less tightly compacted chromatin environment, it may be that H1-deficient chromatin is functionally similar to chromatin with H1t present, at least with respect to permitting spermatogenesis to proceed.     

Structurally divergent histone H1 variants in chromosomes containing highly condensed interphase chromatin

Condensed and late-replicating interphase chromatin in the Dipertan insect Chironomus contains a divergent type of histone H1 with an inserted KAP-KAP repeat that is conserved in single H1 variants of Caenorhabditis elegans and Volvox carteri. H1 peptides comprising the insertion interact specifically with DNA. The Chironomid Glyptotendipes exhibits a corresponding correlation between the presence of condensed chromosome sections and the appearance of a divergent H1 subtype. The centromere regions and other sections of Glyptotendipes barbipes chromosomes are inaccessible to immunodecoration by anti-H2B and anti- H1 antibodies one of which is known to recognize nine different epitopes in all domains of the H1 molecule. Microelectrophoresis of the histones from manually isolated unfixed centromeres revealed the presence of H1 and core histones. H1 genes of G. barpipes were sequenced and found to belong to two groups. H1 II and H1 III are rather similar but differ remarkably from H1 I. About 30% of the deduced amino acid residues were found to be unique to H1 I. Most conspicuous is the insertion, SPAKSPGR, in H1 I that is lacking in H1 II and H1 III and at its position gives rise to the sequence repeat SPAKSPAKSPGR. The homologous H1 I gene in Glyptotendipes salinus encodes the very similar repeat TPAKSPAKSPGR. Both sequences are structurally related to the KAPKAP repeat in H1 I-1 specific for condensed chromosome sites in Chironomus and to the SPKKSPKK repeat in sea urchin sperm H1, lie at almost the same distance from the central globular domain, and could interact with linker DNA in packaging condensed chromatin.     

Telomeric repeats act as nucleosome-disfavouring sequences in vivo

Telomeric DNAs consist of tandem repeats of G-clusters such as TTAGGG and TG_1-3, which are the human and yeast repeat sequences, respectively. In the yeast Saccharomyces cerevisiae, the telomeric repeats are non-nucleosomal, whereas in humans, they are organized in tightly packaged nucleosomes. However, previous in vitro studies revealed that the binding affinities of human and yeast telomeric repeat sequences to histone octamers in vitro were similar, which is apparently inconsistent with the differences in the human and yeast telomeric chromatin structures. To further investigate the relationship between telomeric sequences and chromatin structure, we examined the effect of telomeric repeats on the formation of positioned nucleosomes in vivo by indirect end-label mapping, primer extension mapping and nucleosome repeat analyses, using a defined minichromosome in yeast cells. We found that the human and yeast telomeric repeat sequences both disfavour nucleosome assembly and alter nucleosome positioning in the yeast minichromosome. We further demonstrated that the G-clusters in the telomeric repeats are required for the nucleosome-disfavouring properties. Thus, our results suggest that this inherent structural feature of the telomeric repeat sequences is involved in the functional dynamics of the telomeric chromatin structure.     

58 Packaging trinucleotide repeats: the effect of CAG/CTG repeats on nucleosome formation

In neurological diseases such as fragile X syndrome, spinal and bulbar muscular atrophy, myotonic dystrophy, and Huntington’s disease, the molecular basis of pathogenicity is the presence of an expanded trinucleotide repeat (TNR) tract (Ashley & Warren, 1995). TNRs implicated in many of these diseases are composed of CAG/CTG repeats. For example, in healthy individuals 5–35, CAG/CTG TNR repeats are present in the huntingtin gene. However, individuals with 40 or greater repeats will develop Huntington’s disease (Andrew et al., 1993). We are particularly interested in how these TNR sequences are packaged in chromatin. Recent evaluations of CAG/CTG TNR sequences in our laboratory have demonstrated that the repeats increase the propensity for the DNA sequences to incorporate into nucleosomes, where nucleosomes represent the minimal unit of packaging in chromatin (Volle & Delaney, 2012). In this work, we are interested in determining the minimum number of CAG/CTG repeats required to confer a significant increase in nucleosome incorporation relative to sequences that lack the TNR sequence. By defining the changes imposed on these fundamental interactions by the presence of a CAG/CTG repeat tract, we will gain insight into the possible interactions that allow for the expansion of these TNR tracts.     

Cooperative binding of the globular domains of histones H1 and H5 to DNA.

In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.     

A novel mouse gene family coding for cationic, cysteine-rich peptides. Regulation in small intestine and cells of myeloid origin

Cryptdin is a Paneth cell corticostatin/defensin in the mouse small bowel. To help define the intestinal role of cryptdin, cryptdin-related sequence (CRS) mRNAs have been characterized with respect to developmental regulation, sequence homology, putative coding function, and occurrence in myeloid cells. Cryptdin, CRS1C, and CRS4C mRNAs are transcribed from separate genes, occur at equivalent abundance in small intestine, and appear in the small bowel in concert during the 2nd and 3rd weeks postpartum. Cryptdin and CRS1C mRNAs are not detectable in adult mouse bone marrow, but probes specific for the 5'- or the 3'-untranslated regions of CRS4C mRNA hybridize to a moderately abundant 1.05-kilobase bone marrow mRNA in contrast to a highly abundant 0.75-kilobase mRNA in small intestine. Nucleotide sequences corresponding to the deduced prepro-coding regions of cryptdin, CRS1C, and CRS4C mRNAs contain a highly conserved 200-base pair region of 92% sequence similarity (CSE.2), but the mRNAs are not homologous otherwise. The deduced CRS1C and CRS4C polypeptides are apparent precursors of secreted, cationic, proline- and cysteine-rich peptides that contain Cys-Pro-X repeats. Unlike cryptdin, however, the proposed CRS1C and CRS4C mature peptide regions lack the structural motif characteristic of defensins. Attempts to find homologies between the putative CRS peptides and existing protein sequences have been unsuccessful, leading us to speculate that CRS1C and CRS4C represent a new family of nondefensin antimicrobial peptides in the mouse small bowel.     

Maize Centromere Structure and Evolution: Sequence Analysis of Centromeres 2 and 5 Reveals Dynamic Loci Shaped Primarily by Retrotransposons

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.