Insights into role of bromodomain, testis-specific (Brdt) in acetylated histone H4-dependent chromatin remodeling in mammalian spermiogenesis

Mammalian spermiogenesis is of considerable biological interest especially due to the unique chromatin remodeling events that take place during spermatid maturation. Here, we have studied the expression of chromatin remodeling factors in different spermatogenic stages and narrowed it down to bromodomain, testis-specific (Brdt) as a key molecule participating in chromatin remodeling during rat spermiogenesis. Our immunocytochemistry experiments reveal that Brdt colocalizes with acetylated H4 in elongating spermatids. Remodeling assays showed an acetylation-dependent but ATP-independent chromatin reorganization property of Brdt in haploid round spermatids. Furthermore, Brdt interacts with Smarce1, a member of the SWI/SNF family. We have studied the genomic organization of smarce1 and identified that it has two splice variants expressed during spermatogenesis. The N terminus of Brdt is involved in the recognition of Smarce1 as well as in the reorganization of hyperacetylated round spermatid chromatin. Interestingly, the interaction between Smarce1 and Brdt increases dramatically upon histone hyperacetylation both in vitro and in vivo. Thus, our results indicate this interaction to be a vital step in the chromatin remodeling process during mammalian spermiogenesis.     

Chromosome plasticity in Ctenomys (Rodentia Octodontidae): chromosome 1 evolution and heterochromatin variation

A chromosome 1 (Cr1) pericentric inversion is described in six of seven species in the genus Ctenomys (tuco-tucos) from Uruguay. The inversion was inferred from G-band analyses of subtelocentric Cr1 hypothesised to be derived from the ancestral metacentric condition. Cr1 varies across species in heterochromatin amount and localisation including a metacentric chromosome without positive C-bands in C. torquatus, a subtelocentric chromosome with heterochromatic short arms in C. rionegrensis, and a subtelocentric chromosome negative after C-banding in five of the species analysed here. Pachytene chromosomes from C. rionegrensis, a species with the highest heterochromatin content, and C. torquatus, one of the species with the lowest heterochromatin content, were analysed in order to assess possible mechanisms of heterochromatin evolution. This analysis revealed the presence of three heterochromatic chromocenters in C. rionegrensis where bivalents converge, while in C. torquatus only one chromocenter was observed. In both species, highly repetitive DNA was observed, localised in chromocenters after “in situ” hybridisation. Heterochromatin associated protein M31 was localised in chromocenters of both species after immuno-detection. The spread of heterochromatin in Ctenomys chromosomes could be produced by chromatin exchanges at the chromocenter level. We propose the exchange of this DNA associated proteins between non-homologous chromosomes in pachytene to be the responsible for the spread of heterochromatin through the karyotypes of species like C. rionegrensis     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> LHP1 protein is a component of euchromatin

The HP1 family proteins are involved in several aspects of chromatin function and regulation in Drosophila, mammals and the fission yeast. Here we investigate the localization of LHP1, the unique Arabidopsis thaliana HP1 homolog known at present time, to approach its function. A functional LHP1–GFP fusion protein, able to restore the wild-type phenotype in the lhp1 mutant, was used to analyze the subnuclear distribution of LHP1 in both A. thaliana and Nicotiana tabacum. In A. thaliana interphase nuclei, LHP1 was predominantly located outside the heterochromatic chromocenters. No major aberrations were observed in heterochromatin content or chromocenter organization in lhp1 plants. These data indicate that LHP1 is mainly involved in euchromatin organization in A. thaliana. In tobacco BY-2 cells, the LHP1 distribution, although in foci, slightly differed suggesting that LHP1 localization is determined by the underlying genome organization of plant species. Truncated LHP1 proteins expressed in vivo allowed us to determine the function of the different segments in the localization. The in foci distribution is dependent on the presence of the two chromo domains, whereas the hinge region has some nucleolus-targeting properties. Furthermore, like the animal HP1β and HP1γ subtypes, LHP1 dissociates from chromosomes during mitosis. In transgenic plants expressing the LHP1–GFP fusion protein, two major localization patterns were observed according to cell types suggesting that localization evolves with age or differentiation states. Our results show conversed characteristics of the A. thaliana HP1 homolog with the mammal HP1γ isoform, besides specific plant properties.     

The murine heterochromatin protein M31 is associated with the chromocenter in round spermatids and Is a component of mature spermatozoa

In mature sperm the normal nucleosomal packaging of DNA found in somatic and meiotic cells is transformed into a highly condensed form of chromatin which consists mostly of nucleoprotamines. Although sperm DNA is highly condensed it is nevertheless packaged into a highly defined nuclear architecture which may be organized by the heterochromatic chromocenter. One major component of heterochromatin is the heterochromatin protein 1 which is involved in epigenetic gene silencing. In order to investigate the possible involvement of heterochromatin protein in higher order organization of sperm DNA we studied the localization of the murine homologue of heterochromatin protein 1, M31, during chromatin reorganization in male germ cell differentiation. Each cell type in the testis showed a unique distribution pattern of M31. Colocalization to the heterochromatic regions were found in Sertoli cells, in midstage pachytene spermatocytes, and in round spermatids in which M31 localizes to the centromeric chromocenter. M31 cannot be detected in elongated spermatids or mature spermatozoa immunocytologically, but could be detected in mature spermatozoa by Western blotting. We suggest that M31, a nuclear protein involved in the organization of chromatin architecture, is involved in higher order organization of sperm DNA.     

Two bromodomain proteins functionally interact to recapitulate an essential BRDT-like function in Drosophila spermatocytes

In mammals, the testis-specific bromodomain and extra terminal (BET) protein BRDT is essential for spermatogenesis. In Drosophila, it was recently reported that the tBRD-1 protein is similarly required for male fertility. Interestingly, however, tBRD-1 has two conserved bromodomains in its N-terminus but it lacks an extra terminal (ET) domain characteristic of BET proteins. Here, using proteomics approaches to search for tBRD-1 interactors, we identified tBRD-2 as a novel testis-specific bromodomain protein. In contrast to tBRD-1, tBRD-2 contains a single bromodomain, but which is associated with an ET domain in its C-terminus. Strikingly, we show that tbrd-2 knock-out males are sterile and display aberrant meiosis in a way highly similar to tbrd-1 mutants. Furthermore, these two factors co-localize and are interdependent in spermatocytes. We propose that Drosophila tBRD-1 and tBRD-2 associate into a functional BET complex in spermatocytes, which recapitulates the activity of the single mammalian BRDT-like protein.     

Tet1 regulates epigenetic remodeling of the pericentromeric heterochromatin and chromocenter organization in DNA hypomethylated cells

Pericentromeric heterochromatin (PCH), the constitutive heterochromatin of pericentromeric regions, plays crucial roles in various cellular events, such as cell division and DNA replication. PCH forms chromocenters in the interphase nucleus, and chromocenters cluster at the prophase of meiosis. Chromocenter clustering has been reported to be critical for the appropriate progression of meiosis. However, the molecular mechanisms underlying chromocenter clustering remain elusive. In this study, we found that global DNA hypomethylation, 5hmC enrichment in PCH, and chromocenter clustering of Dnmt1-KO ESCs were similar to those of the female meiotic germ cells. Tet1 is essential for the deposition of 5hmC and facultative histone marks of H3K27me3 and H2AK119ub at PCH, as well as chromocenter clustering. RING1B, one of the core components of PRC1, is recruited to PCH by TET1, and PRC1 plays a critical role in chromocenter clustering. In addition, the rearrangement of the chromocenter under DNA hypomethylated condition was mediated by liquid-liquid phase separation. Thus, we demonstrated a novel role of Tet1 in chromocenter rearrangement in DNA hypomethylated cells.     

DNS-Replikation und Morphologie der X-Chromosomen während der Syntheseperiode bei Microtus agrestis

X-chromosome behaviour of female Microtus agrestis in interphase nuclei with and without large chromocenters was examined in cultured epithelial and fibroblast cells. By means of pulse-labeling, the configuration of the X-chromosomes in these nuclei can be illustrated; staining with pararosaniline-methylgreen seems to be most suitable. According to the replication behaviour, three types of chromatin can be discerned in the X-chromosomes: early replicating euchromatin, late replicating sex chromatin, and very late replicating heterochromatin. In fibroblasts only the sex chromatin forms a single, small chromocenter; in epithelial cells both the sex chromatin and the remaining heterochromatin form large chromocenters. Both types of heterochromatin replicate their DNA in the condensed state. It seems likely that the late replicating segments of the X-chromosomes (sex chromatin and remaining heterochromatin) are condensed in every cell, but they may not always be configurated or even visible as typical chromocenters; these segments could be distributed over a wide range of compact to more or less diffuse superstructures.     

Isolation of the Chromocenter Fraction from Mouse Liver Nuclei

A new method for isolation of the constitutive heterochromatin (chromocenters) from interphase nuclei of mouse liver has been developed. This method allows separation of chromocenters of different size. Chromocenter fractions are essentially free of nucleoli and other contaminants. In contrast to nuclei and nucleoli, the chromocenter fraction is characterized by simpler protein composition, this fraction having a reduced number of proteins (especially high molecular weight proteins). Chromocenters contain all histone fractions; however, the relative proportion of histone H1 is lower and histone H3 is higher than in the total nuclear chromatin. The amount of non-histone proteins of 51, 63, 73, and 180 kD is higher in the chromocenter fraction than in nuclei and nucleoli. The use of immunocytochemistry and immunoblotting methods revealed the presence of the specific kinetochore component, CENP A protein. This suggests tight association of some molecular kinetochore components with chromocenters in the interphase.     

The bromodomain protein LEX-1 acts with TAM-1 to modulate gene expression in C. elegans

In many organisms, repetitive DNA serves as a trigger for gene silencing. However, some gene expression is observed from repetitive genomic regions such as heterochromatin, suggesting mechanisms exist to modulate the silencing effects. From a genetic screen in C. elegans, we have identified mutations in two genes important for expression of repetitive sequences: lex-1 and tam-1. Here we show that lex-1 encodes a protein containing an ATPase domain and a bromodomain. LEX-1 is similar to the yeast Yta7 protein, which maintains boundaries between silenced and active chromatin. tam-1 has previously been shown to encode a RING finger/B-box protein that modulates gene expression from repetitive DNA. We find that lex-1, like tam-1, acts as a class B synthetic multivulva (synMuv) gene. However, since lex-1 and tam-1 mutants have normal P granule localization, it suggests they act through a mechanism distinct from other class B synMuvs. We observe intragenic (interallelic) complementation with lex-1 and a genetic interaction between lex-1 and tam-1, data consistent with the idea that the gene products function in the same biological process, perhaps as part of a protein complex. We propose that LEX-1 and TAM-1 function together to influence chromatin structure and to promote expression from repetitive sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chromatin structural rearrangement during dedifferentiation of protoplasts of <Emphasis Type="Italic">Cucumis sativus</Emphasis> L.

This paper reports on the structural rearrangement of satellite DNA type I repeats and heterochromatin during the dedifferentiation and cell cycling of mesophyll protoplasts of cucumber (Cucumis sativus). These repeats were localized in the telomeric heterochromatin of cucumber chromosomes and in the chromocenters of interphase nuclei. The dramatic reduction of heterochromatin involves decondensation of subtelomeric repeats in freshly isolated protoplasts; however, there are not a great many remarkable changes in the expression profile. In spite of that, reformation of the chromocenters, occurring 48 h after protoplast isolation, is accompanied by recondensation of satellite DNA type I; however, only partial reassembly of these repeats was revealed. In this study, FISH and a flow cytometry assay show a correlation between the partial chromocenter and the repeats reassembly, and with the reentry of cultivated protoplasts into the cell cycle and first cell division. After that, divided cells displayed a higher variability in the expression profile than did leaves’ mesophyll cells and protoplasts.     

Characterization of sequences associated with position-effect variegation at pericentric sites in Drosophila heterochromatin

In a variety of organisms, euchromatic genes brought into juxtaposition with pericentric heterochromatin show position-effect variegation (PEV), a silencing of gene expression in a subset of the cells in which the gene is normally expressed. Previously, a P-element mobilization screen identified transgenic Drosophila stocks showing PEV of an hsp70-white ⁺ reporter gene; transgenes in many of these stocks map to the chromocenter of polytene chromosome. A screen at an elevated temperature identified two stocks that under standard culture temperatures show complete repression of the hsp70-white ⁺ transgene. The transgenes in both cases map to the chromocenter of polytene chromosomes. Different types of middle repetitive elements are adjacent to seven pericentric transgenes; unique sequences are adjacent to two of the perimetric transgenes. All of the transgenes show suppression of PEV in response to a mutation in the gene encoding heterochromatin protein 1 (HP1). This suppression correlates with a more accessible chromatin structure. The results indicate that a pericentric transgene showing PEV can be associated with different types of DNA sequences, while maintaining a common association with the chromosomal protein HP1. Received: 15 January 1998; in revised form: 27 May 1998 / Accepted: 4 September 1998     

Cytological variations in the constitutive heterochromatin of Microtus agrestis

In the field vole, Microtus agrestis, most of the constitutive heterochromatin is localized in the giant chromosomes. A detailed examination of a large number of adult cell types reveals that this chromatin is actually present as a heterochromatic fiber in all interphase nuclei. Depending upon the cell types, however, the fiber shows varying degrees of condensation and folding ranging from a very long and extended fiber to a large compact chromocenter. The number of cell types with giant chromocenters was less commonly observed than those with extended fibers. This explains why some cells were previously thought to be devoid of heterochromatin.—The results of this investigation strongly indicate that constitutive heterochromatin represents a unique nuclear entity.This investigation was supported by NIH funds (Grant No. HD 1962).     

Karyotypanalyse an Hand des Färbungsmusters der Metaphasechromosomen von Cypripedium debile und Trillium kamtschaticum

The somatic metaphase chromosomes of Cypripedium debile and Trillium kamtschaticum are stained differentially by treatment with an acetic orcein-hydrochloric acid mixture: In Cypripedium, the heterochromatic segments stain densely whereas the euchromatic segments are unstained. In Trillium, in contrast, the heterochromatic segments are unstained and euchromatin is stained. Such an inconsistency in stain patterns is considered to stem from different species-specific reactivity of heterochromatin and euchromatin to hydrochloric acid in the staining medium.—In metaphase chromosomes of Cypripedium stained with fast green (Alfert and Geschwind, 1953), the euchromatic segments are stained positively, whereas the heterochromatin, as well as the chromocenters, are unstained. In Trillium, all heterochromatin, euchromatin and chromocenter are stained homogeneously by this method. These results indicate that the heterochromatic segments and chromocenters in Cypripedium are associated with non-histon type proteins, whereas the euchromatic segments in Cypripedium, as well as two different types of the segments and the chromocenters in Trillium, are all bound to histon type proteins. — From these findings, it is concluded that two types of heterochromatin occur in plant materials, though it remains unsettled whether they correspond to - and -heterochromatin as found in Dipteran giant chromosomes.—Karyotype analysis is made on the basis of the differential staining pattern of chromosomes revealed by means of acetic orcein-hydrochloric procedure.     

The PHD domain of Np95 (mUHRF1) is involved in large-scale reorganization of pericentromeric heterochromatin

Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH.     

Multiple bromodomain genes are involved in restricting the spread of heterochromatic silencing at the Saccharomyces cerevisiae HMR-tRNA boundary

The transfer RNA gene downstream from the HMR locus in S. cerevisiae functions as part of a boundary (barrier) element that restricts the spread of heterochromatic gene silencing into the downstream region of chromosome III. A genetic screen for identifying additional genes that, when mutated, allow inappropriate spreading of silencing from HMR through the tRNA gene was performed. YTA7, a gene containing bromodomain and ATPase homologies, was identified multiple times. Previously, others had shown that the bromodomain protein Bdf1p functions to restrict silencing at yeast euchromatin-heterochromatin boundaries; therefore we deleted nonessential bromodomain-containing genes to test their effects on heterochromatin spreading. Deletion of RSC2, coding for a component of the RSC chromatin-remodeling complex, resulted in a significant spread of silencing at HMR. Since the bromodomain of YTA7 lacks a key tyrosine residue shown to be important for acetyllysine binding in other bromodomains, we confirmed that a GST-Yta7p bromodomain fusion was capable of binding to histones in vitro. Epistasis analysis suggests that YTA7 and the HMR-tRNA function independently to restrict the spread of silencing, while RSC2 may function through the tRNA element. Our results suggest that multiple bromodomain proteins are involved in restricting the propagation of heterochromatin at HMR.