Evolution of dosage compensation

In polytene chromosome squashes from the fruit flyDrosophila melanogaster, the single, dosage-compensated X chromosome in males can be distinguished from the autosomes by the presence of an isoform of histone H4 acetylated at lysine 16, H4.Ac16. We have used H4.Ac16 as a marker to examine the evolving relationship between dosage compensation and sex chromosome composition in species ofDrosophila with one (D. melanogaster), two (D. pseudoobscura) or three (D. miranda) identifiable X chromosome arms. In each case, we find that H4.Ac16 is distributed as discrete, closely spaced bands along the entire length of each X chromosome, the only exception being the X2 chromosome ofD. miranda in which a terminal region constituting about 10% of the chromosome by length is not labelled with anti-H4.Ac16 antibodies. We conclude that, with this exception, dosage compensation extends along the X chromosomes of all three species. AsD. pseudoobscura andD. miranda diverged only about 2 Mya, the spread of dosage-compensated loci along X2 has been rapid, suggesting that regional changes rather than piecemeal, gene-by-gene, changes may have been involved.accepted for publication by H. C. Macgregor     

Female-specific hyperacetylation of histone H4 in the chicken Z chromosome

Birds undergo genetic sex determination using a ZW sex chromosome system. Although the avian mechanisms of neither sex determination nor dosage compensation are understood, a female-specific non-coding RNA (MHM) is expressed soon after fertilisation from the single Z chicken chromosome and is likely to have a role in one or both processes. We have now discovered a prominent female-specific modification to the Z chromatin in the region of the MHM locus. We find that chicken chromatin at Zp21, including the MHM locus, is strongly enriched for acetylation of histone H4 at lysine residue 16 in female but not male chromosomes. Interestingly, this specific histone modification is also enriched along the length of the up-regulated Drosophila melanogaster male X chromosome where it plays a vital role in the dosage compensation process.     

Histone deacetylase activity is necessary for chromosome condensation during meiotic maturation in Xenopus laevis

Chromosome condensation is thought to be an essential step for the faithful transmission of genetic information during cellular division or gamete formation. The folding of DNA into metaphase chromosomes and its partition during the cell cycle remains a fundamental cellular process that, at the molecular level, is poorly understood. Particularly, the role of histone deacetylase (HDAC) activities in establishing and maintaining meiotic metaphase chromosome condensation has been little documented. In order to better understand how metaphase chromosome condensation is achieved during meiosis, we explored, in vivo, the consequences of HDAC activities inhibition in a Xenopus oocyte model. Our results show that deacetylase activity plays a crucial role in chromosome condensation. This activity is necessary for correct chromosome condensation since the earlier stages of meiosis, but dispensable for meiosis progression, meiosis exit and mitosis entry. We show that HDAC activity correlates with chromosome condensation, being higher when chromosomes are fully condensed and lower during interphase, when chromosomes are decondensed. In addition, we show that, unlike histone H4, Xenopus maternal histone H3 is stored in the oocyte as a hypoacetylated form and is rapidly acetylated when the oocyte exits meiosis.     

Sex chromosome differentiation revealed by genomic in-situ hybridization

In this work, genomic in-situ hybridization (GISH) was used to study the sex chromosome molecular differentiation on chromosomes of male and female individuals of the isopod crustacean Asellus aquaticus. As a composite hybridization probe, we contemporaneously used male and female whole genomic DNA differently labelled in the presence of an excess of unlabelled DNA of the female homogametic sex. The karyotype of A. aquaticus normally displays eight homomorphic chromosome pairs, but a heteromorphic sex chromosome pair is present in about a quarter of the males of a natural population previously identified by us. GISH did not reveal any sex chromosome molecular differentiation on the male and female homomorphic sex chromosome pair, and the karyotypes of these individuals were equally labelled by the male- and female-derived probe, while the heteromorphic Y chromosome showed a differentially labelled region only with the male-derived probe. This region evidently contains male-specific sequences but, because no similar hybridized region is observed on the male homomorphic chromosome pair, they are probably not important for sex determination but represent a molecular differentiation acquired from the Y chromosome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Roles of histone H3K9 methyltransferases during Drosophila spermatogenesis

Epigenetic regulation of gene expression by covalent modification of histones is important for germ line cell development. In mammals, histone H3 lysine 9 (H3K9)-specific histone methyltransferases (HMTases), such as G9a, SETDB1, and SUV39H, play critical roles, but the contribution of H3K9-specific HMTases in Drosophila remains to be clarified, especially in male sperm. Here, we performed immunocytochemical analyses with a specific antibody to dG9a, Drosophila G9a ortholog, and demonstrated localization in the cytoplasm from the growth to elongation stages of spermatogenesis. In the subsequent early canoe stage, strong dG9a signals were detected exclusively in nuclei, suggesting a regulatory role. However, mono-, di-, and trimethylated H3K9 signals were not extensively decreased in a homozygous dG9a null mutant throughout these stages. In contrast, mono- and trimethylated H3K9 signals were extensively decreased in a heterozygous DmSetdb1 mutant during spermatogenesis, and similar reduction in monomethylated H3K9 signals was observed in a homozygous Su(var)3–9 mutant. Therefore, DmSETDB1 is likely to be mainly responsible for mono- and trimethylation of H3K9 and SU(VAR)3–9 for monomethylation of H3K9 during spermatogenesis. However, the reduced methylation of H3K9 in premeiotic spermatocytes did not influence X–Y chromosome disjunction in male meiosis, suggesting that it may not be critical for spermatogenesis in Drosophila.     

Epe1 recruits BET family bromodomain protein Bdf2 to establish heterochromatin boundaries

Heterochromatin spreading leads to the silencing of genes within its path, and boundary elements have evolved to constrain such spreading. In fission yeast, heterochromatin at centromeres I and III is flanked by inverted repeats termed IRCs, which are required for proper boundary functions. However, the mechanisms by which IRCs prevent heterochromatin spreading are unknown. Here, we identified Bdf2, which is homologous to the mammalian bromodomain and extraterminal (BET) family double bromodomain proteins involved in diverse types of cancers, as a factor required for proper boundary function at IRCs. Bdf2 is enriched at IRCs through its interaction with the boundary protein Epe1. The bromodomains of Bdf2 recognize acetylated histone H4 tails and antagonize Sir2-mediated deacetylation of histone H4K16. Furthermore, abolishing H4K16 acetylation (H4K16ac) with an H4K16R mutation promotes heterochromatin spreading, and mimicking H4K16ac by an H4K16Q mutation blocks heterochromatin spreading at IRCs. Our results thus illustrate a mechanism of establishing chromosome boundaries at specific sites through the recruitment of a factor that protects euchromatic histone modifications. They also reveal a previously unappreciated function of H4K16ac in cooperation with H3K9 methylation to regulate heterochromatin spreading.     

Chromatin of the Barr body: histone and non-histone proteins associated with or excluded from the inactive X chromosome

The Barr body has long been recognized as the cytological manifestation of the inactive X chromosome (Xi) in interphase nuclei. Despite being known for over 50 years, relatively few components of the Barr body have been identified. In this study, we have screened over 30 histone variants, modified histones and non-histone proteins for their association with or exclusion from the Barr body. We demonstrate that, similar to the histone variant macroH2A, heterochromatin protein-1 (HP1), histone H1 and the high mobility group protein HMG-I/Y are elevated at the territory of the Xi in interphase in human cell lines, but only when the Xi chromatin is heteropycnotic, implicating each as a component of the Barr body. Surprisingly, however, virtually all other candidate proteins involved in establishing heterochromatin and gene silencing are notably absent from the Barr body despite being localized generally elsewhere throughout the nucleus, indicating that the Barr body represents a discrete subnuclear compartment that is not freely accessible to most chromatin proteins. A similar dichotomous pattern of association or exclusion describes the spatial relationship of a number of specific histone methylation patterns in relation to the Barr body. Notably, though, several methylated forms of histone H3 that are deficient in Xi chromatin generally are present at a region near the macrosatellite repeat DXZ4, as are the chromatin proteins CTCF and SAP30, indicating a distinctive chromatin state in this region of the Xi. Taken together, our data imply that the Xi adopts a distinct chromatin configuration in interphase nuclei and are consistent with a mechanism by which HP1, through histone H3 lysine-9 methylation, recognizes and assists in maintaining heterochromatin and gene silencing at the human Xi.     

Dosage compensation, the origin and the afterlife of sex chromosomes

Over the past 100 years Drosophila has been developed into an outstanding model system for the study of evolutionary processes. A fascinating aspect of evolution is the differentiation of sex chromosomes. Organisms with highly differentiated sex chromosomes, such as the mammalian X and Y, must compensate for the imbalance in gene dosage that this creates. The need to adjust the expression of sex-linked genes is a potent force driving the rise of regulatory mechanisms that act on an entire chromosome. This review will contrast the process of dosage compensation in Drosophila with the divergent strategies adopted by other model organisms. While the machinery of sex chromosome compensation is different in each instance, all share the ability to direct chromatin modifications to an entire chromosome. This review will also explore the idea that chromosome-targeting systems are sometimes adapted for other purposes. This appears the likely source of a chromosome-wide targeting system displayed by the Drosophila fourth chromosome.     

Heterochromatin in interphase nuclei of Arabidopsis thaliana

The eukaryotic nucleus represents a complex arrangement of heterochromatic and euchromatic domains, each with their specific nuclear functions. Somatic cells of a multicellular organism are genetically identical, yet they may differ completely in nuclear organization and gene expression patterns. Stable changes in gene expression without modifying the sequence are the result of epigenetic changes and include covalent modifications in cytosine residues of DNA and in histone tails giving rise to altered chromatin protein complexes, remodeling of chromatin and changes in chromatin compaction. Large-scale differences in chromatin structure are visible at the microscopic level as euchromatin and heterochromatin. Arabidopsis thaliana chromosomes display a relatively simple distribution of euchromatic and heterochromatic segments overlapping with gene-rich and repeat-rich regions, respectively. Recently, we have shown that Arabidopsis provides a well-defined system to study individual chromosomes and chromatin domains in interphase nuclei as well as the relationship between chromatin condensation and epigenetic mechanisms of gene silencing. This overview focuses on the organization and composition of heterochromatin in Arabidopsis nuclei.     

Genome-wide patterns of histone modifications in fission yeast

We have used oligonucleotide tiling arrays to construct genome-wide high-resolution histone acetylation maps for fission yeast. The maps are corrected for nucleosome density and reveal surprisingly uniform patterns of modifications for five different histone acetylation sites. We found that histone acetylation and methylation patterns are generally polar, i.e. they change as a function of distance from the ATG codon. A typical fission yeast gene shows a distinct peak of histone acetylation around the ATG and gradually decreased acetylation levels in the coding region. The patterns are independent of gene length but dependent on the gene expression levels. H3K9Ac shows a stronger peak near the ATG and is more reduced in the coding regions of genes with high expression compared with genes with low expression levels. H4K16Ac is strongly reduced in coding regions of highly expressed genes. A second microarray platform was used to confirm the 5′ to 3′ polarity effects observed with tiling microarrays. By comparing coding region histone acetylation data in HDAC mutants and wild type, we found that hos2 affects primarily the 5′ regions, sir2 and clr6 affect middle regions, and clr6 affects 3′ regions. Thus, mechanisms involving different HDACs modulate histone acetylation levels to maintain a 5′ to 3′ polarity within the coding regions. Electronic Supplementary Material Supplementary material is available for this article at and accessible for authorised users.