Showing papers in "Chromosoma in 2011"

The ABCs of CENPs

Abstract: Equal distribution of DNA in mitosis requires the assembly of a large proteinaceous ensemble onto the centromeric DNA, called the kinetochore. With few exceptions, kinetochore specification is independent of the DNA sequence and is determined epigenetically by deposition at the centromeric chromatin of special nucleosomes containing an H3-related histone, CENP-A. Onto centromeric CENP-A chromatin is assembled the so-called constitutive centromere-associated network (CCAN) of 16 proteins distributed in several functional groups as follows: CENP-C, CENP-H/CENP-I/CENP-K/, CENP-L/CENP-M/CENP-N, CENP-O/CENP-P/CENP-Q/CENP-R/CENP-U(50), CENP-T/CENP-W, and CENP-S/CENP-X. One role of the CCAN is to recruit outer kinetochore components further, such as KNL1, the Mis12 complex, and the Ndc80 complex (KMN network) to which attach the spindle microtubules with their structural and regulatory proteins. Among the CENPs in CCAN, CENP-C and CENP-T are required in parallel for operational kinetochore specification and spindle attachment. This review presents discussion of the latest structural and functional data on CENP-A and CENPs from the CCAN as well as their interaction with the KMN network.

Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Abstract: Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81–Mms4/EME1, Slx1–Slx4/BTBD12/MUS312, XPF–ERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.

H3.5 is a novel hominid-specific histone H3 variant that is specifically expressed in the seminiferous tubules of human testes.

Abstract: The incorporation of histone variants into chromatin plays an important role for the establishment of particular chromatin states. Six human histone H3 variants are known to date, not counting CenH3 variants: H3.1, H3.2, H3.3 and the testis-specific H3.1t as well as the recently described variants H3.X and H3.Y. We report the discovery of H3.5, a novel non-CenH3 histone H3 variant. H3.5 is encoded on human chromosome 12p11.21 and probably evolved in a common ancestor of all recent great apes (Hominidae) as a consequence of H3F3B gene duplication by retrotransposition. H3.5 mRNA is specifically expressed in seminiferous tubules of human testis. Interestingly, H3.5 has two exact copies of ARKST motifs adjacent to lysine-9 or lysine-27, and lysine-79 is replaced by asparagine. In the Hek293 cell line, ectopically expressed H3.5 is assembled into chromatin and targeted by PTM. H3.5 preferentially colocalizes with euchromatin, and it is associated with actively transcribed genes and can replace an essential function of RNAi-depleted H3.3 in cell growth.

Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence

Abstract: The divergence of lineages leading to extant squamate reptiles (lizards, snakes, and amphisbaenians) and birds occurred about 275 million years ago. Birds, unlike squamates, have karyotypes that are typified by the presence of a number of very small chromosomes. Hence, a number of chromosome rearrangements might be expected between bird and squamate genomes. We used chromosome-specific DNA from flow-sorted chicken (Gallus gallus) Z sex chromosomes as a probe in cross-species hybridization to metaphase spreads of 28 species from 17 families representing most main squamate lineages and single species of crocodiles and turtles. In all but one case, the Z chromosome was conserved intact despite very ancient divergence of sauropsid lineages. Furthermore, the probe painted an autosomal region in seven species from our sample with characterized sex chromosomes, and this provides evidence against an ancestral avian-like system of sex determination in Squamata. The avian Z chromosome synteny is, therefore, conserved albeit it is not a sex chromosome in these squamate species.

DNA double-strand break repair in Caenorhabditis elegans.

Abstract: Faithful repair of DNA double-strand breaks (DSBs) is vital for animal development, as inappropriate repair can cause gross chromosomal alterations that result in cellular dysfunction, ultimately leading to cancer, or cell death. Correct processing of DSBs is not only essential for maintaining genomic integrity, but is also required in developmental programs, such as gametogenesis, in which DSBs are deliberately generated. Accordingly, DSB repair deficiencies are associated with various developmental disorders including cancer predisposition and infertility. To avoid this threat, cells are equipped with an elaborate and evolutionarily well-conserved network of DSB repair pathways. In recent years, Caenorhabditis elegans has become a successful model system in which to study DSB repair, leading to important insights in this process during animal development. This review will discuss the major contributions and recent progress in the C. elegans field to elucidate the complex networks involved in DSB repair, the impact of which extends well beyond the nematode phylum.

Variation in crossover rates across a 3-Mb contig of bread wheat (Triticum aestivum) reveals the presence of a meiotic recombination hotspot.

Abstract: In bread wheat (Triticum aestivum L.), initial studies using deletion lines indicated that crossover (CO) events occur mainly in the telomeric regions of the chromosomes with a possible correlation with the presence of genes. However, little is known about the distribution of COs at the sequence level. To investigate this, we studied in detail the pattern of COs along a contig of 3.110 Mb using two F2 segregating populations (Chinese Spring × Renan (F2-CsRe) and Chinese Spring × Courtot (F2-CsCt)) each containing ~2,000 individuals. The availability of the sequence of the contig from Cs enabled the development of 318 markers among which 23 co-dominant polymorphic markers (11 SSRs and 12 SNPs) were selected for CO distribution analyses. The distribution of CO events was not homogeneous throughout the contig, ranging from 0.05 to 2.77 cM/Mb, but was conserved between the two populations despite very different contig recombination rate averages (0.82 cM/Mb in F2-CsRe vs 0.35 cM/Mb in F2-CsCt). The CO frequency was correlated with the percentage of coding sequence in Cs and with the polymorphism rate between Cs and Re or Ct in both populations, indicating an impact of these two factors on CO distribution. At a finer scale, COs were found in a region covering 2.38 kb, spanning a gene coding for a glycosyl transferase (Hga3), suggesting the presence of a CO hotspot. A non-crossover event covering at least 453 bp was also identified in the same interval. From these results, we can conclude that gene content could be one of the factors driving recombination in bread wheat.

Buffering and the evolution of chromosome-wide gene regulation.

Abstract: Copy number variation (CNV) in terms of aneuploidies of both entire chromosomes and chromosomal segments is an important evolutionary driving force, but it is inevitably accompanied by potentially problematic variations in gene doses and genomic instability. Thus, a delicate balance must be maintained between mechanisms that compensate for variations in gene doses (and thus allow such genomic variability) and selection against destabilizing CNVs. In Drosophila, three known compensatory mechanisms have evolved: a general segmental aneuploidy-buffering system and two chromosome-specific systems. The two chromosome-specific systems are the male-specific lethal complex, which is important for dosage compensation of the male X chromosome, and Painting of fourth, which stimulates expression of the fourth chromosome. In this review, we discuss the origin and function of buffering and compensation using Drosophila as a model.

Immediate unidirectional epigenetic reprogramming of NORs occurs independently of rDNA rearrangements in synthetic and natural forms of a polyploid species Brassica napus.

Abstract: The dynamics of genome modification that occurred from the initial hybridization event to the stabilization of allopolyploid species remains largely unexplored. Here, we studied inheritance and expression of rDNA loci in the initial generations of Brassica napus allotetraploids (2n = 38, AACC) resynthesized from Brassica oleracea (2n = 18, CC) and B. rapa (2n = 20, AA) and compared the patterns to natural forms. Starting already from F1 generation, there was a strong uniparental silencing of B. oleracea genes. The epigenetic reprogramming was accompanied with immediate condensation of C-genome nucleolar organizer region (NOR) and progressive transgeneration hypermethylation of polymerase I promoters, mainly at CG sites. No such changes were observed in the A-genome NORs. Locus loss and gains affecting mainly non-NOR loci after the first allotetraploid meiosis did not influence established functional status of NORs. Collectively, epigenetic and genetic modifications in synthetic lines resemble events that accompanied formation of natural allopolyploid species.

Characterization of CENH3 proteins and centromere-associated DNA sequences in diploid and allotetraploid Brassica species

Abstract: CENH3 is a centromere-specific histone H3 variant and has been used as a marker to identify active centromeres and DNA sequences associated with functional centromere/kinetochore complexes. In this study, up to four distinct CENH3 (BrCENH3) cDNAs were identified in individuals of each of three diploid species of Brassica. Comparison of the BrCENH3 cDNAs implied three related gene families: BrCENH3-A in Brassica rapa (AA), BrCENH3-B in B. nigra (BB), and BrCENH3-C in B. oleracea (CC). Each family encoded a histone fold domain and N-terminal histone tails that vary in length in all three families. The BrCENH3-B cDNAs have a deletion of two exons relative to BrCENH3-A and BrCENH3-C, consistent with the more ancient divergence of the BB genome. Chromatin immunoprecipitation and immunolabeling tests with anti-BrCENH3 antibodies indicated that both centromeric tandem repeats and the centromere-specific retrotransposons of Brassica are directly associated with BrCENH3 proteins. In three allotetraploid species, we find either co-transcription of the BrCENH3 genes of the ancestral diploid species or gene suppression of the BrCENH3 from one ancestor. Although B genome centromeres are occupied by BrCENH3-B in the ancestral species B. nigra, in allotetraploids both BrCENH3-A and BrCENH3-C proteins appear to assemble at these centromeres.

Painting the chromosomes of Brachypodium—current status and future prospects

Abstract: Chromosome painting is one of the most powerful and spectacular tools of modern molecular cytogenetics, enabling complex analyses of nuclear genome structure and evolution. For many years, this technique was restricted to the study of mammalian chromosomes, as it failed to work in plant genomes due mainly to the presence of large amounts of repetitive DNA common to all the chromosomes of the complement. The availability of ordered, chromosome-specific BAC clones of Arabidopsis thaliana containing relatively little repetitive genomic DNA enabled the first chromosome painting in dicotyledonous plants. Here, we show for the first time chromosome painting in three different cytotypes of a monocotyledonous plant—the model grass, Brachypodium distachyon. Possible directions of further detailed studies are proposed, such as the evolution of grass karyotypes, the behaviour of meiotic chromosomes, and the analysis of chromosome distribution at interphase.

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres.

Abstract: Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.

Drosophila CENP-C is essential for centromere identity

Abstract: Centromeres are specialized chromosomal domains that direct mitotic kinetochore assembly and are defined by the presence of CENP-A (CID in Drosophila) and CENP-C. While the role of CENP-A appears to be highly conserved, functional studies in different organisms suggest that the precise role of CENP-C in kinetochore assembly is still under debate. Previous studies in vertebrate cells have shown that CENP-C inactivation causes mitotic delay, chromosome missegregation, and apoptosis; however, in Drosophila, the role of CENP-C is not well-defined. We have used RNA interference depletion in S2 cells to address this question and we find that depletion of CENP-C causes a kinetochore null phenotype, and consequently, the spindle checkpoint, kinetochore–microtubule interactions, and spindle size are severely misregulated. Importantly, we show that CENP-C is required for centromere identity as CID, MEI-S332, and chromosomal passenger proteins fail to localize in CENP-C depleted cells, suggesting a tight communication between the inner kinetochore proteins and centromeres. We suggest that CENP-C might fulfill the structural roles of the human centromere-associated proteins not identified in Drosophila.

Rap1-independent telomere attachment and bouquet formation in mammalian meiosis

Abstract: Attachment of telomeres to the nuclear envelope (NE) and their clustering in a chromosomal bouquet during meiotic prophase I is an evolutionary conserved event that promotes chromosome pairing and recombination. In fission yeast, bouquet formation fails when the telomeric protein Rap1 is absent or when the telomeric protein Taz1 fails to recruit Rap1 to telomeres. The mammalian Rap1 orthologue is a component of the shelterin complex and localises to telomeres through an interaction with a Taz1-like telomeric DNA binding factor, TRF2. Here, we investigated the role of mammalian Rap1 in meiotic telomere attachment and clustering by analysing spermatogenesis in Rap1-deficient mice. The results establish that the meiotic three-dimensional nuclear architecture and recombination are not affected by the absence of Rap1. Furthermore, Rap1-deficient meiotic telomeres assemble the SUN1 nuclear membrane protein, attach to the NE, and undergo bouquet formation indistinguishable from the wild-type setting. Thus, the role of Rap1 in meiosis is not conserved between fission yeast and mammals, suggesting that mammals have alternative modes for connecting telomeres to SUN proteins on the meiotic nuclear envelope.

Compact genomes and complex evolution in the genus Brachypodium

Abstract: The temperate annual grass Brachypodium distachyon is a diploid species with a chromosome base number of 5 It is strikingly different from other Eurasian species of the genus, which are perennial and often polyploid, with the diploids typically having base numbers of 8 or 9 Previously, phylogenies indicated that B distachyon split from the other species early in the evolution of the genus, while its genome sequence revealed that extensive synteny on a chromosomal scale had been maintained with rice, a tropical grass with a base number of 12 Here we show evidence that B distachyon may have a homoploid origin, involving ancestral interspecific hybridisation, although it does not appear to be a component of any of the perennial Eurasian allopolyploids Using a cytogenetic approach, we show that dysploidy in Brachypodium has not followed a simple progression

Deposition, turnover, and release of CENH3 at Arabidopsis centromeres.

Abstract: The kinetochore is a complex multiprotein structure located at centromeres and required for the proper segregation of chromosomes during mitosis and meiosis. An important role in kinetochore assembly and function plays the centromeric histone H3 variant (CENH3). Cell cycle stage of CENH3 deposition to centromeres varies between different organisms. We confirmed by in vivo studies that deposition of Arabidopsis CENH3 takes place at centromeres during G2 and demonstrated that additionally a low turnover of CENH3 occurs along the cell cycle, apparently for replacement of damaged protein. Furthermore, enhanced yellow fluorescent protein (EYFP)-CENH3 of photobleached chromocenters is not replaced by EYFP-CENH3 molecules from unbleached centromeres of the same nucleus, indicating a stable incorporation of CENH3 into centromeric nucleosomes. In differentiated endopolyploid nuclei however, the amount of CENH3 at centromeres declines with age.

Assembly of the transcription machinery: ordered and stable, random and dynamic, or both?

Abstract: The assembly of the transcription machinery is a key step in gene activation, but even basic details of this process remain unclear. Here we discuss the apparent discrepancy between the classic sequential assembly model based mostly on biochemistry and an emerging dynamic assembly model based mostly on fluorescence microscopy. The former model favors a stable transcription complex with subunits that cooperatively assemble in order, whereas the latter model favors an unstable complex with subunits that may assemble more randomly. To confront this apparent discrepancy, we review the merits and drawbacks of the different experimental approaches and list potential biasing factors that could be responsible for the different interpretations of assembly. We then discuss how these biases might be overcome in the future with improved experiments or new techniques. Finally, we discuss how kinetic models for assembly may help resolve the ordered and stable vs. random and dynamic assembly debate.

TCAGG, an alternative telomeric sequence in insects

Abstract: The TTAGG repeat, the only determined telomerase-dependent sequence in the Insecta, is generally reputed to be the canonical telomeric motif within the class. By studying the distribution of telomeric DNAs in 30 coleopteran beetles using Southern hybridization, BAL 31 DNA end-degradation assay and fluorescence in situ hybridization, we showed that arrays built of a TCAGG repeat substitute for (TTAGG)n sequences in all tested species within the superfamily Tenebrionoidea. We also provided the experimental evidence that (TCAGG)n repeats represent the terminal sequences on all chromosomes of the model species Tribolium castaneum. (TCAGG)n repeats are therefore promoted as the first sequence-motif alternative to TTAGG-type chromosome ends in insects. Detection of species negative for both TTAGG and TCAGG reveals that, although widespread, these motifs are not ubiquitous telomeric sequences within the order Coleoptera. In addition, Timarcha balearica proved to be a species that harbors (TTAGG)n repeats, but not at telomeric positions, thus further increasing the complexity of telomeric DNAs. Our experiments discarded CTAGG, CTGGG, TTGGG, and TTAGGG variants as potential replacements in TTAGG/TCAGG-negative species, indicating that chromosome termini of these beetles comprise other form(s) of telomeric sequences and telomere maintenance mechanisms.

The torsional state of DNA within the chromosome

Abstract: Virtually all processes of the genome biology affect or are affected by the torsional state of DNA. Torsional energy associated with an altered twist facilitates or hinders the melting of the double helix, its molecular interactions, and its spatial folding in the form of supercoils. Yet, understanding how the torsional state of DNA is modulated remains a challenging task due to the multiplicity of cellular factors involved in the generation, transmission, and dissipation of DNA twisting forces. Here, an overview of the implication of DNA topoisomerases, DNA revolving motors, and other DNA interactions that determine local levels of torsional stress in bacterial and eukaryotic chromosomes is provided. Particular emphasis is made on the experimental approaches being developed to assess the torsional state of intracellular DNA and its organization into topological domains.

An intranucleolar body associated with rDNA

Abstract: The nucleolus is the subnuclear organelle responsible for ribosome subunit biogenesis and can also act as a stress sensor. It forms around clusters of ribosomal DNA (rDNA) and is mainly organised in three subcompartments, i.e. fibrillar centre, dense fibrillar component and granular component. Here, we describe the localisation of 21 protein factors to an intranucleolar region different to these main subcompartments, called the intranucleolar body (INB). These factors include proteins involved in DNA maintenance, protein turnover, RNA metabolism, chromatin organisation and the post-translational modifiers SUMO1 and SUMO2/3. Increase in the size and number of INBs is promoted by specific types of DNA damage and depends on the functional integrity of the nucleolus. INBs are abundant in nucleoli of unstressed cells during S phase and localise in close proximity to rDNA with heterochromatic features. The data suggest the INB is linked with regulation of rDNA transcription and/or maintenance of rDNA.

Epigenetic profiling of heterochromatic satellite DNA

Abstract: Sugar beet (Beta vulgaris) chromosomes consist of large heterochromatic blocks in pericentromeric, centromeric, and intercalary regions comprised of two different highly abundant DNA satellite families. To investigate DNA methylation at single base resolution at heterochromatic regions, we applied a method for strand-specific bisulfite sequencing of more than 1,000 satellite monomers followed by statistical analyses. As a result, we uncovered diversity in the distribution of different methylation patterns in both satellite families. Heavily methylated CG and CHG (H=A, T, or C) sites occur more frequently in intercalary heterochromatin, while CHH sites, with the exception of CAA, are only sparsely methylated, in both intercalary and pericentromeric/centromeric heterochromatin. We show that the difference in DNA methylation intensity is correlated to unequal distribution of heterochromatic histone H3 methylation marks. While clusters of H3K9me2 were absent from pericentromeric heterochromatin and restricted only to intercalary heterochromatic regions, H3K9me1 and H3K27me1 were observed in all types of heterochromatin. By sequencing of a small RNA library consisting of 6.76 million small RNAs, we identified small interfering RNAs (siRNAs) of 24 nucleotides in size which originated from both strands of the satellite DNAs. We hypothesize an involvement of these siRNAs in the regulation of DNA and histone methylation for maintaining heterochromatin.

Homolog pairing and sister chromatid cohesion in heterochromatin in Drosophila male meiosis I

Abstract: Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.

Nucleocytoplasmic mRNP export is an integral part of mRNP biogenesis

Abstract: Nucleocytoplasmic export and biogenesis of mRNPs are closely coupled. At the gene, concomitant with synthesis of the pre-mRNA, the transcription machinery, hnRNP proteins, processing, quality control and export machineries cooperate to release processed and export competent mRNPs. After diffusion through the interchromatin space, the mRNPs are translocated through the nuclear pore complex and released into the cytoplasm. At the nuclear pore complex, defined compositional and conformational changes are triggered, but specific cotranscriptionally added components are retained in the mRNP and subsequently influence the cytoplasmic fate of the mRNP. Processes taking place at the gene locus and at the nuclear pore complex are crucial for integrating export as an essential part of gene expression. Spatial, temporal and structural aspects of these events have been highlighted in analyses of the Balbiani ring genes.

Characterization of TTAGG telomeric repeats, their interstitial occurrence and constitutively active telomerase in the mealybug Planococcus lilacinus (Homoptera; Coccoidea)

Abstract: We confirmed the occurrence of the insect TTAGG telomeric repeats in the mealybug Planococcus lilacinus, a radiation-resistant coccid, by single primer polymerase chain reaction (PCR) and Southern hybridization. Analysis of Bal31 nuclease-digested DNA by Southern hybridization and chromosomes by FISH suggests that these repeats occur mainly at the ends of the chromosomes. However, sequence analysis of the PCR products of TTAGG-associated sequences from genomic DNA showed their interstitial occurrence and association with certain unrelated low-copy repeats. Because of their shorter length, the interstitial TTAGG sequences were detectable by primed in situ hybridizations but not by FISH. Analysis of chromosomes recovered after irradiation by fluorescent in situ hybridization suggested acquisition of TTAGG repeats at a majority of the healed ends. We also observed mild telomerase activity in unirradiated insects which was further enhanced after irradiation. Taken together, these results suggest that the mealybug has an efficient mechanism of formation of TTAGG repeats at radiation-induced chromosome ends and constitutively active telomerase may be a feature associated with rapid recovery of chromosome ends damaged by ionizing radiation.

Karyotypic polymorphism of the zebra finch Z chromosome.

Abstract: We describe a karyotypic polymorphism on the zebra finch Z chromosome. This polymorphism was discovered because of a difference in the position of the centromere and because it occurs at varying frequencies in domesticated colonies in the USA and Germany and among two zebra finch subspecies. Using DNA fluorescent in situ hybridization to map specific Z genes and measurements of DNA replication, we show that this polymorphism is the result of a large pericentric inversion involving the majority of the chromosome. We sequenced a likely breakpoint for the inversion and found many repetitive sequences. Around the breakpoint, there are numerous repetitive sequences and several copies of PAK3 (p21-activated kinase 3)-related sequences (PAK3Z) which showed testes-specific expression by RT-PCR. Our findings further suggest that the sequenced genome of the zebra finch may be derived from a male heterozygote for the Z chromosome polymorphism. This finding, in combination with regional differences in the frequency of the polymorphism, has important consequences for future studies using zebra finches.

Drosophila melanogaster heterochromatin protein HP1b plays important roles in transcriptional activation and development

Abstract: The condensed heterochromatic domains are known to be associated with transcriptional repression and cell differentiation. Here, we investigate the function of heterochromatin protein HP1b, a member of the HP1 family in Drosophila melanogaster, in transcription and development. Both knockdown and overexpression of HP1b resulted in partial lethality, indicating that HP1b is essential for the normal development. In contrast to the positive role of HP1a in heterochromatin formation, overexpression of HP1b decondensed the pericentromeric heterochromatin and reduced the association of HP1a and H3K9me2 with it, both known markers of pericentric heterochromatin. Interestingly, the structure of the heterochromatic fourth chromosome appeared not to be affected. Further experiments showed that the presence of HP1a partially rescued the lethality caused by HP1b overexpression in males, and it fully rescued the lethality in females. Consistent with this observation, the defective transcription of heterochromatic genes was also partially restored in the presence of HP1a. Overall, this study argues that HP1b counteracts HP1a function both in heterochromatin formation and in the transcriptional regulation of euchromatic genes.

Regulation of DNA replication by chromatin structures: accessibility and recruitment

Abstract: The initiation of DNA replication and the elongation of DNA strands take place in chromatin, a huge compound DNA-protein complex. Although the factors involved in the process of DNA replication have been largely elucidated, the underlying mechanisms that determine their behavior in the context of chromatin have only recently begun to be understood. It has been known that transcription is tightly regulated by the state of chromatin compaction, which governs the accessibility of DNA to trans-acting factors. This process is influenced by several determinants of chromatin structure, including intrinsic nucleosome positioning, the nucleosome remodeling complex, histone post-translational modifiers, and histone- and DNA-binding proteins. Growing evidence indicates that this concept is also applicable to the regulation of DNA replication. In addition, recent studies have demonstrated a distinctive mode of regulation. Some non-histone chromatin-binding proteins have been shown to interact physically with replication factors, thereby facilitating their recruitment at specific chromosomal loci. This type of regulation may allow control of local replication activity without affecting other chromosomal processes.

Histone H3 trimethylation at lysine 9 marks the inactive metaphase X chromosome in the marsupial Monodelphis domestica

Abstract: In somatic cells of female marsupial and eutherian mammals, X chromosome inactivation (XCI) occurs. XCI results in the transcriptional silencing of one of the two X chromosomes and is accompanied by specific covalent histone modifications attributable to the inactive chromatin state. Because data about repressed chromatin of the inactive X chromosome (Xi) in marsupials are sparse, we examined in more detail the distribution of active and inactive chromatin markers on metaphase X chromosomes of an American marsupial, Monodelphis domestica. Consistent with data reported previously both for eutherian and marsupial mammals, we found that the Xi of M. domestica lacks active histone markers—H3K4 dimethylation and H3K9 acetylation. We did not observe on metaphase spreads enrichment of the Xi with H3K27 trimethylation which is involved in XCI in eutherians and was detected on the Xi in the interphase nuclei of mature female M. domestica in an earlier study. Moreover, we found that the Xi of M. domestica was specifically marked with H3K9 trimethylation, which is known to be a component of the Xi chromatin in eutherians and is involved in both marsupials and eutherians in meiotic sex chromosome inactivation which has been proposed as an ancestral mechanism of XCI.

A comparative study of the recombination pattern in three species of Platyrrhini monkeys (primates)

Abstract: Homologous chromosomes exchange genetic information through recombination during meiotic synapsis, a process that increases genetic diversity and is fundamental to sexual reproduction. Meiotic studies in mammalian species are scarce and mainly focused on human and mouse. Here, the meiotic recombination events were determined in three species of Platyrrhini monkeys (Cebus libidinosus, Cebus nigritus and Alouatta caraya) by analysing the distribution of MLH1 foci at the stage of pachytene. Moreover, the combination of immunofluorescence and fluorescent in situ hybridisation has enabled us to construct recombination maps of primate chromosomes that are homologous to human chromosomes 13 and 21. Our results show that (a) the overall number of MLH1 foci varies among all three species, (b) the presence of heterochromatin blocks does not have a major influence on the distribution of MLH1 foci and (c) the distribution of crossovers in the homologous chromosomes to human chromosomes 13 and 21 are conserved between species of the same genus (C. libidinosus and C. nigritus) but are significantly different between Cebus and Alouatta. This heterogeneity in recombination behaviour among Ceboidea species may reflect differences in genetic diversity and genome composition.

The template choice decision in meiosis: is the sister important?

Abstract: Recombination between homologous chromosomes is crucial to ensure their proper segregation during meiosis. This is achieved by regulating the choice of recombination template. In mitotic cells, double-strand break repair with the sister chromatid appears to be preferred, whereas interhomolog recombination is favoured during meiosis. However, in the last year, several studies in yeast have shown the importance of the meiotic recombination between sister chromatids. Although this thinking seems to be new, evidences for sister chromatid exchange during meiosis were obtained more than 50 years ago in non-model organisms. In this mini-review, we comment briefly on the most recent advances in this hot topic and also describe observations which suggest the existence of inter-sister repair during meiotic recombination. For instance, the behaviour of mammalian XY bivalents and that of trivalents in heterozygotes for chromosomal rearrangements are cited as examples. The “rediscovering” of the requirement for the sister template, although it seems to occur at a low frequency, will probably prompt further investigations in organisms other than yeast to understand the complexity of the partner choice during meiosis.

Possible differences in the two Z chromosomes in male chickens and evolution of MHM sequences in Galliformes.

Abstract: The male hypermethylated (MHM) region of the chicken Z chromosome encodes a non-coding RNA that is expressed only in females. The MHM sequence is found only in galliform birds, and Z genes near this region show an unusual degree of dosage compensation between males and females despite the overall low level of dosage compensation in Z chromosome gene expression in birds. Here we report that the MHM locus shows a dramatic sex difference in the configuration of chromatin, open in females and condensed in males, based on DNA fluorescent in situ hybridization of an MHM probe in interphase nuclei. The demethylating agent 5-aza-cytidine causes an asymmetric effect on the two Z chromosomes of males, altering the chromatin configuration, MHM RNA expression, and H4K16Ac modification, suggesting an inequality in the methylation status and chromatin of the two Z chromosomes. We identified numerous MHM-related genomic and RNA sequences that possess a short conserved sequence common to the majority of clones, suggesting the functional importance of the MHM region. Some of the RNA sequences, which like MHM are expressed in females but not in males, are likely to be polyadenylated and have genomic intron/exon structure. The turkey, another galliform bird, has repetitive sequences in the predicted turkey MHM region, raising the question of regional dosage compensation in the turkey as in the chicken.