Chromosome evolution and improved cytogenetic maps of the Y chromosome in cattle, zebu, river buffalo, sheep and goat

Comparative FISH-mapping among Y chromosomes of cattle (Bos taurus, 2n = 60, BTA, submetacentric Y chromosome), zebu (Bos indicus, 2n = 60, BIN, acrocentric Y chromosome but with visible small p-arms), river buffalo (Bubalus bubalis, 2n = 50, BBU, acrocentric Y chromosome), sheep (Ovis aries, 2n = 54, OAR, small metacentric Y chromosome) and goat (Capra hircus, 2n = 60, CHI, Y-chromosome as in sheep) was performed to extend the existing cytogenetic maps and improve the understanding of karyotype evolution of these small chromosomes in bovids. C- and R-banding comparison were also performed and both bovine and caprine BAC clones containing the SRY, ZFY, UMN0504, UMN0301, UMN0304 and DYZ10 loci in cattle and DXYS3 and SLC25A6 in goat were hybridized on R-banded chromosomes by FISH. The main results were the following: (a) Y-chromosomes of all species show a typical distal positive C-band which seems to be located at the same region of the typical distal R-band positive; (b) the PAR is located at the telomeres but close to both R-band positive and ZFY in all species; (c) ZFY is located opposite SRYand on different arms of BTA, BIN, OAR/CHI Y chromosomes and distal (but centromeric to ZFY) in BBU-Y; (d) BTA-Y and BIN-Y differ as a result of a centromere transposition or pericentric inversion since they retain the same gene order along their distal chromosome regions and have chromosome arms of different size; (e) BTA-Y and BBU-Y differ in a pericentric inversion with a concomitant loss or gain of heterochromatin; (f) OAR/CHI-Y differs from BBU-Y for a pericentric inversion with a major loss of heterochromatin and from BTA and BIN for a centromere transposition followed by the loss of heterochromatin.     

Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH)

Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes; (c) absence of hybridization to the ribosomal gene probes; (d) C-band-positive centromeric regions; and (e) euchromatic arms. They differ as follows: (a) the larger B element hybridizes to the centromeric heterochromatin (pMeg-1) probe whereas the smaller B element does not; (b) the amount of C-band-positive material is reduced in the smaller B chromosome relative to that present on the larger B chromosome; and (c) the smaller element is reduced in size by about a third. It is concluded that the larger B chromosome arose as a leftover centromere from centric fusion, whereas the smaller element has a different origin — perhaps as an intact fragment or as an amplified region from the A chromosomes. The presence of euchromatic regions on B chromosomes may account for their survival in the karyotype.This revised version was published online in November 2005 with corrections to the Cover Date.     

The paracentric inversion In(2Rh)PL alters the centromeric organization of chromosome 2 in Drosophila melanogaster

Centromeres are complex structures involved in an evolutionarily conserved function, the correct segregation of chromosomes and chromatids during meiosis and mitosis. The centromere is determined by epigenetic processes that result in a particular nucleosome organization (CEN chromatin) that differs from the rest of the chromatin including the heterochromatin that normally surrounds the centromere in higher organisms. Many of the current models of centromere origin and organization rely on the molecular and cytological characterization of minichromosomes and their derivatives, and on studies on the origin and maintenance of neocentromeres. Here, we describe the peculiar centromere organization observed in In(2Rh)PL, a paracentric D. melanogaster inversion in which the centromere is maintained in its natural context but is directly flanked by a euchromatic domain as a result of the rearrangement. We have identified the breakpoints of the inversion and show that the proximal one is within the centromere region. The data presented suggest that, notwithstanding the loss of all the pericentric 2Rh heterochromatin, the centromere of the In(2Rh)PL chromosome is still active but presents a nucleosomal organization quite different from the organization usually observed in the centromeric region.     

Elimination of shelterin components bypasses RNAi for pericentric heterochromatin assembly

The RNAi pathway is required for heterochromatin assembly at repetitive DNA elements in diverse organisms. In fission yeast, loss of RNAi causes pericentric heterochromatin defects, compromising gene silencing and chromosome segregation. Here we show that deletion of telomere shelterin components restores pericentric heterochromatin and its functions in RNAi mutants. We further isolated a separation-of-function mutant of Poz1 and revealed that defective telomere silencing, but not telomere length control, is critical for bypassing RNAi. Further analyses demonstrated that compromising shelterin-mediated heterochromatin assembly in RNAi mutants releases heterochromatin protein Swi6, which is redistributed to pericentric regions through RNAi-independent heterochromatin assembly pathways. Given the high mobility of Swi6 protein and that increased levels of Swi6 facilitates heterochromatin spreading as well as ectopic heterochromatin assembly, our results suggest that constitutive heterochromatin domains use multiple pathways to form high-affinity platforms to restrain Swi6, thus limiting its availability and avoiding promiscuous heterochromatin formation.     

Local DNA underreplication correlates with accumulation of phosphorylated H2Av in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes

DNA in Drosophila melanogaster polytene chromosomes is known to be locally underreplicated in both pericentric and intercalary heterochromatin. When the SuUR gene is mutant, complete and partial suppression of underreplication are observed in intercalary and pericentric heterochromatin, respectively; in contrast, overexpression of SuUR results in stronger underreplication. Using antibodies against phosphorylated histone H2Av and flies with different levels of SuUR expression, we demonstrated a clear correlation between the extent of underreplication in specific chromosome regions and the accumulation of H2Av phosphorylated at S137 (gamma-H2AX) at the same sites. Phosphorylated H2Av is a well-established marker of DNA double-stranded breaks (DSB). Our data thus argue that DNA underreplication leads to DSBs and that DSBs accumulate as salivary gland cells progress throughout repeated endocycles. We speculate that ligation of free double-stranded DNA termini causes the formation of ectopic contacts between the underreplicated regions in heterochromatin.     

The de novo chromosome 16 translocations of two patients with abnormal phenotypes (mental retardation and epilepsy) disrupt the <Emphasis Type="Italic">A2BP1</Emphasis> gene

The 16p13.3 breakpoints of two de novo translocations of chromosome 16, t(1;16) and t(14;16), were shown by initial mapping studies to have physically adjacent breakpoints. The translocations were ascertained in patients with abnormal phenotypes characterized by predominant epilepsy in one patient and mental retardation in the other. Distamycin/DAPI banding showed that the chromosome 1 breakpoint of the t(1;16) was in the pericentric heterochromatin therefore restricting potential gene disruption to the 16p13.3 breakpoint. The breakpoints of the two translocations were localized to a region of 3.5 and 115 kb respectively and were approximately 900 kb apart. The mapping was confirmed by fluorescence in situ hybridization (FISH) of clones that spanned the breakpoints to metaphase spreads derived from the patients. The mapping data showed both translocations disrupted the ataxin-2-binding protein 1 (A2BP1) gene that encompasses a large genomic region of 1.7 Mb. A2BP1 encodes a protein that is known to interact with the spinocerebellar ataxia type 2 (SCA2) protein. It is proposed that disruption of the A2BP1 gene is a cause of the abnormal phenotype of the two patients. Ninety-six patients with sporadic epilepsy and 96 female patients with mental retardation were screened by SSCP for potential mutations of A2BP1. No mutations were found, suggesting that disruption of the A2BP1 gene is not a common cause of sporadic epilepsy or mental retardation.     

Autosomal location of a new subtype of 1.688 satellite DNA ofDrosophila melanogaster

During the screening of aDrosophila melanogaster YAC library with DNA from the minichromosomeDp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA. Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long. This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays. Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin. We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.accepted for publication by D. Ward     

Histone modification and the control of heterochromatic gene silencing in Drosophila

Covalent modifications of histones index structurally and functionally distinct chromatin domains in eukaryotic nuclei. Drosophila with its polytene chromosomes and developed genetics allows detailed cytological as well as functional analysis of epigenetic histone modifications involved in the control of gene expression pattern during development. All H3K9 mono- and dimethylation together with all H3K27 methylation states and H4K20 trimethylation are predominant marks of pericentric heterochromatin. In euchromatin, bands and interbands are differentially indexed. H3K4 and H3K36 methylation together with H3S10 phosphorylation are predominant marks of interband regions whereas in bands different H3K27 and H4K20 methylation states are combined with acetylation of H3K9 and H3K14. Genetic dissection of heterochromatic gene silencing in position-effect variegation (PEV) by Su(var) and E(var) mutations allowed identification and functional analysis of key factors controlling the formation of heterochromatin. SU(VAR)3-9 association with heterochromatic sequences followed by H3K9 methylation initiates the establishment of repressive SU(VAR)3-9/HP1/SU(VAR)3-7 protein complexes. Differential enzymatic activities of novel point mutants demonstrate that the silencing potential of SU(VAR)3-9 is mainly determined by the kinetic properties of the HMTase reaction. In Su(var)3-9^ptn a significantly enhanced enzymatic activity results in H3K9 hypermethylation, enhanced gene silencing and extensive chromatin compaction. Mutations in factors controlling active histone modification marks revealed the dynamic balance between euchromatin and heterochromatin. Further analysis and definition of Su(var) and E(var) genes in Drosophila will increase our understanding of the molecular hierarchy of processes controlling higher-order structures in chromatin.     

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.     

X chromosome painting in <Emphasis Type="Italic">Microtus</Emphasis>: Origin and evolution of the giant sex chromosomes

Sex chromosomes in species of the genus Microtus present some characteristic features that make them a very interesting group to study sex chromosome composition and evolution. M. cabrerae and M. agrestis have enlarged sex chromosomes (known as ‘giant sex chromosomes’) due to the presence of large heterochromatic blocks. By chromosome microdissection, we have generated probes from the X chromosome of both species and hybridized on chromosomes from six Microtus and one Arvicola species. Our results demonstrated that euchromatic regions of X chromosomes in Microtus are highly conserved, as occurs in other mammalian groups. The sex chromosomes heterochromatic blocks are probably originated by fast amplification of different sequences, each with an independent origin and evolution in each species. For this reason, the sex heterochromatin in Microtus species is highly heterogeneous within species (with different composition for the Y and X heterochromatic regions in M. cabrerae) and between species (as the composition of M. agrestis and M. cabrerae sex heterochromatin is different). In addition, the X chromosome painting results on autosomes of several species suggest that, during karyotypic evolution of the genus Microtus, some rearrangements have probably occurred between sex chromosomes and autosomes.     

Organ identity specification factor WGE localizes to the histone locus body and regulates histone expression to ensure genomic stability in Drosophila

Over‐expression of Winged‐Eye (WGE) in the Drosophila eye imaginal disc induces an eye‐to‐wing transformation. Endogenous WGE is required for organ development, and wge‐deficient mutants exhibit growth arrest at the larval stage, suggesting that WGE is critical for normal growth. The function of WGE, however, remains unclear. Here, we analyzed the subcellular localization of WGE to gain insight into its endogenous function. Immunostaining showed that WGE localized to specific nuclear foci called the histone locus body (HLB), an evolutionarily conserved nuclear body required for S phase‐specific histone mRNA production. Histone mRNA levels and protein levels in cytosolic fractions were aberrantly up‐regulated in wge mutant larva, suggesting a role for WGE in regulating histone gene expression. Genetic analyses showed that wge suppresses position‐effect variegation, and that WGE and a HLB component Mute appears to be synergistically involved in heterochromatin formation. Further supporting a role in chromatin regulation, wge‐deficient mutants showed derepression of retrotransposons and increased γH2Av signals, a DNA damage marker. These findings suggest that WGE is a component of HLB in Drosophila with a role in heterochromatin formation and transposon silencing. We propose that WGE at HLB contributes to genomic stability and development by regulating heterochromatin structure via histone gene regulation.     

The SUUR protein is involved in binding of SU(VAR)3–9 and methylation of H3K9 and H3K27 in chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

In the present work, we found that the SUUR protein is required for the SU(VAR)3–9 enzyme to bind to the salivary gland polytene chromosomes. The SuUR mutation results in loss of SU(VAR)3–9 on the chromosomes, whereas artificial expression of the SuUR gene restores its binding. The SUUR protein is also involved in methylation of the residues H3K9 and H3K27. However, mono-, di-, and tri-methylated forms of H3K9 and H3K27 behave differently in various chromosomal domains in response to the SuUR mutation. Euchromatin and chromosome 4 are almost completely deprived of mono-, di-, and tri-methylation of H3K9. In the chromocenter, mono-methylation is reduced, di-methylation shows no noticeable changes, and tri-methylation is lost. Furthermore, mono- and di-methylation of H3K27 are not influenced by the SuUR mutation, whereas tri-methylation is lost in the chromocenter. Artificial expression of the SuUR gene on the SuUR ^– background restores the pattern of methylated residues characteristic for the wild type.     

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (Hemiptera, Reduviidae)

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4′,6-diamidino-2-phenylindole/actinomycin D and chromomycin A₃/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.     

Comparative Genome Analysis in American Marsupials: Chromosome Banding and In-situ Hybridization

We performed a comparative analysis of the G- and C-banded karyotypes of seven species of didelphid marsupials, representing the three diploid numbers (2n = 14, 18 and 22) known to occur in this family. In addition to a great similarity among karyotypes with the same diploid numbers, we also identified homeologies for all autosomal arms comprising the three karyotypes. Robertsonian rearrangements, pericentric inversions and heterochromatin variation account for the differences among the karyotypes. Interspecific variation in the size of the sex chromosomes is due to differences in heterochromatic content. In-situ hybridization with total genomic DNA revealed considerable conservation of the euchromatic portions of the three karyotypes and indicated divergence of repetitive DNA sequences in autosomal heterochromatin.     

Stable chromosome fission associated with rDNA mobility

A spontaneous chromosome fission in the plantHypochoeris radicata has been characterized by Feulgen staining,in situ hybridization of the rDNA probe pTA71 and silver staining for active nucleolus organizing regions. The parental acrocentric chromosome has no detectable ribosomal genes at the centromere, but both fission derivatives possess active NORs at their centric ends. In fission heterozygotes, pachytene configurations studied by synaptonemal complex spreading show that the ribosomal cistrons form short arms on each telocentric which pair together to form a triradial. The paired short arms are associated with the single nucleolus at pachytene. It is proposed that the origin and stabilization of the fission rearrangement involved transposition of rDNA from the nucleolus organizing region of chromosome 3 into the centromeric region of chromosome 1.