Centromeric Constitutive Heterochromatin Research Articles

Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin

Heterochromatin regulation is critical for genomic stability. Different H3K9 methylation states have been discovered, with distinct roles in heterochromatin formation and silencing. However, how the transition from H3K9me2 to H3K9me3 is controlled is still unclear. Here, we investigate the role of the conserved bromodomain AAA-ATPase, Abo1, involved in maintaining global nucleosome organisation in fission yeast. We identified several key factors involved in heterochromatin silencing that interact genetically with Abo1: histone deacetylase Clr3, H3K9 methyltransferase Clr4, and HP1 homolog Swi6. Cells lacking Abo1 cultivated at 30 °C exhibit an imbalance of H3K9me2 and H3K9me3 in heterochromatin. In abo1∆ cells, the centromeric constitutive heterochromatin has increased H3K9me2 but decreased H3K9me3 levels compared to wild-type. In contrast, facultative heterochromatin regions exhibit reduced H3K9me2 and H3K9me3 levels in abo1∆. Genome-wide analysis showed that abo1∆ cells have silencing defects in both the centromeres and subtelomeres, but not in a subset of heterochromatin islands in our condition. Thus, our work uncovers a role of Abo1 in stabilising directly or indirectly Clr4 recruitment to allow the H3K9me2 to H3K9me3 transition in heterochromatin.

Comparative cytogenetic analysis of marine Palaemon species reveals a X1X1X2X2/X1X2Y sex chromosome system in Palaemon elegans

BackgroundThe maintenance of species and the promotion of speciation are closely related to chromosomal rearrangements throughout evolution. Decapoda represents the most species-rich order among crustaceans and, despite its ecological and economic importance, little is known about decapod karyology. We aim at cytogenetically characterizing two sympatric prawn species.ResultsAnalysis of mitotic metaphases and meiotic diakinesis of the common prawn Palaemon serratus and the rockpool prawn P. elegans, revealed considerable differences between their karyotypes including chromosome numbers and sex determination systems. The cytogenetic data for P. serratus showed a diploid number of 56 and the putative absence of heteromorphic sex chromosomes. However, the diploid chromosome number in P. elegans was 90 for females and 89 for males. The karyotype of the females consisted of the three largest acrocentric pairs and 42 submetacentric and metacentric pairs, while the karyotype of the males comprised a clearly identifiable large metacentric chromosome and two acrocentric pairs as well as the smaller 42 pairs. These results highlight the presence of the X1X1X2X2/X1X2Y multiple sex chromosome system in P. elegans, which constitute the only sexual system for Decapoda reported cytogenetically using modern techniques. The origin of this sex chromosome system is discussed. We hypothesize that the chromosome evolution within the genus could involve several fusion events giving rise to a reduction on the chromosome number in P. serratus. In both species, the major ribosomal genes were located in two chromosome pairs and hybridization signals of the telomeric sequences (TTAGGG)n were visualized at the telomeres of all chromosomes. C-banding revealed that, when present, constitutive heterochromatin had a predominantly telomeric distribution and no centromeric constitutive heterochromatin was observed.ConclusionsAlthough more comparative cytogenetic analyses are needed to clarify our hypotheses, the findings of this work indicate that the prawns of the genus Palaemon represent a promising model among Decapoda representatives to investigate the karyotype evolution and the patterns of sex chromosome differentiation.

Highest Diploid Number Among Gymnotiformes: First Cytogenetic Insights into Rhabdolichops (Sternopygidae).

We report the first comparative cytogenetic analysis of two species from electrogenic fish of genus Rhabdolichops (Sternopygidae, Gymnotiformes): Rhabdolichops troscheli and Rhabdolichops cf eastwardi. R. troscheli has 2n = 54 (fundamental number [FN] = 66), whereas R. cf. eastwardi has 2n = 74 (FN = 78). C-banding revealed centromeric constitutive heterochromatin in both species. Ag-NORs mapped on pair 6 in R. troscheli and pair 30 in R. cf eastwardi. Fluorescense in situ hybridization with 18S rDNA probes confirmed the Ag-NOR staining results and revealed additional (presumably silent) ribosomal genes on pairs 12, 13, 21, 23, 26, and 27 in R. cf eastwardi. 5S rDNA was found on the centromeres of pair 7 in both species. Telomeric probes showed only distal locations. Dispersed signal patterns were obtained using probes for retrotransposons Rex1 and Rex3. Histone H1 and H3 genes were found together on pair 6 in R. cf eastwardi. The high diploid number found in Rhabdolichops suggests that chromosome fission may have contributed to its chromosomal evolution, phylogenetic relationship of the Sternopygidae suggests that this increase in diploid number could be a synapomorphic characteristic of genus Rhabdolichops. Although both species are phylogenetically close related, their karyotype structure has undergone divergent evolutionary directions. All in all, our results strongly suggest that R. cf eastwardi experencied recent intense genome reorganization.

Studies on chromosomal characteristics of Ctenus indicus (Gravely 1931) (Araneae: Ctenidae)

The karyological information gathered for the Indian spiders taxa thus far were cytologically derived from only few species but none for the representatives belonging to the genus Ctenus. Ctenus indicus (Gravely 1931), an Indian ctenid spider was cytogenetically analyzed following conventional, C- and NOR-banding techniques so as to gather substantial data for future course of understanding of karyotypic evolution among spider species. The karyotypic data for Ctenus indicus revealed the complement consisting of (2n = 28) 26AA + X1X2♂ and (2n = 30) 26AA + X1X1X2X2♀ acrocentric chromosomes. A closer scrutiny of meiotic progression disclosed many male pachytenic cells displaying the occurrence of ‘bouquet’ formation. The results of C-banding enabled in identifying centromeric constitutive heterochromatin locales, and in some chromosomes also the distal ends of telomeric regions. Silver nitrate stained NOR-specifications were noticed at the distal telomeric regions of two pairs of chromosomes (#8 and #10) in the complement. Cytological evidence procured from the present study not only adds to the ever-growing list of the spider cytogenetic assessments but also offers as a baseline data towards establishing evolutionary relationships within this important group.

Heterochromatin and molecular characterization of DsmarMITE transposable element in the beetle Dichotomius schiffleri (Coleoptera: Scarabaeidae).

Cytogenetic studies of the Neotropical beetle genus Dichotomius (Scarabaeinae, Coleoptera) have shown dynamism for centromeric constitutive heterochromatin sequences. In the present work we studied the chromosomes and isolated repetitive sequences of Dichotomius schiffleri aiming to contribute to the understanding of coleopteran genome/chromosomal organization. Dichotomius schiffleri presented a conserved karyotype and heterochromatin distribution in comparison to other species of the genus with 2n=18, biarmed chromosomes, and pericentromeric C-positive blocks. Similarly to heterochromatin distributional patterns, the highly and moderately repetitive DNA fraction (C 0 t-1 DNA) was detected in pericentromeric areas, contrasting with the euchromatic mapping of an isolated TE (named DsmarMITE). After structural analyses, the DsmarMITE was classified as a non-autonomous element of the type miniature inverted-repeat transposable element (MITE) with terminal inverted repeats similar to Mariner elements of insects from different orders. The euchromatic distribution for DsmarMITE indicates that it does not play a part in the dynamics of constitutive heterochromatin sequences.

Induction of RNA‐directed DNA methylation upon decondensation of constitutive heterochromatin

Centromeric constitutive heterochromatin is marked by DNA methylation and dimethylated histone H3 Lys 9 (H3K9me2) in Arabidopsis. RNA-directed DNA methylation (RdDM) is a process that uses 24-nucleotide (nt) small interfering RNAs (siRNAs) to induce de novo methylation to its homologous DNA sequences. Despite the presence of centromeric 24-nt siRNAs, mutations in genes required for RdDM do not appreciably influence the methylation of centromeric repeats. The mechanism by which constitutive heterochromatin is protected from RdDM remains puzzling. Here, we report that the vegetative cell nuclei (VN) of the male gametophyte (pollen) invariably undergo extensive decondensation of centromeric heterochromatin and lose centromere identity. VN show greatly reduced H3K9me2, phenocopying nuclei carrying a mutation in the chromatin remodeller DECREASE IN DNA METHYLATION 1 (DDM1). However, unlike the situation in ddm1 nuclei, the decondensed heterochromatin retains dense CG methylation and transcriptional silencing, and, unexpectedly, is subjected to RdDM-dependent hypermethylation in non-CG contexts. These findings reveal two assembly orders of silent heterochromatin and implicate the condensed form in blocking the RdDM machinery.

C-Banded Karyotype and Nucleolar Organizer Regions (NORs) of Marsh Frog, Rana ridibunda (Ranidae: Anura) in Central Anatolia

Summary Karyotype, C-banding and nucleolar organizer regions (NORs) characteristics of nine Rana ridibunda samples collected from Konya (Meram, Beysehir, Hadim) province were examined. The diploid number of chromosomes (2n) and the fundamental number of chromosome arms (FN) were determined as 26 and 52, respectively. All of the chromosomes of this species have centromeric constitutive heterochromatin (C-band). Nucleolar organizer regions (secondary constriction) were determined on the long arm of the no. 8 submetacentric chromosome by using silver-nitrate staining technique.

Cytogenetic Studies on European Brown Hare, Lepus europaeus Pallas, 1778 (Lagomorpha: Leporidae) in Turkey

Summary In this study, the karyotype, C-heterochromatin blocks, and nucleolar organizer regions (NORs) of four specimens of Lepus europaeus from Turkey were examined. The diploid number of chromosomes (2n), the fundamental number of chromosome arms (FN), and the number of autosomal arms (FNa) were determined as 48, 90, and 86, respectively. All of the chromosomes have been determined to be possessed of centromeric constitutive heterochromatin, except of the pair no. 10. The X chromosome had centromeric heterochromatin and Y chromosome had pericentromeric heterochromatin. There were no distal C-bands in any chromosome of samples. The NORs have been determined to be located in the terminal regions of the long arms of three subtelocentric chromosomes (nos. 15, 16, and 18). Any relation was not determined between the localization of the NORs and C-positive regions of autosomes.

Differences in karyotype between two sympatric species of Gymnotus (Gymnotiformes: Gymnotidae) from the eastern amazon of Brazil

In this paper we describe the karyotypes of two sympatric species of Gymnotus from Marajó Island, Pará, in the Eastern Amazon basin of Brazil. One of these species, G. mamiraua, has not previously been identified from this area. Karyotype studies demonstrate that G. cf. carapo Linnaeus, has 2n=42 (30M/SM+12ST/A) and G. mamiraua Albert & Crampton, 2n=54 (50M/SM+4ST). The Nucleolar Organizer Region (NOR) was found in the distal region of the short arm of pair 20 in G. cf. carapo and in the interstitial region of pair 1 of G. mamiraua. Both species have centromeric constitutive heterochromatin (CH) in all chromosomes and some in interstitial or distal regions. In both species, 4´6-Diamidino-2- phenylindole (DAPI) staining has shown banding concordant with C-banding, whereas the Chromomycin A3 (CMA3) staining in both species agrees with the NOR location. The new data and review of cytogenetic data in Gymnotus presented here is concordant with a hypothesis that G. carapo is either a complex of populations, or a complex of morphologically similar cryptic species with distinct karyotypes. These taxa may be reproductively isolated by prezygotic reproductive isolation barriers (such as the structure of the Electric Organ Discharge), or by post-zygotic barriers (such as karyotypic formula). The understanding of the cytogenetic structure of G. carapo, combined with associated studies of signal diversity, will yield insight into the mechanisms underlying the origins and maintenance of high species diversity in Gymnotus.

Differences in karyotype between two sympatric species of Gymnotus (Gymnotiformes: Gymnotidae) from the eastern amazon of Brazil

In this paper we describe the karyotypes of two sympatric species of Gymnotus from Marajó Island, Pará, in the Eastern Amazon basin of Brazil. One of these species, G. mamiraua, has not previously been identified from this area. Karyotype studies demonstrate that G. cf. carapo Linnaeus, has 2n=42 (30M/SM+12ST/A) and G. mamiraua Albert & Crampton, 2n=54 (50M/SM+4ST). The Nucleolar Organizer Region (NOR) was found in the distal region of the short arm of pair 20 in G. cf. carapo and in the interstitial region of pair 1 of G. mamiraua. Both species have centromeric constitutive heterochromatin (CH) in all chromosomes and some in interstitial or distal regions. In both species, 4´6-Diamidino-2- phenylindole (DAPI) staining has shown banding concordant with C-banding, whereas the Chromomycin A3 (CMA3) staining in both species agrees with the NOR location. The new data and review of cytogenetic data in Gymnotus presented here is concordant with a hypothesis that G. carapo is either a complex of populations, or a complex of morphologically similar cryptic species with distinct karyotypes. These taxa may be reproductively isolated by prezygotic reproductive isolation barriers (such as the structure of the Electric Organ Discharge), or by post-zygotic barriers (such as karyotypic formula). The understanding of the cytogenetic structure of G. carapo, combined with associated studies of signal diversity, will yield insight into the mechanisms underlying the origins and maintenance of high species diversity in Gymnotus.

Cytogenetic study of Anopheles albitarsis (Diptera: Culicidae) by C-banding and in situ hybridization

The C-banding pattern and the size and location of the nucleolar organizer regions (NORs) are described for the first time in Brazilian populations of Anopheles (Nyssorhynchus) albitarsis sensu lato. C-banding revealed variation in the size of the centromeric heterochromatic blocks in autosomal chromosomes and in the acrocentric (X) and puntiform (Y) sex chromosomes. Fluorescence in situ hybridization showed that the NORs were located in the pericentromeric region of the sex (XX/XY) chromosomes and that this coincided with the number and location of centromeric constitutive heterochromatin blocks previously revealed by C-banding. The NORs varied in size among the homologues of the three populations. These findings of the populations studied support the hypothesis that the stability of NORs in the A. albitarsis complex is characterized by the presence of clustered and conserved sites in a unique pair of chromosomes.

Karyotype characterization, constitutive heterochromatin and nucleolus organizer regions of Paranaita opima (Coleoptera, Chrysomelidae, Alticinae)

Species of the subtribe Oedionychina not only have a highly uniform diploid number of 2n = 22 (20+X+y) but have the karyotypic peculiarity of possessing extremely large sex chromosomes. We analyzed Paranaita opima embryos and gonadal cells to determine their diploid number, chromosomal morphology, type of sex determination system, constitutive heterochromatin pattern and which chromosomes bear nucleolus organizer regions (NORs). The diploid number of P. opima was 2n = 22 (20+XY/XX) with all chromosomes being metacentric. Chromosome pair 6 showed an interstitial secondary constriction on the short arm. The C-banding technique revealed centromeric constitutive heterochromatin in all chromosomes, which, in pair 6, extended up to the secondary constriction of the short arm, additional C-bands also being present on the Y chromosome. Silver nitrate nucleolar organizer region (Ag-NOR) staining showed NORs on the secondary constriction of pair 6. Fluorochrome analysis with chromomycin A3 (CMA3), 4'-6-diamidino-2-phenylindole (DAPI) and the distamycin A (DA) counterstain showed that the short arm of chromosome pair 6 exhibited a GC-rich block extending from the proximal to the median region, including part of the secondary constriction. The same techniques also showed AT-rich blocks at the centromeric region of all chromosomes and at the terminal region of the short arm of pair 6. The basic karyotype characteristics and C band pattern of P. opima are similar to those described for other species in the subtribe Oedionychina. The pattern of autosomal NORs observed in P. opima corresponds to that registered in the majority of the Chrysomelidae species.

A preliminary note on banded karyotypes of the Perny's long-nosed squirrel Dremomys pernyi (Mammalia, Rodentia) from Taiwan

Abstract The chromosomes of the Perny's long-nosed squirrel Dremomys pernyi owstoni that is endemic subspecies to Taiwan were examined with conventional and Ag-NOR staining and G- and C-banding technique. The diploid chromosome number (2.) and fundamental autosomal arm number (FN) of this squirrel were 38 and 68, respectively. The karyotype consisted of 7 pairs of metacentrics (nos. 1–7), 9 pairs of submetacentrics or subtelocentrics (nos. 8–16), 2 pairs of acrocentrics (nos. 17 and 18), a medium-sized submetacentric X chromosome, and an acrocentric Y chromosome. On the terminal region of short arm of no. 17 chromosomes, the prominent satellite the stalk potion of which essentially corresponded to Ag-NOR was recognized. The no. 18 chromosome was markedly minute acrocentric. In the C-banded karyotype, the all autosomes and X chromosome carried centromeric constitutive heterochromatin (C-heterochromatin), and the Y chromosome appeared to be entirely heterochromatic. Based on the cytogenetical characteristic...

Banded karyotypes of the hairy-footed flying squirrel Belomys (Trogopterus) pearsonii (Mammalia, Rodentia) from Taiwan

Abstract The chromosomes of the hairy-footed flying squirrel Belomys pearsonii from Taiwan were analyzed with conventional and Ag-NOR staining and G- and C-banding technique. The diploid chromosome number (2n) and fundamental autosomal arm number (FN) were 38 and 72, respectively. The karyotype consisted of six pairs of metacentrics (nos. 1–6), twelve pairs of submetacentrics or subtelocentrics (nos. 7–18), an unusually large-sized metacentric X chromosome, and an acrocentric Y chromosome. The satellite structure, which corresponded to the NOR detected by silver-staining, was observed on the long arm of no. 1 chromosomes. All autosomes and the X chromosome had centromeric constitutive heterochromatin (C-heterochromatin), and the long arms of nos. 17 and 18 chromosomes were entirely heterochromaic. By the C-banding technique, the Y chromosome was entirely stained somewhat darkly than euchromatic region, but did not seem to be genuinely heterochromatic. On the basis of the cytogenetical characteristics of s...

Chromosomal characterization of the Formosan striped squirrel Tamiops maritimus formosanus (Mammalia, Rodentia)

Abstract Chromosomes of the Formosan striped squirrel Tamiops maritimus formosanus were analyzed with conventional and silver staining and G- and Cbanding technique. The diploid chromosome number (2n) and fundamental autosomal arm number (FN) were 38 and 68, respectively. Karyotype consisted of seven pairs of metacentrics (nos. 1–7), nine pairs of submetacentrics or subtelocentrics (nos. 8–16), two pairs of acrocentrics (nos. 17 and 18), a medium-sized submetacentric X chromosome, and a small acrocentric Y chromosome. All autosomes and the X chromosome carried centromeric constitutive heterochromatin, but the Y chromosome was entirely euchromatic. In the short arm of no. 17 chromosomes, the prominent satellite structure with its long stalk, which corresponded to the nucleolus organizer region (NOR), was observed, suggesting that it could have been shared with other squirrel species through the evolutionary process.

Karyotype and nuclear DNA content of the Australian lungfish, <i>Neoceratodus forster</i><i>i</i> (Ceratodidae: Dipnoi)

The karyotype of the lungfish, Neoceratodus forsteri, is described as 2n = 54, comprising 3 pairs of very large metacentrics, 1 pair of large submetacentrics, 13 pairs of smaller acrocentrics, and 10 pairs of microchromosomes. In addition to centromeric constitutive heterochromatin, C-bands were located on the arms of almost all of the macrochromosomes. The nuclear DNA content of N. forsteri was confirmed as being high (98.6-111.9 pg/nucleus).

Karyological Study of the Mediterranean Krill Meganyctiphanes norvegica (Euphausiacea)

ABSTRACT The chromosomes of Meganyctiphanes norvegica from the Mediterranean Ligurian Sea were investigated using standard Giemsa, silver staining, and C-banding. The karyotype of both sexes consists of 19 metacentric chromosome pairs of gradually decreasing size. Silver staining of male mitotic and meiotic metaphase showed, in all chromosomes, centromeric regions that appeared to be related to kinetochores rather than to nucleolus-organizer regions. C-banding revealed centromeric constitutive heterochromatin in all chromosomes. No heteromorphic sex differentiation was observed. The karyotype of the krill Meganyctiphanes norvegica is reported here for the first time and will provide a basis for further cytogenetic investigation of species and populations of krill.

Absence of geographic chromosomal variation in the roan and sable antelope and the cytogenetics of a naturally occurring hybrid.

The determination of geographic chromosomal variation in rare or endangered species, or those of special management concern, is important, since geographically defined cytotypes can negatively influence breeding programs involving founders drawn from widely divergent localities. We cytogenetically analyzed specimens of the roan (Hippotragus equinus) and sable antelope (H. niger) collected from widely divergent localities throughout their respective ranges. Each species was characterized by a diploid number of 60 and an invariant karyotype. In contrast to the absence of intraspecific variation, however, the two species differ with respect to centromeric constitutive heterochromatin and numbers of nucleolar organizer regions. These cytogenetic landmarks were subsequently used to verify an anecdotal account of a naturally occurring roan x sable hybrid. The data show that, despite their markedly distinct phenotypes, the roan and sable antelope are nonetheless sufficiently similar genetically to produce viable offspring. Hybridization, although a rare event between these species, is probably partly promoted by behavioral differences which are not always sufficient to prevent mating between them.

Karyotype of Callithrix mauesi (Callitrichidae, Primates) and its relations with those of C. emiliae and C. jacchus.

A study was conducted on the most recently described marmoset species, Callithrix mauesi, and the results obtained were compared to those previously reported for the karyotypes of C. jacchus and C. emiliae. No mechanism of chromosome rearrangement differentiates the karyotypes of C. mauesi (2n = 44) and C. emiliae (2n = 44), which diverge from C. jacchus (2n = 46) by a Robertsonian translocation and a paracentric inversion. C. mauesi, like C. emiliae, presents telomeric constitutive heterochromatin in various chromosomes, forming large heterochromatic blocks in some. This does not occur in C. jacchus, which basically presents centromeric constitutive heterochromatin. The karyotype of C. mauesi differs from that of C. emiliae only by the amount and distribution of this telomeric constitutive heterochromatin. One of the chromosomes presenting a heterochromatic block in C. mauesi is chromosome X, a fact not previously reported in the Order Primates. The present chromosome data show that C. mauesi is closer to C. emiliae than to C. jacchus, in agreement with its inclusion in the C. argentata group. In the present paper, we describe for the first time, at the chromosome level, chimerism between fraternal twins of the same sex (XY/XY), with the heterochromatic block of pair 2 being the marker. © 1994 Wiley-Liss, Inc.

Sequence of centromere separation: role of centromeric heterochromatin.

The late metaphase-early anaphase cells from various tissues of male Mus musculus, M. poschiavinus, M. spretus, M. castaneus, female and male Bos taurus (cattle) and female Myopus schisticolor (wood lemming) were analyzed for centromeres that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y chromosome is the first one to separate, as is the X or Y in the cattle. These sex chromosomes are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late replicating X chromosome appears to separate later than the active X. In the wood lemming the three pairs of autosomes with the least amount of centromeric constitutive heterochromatin separate first. These are followed by the separation of seven pairs of autosomes carrying medium amounts of constitutive heterochromatin. Five pairs of autosomes with the largest amounts of constitutive heterochromatin are the last in the sequence of separation. The sex chromosomes with medium amounts of constitutive heterochromatin around the centromere, and a very large amount of distal heterochromatin, separate among the very late ones but are not the last. These observations assign a specific role to centromeric constitutive heterochromatin and also indicate that nonproximal heterochromatin does not exert control over the sequence in which the centromeres in the genome separate. It appears that qualitative differences among various types of constitutive heterochromatin are as important as quantitative differences in controlling the separation of centromeres.