Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin.

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.

Small chromosomal regions position themselves autonomously according to their chromatin class.

The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (1) gene-rich and SINE-rich euchromatin; (2) gene-poor and LINE/LTR-rich heterochromatin; and (3) gene-depleted and satellite DNA-containing constitutive heterochromatin. We show, using fluorescence in situ hybridization (FISH) and 4C-seq technologies, that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespective of their chromosomal context, thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes.

First cytogenetic record for a species of Otothyropsis Ribeiro, Carvalho & Melo, 2005 (Loricariidae, Hypoptopomatinae)

Hypoptopomatinae is a monophyletic subfamily that includes 147 species, distributed in 20 genera. Otothyropsis is a genus of Hypoptopomatinae, recently described. Here, we provided the first cytogenetic information of Otothyropsis . The specimens were collected from córrego Dourado, a small tributary of rio Iguatemi, which flows into rio Paraná. The specimens of Otothyropsis cf. polyodon were analyzed with respect to diploid number, C-Band and Ag-NOR patterns. The diploid number was 54 chromosomes, distributed in 18 metacentric, 28 submetacentric, and 8 subtelocentric chromosomes, with single Ag-NOR and conspicuous heterochromatic blocks on the short and long arms of the 24th pair of chromosomes. Our study highlights the conservation trend of the diploid number (2n=54) and fundamental number (FN = 108) among the species of Hypoptopomatinae. However, the karyotype formula (18m+28sm+8st) seems to be specific to O. cf. polyodon , considering the other Hypoptopomatinae species already analyzed.

Hypoptopomatinae é uma subfamília monofilética que inclui 147 espécies distribuídas em 20 gêneros, sendo Otothyropsis um gênero recentemente descrito. Aqui, fornecemos a primeira informação citogenética do gênero Otothyropsis . Espécimes foram coletados no córrego Dourado, um pequeno tributário do rio Iguatemi, o qual deságua no rio Paraná. Espécimes de Otothyropsis cf. polyodon foram analisados em relação ao número diploide e padrões de Banda-C e Ag-NOR. O número diploide foi de 54 cromossomos, distribuídos em 18 metacêntricos, 28 submetacêntricos e 8 subtelocêntricos, com Ag-NOR simples e blocos heterocromáticos evidentes no braços curto e longo do par de cromossomos 24. Nosso estudo destaca a tendência de conservação do número diploide (2n=54) e número fundamental (NF=108) entre as espécies de Hypoptopomatinae. Entretanto, a fórmula cariotípica (18m+28sm+8st) parece ser específica para O. cf. polyodon, considerando as outras espécies de Hypoptopomatinae já analisadas.

Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos.

Efficient reprograming of the donor cell genome in nuclear transfer (NT) embryos is linked to the ability of the embryos to sustain full-term development. As the nuclear architecture has recently emerged as a key factor in the regulation of gene expression, we questioned whether early bovine embryos obtained from transfer of cultured fibroblasts into enucleated oocytes would adopt an embryo-like nuclear organization. We studied the dynamics of constitutive heterochromatin in the stages prior to embryonic genome activation by distribution analysis of heterochromatin protein CBX1 (HP1), centromeric proteins CENPA and CENPB, and histone H3 three-methylated at lysine 9. Then we applied descriptive, quantitative, and co-localization analyses. A dramatic reorganization of heterochromatic blocks of somatic donor cells was first observed in the late one-cell stage NT embryos. Then at two- and four-cell stages, we found two types of NT embryos: one displaying noncondensed heterochromatin patches similar to IVF embryos, whereas the second type displayed condensed heterochromatin blocks, normally observed in IVF embryos only after the eight-cell stage. These analyses discriminate for the first time two contrasted types of nuclear organization in NT embryos, which may correspond to different functional states of the nuclei. The relationship with the somatic nucleus reprograming efficiency is discussed.

Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila.

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). This DNA is found in various eukaryotes from yeast to humans, and its levels are elevated by exposure to carcinogens. eccDNA is heterogeneous in size and composed of chromosomal sequences. In this study we used two-dimensional gel electrophoresis to detect and characterize eccDNA in Drosophila. We found eccDNA throughout the fly's life cycle. These molecules comprise up to 10% of the total repetitive DNA content, and their size ranges from <1 kb to >20 kb. The eccDNA population contains circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, and the Suppressor of Stellate. Multimers of centromeric heterochromatin sequences are included in eccDNA as well. Our findings are consistent with the hypothesis that intramolecular homologous recombination between direct tandem repeats is a favorite mechanism for eccDNA formation. The level of eccDNA increased following MMS treatment of wild-type larvae, consistent with phenomena observed in cultured mammalian cells. This shows mutagen-induced eccDNA formation in the context of the whole organism for the first time. Mutations in the genes okra, mus309, and mei41 did not affect eccDNA under normal conditions or following mutagen treatment, implying that eccDNA formation is different from known pathways of DNA repair.

[Heterochromatin, gene position effect and gene silencing]. / Geterokhromatin, éffekt polozheniia gena i geneticheskii sailensing.

Genomes of higher eukaryotes consist of two types of chromatin: euchromatin and heterochromatin. Heterochromatin is densely packed material typically localized in telomeric and pericentric chromosome regions. Euchromatin transferred by chromosome rearrangements in the vicinity of heterochromatin is inactivated and acquires morphological properties of heterochromatin in the case of position effect variegation. One of the X chromosomes in mammal females and all paternal chromosome set in coccides become heterochromatic. The heterochromatic elements of the genome exhibit similar structural properties: genetic inactivation, compaction, late DNA replication at the S stage, and underrepresentation in somatic cells. The genetic inactivation and heterochromatin assembly are underlain by a specific genetic mechanism, silencing, which includes DNA methylation and posttranslational histone modification provided by the complex of nonhistone proteins. The state of silencing is inherited in cell generations. The same molecular mechanisms of silencing shared by all types of heterochromatic regions, be it unique or highly repetitive sequences, suggest the similar organization of these regions. No type of heterochromatin is a permanent structure as they all are formed at the strictly definite stages of early embryogenesis. Based on the bulk of evidence accumulated today, heterochromatin can be regarded as a morphological manifestation of genetic silencing.

Chromosome banding and molecular cytogenetic study of two Mediterranean trachinoid fish species (Teleostei: Trachinidae, Uranoscopidae).

The chromosomes of Echiichthys vipera (Trachinidae) and Uranoscopus scaber (Uranoscopidae) were analyzed by means of various banding methods and fluorescence in situ hybridization (FISH) with telomeric and major rDNA probes, respectively. The karyotype of E. vipera was composed of 48 acrocentric chromosomes and NOR sites, as revealed by all detection methods, were situated pericentromerically on a single pair of middle-sized chromosomes. Blocks of constitutive heterochromatin were present in the pericentromeric regions of all pairs of chromosomes. The karyotype of U. scaber showed three karyomorphs: 2n = 30 (18 m + 12 a/st [m = metacentric, a = acrocentric and st = subtelocentric]), 2n = 28 (20 m + 8 a/st), 2n = 27 (21 m + 6 a/st). NORs, as revealed by FISH, were situated pericentromerically on a single pair of middle-sized chromosomes in spite of Ag-positive signals in the centromeres of all pairs of chromosomes. Robertsonian fusions were hypothesized for observed variation due to invariable number of chromosome arms FN = 48.

Heterochromatic banding patterns on chromosomes of twelve weevil species (Insecta, Coleoptera, Curculionoidea: Apionidae, Curculionidae).

The C-banding patterns of twelve weevil species are presented. The obtained results confirm the existence of two groups of species: with a small or large amount of heterochromatin in the karyotype. The first group comprises seven species (Apionidae: Holotrichapion pisi; Curculionidae: Phyllobius urticae, Ph. pyri, Ph. maculicornis, Tanymecus palliatus, Larinodontes turbinatus, Cionus tuberculosus). In weevils with a small amount of heterochromatin, tiny grains on the nucleus in interphase are visible, afterwards in mitotic and meiotic prophase appearing as dark dots. The absence of C-bands does not indicate a lack of heterochromatin but heterochromatic regions are sometimes so small that the condensation is not visible during the cell cycle. The second group comprises five species (Otiorhynchus niger, O. morio, Polydrusus corruscus, Barypeithes chevrolati, Nedyus quadrimaculatus) which possess much larger heteropicnotic parts of chromosomes visible during all nuclear divisions. The species examined have paracentromeric C-bands on autosomes and the sex chromosome X, except for Otiorhynchus niger, which also has an intercalary bands on one pair of autososomes. All the species examined differ in the size of segments of constitutive heterochromatin. The y heterochromosome is dot-like and wholly euchromatic in all the studied species.

Constitutive heterochromatin in karyotypes of two Cicadidae species from Japan (Cicadoidea, Hemiptera).

Karyotypes of males of cicadas Tibicen bihamatus (Motschulski) and Platypleura kuroiwae Matsumura were studied using C-banding technique. In Tibicen bihamatus two types of C-band distribution were observed. Two chromosome pairs have C-bands at one of the chromosome ends, while in the other, including the sex chromosome, C-heterochromatin blocks occurred at both ends. Platypleura kuroiwae has a smaller amount of C-heterochromatin located as small subterminal blocks. The intercalar C-bands were seen in the early spermatogonial metaphase chromosomes.