Research Article |
Corresponding author: Clóvis C. Motta-Neto ( mottaneto.cc@gmail.com ) Academic editor: Eugene Krysanov
© 2018 Clóvis C. Motta-Neto, André Marques, Gideão W.W.F. Costa, Marcelo B. Cioffi, Luiz A.C. Bertollo, Rodrigo X. Soares, Kátia C. Scortecci, Roberto F. Artoni, Wagner F. Molina.
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Citation:
Motta-Neto CC, Marques A, Costa GWWF, Cioffi MB, Bertollo LAC, Soares RX, Scortecci KC, Artoni RF, Molina WF (2018) Differential hypomethylation of the repetitive Tol2/Alu-rich sequences in the genome of Bodianus species (Labriformes, Labridae). Comparative Cytogenetics 12(2): 145-162. https://doi.org/10.3897/CompCytogen.v12i2.21830
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Representatives of the order Labriformes show karyotypes of extreme conservatism together with others with high chromosomal diversification. However, the cytological characterization of epigenetic modifications remains unknown for the majority of the species. In the family Labridae, the most abundant fishes on tropical reefs, the genomes of the genus Bodianus Bloch, 1790 have been characterized by the occurrence of a peculiar chromosomal region, here denominated BOD. This region is exceptionally decondensed, heterochromatic, argentophilic, GC-neutral and, in contrast to classical secondary constrictions, shows no signals of hybridization with 18S rDNA probes. In order to characterize the BOD region, the methylation pattern, the distribution of Alu and Tol2 retrotransposons and of 18S and 5S rDNA sites, respectively, were analyzed by Fluorescence In Situ Hybridization (FISH) on metaphase chromosomes of two Bodianus species, B. insularis Gomon & Lubbock, 1980 and B. pulchellus (Poey, 1860). Immunolocalization of the 5-methylcytosine revealed hypermethylated chromosomal regions, dispersed along the entire length of the chromosomes of both species, while the BOD regions exhibited a hypomethylated pattern. Hypomethylation of the BOD region is associated with the precise co-location of Tol2 and Alu elements, suggesting their active participation in the regulatory epigenetic process. This evidence underscores a probable differential methylation action during the cell cycle, as well as the role of Tol2/Alu elements in functional processes of fish genomes.
Fish cytogenetics, Methylation, BOD region, pseudo-NORs, Mobile elements, Repetitive DNA
Genomes of some representatives of Labriformes families carry preferential chromosomal rearrangements (
The representatives of the tribe Hypsigenyini exhibit relatively symmetrical karyotypes, with 2n = 48 and high fundamental number (NF) values as compared to other ones (
Molecular analyses have significantly widened the knowledge of the genomic organization and epigenetic modeling of the chromatin, particularly with respect to histone modifications of the euchromatin and heterochromatin (
Although the knowledge of the methylation patterns is growing among vertebrates, it is still restricted in fishes, especially in relation to repetitive DNA regions (transcriptional and non-transcriptional), which are apparently limited to the heterochromatic regions and sex chromosomes (
In this study, we analyzed the DNA methylation pattern in the metaphase chromosomes of B. pulchellus and B. insularis, phylogenetically very close species (
Individuals of Bodianus pulchellus (n = 6, all immature individuals) from Bahia State (12°58'20"S, 38°31'05"W), on the northeastern Brazilian coast, and B. insularis (n = 5, 2 males and 3 immature individuals) from São Pedro and Paulo Archipelago (0°55'19"N, 29°21'44"W), were used in cytogenetic analyses. The individuals were collected under authorization provided by the Chico Mendes Institute of Biodiversity Conservation (ICMBIO/SISBIO) (license #02001.001902/06-82) and all experimental procedures followed the rules of the Animal Ethics Committee of the Federal University of Rio Grande do Norte (protocol 044/2015).
Mitosis stimulation followed the protocols developed by
FISH was performed according to
The DNA methylation patterns in the metaphase chromosomes were detected through binding analysis of the monoclonal antibody to 5-methylcytosine. Indirect immunodetection of the methylated DNA was conducted according to
The chromosomes were categorized as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a), according to
Bodianus pulchellus and B. insularis have diploid chromosome number 2n=48 and identical karyotypes, composed of 4m+12sm+14st+18a chromosomes, NF value 78. As previously described for these species (
Metaphase chromosomes of the species B. insularis (a, c, e) and B. pulchellus (b, d); the chromosome pairs bearing the BOD region are identified with arrows and highlighted in the boxes a, b 18S (red signals) and 5S rDNA FISH (green signals). In the boxes, the argentophilic pattern showed in the BOD regions and the DAPI staining pattern, respectively c, d Distribution of the Tol2 element in the chromosomes. An accumulation of Tol2 sequences is perceptible in the BOD regions e - Distribution of the Alu transposable element on the chromosomes of B. insularis. Scale bar: 5μm.
Metaphase chromosomes of the species Bodianus pulchellus (a, b) and Bodianus insularis (c, d) after DAPI staining (left) and sequential immunodetection of methylated sites with the monoclonal antibody 5mC (right). The chromosome pairs bearing the BOD region are denoted with arrows and highlighted in the boxes. Scale bar: 5μm.
Ag-NOR sites were located in the terminal region of the pair No. 9 in karyotypes of both species (Fig.
Double-FISH with 5S and 18S rDNA probes revealed a non-syntenic location for these ribosomal sites. The 18S rDNA sites were exclusively located in the terminal regions on the p arms of the pair No. 9, corresponding with the Ag-NOR signals. No hybridization signals were detected in the BOD regions of both species (Fig.
The hybridization with the transposable element Alu was only performed in B. insularis, while Tol2 mapping was performed in both species. These sequences exhibited a similar distribution pattern in the chromosomes preferentially located in the terminal regions of the chromosomes and particularly accumulated in the BOD one (Fig.
The hybridization signals with (TTAGGG)n probes were variable, with the majority having the same size, besides some chromosomes showed no detectable signals (data not shown). Immunostaining with 5 mC revealed that most metaphase chromosomes of the two species were hypermethylated (Fig.
All results were summarized in the ideogram of the Figure
The heterochromatic regions of fish genomes have been the target of intense investigation. Although displaying variations in the amount and distribution on chromosomes, heterochromatin can harbor a diversified panel of collocated sequences, whereby the effects of this interaction under gene regulation and dispersion pattern need to be better investigated (
Argentophilic decondensed regions in vertebrates are often related to NOR sites (
In several vertebrate species, including fishes, argentophilic sites not associated with rDNA sites, known as pseudo-NORs, were already described (
Pseudo-NORs used to mimic real NORs in several aspects, as they can remain decondensed during mitosis when the transcription is inactivated and the nucleolus is broken down, forming novel silver positive secondary constrictions (
Representatives of Bodianus display karyotypes with a larger number of biarmed chromosomes when compared to those of other Labridae genera (
In genomes of some fish species, a high chromosome dynamism has been identified for Tol2 elements, which can be situated in different genomic regions (
Alu elements concentrate huge amounts of CpG islands that are genomic regions that contain a high frequency of CpG dinucleotides, commonly representing promoters, which are usually located in GC dense regions. CpG islands tend to be hypomethylated allowing an open chromatin organization and facilitating neighboring gene expression (
Despite the fact that a significant part of the genome of some organisms is composed of repetitive DNA sequences, their origins, dispersion and functional interaction remain largely unknown (
The immunolocalization of 5-methylcytosine in the metaphase chromosomes of the two Bodianus species revealed a primarily hypermethylated pattern, despite the striking contrast observed in the BOD and the centromeric regions, both notably hypomethylated. In general, centromeric regions exhibit particular epigenetic characteristics, including DNA hypermethylation. The presence of hypomethylated regions in the centromeres of some chromosome pairs of the Bodianus species demonstrates an uncommon and likely functional condition of these regions, which are closely associated with the chromosome segregation process.
DNA methylation is considered a controlling mechanism of gene expression, including the ribosomal ones (
In some Perciformes species, Tol2 elements are distributed along the chromosomes and distinctly associated with 18S rDNA sequences (
It is known that DNA methylation may limit the dispersion of various transposable elements in a number of genomes (
DNA methylation is one of the epigenetic processes that has modulated the molecular evolution of life, but its influence in karyotype evolution and interaction in the structural chromosome regions are little known, especially for fish species. The use of monoclonal antibodies in cytogenetic study of Bodianus species provided an overview of the methylation pattern of metaphase chromosomes, with sufficient resolution to characterize the peculiar BOD regions. The complex composition of the BOD chromatin suggests that it is a pseudo-NOR containing a relict sequence of an ancestor rDNA. The DNA organization of such region provided evidence of its functional dynamics, possibly in the transcriptional control of Tol2 and Alu elements. In this sense, the methylation process, associated with the dispersion control of the transposable elements, may have played a particular active role in the evolutionary process of Bodianus species.