Cesar Martins

B chromosomes: from cytogenetics to systems biology

Though hundreds to thousands of reports have described the distribution of B chromosomes among diverse eukaryote groups, a comprehensive theory of their biological role has not yet clearly emerged. B chromosomes are classically understood as a sea of repetitive DNA sequences that are poor in genes and are maintained by a parasitic-drive mechanism during cell division. Recent developments in high-throughput DNA/RNA analyses have increased the resolution of B chromosome biology beyond those of classical and molecular cytogenetic methods; B chromosomes contain many transcriptionally active sequences, including genes, and can modulate the activity of autosomal genes. Furthermore, the most recent knowledge obtained from omics analyses, which is associated with a systemic view, has demonstrated that B chromosomes can influence cell biology in a complex way, possibly favoring their own maintenance and perpetuation.

The opsin genes of amazonian cichlids

Vision is a critical sense for organismal survival with visual sensitivities strongly shaped by the environment. Some freshwater fishes with a Gondwanan origin are distributed in both South American rivers including the Amazon and African rivers and lakes. These different habitats likely required adaptations to murky and clear environments. In this study, we compare the molecular basis of Amazonian and African cichlid fishes’ visual systems. We used next‐generation sequencing of genomes and retinal transcriptomes to examine three Amazonian cichlid species. Genome assemblies revealed six cone opsin classes (SWS1, SWS2B, SWS2A, RH2B, RH2A and LWS) and rod opsin (RH1). However, the functionality of these genes varies across species with different pseudogenes found in different species. Our results support evidence of an RH2A gene duplication event that is shared across both cichlid groups, but which was probably followed by gene conversion. Transcriptome analyses show that Amazonian species mainly express three opsin classes (SWS2A, RH2A and LWS), which likely are a good match to the long‐wavelength‐oriented light environment of the Amazon basin. Furthermore, analysis of amino acid sequences suggests that the short‐wavelength‐sensitive genes (SWS2B, SWS2A) may be under selective pressures to shift their spectral properties to a longer‐wavelength visual palette. Our results agree with the ‘sensitivity hypothesis’ where the light environment causes visual adaptation. Amazonian cichlid visual systems are likely adapting through gene expression, gene loss and possibly spectral tuning of opsin sequences. Such mechanisms may be shared across the Amazonian fish fauna.

Sequence analyses and chromosomal distribution of the Tc1/Mariner element in Parodontidae fish (Teleostei: Characiformes)

Transposable elements are able to move along eukaryotic genomes. They are divided into two classes according to their transposition intermediate: RNA (class I or retrotransposons) or DNA (class II or DNA transposons). Most of these sequences are inactive or non-autonomous in eukaryotic genomes. Inactivate transposons can accumulate mutations at neutral rates until losing their molecular identity. They may either be eliminated from the genome or take on different molecular functions. Transposable elements may also participate in the differentiation of sex chromosomes. Therefore, the structural variations and nucleotide similarity of Tc1/Mariner sequences were analyzed along with their potential participation in the differentiation processes of sex chromosomes in the genomes of Parodontidae fish. All Parodontidae species presented non-autonomous copies of Tc1/Mariner with structural variation, different levels of deterioration (genetic distance), and variations in insertion and deletion patterns. The physical mapping of Tc1/Mariner on chromosomes revealed dispersed signals in euchromatins, with small accumulations in terminal regions and in the sex chromosomes. The gene dosage ratios indicated copy number variations of Tc1/Mariner among the genomes and high transposase open reading frame deterioration in Parodon hilarii and Parodon pongoensis genomes. This transposon presented transcriptional activity in gonads, but there was no significant difference between sexes. This may indicate non-functional protein expression or may correspond to DNA binding proteins derived from Tc1/Mariner. Thus, our results show Tc1/Mariner inactivation along with a diversity in Parodontidae genomes and its participation in the differentiation of the W sex chromosome.

MicroRNA-10 modulates Hox genes expression during Nile tilapia embryonic development

Hox gene clusters encode a family of transcription factors that govern anterior-posterior axis patterning during embryogenesis in all bilaterian animals. The time and place of Hox gene expression are largely determined by the relative position of each gene within its cluster. Furthermore, Hox genes were shown to have their expression fine-tuned by regulatory microRNAs (miRNAs). However, the mechanisms of miRNA-mediated regulation of these transcription factors during fish early development remain largely unknown. Here we have profiled three highly expressed miR-10 family members of Nile tilapia at early embryonic development, determined their genomic organization as well as performed functional experiments for validation of target genes. Quantitative analysis during developmental stages showed miR-10 family expression negatively correlates with the expression of HoxA3a, HoxB3a and HoxD10a genes, as expected for bona fide miRNA-mRNA interactions. Moreover, luciferase assays demonstrated that HoxB3a and HoxD10a are targeted by miR-10b-5p. Overall, our data indicate that the miR-10 family directly regulates members of the Hox gene family during Nile tilapia embryogenesis.

Uncovering the evolutionary history of neo-XY sex chromosomes in the grasshopper Ronderosia bergii (Orthoptera, Melanoplinae) through satellite DNA analysis

BackgroundNeo-sex chromosome systems arose independently multiple times in evolution, presenting the remarkable characteristic of repetitive DNAs accumulation. Among grasshoppers, occurrence of neo-XY was repeatedly noticed in Melanoplinae. Here we analyzed the most abundant tandem repeats of R. bergii (2n = 22, neo-XY♂) using deep Illumina sequencing and graph-based clustering in order to address the neo-sex chromosomes evolution.ResultsThe analyses revealed ten families of satDNAs comprising about ~1% of the male genome, which occupied mainly C-positive regions of autosomes. Regarding the sex chromosomes, satDNAs were recorded within centromeric or interstitial regions of the neo-X chromosome and four satDNAs occurred in the neo-Y, two of them being exclusive (Rber248 and Rber299). Using a combination of probes we uncovered five well-defined cytological variants for neo-Y, originated by multiple paracentric inversions and satDNA amplification, besides fragmented neo-Y. These neo-Y variants were distinct in frequency between embryos and adult males.ConclusionsThe genomic data together with cytogenetic mapping enabled us to better understand the neo-sex chromosome dynamics in grasshoppers, reinforcing differentiation of neo-X and neo-Y and revealing the occurrence of multiple additional rearrangements involved in the neo-Y evolution of R. bergii. We discussed the possible causes that led to differences in frequency for the neo-Y variants between embryos and adults. Finally we hypothesize about the role of DNA satellites in R. bergii as well as putative historical events involved in the evolution of the R. bergii neo-XY.

Centromeric enrichment of LINE-1 retrotransposons and its significance for the chromosome evolution of Phyllostomid bats

Despite their ubiquitous incidence, little is known about the chromosomal distribution of long interspersed elements (LINEs) in mammalian genomes. Phyllostomid bats, characterized by lineages with distinct trends of chromosomal evolution coupled with remarkable ecological and taxonomic diversity, represent good models to understand how these repetitive sequences contribute to the evolution of genome architecture and its link to lineage diversification. To test the hypothesis that LINE-1 sequences were important modifiers of bat genome architecture, we characterized the distribution of LINE-1-derived sequences on genomes of 13 phyllostomid species within a phylogenetic framework. We found massive accumulation of LINE-1 elements in the centromeres of most species: a rare phenomenon on mammalian genomes. We hypothesize that expansion of these elements has occurred early in the radiation of phyllostomids and recurred episodically. LINE-1 expansions on centromeric heterochromatin probably spurred chromosomal change before the radiation of phyllostomids into the extant 11 subfamilies and contributed to the high degree of karyotypic variation observed among different lineages. Understanding centromere architecture in a variety of taxa promises to explain how lineage-specific changes on centromere structure can contribute to karyotypic diversity while not disrupting functional constraints for proper cell division.

The satellite DNA AflaSAT-1 in the A and B chromosomes of the grasshopper Abracris flavolineata

BackgroundSatellite DNAs (satDNAs) are organized in repetitions directly contiguous to one another, forming long arrays and composing a large portion of eukaryote genomes. These sequences evolve according to the concerted evolution model, and homogenization of repeats is observed at the intragenomic level. Satellite DNAs are the primary component of heterochromatin, located primarily in centromeres and telomeres. Moreover, satDNA enrichment in specific chromosomes has been observed, such as in B chromosomes, that can provide clues about composition, origin and evolution of this chromosome. In this study, we isolated and characterized a satDNA in A and B chromosomes of Abracris flavolineata by integrating cytogenetic, molecular and genomics approaches at intra- and inter-population levels, with the aim to understand the evolution of satDNA and composition of B chromosomes.ResultsAflaSAT-1 satDNA was shared with other species and in A. flavolineata, was associated with another satDNA, AflaSAT-2. Chromosomal mapping revealed centromeric blocks variable in size in almost all chromosomes (except pair 11) of A complement for both satDNAs, whereas for B chromosome, only a small centromeric signal occurred. In distinct populations, variable number of AflaSAT-1 chromosomal sites correlated with variability in copy number. Instead of such variability, low sequence diversity was observed in A complement, but monomers from B chromosome were more variable, presenting also exclusive mutations. AflaSAT-1 was transcribed in five tissues of adults in distinct life cycle phases.ConclusionsThe sharing of AflaSAT-1 with other species is consistent with the library hypothesis and indicates common origin in a common ancestor; however, AflaSAT-1 was highly amplified in the genome of A. flavolineata. At the population level, homogenization of repeats in distinct populations was documented, but dynamic expansion or elimination of repeats was also observed. Concerning the B chromosome, our data provided new information on the composition in A. flavolineata. Together with previous results, the sequences of heterochromatic nature were not likely highly amplified in the entire B chromosome. Finally, the constitutive transcriptional activity suggests a possible unknown functional role, which should be further investigated.

Genome-wide microRNA screening in Nile tilapia reveals pervasive isomiRs’ transcription, sex-biased arm switching and increasing complexity of expression throughout development

MicroRNAs (miRNAs) are key regulators of gene expression in multicellular organisms. The elucidation of miRNA function and evolution depends on the identification and characterization of miRNA repertoire of strategic organisms, as the fast-evolving cichlid fishes. Using RNA-seq and comparative genomics we carried out an in-depth report of miRNAs in Nile tilapia (Oreochromis niloticus), an emergent model organism to investigate evo-devo mechanisms. Five hundred known miRNAs and almost one hundred putative novel vertebrate miRNAs have been identified, many of which seem to be teleost-specific, cichlid-specific or tilapia-specific. Abundant miRNA isoforms (isomiRs) were identified with modifications in both 5p and 3p miRNA transcripts. Changes in arm usage (arm switching) of nine miRNAs were detected in early development, adult stage and even between male and female samples. We found an increasing complexity of miRNA expression during ontogenetic development, revealing a remarkable synchronism between the rate of new miRNAs recruitment and morphological changes. Overall, our results enlarge vertebrate miRNA collection and reveal a notable differential ratio of miRNA arms and isoforms influenced by sex and developmental life stage, providing a better picture of the evolutionary and spatiotemporal dynamics of miRNAs.

Dimerization and Transactivation Domains as Candidates for Functional Modulation and Diversity of Sox9.

Sox9 plays an important role in a large variety of developmental pathways in vertebrates. It is composed of three domains: high-mobility group box (HMG box), dimerization (DIM) and transactivation (TAD). One of the main processes for regulation and variability of the pathways involving Sox9 is the self-gene expression regulation of Sox9. However, the subsequent roles of the Sox9 domains can also generate regulatory modulations. Studies have shown that TADs can bind to different types of proteins and its function seems to be influenced by DIM. Therefore, we hypothesized that both domains are directly associated and can be responsible for the functional variability of Sox9. We applied a method based on a broad phylogenetic context, using sequences of the HMG box domain, to ensure the homology of all the Sox9 copies used herein. The data obtained included 4,921 sequences relative to 657 metazoan species. Based on coevolutionary and selective pressure analyses of the Sox9 sequences, we observed coevolutions involving DIM and TADs. These data, along with the experimental data from literature, indicate a functional relationship between these domains. Moreover, DIM and TADs may be responsible for the functional plasticity of Sox9 because they are more tolerant for molecular changes (higher Ka/Ks ratio than the HMG box domain). This tolerance could allow a differential regulation of target genes or promote novel targets during transcriptional activation. In conclusion, we suggest that DIM and TADs functional association may regulate differentially the target genes or even promote novel targets during transcription activation mediated by Sox9 paralogs, contributing to the subfunctionalization of Sox9a and Sox9b in teleosts.

21st International Chromosome Conference-Foz do Iguaçu, Brazil.

International Chromosome Conferences (ICC) can be traced back to the first Oxford Chromosome Conference, organized in 1964 by C.D. Darlington and K.R. Lewis. After evolving into a series of key international conferences, the 21st edition of the ICC series will for the first time be held in South America, specifically in beautiful Foz do Iguaçu, Brazil, July 10–13, 2016. Organized by Cesar Martins (Brazil), Andrea Pedrosa-Harand (Brazil), Andreas Houben (Germany), Beth Sullivan (USA), Lúcia Martelli (Brazil), and Rachel O’Neill (USA), this meeting will be attended by many renowned speakers and scientists from all around the world (http://www.ibb.unesp.br/icc2016). Attesting to the importance of ICC for the broad chromosome community, Chromosomawill publish a series of review articles dedicated to the 21st edition of this conference. The first article by Benavente and colleagues is published in this issue (Fraune et al. 2016) and focuses on the evolution of the mammalian synaptonemal complex. In the next few months, additional ICC-related review articles will continue to highlight recent progress as well as the development of exciting new technologies in chromosome research. Stay tuned!