Sequence Composition Underlying Centromeric and Heterochromatic Genome Compartments of the Pacific Oyster Crassostrea gigas.

Segments of the genome enriched in repetitive sequences still present a challenge and are omitted in genome assemblies. For that reason, the exact composition of DNA sequences underlying the heterochromatic regions and the active centromeres are still unexplored for many organisms. The centromere is a crucial region of eukaryotic chromosomes responsible for the accurate segregation of genetic material. The typical landmark of centromere chromatin is the rapidly-evolving variant of the histone H3, CenH3, while DNA sequences packed in constitutive heterochromatin are associated with H3K9me3-modified histones. In the Pacific oyster Crassostrea gigas we identified its centromere histone variant, Cg-CenH3, that shows stage-specific distribution in gonadal cells. In order to investigate the DNA composition of genomic regions associated with the two specific chromatin types, we employed chromatin immunoprecipitation followed by high-throughput next-generation sequencing of the Cg-CenH3- and H3K9me3-associated sequences. CenH3-associated sequences were assigned to six groups of repetitive elements, while H3K9me3-associated-ones were assigned only to three. Those associated with CenH3 indicate the lack of uniformity in the chromosomal distribution of sequences building the centromeres, being also in the same time dispersed throughout the genome. The heterochromatin of C. gigas exhibited general paucity and limited chromosomal localization as predicted, with H3K9me3-associated sequences being predominantly constituted of DNA transposons.

Terminal-Repeat Retrotransposons in Miniature (TRIMs) in bivalves.

Terminal repeat retrotransposons in miniature (TRIMs) are small non-autonomous LTR retrotransposons consisting of two terminal direct repeats surrounding a short internal domain. The detection and characterization of these elements has been mainly limited to plants. Here we present the first finding of a TRIM element in bivalves, and among the first known in the kingdom Animalia. Class Bivalvia has high ecological and commercial importance in marine ecosystems and aquaculture, and, in recent years, an increasing number of genomic studies has addressed to these organisms. We have identified biv-TRIM in several bivalve species: Donax trunculus, Ruditapes decussatus, R. philippinarum, Venerupis corrugata, Polititapes rhomboides, Venus verrucosa, Dosinia exoleta, Glycymeris glycymeris, Cerastoderma edule, Magallana gigas, Mytilus galloprovincialis. biv-TRIM has several characteristics typical for this group of elements, exhibiting different variations. In addition to canonically structured elements, solo-TDRs and tandem repeats were detected. The presence of this element in the genome of each species is <1%. The phylogenetic analysis showed a complex clustering pattern of biv-TRIM elements, and indicates the involvement of horizontal transfer in the spreading of this element.

Chromatin remodeling in Drosophila preblastodermic embryo extract.

Chromatin is known to undergo extensive remodeling during nuclear reprogramming. However, the factors and mechanisms involved in this remodeling are still poorly understood and current experimental approaches to study it are not best suited for molecular and genetic analyses. Here we report on the use of Drosophila preblastodermic embryo extracts (DREX) in chromatin remodeling experiments. Our results show that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming, such as rapid binding of the germline specific linker histone dBigH1 variant to somatic chromatin, heterochromatin reorganization, changes in the epigenetic state of chromatin, and nuclear lamin disassembly. These results raise the possibility of using the powerful tools of Drosophila genetics for the analysis of chromatin changes associated with this essential process.

Distribution of DTHS3 satellite DNA across 12 bivalve species.

In this study, characterization of DTHS3 satellite DNA (satDNA) was further expanded within the class Bivalvia. Monomer variants of DTHS3 satDNA were compared in 12 bivalve species belonging to two different subclasses, Heterodonta and Pteriomorphia. This satDNA, whose age is estimated to a minimum of 516 Ma, is contributing to the concept of the dual character of satDNA sequences: their sequence preservation throughout long evolutionary periods and generation of species-specific variants of the same satDNA family.

Mutual interactions between telomere heterogeneity and cell culture growth dynamics shape stochasticity of cell aging.

Mathematical modeling and computational simulations are often used to explain the stochastic nature of cell aging. The models published thus far are based on the molecular mechanisms of telomere biology and how they dictate the dynamics of cell culture proliferation. However, the influence of cell growth conditions on telomere dynamics has been widely overlooked. These conditions include interactions with surrounding cells through contact inhibition, gradual accumulation of non-dividing cells, culture propagation and other cell culture maintenance factors. In order to follow the intrinsic growth dynamics of normal human fibroblasts we employed the fluorescent dye DiI and FACS analysis which can distinguish cells that undergo different numbers of divisions within culture. We observed rapid generation of cell subpopulations undergoing from 0 to 9 divisions within growing cultures at each passage analyzed. These large differences in number of divisions among individual cells guarantee a strong impact on generation of telomere length heterogeneity in normal cell cultures and suggest that culture conditions should be included in future modeling of cell senescence.

Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae).

Tandemly repeated DNAs usually constitute significant portions of eukaryotic genomes. In bivalves, however, repetitive DNAs are habitually not widespread. In our search for abundant repetitive DNAs in trough shells, we discovered a novel satellite DNA, SSUsat, which constitutes at least 1.3% of the genome of Spisula subtruncata. As foreseen by the satellite DNA library hypothesis, we confirmed that this satellite DNA is also present in two other Mactridae species, showing a highly conserved nucleotide sequence together with a dramatic diminution in the number of repeats. Predominantly located at the G + C-rich intercalary heterochromatin of S. subtruncata, SSUsat displays several DNA methylation peculiarities. The level of methylation of SSUsat is high (3.38%) in comparison with bivalve standards and triplicates the mean of the S. subtruncata genome (1.13%). Methylation affects not only the cytosines in CpG dinucleotides but also those in CHH and CHG trinucleotides, a feature common in plants but scarce and without any clear known relevance in animals. SSUsat segments enriched in methylated cytosines partly overlap those showing higher sequence conservation. The presence of a chromosome pair showing an accumulation of markedly under-methylated SSUsat monomers additionally indicates that the methylation processes that shape repetitive genome compartments are quite complex.

Two new miniature inverted-repeat transposable elements in the genome of the clam Donax trunculus.

Repetitive sequences are important components of eukaryotic genomes that drive their evolution. Among them are different types of mobile elements that share the ability to spread throughout the genome and form interspersed repeats. To broaden the generally scarce knowledge on bivalves at the genome level, in the clam Donax trunculus we described two new non-autonomous DNA transposons, miniature inverted-repeat transposable elements (MITEs), named DTC M1 and DTC M2. Like other MITEs, they are characterized by their small size, their A + T richness, and the presence of terminal inverted repeats (TIRs). DTC M1 and DTC M2 are 261 and 286 bp long, respectively, and in addition to TIRs, both of them contain a long imperfect palindrome sequence in their central parts. These elements are present in complete and truncated versions within the genome of the clam D. trunculus. The two new MITEs share only structural similarity, but lack any nucleotide sequence similarity to each other. In a search for related elements in databases, blast search revealed within the Crassostrea gigas genome a larger element sharing sequence similarity only to DTC M1 in its TIR sequences. The lack of sequence similarity with any previously published mobile elements indicates that DTC M1 and DTC M2 elements may be unique to D. trunculus.

Adjacent sequences disclose potential for intra-genomic dispersal of satellite DNA repeats and suggest a complex network with transposable elements.

BACKGROUND: Satellite DNA (satDNA) sequences are typically arranged as arrays of tandemly repeated monomers. Due to the similarity among monomers, their organizational pattern and abundance, satDNAs are hardly accessible to structural and functional studies and still represent the most obscure genome component. Although many satDNA arrays of diverse length and even single monomers exist in the genome, surprisingly little is known about transition from satDNAs to other sequences. Studying satDNA monomers at junctions and identifying DNA sequences adjacent to them can help to understand the processes that (re)distribute satDNAs and significance that evolution of these sequence elements might have in creating the genomic landscape. RESULTS: We explored sets of randomly selected satDNA-harboring genomic fragments in four mollusc species to examine satDNA transition sites, and the nature of adjacent sequences. All examined junctions are characterized by abrupt transitions from satDNAs to other sequences. Among them, junctions of only one examined satDNA mapped non-randomly (within the palindrome), indicating that well-defined sequence feature is not a necessary prerequisite in the junction formation. In the studied sample, satDNA flanking sequences can be roughly classified into two groups. The first group is composed of anonymous DNA sequences which occasionally include short segments of transposable elements (TEs) as well as segments of other satDNA sequences. In the second group, satDNA repeats and the array flanking sequences are identified as parts of TEs of the Helitron superfamily. There, some array flanking regions hold fragmented satDNA monomers alternating with anonymous sequences of comparable length as missing monomer parts, suggesting a process of sequence reorganization by a mechanism able to excise short monomer parts and replace them with unrelated sequences. CONCLUSIONS: The observed architecture of satDNA transition sites can be explained as a result of insertion and/or recombination events involving short arrays of satDNA monomers and TEs, in combination with hypothetical transposition-related ability of satDNA monomers to be shuffled independently in the genome. We conclude that satDNAs and TEs can form a complex network of sequences which essentially share the propagation mechanisms and in synergy shape the genome.

RUDI, a short interspersed element of the V-SINE superfamily widespread in molluscan genomes.

Short interspersed elements (SINEs) are non-autonomous retrotransposons that are widespread in eukaryotic genomes. They exhibit a chimeric sequence structure consisting of a small RNA-related head, an anonymous body and an AT-rich tail. Although their turnover and de novo emergence is rapid, some SINE elements found in distantly related species retain similarity in certain core segments (or highly conserved domains, HCD). We have characterized a new SINE element named RUDI in the bivalve molluscs Ruditapes decussatus and R. philippinarum and found this element to be widely distributed in the genomes of a number of mollusc species. An unexpected structural feature of RUDI is the HCD domain type V, which was first found in non-amniote vertebrate SINEs and in the SINE from one cnidarian species. In addition to the V domain, the overall sequence conservation pattern of RUDI elements resembles that found in ancient AmnSINE (~310 Myr old) and Au SINE (~320 Myr old) families, suggesting that RUDI might be among the most ancient SINE families. Sequence conservation suggests a monophyletic origin of RUDI. Nucleotide variability and phylogenetic analyses suggest long-term vertical inheritance combined with at least one horizontal transfer event as the most parsimonious explanation for the observed taxonomic distribution.

Structural and functional liaisons between transposable elements and satellite DNAs.

Transposable elements (TEs) and satellite DNAs (satDNAs) are typically identified as major repetitive DNA components in eukaryotic genomes. TEs are DNA segments able to move throughout a genome while satDNAs are tandemly repeated sequences organized in long arrays. Both classes of repetitive sequences are extremely diverse, and many TEs and satDNAs exist within a genome. Although they differ in structure, genomic organization, mechanisms of spread, and evolutionary dynamics, TEs and satDNAs can share sequence similarity and organizational patterns, thus indicating that complex mutual relationships can determine their evolution, and ultimately define roles they might have on genome architecture and function. Motivated by accumulating data about sequence elements that incorporate features of both TEs and satDNAs, here we present an overview of their structural and functional liaisons.

Tandem repeat-containing MITEs in the clam Donax trunculus.

Two distinct classes of repetitive sequences, interspersed mobile elements and satellite DNAs, shape eukaryotic genomes and drive their evolution. Short arrays of tandem repeats can also be present within nonautonomous miniature inverted repeat transposable elements (MITEs). In the clam Donax trunculus, we characterized a composite, high copy number MITE, named DTC84. It is composed of a central region built of up to five core repeats linked to a microsatellite segment at one array end and flanked by sequences holding short inverted repeats. The modular composition and the conserved putative target site duplication sequence AA at the element termini are equivalent to the composition of several elements found in the cupped oyster Crassostrea virginica and in some insects. A unique feature of D. trunculus element is ordered array of core repeat variants, distinctive by diagnostic changes. Position of variants in the array is fixed, regardless of alterations in the core repeat copy number. Each repeat harbors a palindrome near the junction with the following unit, being a potential hotspot responsible for array length variations. As a consequence, variations in number of tandem repeats and variations in flanking sequences make every sequenced element unique. Core repeats may be thus considered as individual units within the MITE, with flanking sequences representing a "cassette" for internal repeats. Our results demonstrate that onset and spread of tandem repeats can be more intimately linked to processes of transposition than previously thought and suggest that genomes are shaped by interplays within a complex network of repetitive sequences.