Biology subjects

Timoshevskiy, V. A.

Publications and source records attributed to Timoshevskiy, V. A..

5 recordsLinked to original sources

bioRxiv2026

Dissecting functional elements in giant genomes using the first generation of high quality salamander assemblies

Salamanders are an enduring and historically significant group that helped originate multiple scientific fields of study, including regeneration, cytogenetics, experimental embryology, cloning, evolutionary developmental biology and evolution of extreme genome size. We generated a high-quality, single haplotype assembly for the 32 Gb axolotl (Ambystoma mexicanum) genome using an F1 hybrid between axolotl and a divergent relative the tiger salamander (A. tigrinum). Using multi-genome alignments to other salamanders that meet VGP metrics, we leveraged strong conservation of karyotypic structure to identify >523,000 highly conserved non-coding elements within massively expanded intronic and intergenic regions, the vast majority of which are unique to the salamander lineage. Orthogonal measures of chromatin state and enhancer activity confirmed that many conserved elements correspond to cis-regulatory sequences and identified several thousand that are utilized during regeneration. The conserved elements identified here will enable robust functional genomic analyses of tissue regeneration and other traits that uniquely empower salamander models in biological research.

genomics↗

bioRxiv2026

Functional Analyses of Histone Methyltransferases in Sea Lamprey Embryos Undergoing Programmed DNA Elimination

During early embryogenesis, the sea lamprey (Petromyzon marinus) undergoes a dramatic form of genome reprogramming wherein specific chromosomes are selectively eliminated from somatic progenitor cells. These programmatic elimination events effectively silence all genes on these chromosomes in all somatic cells. Previous studies in lamprey and other eliminating species have shown that epigenetic silencing marks are enriched on germline-specific chromosomes during programmed elimination. These silencing marks include the histone marks H3K9me3 and H4K20me3, which are respectively deposited by KMT1A/SUV39 and KMT5/SUV420 methyltransferases. To test whether lamprey homologs of these methyltransferases contribute to deposition of silencing marks on eliminated (micronucleated) chromatin and whether these marks contribute to the highly coordinated process of DNA elimination in sea lamprey, we used Cas9 gene editing, lightsheet imaging and RNA sequencing to investigate their potential roles in DNA elimination and more generally during early development. Analysis of knockout embryos for four histone methyltransferases show that these genes contribute to the deposition of repressive histone marks on elimination micronuclei, but are not essential for programmed DNA elimination per se. Analysis of later embryogenic stages suggests that these marks may contribute to interim silencing of germline-specific chromosomes, and reveals major impacts on post-blastula survival and development.

evolutionary biology↗

bioRxiv2019

Content of and interactions between repetitive elements in programmatically eliminated chromosomes of the sea lamprey (Petromyzon marinus)

The sea lamprey (Petromyzon marinus) is one of few vertebrate species that is known to reproducibly eliminate large fractions of its genome during normal embryonic development. In lamprey, elimination events are initiated at the 6th embryonic cleavage and result in the loss of [~]20% of an embryos genomic DNA from essentially all somatic cell lineages (these same sequences are retained in the germline). This germline-specific DNA is lost in the form of large fragments, including entire chromosomes, and available evidence suggests that DNA elimination acts as a permanent silencing mechanism that prevents the somatic expression of a specific subset of \"germline\" genes. However, reconstruction of eliminated regions has proven challenging due to the complexity of the lamprey karyotype (84 small pairs of somatic chromosomes and [~]100 pairs of germline chromosomes), the exceedingly high repeat content of the genome and even higher repeat content of eliminated fragments.\n\nWe applied an integrative approach aimed at further characterization of the large-scale structure of eliminated segments, including: 1) in silico identification of germline-enriched repeats; mapping the chromosomal location of specific repetitive sequences in germline metaphases, and verification of repeat specificity to eliminated chromosomes by 3D DNA/DNA-hybridization to embryonic lagging anaphases. Our integrative approach resulted in the discovery of multiple highly abundant repetitive elements that are found exclusively on the eliminated (germline-specific) chromosomes which in turn permitted the identification of 12 individual chromosomes that are programmatically eliminated during early embryogenesis. The fidelity of germline-specific repetitive elements and their distinctive patterning in elimination anaphases are taken as evidence that these sequences might contribute to the specific targeting of chromosomes for elimination and possibly in molecular interactions that mediate their decelerated poleward movement in chromosome elimination anaphases, isolation from the primary nuclei and eventual degradation.\n\nAUTHOR SUMMARYEpigenetic silencing methods provide a means of precisely restricting gene expression while maintaining the integrity of the genomic template that encodes this information, and are employed by diverse species throughout the tree of life. Programmed genome rearrangement (PGR) represents a parallel approach that maintains genome integrity across generations but alters the genomes of cells within an organism. To better resolve elimination events that take place during PGR in the sea lamprey (one of few vertebrate species known to undergo large scale PGR) we sought to identify sequences that define specific eliminated chromosomes. Using computational predictions and cytogenetic validation, we identified six new repetitive elements that are restricted to the eliminated chromosomes and permit the identification of twelve distinct eliminated chromosomes. Analysis of these repeats in meiotic testes and in embryos sampled during the process of elimination shows that these repeats localize to specific subcellular regions, and suggest a potential role of these repetitive elements in targeting chromosomes for silencing via elimination.

genetics↗

bioRxiv2018

A Chromosome-Scale Assembly of the Enormous (32 Gb) Axolotl Genome

The axolotl (Ambystoma mexicanum) provides critical models for studying regeneration, evolution and development. However, its large genome (~32 gigabases) presents a formidable barrier to genetic analyses. Recent efforts have yielded genome assemblies consisting of thousands of unordered scaffolds that resolve gene structures, but do not yet permit large scale analyses of genome structure and function. We adapted an established mapping approach to leverage dense SNP typing information and for the first time assemble the axolotl genome into 14 chromosomes. Moreover, we used fluorescence in situ hybridization to verify the structure of these 14 scaffolds and assign each to its corresponding physical chromosome. This new assembly covers 27.3 gigabases and encompasses 94% of annotated gene models on chromosomal scaffolds. We show the assemblys utility by resolving genome-wide orthologies between the axolotl and other vertebrates, identifying the footprints of historical introgression events that occurred during the development of axolotl genetic stocks, and precisely mapping several phenotypes including a large deletion underlying the cardiac mutant. This chromosome-scale assembly will greatly facilitate studies of the axolotl in biological research.

genomics↗

bioRxiv2018

Miniscule differences between the sex chromosomes in the giant genome of a salamander, Ambystoma mexicanum

In the Mexican axolotl (Ambystoma mexicanum) sex is known to be determined by a single Mendelian factor, yet the sex chromosomes of this model salamander do not exhibit morphological differentiation that is typical of many vertebrate taxa that possess a single sex-determining locus. Differentiated sex chromosomes are thought to evolve rapidly in the context of a Mendelian sex-determining gene and, therefore, undifferentiated chromosomes provide an exceptional opportunity to reconstruct early events in sex chromosome evolution. Whole chromosome sequencing, whole genome resequencing (48 individuals from a backcross of axolotl and tiger salamander) and in situ hybridization were used to identify a homomorphic chromosome that carries an A. mexicanum sex determining factor and identify sequences that are present only on the W chromosome. Altogether, these sequences cover ~300 kb, or roughly 1/100,000th of the ~32 Gb genome. Notably, these W-specific sequences also contain a recently duplicated copy of the ATRX gene: a known component of mammalian sex-determining pathways. This gene (designated ATRW) is one of the few functional (non-repetitive) genes in the chromosomal segment and maps to the tip of chromosome 9 near the marker E24C3, which was previously found to be linked to the sex-determining locus. These analyses provide highly predictive markers for diagnosing sex in A. mexicanum and identify ATRW as a strong candidate for the primary sex determining locus or alternately a strong candidate for a recently acquired, sexually antagonistic gene.\n\nAUTHOR SUMMARYSex chromosomes are thought to follow fairly stereotypical evolutionary trajectories that result in differentiation of sex-specific chromosomes. In the salamander A. mexicanum (the axolotl), sex is determined by a single Mendelian locus, yet the sex chromosomes are essentially undifferentiated, suggesting that these sex chromosomes have recently acquired a sex locus and are in the early stages of differentiating. Although Mendelian sex determination was first reported for the axolotl more than 70 years ago, no sex-specific sequences have been identified for this important model species. Here, we apply new technologies and approaches to identify and validate a tiny region of female-specific DNA within the gigantic genome of the axolotl (1/100,000th of the genome). This region contains a limited number of genes, including a duplicate copy of the ATRX gene which, has been previously shown to contribute to mammalian sex determination. Our analyses suggest that this gene, which we refer to as ATRW, evolved from a recent duplication and presents a strong candidate for the primary sex determining factor of the axolotl, or alternately a recently evolved sexually antagonistic gene.

evolutionary biology↗