Biology subjects

Soltys, V.

Publications and source records attributed to Soltys, V..

6 recordsLinked to original sources

bioRxiv2024

Genetic determinants of distinct CD8+ α/β-TCR repertoires in the genus Mus

The adaptive immune systems efficacy relies on the diversity of T cell receptors and the ability to distinguish between self and foreign antigens. Analysis of the paired heterodimeric {beta}-TCR chains of individual T cells requires single-cell resolution, but existing single-cell approaches offer limited coverage of the vast TCR repertoire diversity. Here we introduce CITR-seq, a novel, instrument-free, high-throughput method for single-cell TCR sequencing with >88% {beta}-TCR pairing precision. We analyzed the TCR repertoires of CD8+ T cells originated from 32 inbred mice using CITR-seq, comprising four evolutionary divergent sister species and their F1 hybrids. Overall, we identified more than 5 million confidently paired TCRs. We found that V(D)J gene usage patterns are highly specific to the genotype and that V{beta}-gene usage is strongly impacted by thymic selection. Using F1 hybrids, we show that differences in gene segment usage across species are likely caused by cis-acting factors prior to thymic selection, which imposed strong allelic biases. At the greatest divergence, this led to increased rates of TCR depletion through rejection of particular V{beta}-genes. TCR repertoire overlap analysis across all mice revealed that sharing of identical paired CDR3 amino acid motifs is four times more frequent than predicted by random pairing of TCR and TCR{beta} chains, with significantly increased sharing rates among related individuals. Collectively, we show that beyond the stochastic nature of TCR repertoire generation, genetic factors contribute significantly to the shape of an individuals repertoire.

genomics↗

bioRxiv2024

Distinct evolution at TCRα and TCRβ loci in the genus Mus

T cells recognize an immense spectrum of pathogens to initiate immune responses by means of a large repertoire of T cell receptors (TCRs) that arise from somatic rearrangements of variable, diversity and joining gene segments at the TCR loci. These gene segments have emerged from a limited number of ancestral genes through a series of gene duplication events, resulting in a greatly variable number of such genes across different species. Apart from the complete V(D)J gene annotations in the human and mouse reference assemblies, little is known about the structure of TCR loci in other species. Here, we performed a comprehensive comparison of the TCR and TCR{beta} gene segment clusters in mice and three of its closely related sister species. We show that the TCR variable gene cluster is frequently rearranged, leading to deletions and sequence inversions in this region. The resulting complexity of TCR loci severely complicates the assembly of these loci and the annotation of gene segments. By jointly utilizing genomic and transcriptomic data, we show that in Mus musculus castaneus the variable gene cluster at the locus has undergone a recent major locus contraction, leading to the loss of 74 variable gene segments. Additionally, we validated the expression of functional variable genes, including atypical ones with inverted orientation relative to other such segments. Disentangling the fine-scale structure of TCR loci in different species can provide valuable insights in the evolution and diversity of TCR repertoires.

genomics↗

bioRxiv2024

Copy number normalization distinguishes differential signals driven by copy number differences in ATAC-seq and ChIP-seq

A common objective across ATAC-seq and ChIP-seq analyses is to identify differential signals across contrasted conditions. However, in differential analyses, the impact of copy number variation is often overlooked. Here, we demonstrated copy number differences among samples could drive, if not dominate, differential signals. To address this, we propose a pipeline featuring copy number normalization. By comparing the averaged signal per gene copy, it effectively segregates differential signals driven by copy number differences from other factors. Further applying it to Down syndrome, we unveiled distinct dosage-dependent and -independent changes on chromosome 21. Thus, we recommend normalization as a general approach.

genomics↗

bioRxiv2024

Flexible and high-throughput simultaneous profiling of gene expression and chromatin accessibility in single cells

Gene regulation underpins development and is an intricate biological process involving transcription, typically at promoters within accessible chromatin. To understand cell-type specific regulatory networks, the ability to capture both transcription and chromatin accessibility simultaneously is crucial. However, joint measurements are technically challenging and current methodologies still face adoption challenges. Here, we present easySHARE-seq, an improvement on SHARE-seq, for the simultaneous measurement of ATAC- and RNA-seq in single cells. We address several limitations of the previous method by improving the barcode and streamlining the protocol. As a result, easySHARE-seq libraries have a usable sequence of up to 300bp (+200bp increase), making it suitable for e.g. investigation of allele-specific signals or variant discovery. Furthermore, easySHARE-seq libraries do not require a dedicated sequencing run thus saving costs. We applied easySHARE-seq to murine liver nuclei and recovered 19,664 nuclei with joint chromatin and expression profiles. By benchmarking against other combinatorial indexing-based techniques, we showed we can recover over 1.5 fold more transcripts per cell while retaining high scalability and low cost. To showcase our method, we identified cell types, exploited the multiomic measurements to link cis-regulatory elements to their target genes and investigated liver-specific micro-scale changes. We conclude that easySHARE-seq improves upon previous methods and can produce high-quality multiomic datasets. We expect it to be applicable to a wide range of study designs.

genomics↗

bioRxiv2023

Nuclear dualism without extensive DNA elimination in the ciliate Loxodes magnus

Ciliates are unicellular eukaryotes with two distinct kinds of nuclei in each cell: transcriptionally active somatic macronuclei (MAC) and silent germline micronuclei (MIC). In the best-studied model species, both nuclei can divide asexually, but only germline MICs participate in meiosis, karyogamy, and development into new MACs. During MIC-to-MAC development, thousands of mobile element relics in the germline, called internally eliminated sequences (IESs), are excised. This genome editing enables IESs to persist by shielding them from somatic natural selection. Editing itself is a costly, time-consuming process, hypothetically maintained by evolutionary addiction. Loxodes magnus and its relatives (class Karyorelictea) are cytologically unusual because their MACs do not divide asexually, but must develop anew from mitotically generated MIC copies every cell division. Here, we report that Loxodes genome development is also unconventional. We found no canonical germline-limited IESs in Loxodes despite careful purification and long-read sequencing of MICs and MACs. The k-mer content of these nuclei overlapped, and indels found by read mapping were consistent with allele variants rather than IESs. Two other hallmarks of genome editing--domesticated DDE-family transposases and editing-associated small RNAs--were also absent. Nonetheless, histone marks, nucleosome and DNA N6-methyladenosine distributions in vegetative Loxodes cells are consistent with actively transcribed MACs and inactive MICs, like other ciliates. Both genomes, not only the MIC, were large and replete with retrotransposon sequences. Given the costs associated with genome editing, we hypothesize that karyorelicteans like Loxodes have lost or streamlined editing during MIC-to-MAC development, and have found a way out of the addictive cycle.

genomics↗

bioRxiv2021

Genetic studies of human-chimpanzee divergence using stem cell fusions

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee auto-tetraploids and allo-tetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or auto-tetraploid iPSCs. Analysis of gene expression patterns in inter-specific allo-tetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves, and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allo-tetraploid cells. We successfully generated derivative cells with nested deletions or inter-specific recombination on the X chromosome. These studies identify a long distance cis-regulatory domain of the Fragile X-associated gene (FMR1), confirm an important role for the X chromosome in trans-regulation of other expression differences, and illustrate the potential of this system for more detailed mapping of the molecular basis of human and chimpanzee evolution. Significance StatementComparative studies of humans and chimpanzees have revealed many anatomical, physiological, behavioral, and molecular differences. However, it has been challenging to map these differences to particular chromosome regions. Here, we develop a genetic approach in fused stem cell lines that makes it possible to map human-chimpanzee molecular and cellular differences to specific regions of the genome. We illustrate this approach by mapping chromosome regions responsible for species-specific gene expression differences in fused tetraploid cells. This approach is general, and could be used in the future to map the genomic changes that control many other humanchimpanzee differences in various cell types or organoids in vitro.

evolutionary biology↗