Y-chromosome clonal random expression produces mosaics in male mice
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ABSTRACT:
PROVIDER: PRJNA1129861 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29653163_1.fastq.gz | Fastqsanger.gz | |||
| SRR29653163_2.fastq.gz | Fastqsanger.gz | |||
| SRR29653164_1.fastq.gz | Fastqsanger.gz | |||
| SRR29653164_2.fastq.gz | Fastqsanger.gz | |||
| SRR29653165_1.fastq.gz | Fastqsanger.gz |
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Project description:Monoallelic expression of X-chromosome inactivation (XCI) genes produces female mosaicism, but it is unknown whether chromosome Y (chrY) mosaicism exists in males. ChrY contains some ubiquitously expressed and evolutionary conserved dosage-sensitive regulator genes, including neighbouring Kdm5d, Ddx3y, Uty, and (in mice) Eif2s3y, that have on chrX counterparts that escape XCI. Here, we have produced a transgenic mice line containing an EGFP gene between Uty and Ddx3y genes. These males have mosaic (tortoiseshell) green fluorescence in the skin, similar to females carrying one copy (hemizygous) of an X-linked EGFP transgene owing to inactivation of one chrX, and show clonal inheritance of the inactive state through cell division. The skin tortoiseshell expression of the Y-GFP indicates that the choice of which chrY remains active is stably maintained and clonally inherited. We have produced 3 Sertoli lines from Y-GFP transgenic male, and we have separated by flow cytometry size homogeneous GFP-positive and GFP-negative cells. To analyse the transcriptomic profile and evaluate whether genes from chrY follow the same EGFP expression pattern and explore the regulatory mechanism of the chrY we performed RNA-seq. The results indicate that GFP-positive and GFP-negative cells have considerable differences in their expression levels at chrY, chrX and autosomal chromosomes. Concretely, 18 chrY genes show under expression in GFP-negative cells, including Ddx3y, Zfy1, Zfy2, Usp9y, Uba1y and 10 genes from the Rbmy family. Moreover, the differences in expression observed brings out the influence of chromosome Y expression on the expression of the rest of the autosomal genes.
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Project description:Random monoallelic expression is defined by the allele-specific expression of genes, and by the fact that for an individual cell this monoallelic expression is neither obligate nor necessarily coordinated with the allelic expression in other cells. In order to find novel examples of random monoallelic expression in mouse, we did a transcriptome-wide survey of allele-specific gene expression in two different immortalized cell types. Lymphoblast cell lines and fibroblast cell lines were established (both clonal and nonclonal) and were used as a source of both nuclear RNA and genomic DNA. These samples were assessed for allele-specific gene expression using a custom-designed Mouse SNP Chip. A large number of genes (over 10% of those that were assessed in lymphoblast clones) displayed random monoallelic expression.
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Project description:X chromosome inactivation (XCI) is a process that compensates X-linked gene dosage in mammalian female cells. The silencing of a randomly selected chromosome is accompanied by dramatic three-dimensional (3D) reorganization across the entire chromosome. To investigate the 4D chromatin dynamics during early inactivation stages, we applied the multi-omics sequencing technique HiRES, which simultaneously detects 3D genome and transcriptome in single cells, in a mouse embryonic stem cell line with induced random XCI. The 3D genome and transcriptome dual-omics data allowed us to identify XCI lineages at single-cell resolution. We characterized multiple layers of chromosome reorganization during random XCI and discovered a transient structural inactivation state for both X chromosomes resulted from biallelic Xist expression. By constructing single-cell inactivation trajectories, we found that a vast majority of chromatin remodeling either accompanied or followed gene silencing. Further analysis of interaction decay kinetics revealed that TAD attenuation began from loss of interactions on TAD anchors. This study thus drew a detailed depiction of fine-scale chromatin reorganization during the initiation of random XCI.
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Project description:Random monoallelic expression is defined by the allele-specific expression of genes, and by the fact that for an individual cell this monoallelic expression is neither obligate nor necessarily coordinated with the allelic expression in other cells. In order to find novel examples of random monoallelic expression in mouse, we did a transcriptome-wide survey of allele-specific gene expression in two different immortalized cell types. Lymphoblast cell lines and fibroblast cell lines were established (both clonal and nonclonal) and were used as a source of both nuclear RNA and genomic DNA. These samples were assessed for allele-specific gene expression using a custom-designed Mouse SNP Chip. A large number of genes (over 10% of those that were assessed in lymphoblast clones) displayed random monoallelic expression. For each cell line, two replicate samples of ds-cDNA were assessed for monoallelic expression, while genomic DNA was assessed as a control for possible LOH events. Nonclonal samples were used as controls for cis-acting allelic bias.
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Project description:To ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is a model for cellular decision making and epigenetic gene regulation. The random cell-autonomous nature of XCI however has so far prevented its chromosome-wide investigation in its endogenous context without genetic perturbation. Here we use allele-specific single-cell RNA-sequencing to assess the onset of random XCI with high temporal resolution in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. We find that XCI is initiated on both chromosomes in a subset of cells, but reverted to the final mono-allelic state once gene silencing is established. By exploiting the heterogeneity of XCI onset, we identify Nanog as its main trigger and discover additional genes that might control upregulation of Xist, the master regulator of XCI. Finally, we show that genetic variation between different mouse strains modulates the XCI process at multiple levels, and validate these findings through an orthogonal experimental approach. We thus draw a detailed picture of how XCI is initiated and the experimental and computational strategies we have developed here will now allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.
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