Studying X chromosome inactivation in the single-cell genomic era

2018 ◽  
Vol 46 (3) ◽  
pp. 577-586 ◽  
Author(s):  
Andrew Keniry ◽  
Marnie E. Blewitt
Keyword(s):  
Single Cell ◽  
X Chromosome ◽  
Single Cells ◽  
Transcriptional Silencing ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
The Novel ◽  
Ideal Model ◽  
Single Cell Genomics ◽  
Silent State

Single-cell genomics is set to revolutionise our understanding of how epigenetic silencing works; by studying specific epigenetic marks or chromatin conformations in single cells, it is possible to ask whether they cause transcriptional silencing or are instead a consequence of the silent state. Here, we review what single-cell genomics has revealed about X chromosome inactivation, perhaps the best characterised mammalian epigenetic process, highlighting the novel findings and important differences between mouse and human X inactivation uncovered through these studies. We consider what fundamental questions these techniques are set to answer in coming years and propose that X chromosome inactivation is an ideal model to study gene silencing by single-cell genomics as technical limitations are minimised through the co-analysis of hundreds of genes.

Impact of Xist RNA on chromatin modifications and transcriptional silencing maintenance at different stages of imprinted X chromosome inactivation in vole Microtus levis

Chromosoma ◽  
2017 ◽  
Vol 127 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Alexander I. Shevchenko ◽  
Elena V. Grigor’eva ◽  
Sergey P. Medvedev ◽  
Irina S. Zakharova ◽  
Elena V. Dementyeva ◽  
...  
Keyword(s):  
X Chromosome ◽  
Transcriptional Silencing ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Chromatin Modifications ◽  
Xist Rna ◽  
Microtus Levis

scholarly journals Single Cell Expression Data Reveal Human Genes that Escape X-Chromosome Inactivation

2016 ◽  
Author(s):  
Kerem Wainer-Katsir ◽  
Michal Linial
Keyword(s):  
X Chromosome ◽  
Direct Method ◽  
Single Cells ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
P Value ◽  
Expression Data ◽  
Rna Seq ◽  
Specific Expression ◽  
Biallelic Expression

ABSTRACTSex chromosomes pose an inherent genetic imbalance between genders. In mammals, one of the female’s X-chromosomes undergoes inactivation (Xi). Indirect measurements estimate that about 20% of Xi genes completely or partially escape inactivation. The identity of these escapee genes and their propensity to escape inactivation remain unsolved. A direct method for identifying escapees was applied by quantifying differential allelic expression from single cells. RNA-Seq fragments were assigned to informative SNPs which were labeled by the appropriate parental haplotype. This method was applied for measuring allelic specific expression from Chromosome-X (ChrX) and an autosomal chromosome as a control. We applied the protocol for measuring biallelic expression from ChrX to 104 primary fibroblasts. Out of 215 genes that were considered, only 13 genes (6%) were associated with biallelic expression. The sensitivity of escapees' identification was increased by combining SNP mapping for parental diploid genomes together with RNA-Seq from clonal single cells (25 lymphoblasts). Using complementary protocols, referred to as strict and relaxed, we confidently identified 25 and 31escapee genes, respectively. When pooled versions of 30 and 100 cells were used, <50% of these genes were revealed. We assessed the generality of our protocols in view of an escapee catalog compiled from indirect methods. The overlap between the escapee catalog and the genes’ list from this study is statistically significant (P-value of E-07). We conclude that single cells’ expression data are instrumental for studying X-inactivation with an improved sensitivity. Finally, our results support the emerging notion of the non-deterministic nature of genes that escape X-chromosome inactivation.

scholarly journals Integrated analysis of Xist upregulation and X-chromosome inactivation with single-cell and single-allele resolution

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guido Pacini ◽  
Ilona Dunkel ◽  
Norbert Mages ◽  
Verena Mutzel ◽  
Bernd Timmermann ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Single Cell ◽  
X Chromosome ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Cellular Heterogeneity ◽  
Integrated Analysis ◽  
X Chromosomes ◽  
Multiple Levels

AbstractTo 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 initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and gene silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify putative Xist regulators. Moreover, we show that transient Xist upregulation from both X chromosomes results in biallelic gene silencing right before transitioning to the monoallelic state, confirming a prediction of the stochastic model of XCI. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known X-controlling element (Xce) effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

scholarly journals X-chromosome dosage compensation dynamics in human early embryos

2020 ◽  
Author(s):  
Kevin Huang ◽  
Qiao Zeng ◽  
Yun Feng ◽  
Youjin Hu ◽  
Qin An ◽  
...  
Keyword(s):  
Single Cell ◽  
X Chromosome ◽  
Dosage Compensation ◽  
Cell Mass ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Dosage Balance ◽  
Sister Cells

AbstractIn mammals, female cells are obliged to inactivate one of two X chromosomes to achieve dosage parity with the single X chromosome in male cells, and it is also thought that the single active X chromosome is increased 2-fold to achieve dosage balance with two sets of autosomes (X:A ratio = 1, or Ohno’s hypothesis). However, the ontogeny of X-chromosome inactivation and augmentation of the single active X remains unclear during human embryogenesis. Here, we perform single-cell RNA-seq analysis to examine the timing of X:A balancing and X-inactivation (XCI) in pre- and peri-implantation human embryos up to fourteen days in culture. We find that X-chromosome gene expression in both male and female preimplantation embryos is approximately balanced with autosomes (X:A ratio = 1) after embryonic genome activation (EGA) and persists through fourteen days in vitro. Cross-species analysis of preimplantation embryo also show balanced X:A ratio within the first few days of development. By single-cell mRNA SNP profiling, we find XCI beginning in day 6-7 blastocyst embryos, but does not affect X:A dosage balance. XCI is most evident in trophoectoderm (TE) cells, but can also be observed in a small number of inner cell mass (ICM)-derived cells including primitive endoderm (PE) and epiblast (EPI) cells. Analysis between individual XaXa and XaXi sister cells from the same embryo reveals random XCI and persistently balanced X:A ratio, including sister cells transitioning between XaXa and XaXi states. We therefore conclude that the male X-chromosome undergoes X chromosome augmentation prior to the simultaneous X-chromosome inactivation and augmentation in females. Together, our data demonstrate an evolutionally conserved model of X chromosome dosage compensation in humans and other mammalian species.

scholarly journals Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading

2020 ◽  
Author(s):  
Lisa Rodermund ◽  
Heather Coker ◽  
Roel Oldenkamp ◽  
Guifeng Wei ◽  
Joseph Bowness ◽  
...  
Keyword(s):  
X Chromosome ◽  
Dynamic Properties ◽  
Single Cells ◽  
Super Resolution ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Rna Molecules ◽  
Xist Rna

AbstractXist RNA directs the process of X-chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, enabling us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation, and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a distinct function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique dynamic properties of Xist RNA.One Sentence SummaryVisualizing Xist RNA dynamics in single cells during X chromosome inactivation

scholarly journals Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation

2016 ◽  
Vol 26 (10) ◽  
pp. 1342-1354 ◽  
Author(s):  
Geng Chen ◽  
John Paul Schell ◽  
Julio Aguila Benitez ◽  
Sophie Petropoulos ◽  
Marlene Yilmaz ◽  
...  
Keyword(s):  
Single Cell ◽  
X Chromosome ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation
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