Single Cell Analysis Reveals Partial Reactivation of X-chromosome Instead of Chromosome-wide Dampening in Naïve Human Pluripotent Stem Cells

AbstractRecently, a unique form of X-chromosome dosage compensation has been demonstrated in human preimplantation embryos, which happens through the dampening of X-linked gene expression from both X-chromosomes. Subsequently, X-chromosome dampening has also been demonstrated in female human pluripotent stem cells (hPSCs) during the transition from primed to naïve state. However, the existence of dampened X-chromosomes remains controversial in both embryos and hPSCs. Specifically, in preimplantation embryos it has been shown that there is inactivation of X-chromosome instead of dampening. Here, we have performed allelic analysis of X-linked genes at the single cell level in hPSCs and found that there is partial reactivation of the inactive X-chromosome instead of chromosome-wide dampening upon conversion from primed to naïve state. In addition, our analysis suggests that the reduced X-linked gene expression in naïve hPSCs might be the consequence of erasure of active X-chromosome upregulation.

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Single-Cell Analysis Reveals Partial Reactivation of X Chromosome instead of Chromosome-wide Dampening in Naive Human Pluripotent Stem Cells

Stem Cell Reports ◽

10.1016/j.stemcr.2020.03.027 ◽

2020 ◽

Vol 14 (5) ◽

pp. 745-754 ◽

Cited By ~ 4

Author(s):

Susmita Mandal ◽

Deepshikha Chandel ◽

Harman Kaur ◽

Sudeshna Majumdar ◽

Maniteja Arava ◽

...

Keyword(s):

Stem Cells ◽

Single Cell ◽

X Chromosome ◽

Pluripotent Stem Cells ◽

Single Cell Analysis ◽

Human Pluripotent Stem Cells ◽

Cell Analysis

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Single-Cell Gene Expression Profiles Define Self-Renewing, Pluripotent, and Lineage Primed States of Human Pluripotent Stem Cells

Stem Cell Reports ◽

10.1016/j.stemcr.2014.04.014 ◽

2014 ◽

Vol 2 (6) ◽

pp. 881-895 ◽

Cited By ~ 60

Author(s):

Shelley R. Hough ◽

Matthew Thornton ◽

Elizabeth Mason ◽

Jessica C. Mar ◽

Christine A. Wells ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Single Cell ◽

Pluripotent Stem Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Human Pluripotent Stem Cells ◽

Cell Gene Expression ◽

Cell Gene

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Engineering at the microscale: A step towards single-cell analysis of human pluripotent stem cells

Biotechnology Journal ◽

10.1002/biot.201500307 ◽

2015 ◽

Vol 10 (10) ◽

pp. 1511-1512

Author(s):

Tiago G. Fernandes

Keyword(s):

Stem Cells ◽

Single Cell ◽

Pluripotent Stem Cells ◽

Single Cell Analysis ◽

Human Pluripotent Stem Cells ◽

Cell Analysis

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Identification of stable reference genes in differentiating human pluripotent stem cells

Physiological Genomics ◽

10.1152/physiolgenomics.00130.2014 ◽

2015 ◽

Vol 47 (6) ◽

pp. 232-239 ◽

Cited By ~ 11

Author(s):

Gustav Holmgren ◽

Nidal Ghosheh ◽

Xianmin Zeng ◽

Yalda Bogestål ◽

Peter Sartipy ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Gene Expression Data ◽

Pluripotent Stem Cells ◽

Reference Genes ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

Expression Data ◽

Large Variability ◽

The Stability

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.

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The Role of RNA Polymerase II Contiguity and Long-Range Interactions in the Regulation of Gene Expression in Human Pluripotent Stem Cells

Stem Cells International ◽

10.1155/2019/1375807 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Livia Eiselleova ◽

Viktor Lukjanov ◽

Simon Farkas ◽

David Svoboda ◽

Karel Stepka ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Long Range ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Transcription Factories ◽

Long Range Interactions ◽

Rnap Ii

The eukaryotic nucleus is a highly complex structure that carries out multiple functions primarily needed for gene expression, and among them, transcription seems to be the most fundamental. Diverse approaches have demonstrated that transcription takes place at discrete sites known as transcription factories, wherein RNA polymerase II (RNAP II) is attached to the factory and immobilized while transcribing DNA. It has been proposed that transcription factories promote chromatin loop formation, creating long-range interactions in which relatively distant genes can be transcribed simultaneously. In this study, we examined long-range interactions between the POU5F1 gene and genes previously identified as being POU5F1 enhancer-interacting, namely, CDYL, TLE2, RARG, and MSX1 (all involved in transcriptional regulation), in human pluripotent stem cells (hPSCs) and their early differentiated counterparts. As a control gene, RUNX1 was used, which is expressed during hematopoietic differentiation and not associated with pluripotency. To reveal how these long-range interactions between POU5F1 and the selected genes change with the onset of differentiation and upon RNAP II inhibition, we performed three-dimensional fluorescence in situ hybridization (3D-FISH) followed by computational simulation analysis. Our analysis showed that the numbers of long-range interactions between specific genes decrease during differentiation, suggesting that the transcription of monitored genes is associated with pluripotency. In addition, we showed that upon inhibition of RNAP II, long-range associations do not disintegrate and remain constant. We also analyzed the distance distributions of these genes in the context of their positions in the nucleus and revealed that they tend to have similar patterns resembling normal distribution. Furthermore, we compared data created in vitro and in silico to assess the biological relevance of our results.

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