Gene Expression In Pluripotent Cells Research Articles

Large-Scale Profiling Reveals the Influence of Genetic Variation on Gene Expression in Human Induced Pluripotent Stem Cells

In this study, we used whole-genome sequencing and gene expression profiling of 215 human induced pluripotent stem cell (iPSC) lines from different donors to identify genetic variants associated with RNA expression for 5,746 genes. We were able to predict causal variants for these expression quantitative trait loci (eQTLs) that disrupt transcription factor binding and validated a subset of them experimentally. We also identified copy-number variant (CNV) eQTLs, including some that appear to affect gene expression by altering the copy number of intergenic regulatory regions. In addition, we were able to identify effects on gene expression of rare genic CNVs and regulatory single-nucleotide variants and found that reactivation of gene expression on the X chromosome depends on gene chromosomal position. Our work highlights the value of iPSCs for genetic association analyses and provides a unique resource for investigating the genetic regulation of gene expression in pluripotent cells.

SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells.

The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor.

Analysis of a transgenic Oct4 enhancer reveals high fidelity long-range chromosomal interactions.

Genome structure or nuclear organization has fascinated researchers investigating genome function. Recently, much effort has gone into defining relationships between specific genome structures and gene expression in pluripotent cells. We previously analyzed chromosomal interactions of the endogenous Oct4 distal enhancer in pluripotent cells. Here, we derive ES and iPS cells from a transgenic Oct4 distal enhancer reporter mouse. Using sonication-based Circularized Chromosome Conformation Capture (4C) coupled with next generation sequencing, we determined and compared the genome-wide interactome of the endogenous and transgenic Oct4 distal enhancers. Integrative genomic analysis indicated that the transgenic enhancer binds to a similar set of loci and shares similar key enrichment profiles with its endogenous counterpart. Both the endogenous and transgenic Oct4 enhancer interacting loci were enriched in the open nucleus compartment, which is associated with active histone marks (H3K4me1, H3K27ac, H3K4me3 and H3K9ac), active cis-regulatory sequences (DNA hypersensitivity sites (DHS)), 5-hydroxymethylcytosine (5-hmc), and early DNA replication domains. In addition, binding of some pluripotency-related transcription factors was consistently enriched in our 4C sites, and genes in those sites were generally more highly expressed. Overall, our work reveals critical features that may function in gene expression regulation in mouse pluripotent cells.

Potential roles of 5´ UTR and 3´ UTR regions in post-trans-criptional regulation of mouse Oct4 gene in BMSC and P19 cells.

OCT4 is a transcription factor required for pluripotency during early embryogenesis and the maintenance of identity of embryonic stem cells and pluripotent cells. Therefore, the effective expression regulation of this gene is highly critical. UTR regions are of great significance to gene regulation. In this study, we aimed to investigate the potential regulatory role played by 5´UTR and 3´UTR of the Oct4 gene in mouse BMSC and P19 cells. The Oct4 5´UTR and 3´UTR sequences were cloned into pGL3 luciferase plasmid which led to the generation of pGL3 5´-UTR, pGL3 5´&3´-UTRs and pGL3 3´-UTR vectors. The vectors were transfected into BMSC and P19 cells followed by luciferase assay. The assay of luciferase expression exhibited a direct link between the presence of Oct4 3´- UTR and the decrease of luciferase count in both cell lines; whereas 5´UTR indicated diverse behaviors in two cells. This discrepancy could be explained in view of the difference of cellular contexts in which the Oct4 UTRs act. This study sheds some light on the role of UTR regions of mouse Oct4 in regulating post-transcriptional gene expression in pluripotent cells. These data represent potential to be used for the development of novel therapeutic approaches for a variety of malignancies.

Single-Cell Analysis Reveals that Expression of Nanog Is Biallelic and Equally Variable as that of Other Pluripotency Factors in Mouse ESCs

The homeodomain transcription factor Nanog is a central part of the core pluripotency transcriptional network and plays a critical role in embryonic stem cell (ESC) self-renewal. Several reports have suggested that Nanog expression is allelically regulated and that transient downregulation of Nanog in a subset of pluripotent cells predisposes them toward differentiation. Using single-cell gene expression analyses combined with different reporters for the two alleles of Nanog, we show that Nanog is biallelically expressed in ESCs independently of culture condition. We also show that the overall variation in endogenous Nanog expression in ESCs is very similar to that of several other pluripotency markers. Our analysis suggests that reporter-based studies of gene expression in pluripotent cells can be significantly influenced by the gene-targeting strategy and genetic background employed.