Human pluripotent stem cells (hPSCs) are an important player in disease modeling and regenerative medicine. Nonetheless, multiple studies uncovered their inherent genetic instability upon prolonged ...culturing, where specific chromosomal aberrations provide cells with a growth advantage. These positively selected modifications have dramatic effects on multiple cellular characteristics. Epigenetic aberrations also possess the potential of changing gene expression and altering cellular functions. In the current study we assessed the landscape of DNA methylation aberrations during prolonged culturing of hPSCs, and defined a set of genes which are recurrently hypermethylated and silenced. We further focused on one of these genes, testis-specific Y-encoded like protein 5 (TSPYL5), and demonstrated that when silenced, differentiation-related genes and tumor-suppressor genes are downregulated, while pluripotency- and growth promoting genes are upregulated. This process is similar to the hypermethylation-mediated inactivation of certain genes during tumor development. Our analysis highlights the existence and importance of recurrent epigenetic aberrations in hPSCs during prolonged culturing.
Genomic instability has profound effects on cellular phenotypes. Studies have shown that pluripotent cells with abnormal karyotypes may grow faster, differentiate less and become more resistance to ...apoptosis. Previously, we showed that microarray gene expression profiles can be utilized for the analysis of chromosomal aberrations by comparing gene expression levels between normal and aneuploid samples. Here we adopted this method for RNA-Seq data and present eSNP-Karyotyping for the detection of chromosomal aberrations, based on measuring the ratio of expression between the two alleles. We demonstrate its ability to detect chromosomal gains and losses in pluripotent cells and their derivatives, as well as meiotic recombination patterns. This method is advantageous since it does not require matched diploid samples for comparison, is less sensitive to global expression changes caused by the aberration and utilizes already available gene expression profiles to determine chromosomal aberrations.
Dosage compensation of sex-chromosome gene expression between male and female mammals is achieved via X chromosome inactivation (XCI) by employing epigenetic modifications to randomly silence one X ...chromosome during early embryogenesis. Human pluripotent stem cells (hPSCs) were reported to present various states of XCI that differ according to the expression of the long non-coding RNA XIST and the degree of X chromosome silencing. To obtain a comprehensive perspective on XCI in female hPSCs, we performed a large-scale analysis characterizing different XCI parameters in more than 700 RNA high-throughput sequencing samples. Our findings suggest differences in XCI status between most published samples of embryonic stem cells (ESCs) and induced PSCs (iPSCs). While the majority of iPSC lines maintain an inactive X chromosome, ESC lines tend to silence the expression of XIST and upregulate distal chromosomal regions. Our study highlights significant epigenetic heterogeneity within hPSCs, which may bear implications for their use in research and regenerative therapy.
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•The status of XCI is analyzed in more than 700 samples of human PSCs•XIST is expressed in most human iPSCs and silenced in the majority of ESCs•XIST silencing leads to overexpression of genes at distal X chromosomal regions•Erosion of XCI is prevalent in human ESCs but not in other female cell lines
Bar et al. perform a large-scale analysis of X chromosome inactivation (XCI) in over 700 samples of human pluripotent stem cells (PSCs). Erosion of XCI involves stable silencing of XIST and partial overexpression of distal X-linked genes and is prevalent in embryonic stem cells, but not in most induced PSCs.
Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), we knocked down the miRNA ...biogenesis pathway in Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify miRNA-mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs-clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, we exploited circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 3' untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 3' UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes.
Due to their unique cellular features, pluripotent stem cells (PSCs) acquire chromosomal aberrations at a rather high frequency during their growth in culture. Analysis of chromosomal integrity ...should be routinely performed and usually is done at the DNA level of the cells. RNA sequencing (RNA-Seq) has recently become the basic tool for transcriptional studies. Therefore, methods that utilize this already available data to inspect the genomic integrity are very valuable. In this chapter, we provide a practical guide to implement methods of detection of chromosomal aberrations, which are based on RNA-Seq data. The expression-based karyotyping (e-Karyotyping) method is based on global gene expression analysis, while the expressed-SNP-karyotyping (eSNP-Karyotyping) method is based on changes in the ratio between alleles.
The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. Here we show ...that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall our results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.
Pluripotent stem cells (PSCs) must maintain their proper genomic content in order to preserve appropriate self-renewal and differentiation capacities. However, their prolonged in vitro propagation, ...as well as the environmental culture conditions, present serious challenges to genome maintenance. Recent work has been focused on potential means to alleviate the genomic insults experienced by PSCs, and to detect them as soon as they arise, in order to prevent the detrimental consequences of these genomic aberrations on PSC application in basic research and regenerative medicine.
Human pluripotent stem cells (hPSCs) acquire genetic changes during their propagation in culture that can affect their use in research and future therapies. To identify the key genes involved in ...selective advantage during culture adaptation and tumorigenicity of hPSCs, we generated a genome-wide screening system for genes and pathways that provide a growth advantage either in vitro or in vivo. We found that hyperactivation of the RAS pathway confers resistance to selection with the hPSC-specific drug PluriSIn-1. We also identified that inactivation of the RHO-ROCK pathway gives growth advantage during culture adaptation. Last, we demonstrated the importance of the PI3K-AKT and HIPPO pathways for the teratoma formation process. Our screen revealed key genes and pathways relevant to the tumorigenicity and survival of hPSCs and should thus assist in understanding and confronting their tumorigenic potential.
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•Large-scale analysis of genes and pathways involved in growth and survival of hPSCs•Activation of the RAS pathways confers enhanced resistance to PluriSIn-1 treatment•Inactivation of the RHO-ROCK pathway gives selective growth advantage to hPSCs•The PI3K-AKT and HIPPO pathways are involved in the process of teratoma formation
Cell Biology; Stem Cells Research; Omics; Genomics
Neural cells can be derived either from pluripotent or adult stem cells via differentiation or by transdifferentiation from other cell types with the aid of tissue regulators. We compared the ...chromosomal stability of over 500 neural cell samples from human and mouse with virtual karyotyping (e-karyotyping). We detected notable genomic instability in cells derived from pluripotent or adult stem cells, but surprisingly, transdifferentiated cells seemed more chromosomally stable, except if they were reprogrammed using pluripotency factors.
How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold ...extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing.
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