Global Characterization of X Chromosome Inactivation in Human Pluripotent Stem Cells

Summary: 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. : 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. Keywords: X inactivation, human embryonic stem cells, human induced pluripotent stem cells, XIS

Culture-induced recurrent epigenetic aberrations in human pluripotent stem cells

<div><p>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 (<i>TSPYL5</i>), 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.</p></div

Recurrent hypermethylation during culturing occurs at regulatory CpG Islands.

<p><b>(A)</b> Venn diagram defining 10 genes that undergo hypermethylation and silencing in both analyzed datasets. <b>(B)</b> Expression changes between high passage and low passage hESCs in large expression database of samples with given passage. Shown are the average values of each probe in each group. <b>(C)</b> Examples of methylation levels of six probes in different samples from different passages. In red are the linear regression lines. The slopes of the trend lines are presented above each graph. <b>(D)</b> Histogram describing the distribution of the methylation slopes of all the methylation probes. The methylation sloped of the probes of the 10 candidate genes are shown. <b>(E-F)</b> Variation in expression levels (E) and methylation values (F) in data sets of multiple hESCs without known passage number. Methylation probes shown are only those located in CpG islands.</p

Changes in expression and methylation during prolonged culturing of hPSCs.

<p><b>(A)</b> Hierarchical clustering of expression patterns (upper panel) and methylation patterns (lower panel) of the same samples. Number shown are the passage number of each sample. Clustering was performed using Pearson correlation and complete linkage. <b>(B)</b> Scatter plots of expression and methylation differences between high passage and low passage samples of data from Nazor et al[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006979#pgen.1006979.ref015" target="_blank">15</a>]. Values represent the change between the averages of the high passage group and the low passage group. Colors indicate the correlation between the expression and methylation. Vertical red lines represent expression change of 1.5 fold. Horizontal red lines indicated methylation change of 0.2. <b>(C)</b> Expression and methylation heatmaps of the probes that showed hypermethylation and reduced expression at the high passage samples in data from Nazor et al[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006979#pgen.1006979.ref015" target="_blank">15</a>]. The triangle to the right of each heatmap represents the increasing passage number, blue denotes the low passage samples and red denotes the high passage sample. <b>(D and E)</b> Same as (B and C) but for data from Mallon et al[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006979#pgen.1006979.ref017" target="_blank">17</a>].</p

<i>TSPYL5</i> undergoes hyper methylation and silencing in culture.

<p><b>(A)</b> Schematic representation of <i>TSPYL5</i> gene. <b>(B)</b> Western blot analysis for TSPYL5 of samples from multiple hPSCs cell lines at different passages. The plot at the right panel represents the TSPYL5 expression state of each sample as detected in the western blot analysis. <b>(C)</b> Analysis of methylation levels using the methylation sensitive enzyme McrBC. Shown are differences between quantitative-PCR (qPCR) results of digested and undigested DNA. Blue samples express <i>TSPYL5</i> while red samples do not. Samples of the same cell line are connected with dashed line. <b>(D)</b> <i>TSPYL5</i> expression and methylation changes during continuous passaging of the WA09 cell line. Samples were grown either on extracellular matrix or on MEFs, and passaged mechanically or enzymatically. <b>(E)</b> Changes in <i>TSPYL5</i> Expression and methylation levels in pES6 cells after 5 days treatment with 5-AZA-dC.</p

A role for microRNAs in the Drosophila circadian clock

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