Co-transcriptional genome surveillance by HUSH is coupled to termination machinery.

The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.

Epigenetic aberrations in human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are being increasingly utilized worldwide in investigating human development, and modeling and discovering therapies for a wide range of diseases as well as a source for cellular therapy. Yet, since the first isolation of human embryonic stem cells (hESCs) 20 years ago, followed by the successful reprogramming of human-induced pluripotent stem cells (hiPSCs) 10 years later, various studies shed light on abnormalities that sometimes accumulate in these cells in vitro Whereas genetic aberrations are well documented, epigenetic alterations are not as thoroughly discussed. In this review, we highlight frequent epigenetic aberrations found in hPSCs, including alterations in DNA methylation patterns, parental imprinting, and X chromosome inactivation. We discuss the potential origins of these abnormalities in hESCs and hiPSCs, survey the different methods for detecting them, and elaborate on their potential consequences for the different utilities of hPSCs.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.

Differentiation of uniparental human embryonic stem cells into granulosa cells reveals a paternal contribution to gonadal development.

Genomic imprinting underlies the mammalian requirement for sexual reproduction. Nonetheless, the relative contribution of the two parental genomes during human development is not fully understood. Specifically, a fascinating question is whether the formation of the gonad, which holds the ability to reproduce, depends on equal contribution from both parental genomes. Here, we differentiated androgenetic and parthenogenetic human pluripotent stem cells (hPSCs) into ovarian granulosa-like cells (GLCs). We show that in contrast to biparental and androgenetic cells, parthenogenetic hPSCs present a reduced capacity to differentiate into GLCs. We further identify the paternally expressed gene IGF2 as the most upregulated imprinted gene upon differentiation. Remarkably, while IGF2 knockout androgenetic cells fail to differentiate into GLCs, the differentiation of parthenogenetic cells supplemented with IGF2 is partly rescued. Thus, our findings unravel a surprising essentiality of genes that are only expressed from the paternal genome to the development of the female reproductive system.

Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells.

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.

Global Characterization of X Chromosome Inactivation in Human Pluripotent Stem Cells.

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.

Mice from Same-Sex Parents: CRISPRing Out the Barriers for Unisexual Reproduction.

Genomic imprinting results in the molecular and functional inequality of maternal and paternal alleles, precluding mammalian unisexual development. In this issue of Cell Stem Cell, Li et al. (2018) employ sophisticated manipulations of gametes and engineered haploid embryonic stem cells to successfully generate both all-maternal and all-paternal mice, effectively overcoming the roadblocks of imprinting.

Large-Scale Analysis of Loss of Imprinting in Human Pluripotent Stem Cells.

The parent-specific monoallelic expression of imprinted genes is controlled by DNA methylation marks that are established differentially in the germline. Perturbation of these marks leads to loss of imprinting (LOI), which is associated with developmental disorders and malignancy and may also obstruct applications of human pluripotent stem cells (hPSCs). Previous studies of LOI in hPSCs were performed on relatively small numbers of cell lines, often leading to conflicting conclusions regarding imprinting stability. Here, we chart the landscape of LOI in hPSCs by applying a large-scale analysis of allele-specific RNA-seq data from more than 270 hPSC samples. We show that reprogrammed hPSCs acquire higher levels of LOI compared with embryonic stem cells and that LOI can pre-exist in their somatic cells of origin. Furthermore, different imprinted genes vary with respect to LOI incidence, surprisingly revealing that those controlled paternally are more prone to disruption. Our findings emphasize the importance of inspecting the imprinting status of hPSCs.

Differentiation of human parthenogenetic pluripotent stem cells reveals multiple tissue- and isoform-specific imprinted transcripts.

Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5' RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes.