The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used
Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for ...target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.
Epstein-Barr virus (EBV) establishes latent infection and is associated with tumors, such as Burkitt lymphoma, nasopharyngeal carcinoma, and gastric cancers. We recently reported that EBV(+) gastric ...cancer shows an EBV(+)/extensively high-methylation epigenotype, and in vitro EBV infection induces extensive DNA methylation with gene repression within 18 weeks. On the basis of the absence of both EBV and high-methylation accumulation in the surrounding mucosa of EBV(+) gastric cancer, it is suggested that an EBV-infected cell acquires extensive methylation to silence multiple tumor suppressor genes in a short time period and transforms into cancer cells, not forming a precancerous field with EBV infection or methylation accumulation. The methylation mechanism induced by EBV infection has not been fully clarified. Differences in EBV genome methylation that are dependent on a different latency status or other epigenomic alterations, such as 3-dimensional conformation and histone modification, may affect host genome methylation. Expressions of viral proteins and small RNAs are also different depending on latency status, and some viral proteins might trigger DNA methylation by inducing DNA methyltransferase overexpression. In this review, we discuss these roles of EBV infection in driving tumorigenesis and their possible association with aberrant DNA methylation.
Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by ...UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
Hepatoblastoma (HB) is the most common pediatric liver malignancy; however, hereditary predisposition and acquired molecular aberrations related to HB clinicopathological diversity are not well ...understood. Here, we perform an integrative genomic profiling of 163 pediatric liver tumors (154 HBs and nine hepatocellular carcinomas) based on the data acquired from a cohort study (JPLT-2). The total number of somatic mutations is precious low (0.52/Mb on exonic regions) but correlated with age at diagnosis. Telomerase reverse transcriptase (TERT) promoter mutations are prevalent in the tween HBs, selective in the transitional liver cell tumor (TLCT, > 8 years old). DNA methylation profiling reveals that classical HBs are characterized by the specific hypomethylated enhancers, which are enriched with binding sites for ASCL2, a regulatory transcription factor for definitive endoderm in Wnt-pathway. Prolonged upregulation of ASCL2, as well as fetal-liver-like methylation patterns of IGF2 promoters, suggests their "cell of origin" derived from the premature hepatoblast, similar to intestinal epithelial cells, which are highly proliferative. Systematic molecular profiling of HB is a promising approach for understanding the epigenetic drivers of hepatoblast carcinogenesis and deriving clues for risk stratification.
We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase ...(Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.
In most eukaryotes, constitutive heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), is enriched on repetitive DNA, such as pericentromeric repeats and transposons. Furthermore, ...repetitive transgenes also induce heterochromatin formation in diverse model organisms. However, the mechanisms that promote heterochromatin formation at repetitive DNA elements are still not clear. Here, using fission yeast, we show that tandemly repeated mRNA genes promote RNA interference (RNAi)-mediated heterochromatin formation in cooperation with an antisilencing factor, Epe1. Although the presence of tandemly repeated genes itself does not cause heterochromatin formation, once complementary small RNAs are artificially supplied in
, the RNAi machinery assembled on the repeated genes starts producing cognate small RNAs in
to autonomously maintain heterochromatin at these sites. This "repeat-induced RNAi" depends on the copy number of repeated genes and Epe1, which is known to remove H3K9me and derepress the transcription of genes underlying heterochromatin. Analogous to repeated genes, the DNA sequence underlying constitutive heterochromatin encodes widespread transcription start sites (TSSs), from which Epe1 activates ncRNA transcription to promote RNAi-mediated heterochromatin formation. Our results suggest that when repetitive transcription units underlie heterochromatin, Epe1 generates sufficient transcripts for the activation of RNAi without disruption of heterochromatin.
Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling and ...clarify distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into adaptive and failing cardiomyocytes, and p53 signaling is specifically activated in late hypertrophy. Cardiomyocyte-specific p53 deletion shows that cardiomyocyte remodeling is initiated by p53-independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent mitochondrial inhibition, morphological elongation, and heart failure gene program activation. Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptional signatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and functional phenotypes.
Melanoma is one of the most aggressive types of cancer wherein resistance to treatment prevails. Therefore, it is important to discover novel molecular targets of melanoma progression as potential ...treatments. Here we show that paired-like homeodomain transcription factor 1 (PITX1) plays a crucial role in the inhibition of melanoma progression through regulation of SRY-box transcription factors (SOX) gene family mRNA transcription. Overexpression of PITX1 in melanoma cell lines resulted in a reduction in cell proliferation and an increase in apoptosis. Additionally, analysis of protein levels revealed an antagonistic cross-regulation between SOX9 and SOX10. Interestingly, PITX1 binds to the SOX9 promoter region as a positive regulatory transcription factor; PITX1 mRNA expression levels were positively correlated with SOX9 expression, and negatively correlated with SOX10 expression in melanoma tissues. Furthermore, transcription of the long noncoding RNA (lncRNA), survival-associated mitochondrial melanoma-specific oncogenic noncoding RNA (SAMMSON), was decreased in PITX1-overexpressing cells. Taken together, the findings in this study indicate that PITX1 may act as a negative regulatory factor in the development and progression of melanoma via direct targeting of the SOX signaling.
Tissue fibrosis and organ dysfunction are hallmarks of age-related diseases including heart failure, but it remains elusive whether there is a common pathway to induce both events. Through ...single-cell RNA-seq, spatial transcriptomics, and genetic perturbation, we elucidate that high-temperature requirement A serine peptidase 3 (Htra3) is a critical regulator of cardiac fibrosis and heart failure by maintaining the identity of quiescent cardiac fibroblasts through degrading transforming growth factor-β (TGF-β). Pressure overload downregulates expression of Htra3 in cardiac fibroblasts and activated TGF-β signaling, which induces not only cardiac fibrosis but also heart failure through DNA damage accumulation and secretory phenotype induction in failing cardiomyocytes. Overexpression of Htra3 in the heart inhibits TGF-β signaling and ameliorates cardiac dysfunction after pressure overload. Htra3-regulated induction of spatio-temporal cardiac fibrosis and cardiomyocyte secretory phenotype are observed specifically in infarct regions after myocardial infarction. Integrative analyses of single-cardiomyocyte transcriptome and plasma proteome in human reveal that IGFBP7, which is a cytokine downstream of TGF-β and secreted from failing cardiomyocytes, is the most predictable marker of advanced heart failure. These findings highlight the roles of cardiac fibroblasts in regulating cardiomyocyte homeostasis and cardiac fibrosis through the Htra3-TGF-β-IGFBP7 pathway, which would be a therapeutic target for heart failure.
Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are in a high-flux metabolic state, with a high dependence on threonine catabolism. However, little is known regarding ...amino acid metabolism in human ESCs/iPSCs. We show that human ESCs/iPSCs require high amounts of methionine (Met) and express high levels of enzymes involved in Met metabolism. Met deprivation results in a rapid decrease in intracellular S-adenosylmethionine (SAM), triggering the activation of p53-p38 signaling, reducing NANOG expression, and poising human iPSC/ESCs for differentiation, follow by potentiated differentiation into all three germ layers. However, when exposed to prolonged Met deprivation, the cells undergo apoptosis. We also show that human ESCs/iPSCs have regulatory systems to maintain constant intracellular Met and SAM levels. Our findings show that SAM is a key regulator for maintaining undifferentiated pluripotent stem cells and regulating their differentiation.
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•SAM is essential for the maintenance of pluripotent stem cells•Decrease in SAMi by methionine deprivation led to p53-p38 signaling activation•Short methionine deprivation poises human ESCs/iPSCs for differentiation•Prolonged methionine deprivation induces apoptosis of pluripotent stem cells
Shiraki et al. show that human iPSC/ESCs rely on methionine metabolism to maintain pluripotency and self-renewal via the methionine intermediate SAM. Methionine deprivation reduces SAM levels, which primes iPSC/ESCs toward differentiation and ultimately triggers cell death. Differences in SAM dependence improve differentiation efficiency by selectively eliminating undifferentiated cells.
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