The totipotent 2C-like state safeguards genomic stability of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) sporadically transition to a transient totipotent state that resembles blastomeres of the two-cell (2C) embryo stage, which has been proposed to contribute to exceptional genomic stability, one of the key features of mESCs. However, the biological significance of the rare population of 2C-like cells (2CLCs) in ESC cultures remains to be tested. Here we generated an inducible reporter cell system for specific elimination of 2CLCs from the ESC cultures to disrupt the equilibrium between ESCs and 2CLCs. We show that removing 2CLCs from the ESC cultures leads to dramatic accumulation of DNA damage, genomic mutations, and rearrangements, indicating impaired genomic instability. Furthermore, 2CLCs removal results in increased apoptosis and reduced proliferation of mESCs in both serum/LIF and 2i/LIF culture conditions. Unexpectedly, p53 deficiency results in defective response to DNA damage, leading to early accumulation of DNA damage, micronuclei, indicative of genomic instability, cell apoptosis, and reduced self-renewal capacity of ESCs when devoid of 2CLCs in cultures. Together, our data reveal that transition to the privileged 2C-like state is a major component of the intrinsic mechanisms that maintain the exceptional genomic stability of mESCs for long-term self-renewal.

Genetic Studies on Mammalian DNA Methyltransferases.

Cytosine methylation at the C5-position-generating 5-methylcytosine (5mC)-is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60-80%) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation and genetic alterations in enzymes and regulators involved in DNA methylation are associated with various human diseases, including cancer and developmental disorders. In mammals, DNA methylation is mediated by two families of DNA methyltransferases (Dnmts), namely Dnmt1 and Dnmt3 proteins. Over the last three decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and human diseases.

Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse.

Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression.

Genetic Studies on Mammalian DNA Methyltransferases.

Cytosine methylation at the C5-position, generating 5-methylcytosine (5mC), is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60-80 %) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation levels and patterns are associated with various human diseases, including cancer and developmental disorders. DNA methylation is mediated by three active DNA methyltransferases (Dnmts), namely, Dnmt1, Dnmt3a, and Dnmt3b, in mammals. Over the last two decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and X-chromosome inactivation.

Roles for Histone Acetylation in Regulation of Telomere Elongation and Two-cell State in Mouse ES Cells.

Mammalian telomeres and subtelomeres are marked by heterochromatic epigenetic modifications, including repressive DNA methylation and histone methylation (e.g., H3K9me3 and H4K20me3). Loss of these epigenetic marks results in increased rates of telomere recombination and elongation. Other than these repressive epigenetic marks, telomeric and subtelomeric H3 and H4 are underacetylated. Yet, whether histone acetylation also regulates telomere length has not been directly addressed. We thought to test the effects of histone acetylation levels on telomere length using histone deacetylase (HDAC) inhibitor (sodium butyrate, NaB) that mediates histone hyperacetylation and histone acetyltransferase (HAT) inhibitor (C646) that mediates histone hypoacetylation. We show that histone hyperacetylation dramatically elongates telomeres in wild-type ES cells, and only slightly elongates telomeres in Terc(-/-) ES cells, suggesting that Terc is involved in histone acetylation-induced telomere elongation. In contrast, histone hypoacetylation shortens telomeres in both wild-type and Terc(-/-) ES cells. Additionally, histone hyperacetylation activates 2-cell (2C) specific genes including Zscan4, which is involved in telomere recombination and elongation, whereas histone hypoacetylation represses Zscan4 and 2C genes. These data suggest that histone acetylation levels affect the heterochromatic state at telomeres and subtelomeres, and regulate gene expression at subtelomeres, linking histone acetylation to telomere length maintenance.

The ICF syndrome protein CDCA7 harbors a unique DNA-binding domain that recognizes a CpG dyad in the context of a non-B DNA.

CDCA7 , encoding a protein with a C-terminal cysteine-rich domain (CRD), is mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a disease related to hypomethylation of juxtacentromeric satellite DNA. How CDCA7 directs DNA methylation to juxtacentromeric regions is unknown. Here, we show that the CDCA7 CRD adopts a unique zinc-binding structure that recognizes a CpG dyad in a non-B DNA formed by two sequence motifs. CDCA7, but not ICF mutants, preferentially binds the non-B DNA with strand-specific CpG hemi-methylation. The unmethylated sequence motif is highly enriched at centromeres of human chromosomes, whereas the methylated motif is distributed throughout the genome. At S phase, CDCA7, but not ICF mutants, is concentrated in constitutive heterochromatin foci, and the formation of such foci can be inhibited by exogenous hemi-methylated non-B DNA bound by the CRD. Binding of the non-B DNA formed in juxtacentromeric regions during DNA replication provides a mechanism by which CDCA7 controls the specificity of DNA methylation.

Zscan4 Contributes to Telomere Maintenance in Telomerase-Deficient Late Generation Mouse ESCs and Human ALT Cancer Cells.

Proper telomere length is essential for indefinite self-renewal of embryonic stem (ES) cells and cancer cells. Telomerase-deficient late generation mouse ES cells and human ALT cancer cells are able to propagate for numerous passages, suggesting telomerase-independent mechanisms responding for telomere maintenance. However, the underlying mechanisms ensuring the telomere length maintenance are unclear. Here, using late generation telomerase KO (G4 Terc-/-) ESCs as a model, we show that Zscan4, highly upregulated in G4 Terc-/- ESCs, is responsible for the prolonged culture of these cells with stably short telomeres. Mechanistically, G4 Terc-/- ESCs showed reduced levels of DNA methylation and H3K9me3 at Zscan4 promoter and subtelomeres, which relieved the expression of Zscan4. Similarly, human ZSCAN4 was also derepressed by reduced H3K9me3 at its promoter in ALT U2 OS cells, and depletion of ZSCAN4 significantly shortened telomeres. Our results define a similar conserved pathway contributing to the telomere maintenance in telomerase-deficient late generation mESCs and human ALT U2OS cancer cells.

Expanding the Toolbox and Targets for Gene Editing.

Genome editing holds great promise for treating a range of human genetic diseases. While emerging clustered regularly interspaced short-palindromic repeats (CRISPR) technologies allow editing of the nuclear genome, it is still not possible to precisely manipulate mitochondrial DNA (mtDNA). Here, we summarize past developments and recent advances in nuclear and mitochondrial genome editing.

Chemical combinations potentiate human pluripotent stem cell-derived 3D pancreatic progenitor clusters toward functional ß cells.

Human pluripotent stem cell (hPSC)-derived pancreatic ß cells are an attractive cell source for treating diabetes. However, current derivation methods remain inefficient, heterogeneous, and cell line dependent. To address these issues, we first devised a strategy to efficiently cluster hPSC-derived pancreatic progenitors into 3D structures. Through a systematic study, we discovered 10 chemicals that not only retain the pancreatic progenitors in 3D clusters but also enhance their potentiality towards NKX6.1+/INS+ ß cells. We further systematically screened signaling pathway modulators in the three steps from pancreatic progenitors toward ß cells. The implementation of all these strategies and chemical combinations resulted in generating ß cells from different sources of hPSCs with high efficiency. The derived ß cells are functional and can reverse hyperglycemia in mice within two weeks. Our protocol provides a robust platform for studying human ß cells and developing hPSC-derived ß cells for cell replacement therapy.

Zscan4 Inhibits Maintenance DNA Methylation to Facilitate Telomere Elongation in Mouse Embryonic Stem Cells.

Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell (2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs.

The Arginine Methyltransferase PRMT6 Regulates DNA Methylation and Contributes to Global DNA Hypomethylation in Cancer.

DNA methylation plays crucial roles in chromatin structure and gene expression. Aberrant DNA methylation patterns, including global hypomethylation and regional hypermethylation, are associated with cancer and implicated in oncogenic events. How DNA methylation is regulated in developmental and cellular processes and dysregulated in cancer is poorly understood. Here, we show that PRMT6, a protein arginine methyltransferase responsible for asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a), negatively regulates DNA methylation and that PRMT6 upregulation contributes to global DNA hypomethylation in cancer. Mechanistically, PRMT6 overexpression impairs chromatin association of UHRF1, an accessory factor of DNMT1, resulting in passive DNA demethylation. The effect is likely due to elevated H3R2me2a, which inhibits the interaction between UHRF1 and histone H3. Our work identifies a mechanistic link between protein arginine methylation and DNA methylation, which is disrupted in cancer.

Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells.

Mouse embryonic stem cell (ESC) cultures exhibit a heterogeneous mixture of metastable cells sporadically entering the 2-cell (2C)-embryo-like state, critical for ESC potency. One of 2-cell genes, Zscan4, has been shown to be responsible for telomere maintenance, genomic stability and pluripotency of mouse ESCs. Functions of other 2C-genes in ESCs remain elusive. Here we show that 2C-genes Tcstv1 and Tcstv3 play a role in regulation of telomere lengths. Overexpression or knockdown Tcstv1 and Tcstv3 does not immediately affect proliferation, pluripotency and differentiation in vitro of ESCs. However, ectopic expression of Tcstv1 or Tcstv3 results in telomere elongation, whereas Tcstv1/3 knockdown shortens telomeres of ESCs. Overexpression of Tcstv1 or Tcstv3 does not alter telomere stability. Furthermore, Tcstv1 can increase Zscan4 protein levels and telomere recombination by telomere sister chromatid exchange (T-SCE). Depletion of Tcstv1/3 reduces Zscan4 protein levels. Together, Tcstv1 and Tcstv3 are involved in telomere maintenance that is required for long-term self-renewal of mouse ESCs. Our data also suggests that Tcstv1/3 may co-operate and stabilize Zscan4 protein but the molecular bases remain to be determined.

Tet Enzymes Regulate Telomere Maintenance and Chromosomal Stability of Mouse ESCs.

Ten-eleven translocation (Tet) family proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. We show that mouse embryonic stem cells (ESCs) depleted of Tet1 and/or Tet2 by RNAi exhibit short telomeres and chromosomal instability, concomitant with reduced telomere recombination. Tet1 and Tet2 double-knockout ESCs also display short telomeres but to a lesser extent. Notably, Tet1/2/3 triple-knockout ESCs show heterogeneous telomere lengths and increased frequency of telomere loss and chromosomal fusion. Mechanistically, Tets depletion or deficiency increases Dnmt3b and decreases 5hmC levels, resulting in elevated methylation levels at sub-telomeres. Consistently, knockdown of Dnmt3b or addition of 2i (MAPK and GSK3ß inhibitors), which also inhibits Dnmt3b, reduces telomere shortening, partially rescuing Tet1/2 deficiency. Interestingly, Tet1/2 double or Tet1/2/3 triple knockout in ESCs consistently upregulates Zscan4, which may counteract telomere shortening. Together, Tet enzymes play important roles in telomere maintenance and chromosomal stability of ESCs by modulating sub-telomeric methylation levels.

Drp1 is dispensable for mitochondria biogenesis in induction to pluripotency but required for differentiation of embryonic stem cells.

Mature mitochondria with high oxidative phosphorylation undergo fission and fusion and morphogenesis to become immature mitochondria during induced pluripotent stem (iPS) induction from somatic cells. Dynamin-related protein 1 (Drp1) is involved in mitochondria fission and biogenesis in somatic cells. We tested the role of Drp1 in the induction and maintenance of pluripotency. We show that Drp1 band shift occurs in embryonic stem cells (ESCs) and iPS cells (iPSCs) induced from fibroblasts, in association with mitochondrial morphogenesis. However, knockdown of Drp1 by shRNA does not abrogate mitochondria morphogenesis and induction of iPSCs from fibroblasts. Also, knockdown of Drp1 affects neither mitochondria fission and function as shown by normal mitochondrial membrane potential, nor proliferation and pluripotency of ESCs. Nonetheless, Drp1 knockdown negatively influences terminal differentiation of ESCs, particularly in the lineage of neurogenesis in vitro and in vivo, coincident with delayed reduction of Oct4 and Nanog during mid-differentiation. Our data suggest that Drp1 is not critical for mitochondria biogenesis in stem cell proliferation but it is required for neurogenesis likely by downregulation of pluripotency-associated genes Nanog and Oct4. ESC differentiation model could be used to model role of Drp1 in neuron development and diseases.

Rif1 maintains telomere length homeostasis of ESCs by mediating heterochromatin silencing.

Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.

Roles for Tbx3 in regulation of two-cell state and telomere elongation in mouse ES cells.

Mouse embryonic stem (ES) cell cultures exhibit heterogeneity and recently are discovered to sporadically enter the 2-cell (2C)-embryo state, critical for ES potency. Zscan4 could mark the sporadic 2C-state of ES cells. However, factors that regulate the Zscan4(+)/2C state remain to be elucidated. We show that Tbx3 plays a novel role in regulation of Zscan4(+)/2C state. Tbx3 activates 2-cell genes including Zscan4 and Tcstv1/3, but not vise versa. Ectopic expression of Tbx3 results in telomere elongation, consistent with a role for Zscan4 in telomere lengthening. Mechanistically, Tbx3 decreases Dnmt3b and increases Tet2 protein levels, and reduces binding of Dnmt3b to subtelomeres, resulting in reduced DNA methylation and derepression of genes at subtelomeres, e.g. Zscan4. These data suggest that Tbx3 can activate Zscan4(+)/2C state by negative regulation of DNA methylation at repeated sequences, linking to telomere maintenance and self-renewal of ES cells.

Reprogramming- and pluripotency-associated membrane proteins in mouse stem cells revealed by label-free quantitative proteomics.

Induced pluripotent stem cells (iPSCs), derived from somatic cells and functionally very similar to embryonic stem cells (ESCs), are at the center stage of intense research in regenerative medicine. We carried out the first membrane proteomic profiling of mouse iPSCs, in comparison with ESCs and adult mouse tail tip fibroblasts (TTFs) from which iPSCs were generated. Using a proteomic workflow combining membrane fractionation, SDS-PAGE separation and nanoUPLC-MS(E) technology, we identified 673, 679 and 682 non-redundant proteins from mouse iPSC, ESC and TTF membrane fractions, respectively. Label-free quantitation revealed 155 reprogramming-associated and 128 pluripotency-associated transmembrane proteins. Furthermore, a small group of 23 membrane proteins mainly involved in amino acid/glucose/ion transport, membrane fusion and vesicular trafficking were found potentially regulated between miPSCs and mESCs. Expression changes of selected proteins were verified by qPCR, western blot and/or immunofluorescence analyses in a wider array of cell types. Notably, epithelial cell adhesion molecules, glucose transporters 1 and 3, transferrin receptor and several nuclear membrane-associated components were highly expressed in both iPSCs and ESCs, relative to TTFs. Moreover, knock-down of glucose transporter 3 in ESCs impaired the beating function of ESC-derived cardiomyocytes, suggesting its potential role in mediating stem cell differentiation. BIOLOGICAL SIGNIFICANCE: This study constitutes a membrane proteomic resource for murine iPSCs and ESCs, and offers a comparison between pluripotent stem cells and fibroblasts in the proteomic landscape. An integrated proteomics platform combining technologies of membrane fractionation, LC-MS(E) analysis and label-free quantitation was developed to identify membrane proteins with their abundances related to reprogramming of fibroblasts or maintenance of stem cell pluripotency. The high similarity in the membrane proteomic patterns between iPSCs and ESCs strengthens the usefulness of iPSCs in biomedical research and therapeutic application. Moreover, we found a small subset of membrane proteins potentially regulated between miPSCs and mESCs. This membrane proteomic resource of pluripotent stem cells would be expected to inspire further investigations leading to discovery of new regulatory factors or membrane markers for reprogramming and pluripotency.

Influences of lamin A levels on induction of pluripotent stem cells.

Lamin A is an inner nuclear membrane protein that maintains nuclear structure integrity, is involved in transcription, DNA damage response and genomic stability, and also links to cell differentiation, senescence, premature aging and associated diseases. Induced pluripotent stem (iPS) cells have been successfully generated from various types of cells and used to model human diseases. It remains unclear whether levels of lamin A influence reprogramming of somatic cells to pluripotent states during iPS induction. Consistently, lamin A is expressed more in differentiated than in relatively undifferentiated somatic cells, and increases in expression levels with age. Somatic cells with various expression levels of lamin A differ in their dynamics and efficiency during iPS cell induction. Cells with higher levels of lamin A show slower reprogramming and decreased efficiency to iPS cells. Furthermore, depletion of lamin A by transient shRNA accelerates iPS cell induction from fibroblasts. Reduced levels of lamin A are associated with increased expression of pluripotent genes Oct4 and Nanog, and telomerase genes Tert and Terc. On the contrary, overexpression of lamin A retards somatic cell reprogramming to iPS-like colony formation. Our data suggest that levels of lamin A influence reprogramming of somatic cells to pluripotent stem cells and that artificial silencing of lamin A facilitates iPS cell induction. These findings may have implications in enhancing rejuvenation of senescent or older cells by iPS technology and manipulating lamin A levels.

Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells.

Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc(-/-) and Terc(+/-)) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.