Abstract
Human pluripotent stem cell (hPSC)-derived pancreatic β 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+ β cells. We further systematically screened signaling pathway modulators in the three steps from pancreatic progenitors toward β cells. The implementation of all these strategies and chemical combinations resulted in generating β cells from different sources of hPSCs with high efficiency. The derived β cells are functional and can reverse hyperglycemia in mice within two weeks. Our protocol provides a robust platform for studying human β cells and developing hPSC-derived β cells for cell replacement therapy.
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.
Display omitted
•2C-like mESCs show global DNA hypomethylation•Zscan4 is responsible for DNA demethylation in 2C-like mESCs•Zscan4 induces Uhrf1-dependent ubiquitination and degradation of Uhrf1 and Dnmt1•Blocking DNA demethylation prevents Zscan4-mediated telomere elongation
Mouse embryonic stem cells sporadically convert to a transient totipotent (2C-like) state in which shortened telomeres are extended dependent on Zscan4. Dan et al. demonstrate that Zscan4 facilitates telomere elongation by inducing Uhrf1-dependent Uhrf1 and Dnmt1 degradation, leading to global DNA demethylation.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Display omitted
•PRMT6 overexpression induces histone H3R2me2a and DNA hypomethylation in mESCs•Uhrf1 association with chromatin is impaired in mESCs overexpressing PRMT6•PRMT6 upregulation correlates with DNA hypomethylation in cancer cells•PRMT6 depletion or inhibition restores DNA methylation in MCF7 cells
Veland et al. find that PRMT6, an arginine methyltransferase responsible for histone H3 arginine 2 (H3R2) methylation, negatively regulates maintenance DNA methylation by impairing UHRF1 recruitment to chromatin. The authors also find that PRMT6 upregulation contributes to global DNA hypomethylation in cancer cells.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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.
Display omitted
•Rif1 maintains telomere length homeostasis and chromosomal stability of ESCs•Rif1 suppresses Zscan4 to prevent hyper telomere recombination•Rif1 interacts with and stabilizes the H3K9 methylation complex•Rif1 facilitates epigenetic silencing at subtelomeric heterochromatin
Dan et al. uncover a role for telomere-associated protein Rif1 in the maintenance of telomere length homeostasis and genomic stability in mouse ESCs and during early embryonic development. Rif1 modulates sporadic Zscan4 expression, important for telomere elongation by recombination, by stabilizing subtelomeric heterochromatin and thereby prevents hyper telomere recombination.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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 GSK3β 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.
Display omitted
•Tet enzymes maintain telomere length•Tet enzymes maintain chromosomal stability of ESCs•Dnmt3b and 5hmC are involved in Tet-signaling-mediated telomere maintenance•Excessive Zscan4 and Dnmt3b lead to heterogeneous telomere elongation and loss
Sub-telomeric DNA methylation negatively regulates telomere recombination and telomere length. Here, Yang et al. report that Tet enzymes maintain telomere length and chromosomal stability by modulating both Dnmt3b expression and methylation levels.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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
, 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.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
PDF
