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• W3113714347 abstract "•Depletion of Dcaf11 leads to reduced telomeres in embryos and ESCs•Dcaf11-deficient mice exhibit telomere erosion and compromised HSC activity•Dcaf11 targets Kap1 for ubiquitination-mediated degradation•Dcaf11 activates a distal enhancer of Zscan4 via the release of Kap1 Telomeres play vital roles in ensuring chromosome stability and are thus closely linked with the onset of aging and human disease. Telomeres undergo extensive lengthening during early embryogenesis. However, the detailed molecular mechanism of telomere resetting in early embryos remains unknown. Here, we show that Dcaf11 (Ddb1- and Cul4-associated factor 11) participates in telomere elongation in early embryos and 2-cell-like embryonic stem cells (ESCs). The deletion of Dcaf11 in embryos and ESCs leads to reduced telomere sister-chromatid exchange (T-SCE) and impairs telomere lengthening. Importantly, Dcaf11-deficient mice exhibit gradual telomere erosion with successive generations, and hematopoietic stem cell (HSC) activity is also greatly compromised. Mechanistically, Dcaf11 targets Kap1 (KRAB-associated protein 1) for ubiquitination-mediated degradation, leading to the activation of Zscan4 downstream enhancer and the removal of heterochromatic H3K9me3 at telomere/subtelomere regions. Our study therefore demonstrates that Dcaf11 plays important roles in telomere elongation in early embryos and ESCs through activating Zscan4. Telomeres play vital roles in ensuring chromosome stability and are thus closely linked with the onset of aging and human disease. Telomeres undergo extensive lengthening during early embryogenesis. However, the detailed molecular mechanism of telomere resetting in early embryos remains unknown. Here, we show that Dcaf11 (Ddb1- and Cul4-associated factor 11) participates in telomere elongation in early embryos and 2-cell-like embryonic stem cells (ESCs). The deletion of Dcaf11 in embryos and ESCs leads to reduced telomere sister-chromatid exchange (T-SCE) and impairs telomere lengthening. Importantly, Dcaf11-deficient mice exhibit gradual telomere erosion with successive generations, and hematopoietic stem cell (HSC) activity is also greatly compromised. Mechanistically, Dcaf11 targets Kap1 (KRAB-associated protein 1) for ubiquitination-mediated degradation, leading to the activation of Zscan4 downstream enhancer and the removal of heterochromatic H3K9me3 at telomere/subtelomere regions. Our study therefore demonstrates that Dcaf11 plays important roles in telomere elongation in early embryos and ESCs through activating Zscan4. Telomeres reside at the end of chromosomes and play essential roles in maintenance of genomic stability (Blasco, 2005Blasco M.A. Telomeres and human disease: ageing, cancer and beyond.Nat. Rev. Genet. 2005; 6: 611-622Crossref PubMed Scopus (1157) Google Scholar). Telomeres shorten with increasing age, which is closely associated with aging syndrome. Telomere length is reset during early embryogenesis (Liu et al., 2007Liu L. Bailey S.M. Okuka M. Muñoz P. Li C. Zhou L. Wu C. Czerwiec E. Sandler L. Seyfang A. et al.Telomere lengthening early in development.Nat. Cell Biol. 2007; 9: 1436-1441Crossref PubMed Scopus (264) Google Scholar; Schaetzlein et al., 2004Schaetzlein S. Lucas-Hahn A. Lemme E. Kues W.A. Dorsch M. Manns M.P. Niemann H. Rudolph K.L. Telomere length is reset during early mammalian embryogenesis.Proc. Natl. Acad. Sci. USA. 2004; 101: 8034-8038Crossref PubMed Scopus (162) Google Scholar). Significant telomere lengthening occurs within the first few cell cycles, while telomerase activity remains low in early cleavage embryos. Coincidently, telomere sister-chromatid exchange (T-SCE) is extensive, and the DNA recombination protein RAD50 is colocalized with TRF1 (Liu et al., 2007Liu L. Bailey S.M. Okuka M. Muñoz P. Li C. Zhou L. Wu C. Czerwiec E. Sandler L. Seyfang A. et al.Telomere lengthening early in development.Nat. Cell Biol. 2007; 9: 1436-1441Crossref PubMed Scopus (264) Google Scholar). These unique features are distinct from the telomerase-based telomere elongation mechanism. Thus, early embryos adopt a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT) to elongate telomeres. Due to the scarcity of embryonic material, the molecular details of telomere elongation in early embryos remain enigmatic. Studies of the ALT mechanism in embryonic stem cells (ESCs) indicate Zscan4 gene cluster as a critical regulator in telomere extension (Zalzman et al., 2010Zalzman M. Falco G. Sharova L.V. Nishiyama A. Thomas M. Lee S.L. Stagg C.A. Hoang H.G. Yang H.T. Indig F.E. et al.Zscan4 regulates telomere elongation and genomic stability in ES cells.Nature. 2010; 464: 858-863Crossref PubMed Scopus (264) Google Scholar). Zscan4 gene cluster is transiently expressed in 2-cell embryos and a rare portion of ESCs (Falco et al., 2007Falco G. Lee S.L. Stanghellini I. Bassey U.C. Hamatani T. Ko M.S. Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells.Dev. Biol. 2007; 307: 539-550Crossref PubMed Scopus (164) Google Scholar; Zalzman et al., 2010Zalzman M. Falco G. Sharova L.V. Nishiyama A. Thomas M. Lee S.L. Stagg C.A. Hoang H.G. Yang H.T. Indig F.E. et al.Zscan4 regulates telomere elongation and genomic stability in ES cells.Nature. 2010; 464: 858-863Crossref PubMed Scopus (264) Google Scholar). In ESCs, the transient activation of Zscan4 is associated with rapid telomere elongation in a telomerase-independent manner (Zalzman et al., 2010Zalzman M. Falco G. Sharova L.V. Nishiyama A. Thomas M. Lee S.L. Stagg C.A. Hoang H.G. Yang H.T. Indig F.E. et al.Zscan4 regulates telomere elongation and genomic stability in ES cells.Nature. 2010; 464: 858-863Crossref PubMed Scopus (264) Google Scholar). Zscan4-positive ESCs exhibit increased telomere recombination and global derepression of heterochromatin (Akiyama et al., 2015Akiyama T. Xin L. Oda M. Sharov A.A. Amano M. Piao Y. Cadet J.S. Dudekula D.B. Qian Y. Wang W. et al.Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells.DNA Res. 2015; 22: 307-318Crossref PubMed Scopus (47) Google Scholar; Ko, 2016Ko M.S. Zygotic Genome Activation Revisited: Looking Through the Expression and Function of Zscan4.Curr. Top. Dev. Biol. 2016; 120: 103-124Crossref PubMed Scopus (32) Google Scholar). Zscan4 is capable of forming complexes with epigenetic modifiers, indicating its role in epigenetic regulation (Akiyama et al., 2015Akiyama T. Xin L. Oda M. Sharov A.A. Amano M. Piao Y. Cadet J.S. Dudekula D.B. Qian Y. Wang W. et al.Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells.DNA Res. 2015; 22: 307-318Crossref PubMed Scopus (47) Google Scholar). Zscan4 can be reactivated in response to telomere shortening and DNA damage (Nakai-Futatsugi and Niwa, 2016Nakai-Futatsugi Y. Niwa H. Zscan4 Is Activated after Telomere Shortening in Mouse Embryonic Stem Cells.Stem Cell Reports. 2016; 6: 483-495Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In ESCs, the continuous expression of Zscan4 is linked with telomere hyper-recombination and potential genomic instability (Dan et al., 2014Dan J. Liu Y. Liu N. Chiourea M. Okuka M. Wu T. Ye X. Mou C. Wang L. Wang L. et al.Rif1 maintains telomere length homeostasis of ESCs by mediating heterochromatin silencing.Dev. Cell. 2014; 29: 7-19Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Furthermore, failure of activation or silence of Zscan4 in 2-cell embryos leads to development retardation (Falco et al., 2007Falco G. Lee S.L. Stanghellini I. Bassey U.C. Hamatani T. Ko M.S. Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells.Dev. Biol. 2007; 307: 539-550Crossref PubMed Scopus (164) Google Scholar). Therefore, the Zscan4 expression needs to be tightly controlled. However, the upstream molecular mechanism of Zscan4 reactivation remains to be elusive. To dissect the molecular details of ALT-mediated telomere elongation in early embryos, we performed a screen in ESCs and identified Ddb1- and Cul4-associated factor 11 (Dcaf11) as an essential factor. Depletion of Dcaf11 led to reduced telomere sister-chromatid exchange (T-SCE) and impaired telomere lengthening through a telomerase-independent manner in embryos and ESCs. Overexpression of Dcaf11 facilitated somatic cell reprogramming and promoted telomere elongation in pluripotent stem cells. Dcaf11-deficient mice exhibited gradual telomere erosion with successive generations and compromised hematopoietic stem cells (HSCs) activity. Zscan4 is the major downstream effector responsible for Dcaf11-induced telomere extension, and Dcaf11 targets Kap1 for ubiquitination-mediated degradation, leading to the activation of Zscan4 and telomere elongation. Furthermore, we identified a distal enhancer of Zscan4, which is activated by Dcaf11 in a direct manner via the release of Kap1. At telomere region, Dcaf11 reduces the occupancy of Kap1 and heterochromatic H3K9me3. Thus, Dcaf11 plays an essential role in ALT-mediated telomere elongation in early embryos and ESCs. Telomeres undergo extensive elongation in a telomerase-independent manner during early embryo development (Liu et al., 2007Liu L. Bailey S.M. Okuka M. Muñoz P. Li C. Zhou L. Wu C. Czerwiec E. Sandler L. Seyfang A. et al.Telomere lengthening early in development.Nat. Cell Biol. 2007; 9: 1436-1441Crossref PubMed Scopus (264) Google Scholar, Schaetzlein et al., 2004Schaetzlein S. Lucas-Hahn A. Lemme E. Kues W.A. Dorsch M. Manns M.P. Niemann H. Rudolph K.L. Telomere length is reset during early mammalian embryogenesis.Proc. Natl. Acad. Sci. USA. 2004; 101: 8034-8038Crossref PubMed Scopus (162) Google Scholar). Zscan4+ ESCs share many similar features with early cleavage embryos, including extensive telomere elongation independent of telomerase, increased T-SCE, and recruitment of homologous recombination proteins to telomeres (Liu et al., 2007Liu L. Bailey S.M. Okuka M. Muñoz P. Li C. Zhou L. Wu C. Czerwiec E. Sandler L. Seyfang A. et al.Telomere lengthening early in development.Nat. Cell Biol. 2007; 9: 1436-1441Crossref PubMed Scopus (264) Google Scholar, Zalzman et al., 2010Zalzman M. Falco G. Sharova L.V. Nishiyama A. Thomas M. Lee S.L. Stagg C.A. Hoang H.G. Yang H.T. Indig F.E. et al.Zscan4 regulates telomere elongation and genomic stability in ES cells.Nature. 2010; 464: 858-863Crossref PubMed Scopus (264) Google Scholar). Due to the scarcity of early embryos, we utilized Zscan4+ ESCs as a model to identify factors involved in telomere elongation during early embryogenesis. Zscan4 expression is dynamic and transient in mouse ESCs, and only 1%–5% of ESCs enter a Zscan4-positive state at a given time (Zalzman et al., 2010Zalzman M. Falco G. Sharova L.V. Nishiyama A. Thomas M. Lee S.L. Stagg C.A. Hoang H.G. Yang H.T. Indig F.E. et al.Zscan4 regulates telomere elongation and genomic stability in ES cells.Nature. 2010; 464: 858-863Crossref PubMed Scopus (264) Google Scholar). To trace Zscan4 expression, we generated an ESC line stably transfected with a Zscan4::EGFP transgene containing a Zscan4 promoter-driven EGFP fluorescent reporter as previously described (Figure S1A) (Zalzman et al., 2010Zalzman M. Falco G. Sharova L.V. Nishiyama A. Thomas M. Lee S.L. Stagg C.A. Hoang H.G. Yang H.T. Indig F.E. et al.Zscan4 regulates telomere elongation and genomic stability in ES cells.Nature. 2010; 464: 858-863Crossref PubMed Scopus (264) Google Scholar). We first utilized our previous proteomic data of mouse pre-implantation development (Gao et al., 2017Gao Y. Liu X. Tang B. Li C. Kou Z. Li L. Liu W. Wu Y. Kou X. Li J. et al.Protein Expression Landscape of Mouse Embryos during Pre-implantation Development.Cell Rep. 2017; 21: 3957-3969Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar; Wang et al., 2010Wang S. Kou Z. Jing Z. Zhang Y. Guo X. Dong M. Wilmut I. Gao S. Proteome of mouse oocytes at different developmental stages.Proc. Natl. Acad. Sci. USA. 2010; 107: 17639-17644Crossref PubMed Scopus (165) Google Scholar) to select potential positive activators according to Gene Ontology (GO) analysis and an expression pattern that was coincident with extensive telomere elongation in early cleavage embryos (Figures S1B and S1C) (Liu et al., 2007Liu L. Bailey S.M. Okuka M. Muñoz P. Li C. Zhou L. Wu C. Czerwiec E. Sandler L. Seyfang A. et al.Telomere lengthening early in development.Nat. Cell Biol. 2007; 9: 1436-1441Crossref PubMed Scopus (264) Google Scholar). An RNAi screen was performed to identify factors associated with Zscan4 activation. Short hairpin RNAs (shRNAs) that targeted Dux, which has been implicated in driving zygotic genome activation (ZGA) genes expression, including Zscan4, as a positive control and a non-targeting shRNA as a negative control were included. The ability of the candidate genes to activate the ALT mechanism was tested by flow cytometry via analyzing the expression of the Zscan4::EGFP reporter (Figure 1A). Among the 12 candidates, knockdown of Dcaf11 led to the most significant reduction of Zscan4+ ESCs (Figure 1B). To validate the result, we generated ESCs deficient for Dcaf11 using CRISPR-Cas9 gene targeting in Zscan4+ ESCs. Deletion of Dcaf11 was confirmed by western blot analysis (Figure S1D). We also generated stable Dcaf11 overexpression (OE) ESC lines under the control of doxycycline (Figure S1D). The increased expression level of Dcaf11 in OE ESCs was confirmed by western blot analysis (Figure S1D). Deletion of Dcaf11 in ESCs did not change the typical compact cell colonies or the expression of pluripotent genes (Figure S1E). Flow cytometry analysis revealed that Dcaf11 overexpression significantly increased the population of Zscan4+ cells by 3- to 17-fold, while deletion of Dcaf11 resulted in a marked loss of Zscan4+ cells (Figures 1C–1E), which was consistently reflected in the expression of Zscan4 by qRT-PCR (Figure S1F). To further investigate the transcriptome impacts following the loss of Dcaf11, we performed RNA sequencing (RNA-seq) of Dcaf11−/− ESCs. Deletion of Dcaf11 led to the upregulation of 635 genes and the downregulation of 935 genes compared with those of wild-type (WT) ESCs (fold change [FC] >2 or FC <−2; false discovery rate [FDR] <0.05) (Figure 1F). Markers of pluripotency-associated genes or three germ layers were unaffected (Data S1). There is a markedly enrichment of Zscan4+-specific genes among the downregulated genes (Figures 1F and 1G; Data S1). Besides Zscan4 gene clusters, other 2-cell embryo (2C) genes such as Dux, Eif1a-like gene clusters (Gm2016, Gm2022, Gm8300), and Tdpoz genes (Tdpoz3, Tdpoz4) are also significantly downregulated upon Dcaf11 depletion (Data S1). Analysis of the repetitive proportion of the genome also revealed a markedly downregulation of the MERVL endogenous retrovirus (Figure 1H). In addition, overexpression of Dcaf11 in MEFs could not reactivate Zscan4 expression (Figure S1G). This observation confirms that Zscan4 activation by Dcaf11 is ESCs specific. Thus, Dcaf11 is required for Zscan4 activation in ESCs. Next, we investigated the role of Dcaf11 during mouse early embryonic development. We first generated Dcaf11 knockout mice by using the CRISPR-Cas9 system. Based on our previous proteomic data, Dcaf11 is highly expressed in zygotes and 2-cell embryos and is gradually downregulated in the following embryonic developmental stages (Figure S2A). Dcaf11 deficiency led to a mild decrease in 2- to 4-cell embryos (Figure 2A). A subset of Dcaf11-depleted embryos arrested at the 2- to 4-cell stage and showed signs of fragmentation (Figure 2B). Since Dcaf11 is required for activation of a subset of 2C genes in ESCs, we next investigated whether Dcaf11 participated in zygotic genome activation in 2-cell embryos. RNA-seq analysis of Dcaf11-deficient 2-cell embryos showed the upregulation of 612 genes and downregulation of 1325 genes (Figure 2C, Data S2) (FC >2 or FC <−2; FDR <0.05). The downregulated genes were enriched at the zygote and 2-cell stage, while the upregulated genes were expressed from the mid-2-cell stage and progressively upregulated afterward during embryogenesis (Figures 2D and 2E). Among the downregulated genes, 371 genes were 2-cell embryo-specific genes, including Zscan4 gene families, Prame-family genes, and Eif-a-like genes, while only 27 2-cell embryo-specific genes were upregulated (Figures 2F, S2B, and S2C; Data S2). We also performed RNA-seq of Dcaf11-deficient embryos arrested at 2-cell stage. Arrested Dcaf11-deficient 2-cell embryos exhibited a more severe defect of activating 2-cell embryo-specific genes (Figures S2D–S2F; Data S3). Many retrotransposons are reactivated in 2-cell embryos (Macfarlan et al., 2012Macfarlan T.S. Gifford W.D. Driscoll S. Lettieri K. Rowe H.M. Bonanomi D. Firth A. Singer O. Trono D. Pfaff S.L. Embryonic stem cell potency fluctuates with endogenous retrovirus activity.Nature. 2012; 487: 57-63Crossref PubMed Scopus (590) Google Scholar). Next, we examined the repetitive portion of the transcriptome. Deletion of Dcaf11 resulted in more downregulation than upregulation of retrotransposons (Figures 2G and S2G; Data S2). MERVL acts as an alternative promoter to drive 2C genes during ZGA (Peaston et al., 2004Peaston A.E. Evsikov A.V. Graber J.H. de Vries W.N. Holbrook A.E. Solter D. Knowles B.B. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos.Dev. Cell. 2004; 7: 597-606Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Notably, Dcaf11 depletion resulted in minimal changes in the MERVL expression level (Figure S2H). Collectively, Dcaf11 contributes to the activation of the 2-cell transcriptional program during zygotic genome activation in 2-cell embryos. Deletion of Dcaf11 led to downregulation of Zscan4 in early embryos and ESCs. Since Zscan4 plays a central role in ALT-mediated telomere elongation, we speculated that Dcaf11 may participate in telomere elongation. We performed quantitative fluorescent in situ hybridization (Q-FISH) to investigate the contribution of Dcaf11 in telomere resetting during early embryo development. In agreement with a previous report (Liu et al., 2007Liu L. Bailey S.M. Okuka M. Muñoz P. Li C. Zhou L. Wu C. Czerwiec E. Sandler L. Seyfang A. et al.Telomere lengthening early in development.Nat. Cell Biol. 2007; 9: 1436-1441Crossref PubMed Scopus (264) Google Scholar), telomeres were markedly elongated in WT embryos, while Dcaf11 deletion led to impaired telomere elongation, especially in the 2- to 4-cell stage (Figures 3A–3D). Consistent with previous findings (Liu et al., 2007Liu L. Bailey S.M. Okuka M. Muñoz P. Li C. Zhou L. Wu C. Czerwiec E. Sandler L. Seyfang A. et al.Telomere lengthening early in development.Nat. Cell Biol. 2007; 9: 1436-1441Crossref PubMed Scopus (264) Google Scholar), almost all the chromosomes of WT 2-cell embryos exhibited T-SCE at their ends as detected by chromosome orientation-FISH (CO-FISH) (Figures 3E and 3F). In contrast, T-SCE significantly decreased in Dcaf11−/− 2-cell embryos (Figures 3E and 3F). It has been previously reported that telomerase activity was low in early cleavage embryos and significantly increased in blastocysts and ESCs (Liu et al., 2007Liu L. Bailey S.M. Okuka M. Muñoz P. Li C. Zhou L. Wu C. Czerwiec E. Sandler L. Seyfang A. et al.Telomere lengthening early in development.Nat. Cell Biol. 2007; 9: 1436-1441Crossref PubMed Scopus (264) Google Scholar). To investigate whether increased telomerase could compensate for telomere loss inherited from early embryos, we established ESCs from Dcaf11−/− blastocysts. Dcaf11−/− ESCs exhibited the typical morphology of pluripotent stem cells with a compact appearance and a well-defined border (Figure S3A). Immunofluorescence staining indicated the expression of pluripotent markers in Dcaf11−/− ESCs (Figure S3B). However, in the absence of Dcaf11, the derivation efficiency markedly decreased compared with the WT counterparts (Figure S3C). In addition, the percentage of Zscan4+ cells significantly decreased in Dcaf11−/− ESCs (Figure S3D). The resulting Dcaf11−/− ESCs displayed an approximately 50% decrease in telomere length compared with WT ESCs, supported by telomere Q-FISH (Figure 3G). Telomere length gradually decreased in ESCs derived from Dcaf11−/− mice with late generations (Figure 3G). Importantly, chromosome aberrations were observed in ESCs derived from G3 Dcaf11−/− mice, as evidenced by the increased frequency of telomere loss and end-to-end fusions (Figures 3H and 3I). To assess whether Dcaf11 participates in telomere maintenance in ESCs during long-term culture, we depleted Dcaf11 using the CRISPR-Cas9 system. Telomere length decreased markedly by 30%–40% in 10 passages following Dcaf11 deletion (Figures S3E–S3G). In addition, the frequency of T-SCE significantly decreased in Dcaf11−/− ESCs (Figure S3H). To test whether telomere deficiency caused by Dcaf11 depletion was associated with telomerase, we next performed a telomeric repeat amplification protocol (TRAP) assay to measure telomerase activity (Figure S3I). As shown in Figure S3I, Dcaf11 depletion did not cause significant change of telomerase activity. To further validate that Dcaf11 could act in a telomerase-independent manner, we overexpressed Dcaf11 in G3 Terc−/− ESCs. Dcaf11 overexpression (OE) significantly upregulates the expression levels of Zscan4c and Zscan4d in G3 Terc−/− ESCs (Figures S3J and S3K). Of noted, Dcaf11 OE in 10 passages leads to significantly elongated telomeres and reduced telomere free ends in G3 Terc−/− ESCs (Figures S3L–S3N). These data together indicate that Dcaf11 is required for telomere elongation in early embryos and telomere maintenance in ESCs in a telomerase-independent manner. Given that Zscan4 dramatically enhances the reprogramming efficiency and improves the quality of iPSCs (Jiang et al., 2013Jiang J. Lv W. Ye X. Wang L. Zhang M. Yang H. Okuka M. Zhou C. Zhang X. Liu L. Li J. Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation.Cell Res. 2013; 23: 92-106Crossref PubMed Scopus (103) Google Scholar), we next explored the role of Dcaf11 in iPSC generation. Dcaf11 is highly expressed in pluripotent stem cells and gradually upregulated in the OSKM-induced reprogramming process (Figure S4A). To confirm the role of Dcaf11 in iPSC induction, we exogenously expressed Dcaf11 in Oct4_IRES_GFP/Rosa26-M2rtTA mouse embryonic fibroblast (MEF) cells carrying the tetO-OSKM transgene and Oct4-GFP/Rosa26-M2rtTA (Carey et al., 2010Carey B.W. Markoulaki S. Beard C. Hanna J. Jaenisch R. Single-gene transgenic mouse strains for reprogramming adult somatic cells.Nat. Methods. 2010; 7: 56-59Crossref PubMed Scopus (438) Google Scholar). Dcaf11 OE enhanced the cell-proliferative capacity during the reprogramming process in a manner similar to Zscan4 OE (Figures 4A and S4B). Furthermore, ectopic expression of Dcaf11 resulted in a significant increase in Oct4-GFP-positive cells and a dramatic increase in iPSC colonies (Figures 4B–4D). The OSKM plus Dcaf11-iPSCs exhibited expression of pluripotent genes comparable with that of ESCs (Figure 4E). Furthermore, a chimera formation assay was performed, whereby the OSKM plus Dcaf11-iPSC lines could integrate into the gonads of the chimeric mice (Figures S4C and S4D). Next, we derived Dcaf11−/− MEFs from Dcaf11-deficient mice and infected Dcaf11−/− MEF cells with a doxycycline-inducible lentiviral construct expressing OSKM. Dcaf11 deletion caused a dramatic reduction of iPSC colonies (Figure S4E), suggesting that endogenous Dcaf11 is required for efficient iPSC generation. Notably, ectopic expression of Dcaf11 caused a marked increase in telomere length in iPSCs, as indicated by the T/S ratio (Figure 4F). To investigate the role of Dcaf11 in the promotion of the ALT mechanism during iPSC induction, we further reprogrammed somatic cells in the absence of telomerase. Telomere defects caused by inactivation of telomerase greatly impaired reprogramming efficiency as previously reported (Marion et al., 2009Marion R.M. Strati K. Li H. Tejera A. Schoeftner S. Ortega S. Serrano M. Blasco M.A. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells.Cell Stem Cell. 2009; 4: 141-154Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Notably, ectopic expression of Dcaf11 greatly enhanced iPSC colonies induced from Terc−/− somatic cells (Figure 4G). In addition, the proliferation crisis was alleviated by Dcaf11, as evidenced by β-galactosidase staining (Figure 4H). The resulting iPSCs exhibited markedly increased telomere length and chromosomal stability compared with control iPSCs although normal karyotype percentage was not significantly changed (Figures 4I, 4J, and S4F). In agreement with the improved telomere function, the developmental potential of Terc−/− iPSCs was also greatly improved by Dcaf11 overexpression (Figures 4K, S4G, and S4H). Taken together, these results demonstrated that Dcaf11 promotes telomere elongation during iPSC production in a telomerase-independent manner. Mouse tail-tip fibroblasts derived from the second and the fourth generations (G2 and G4) of Dcaf11 KO mice exhibited gradually telomere loss with increasing generations (Figures 5A). G2 Dcaf11 KO mice showed no overt phenotypes relative to Dcaf11+/+ mice in terms of differentiation status among hematological systems, including peripheral blood (PB) and bone marrow (BM) (Figures S5A and S5B). Absolute numbers of various primitive hematopoietic cells (LT-HSCs, ST-HSCs, MPPs, and progenitors) were also indistinguishable in Dcaf11+/+ and G2 Dcaf11 KO BM (Figures S5C and S5D). Thus, Dcaf11 is dispensable for steady-state hematopoiesis in G2 Dcaf11 KO mice. However, there was a very short telomere length in the HSCs of the G4 Dcaf11 KO mice compared with the HSCs of WT mice (Figure 5B). G4 Dcaf11 KO mice showed different phenotypes relative to G2 Dcaf11 KO and WT mice in terms of differentiation status among hematological systems, including PB and BM (Figures 5C and 5D). The frequencies of LT-HSCs, ST-HSCs, and CLP were also decreased in G4 Dcaf11 KO BM compared with G2 Dcaf11 KO and WT mice (Figures 5D and 5E). These results support the notion that Dcaf11 deletion regulates hematopoiesis maintenance and HSC function dependent on telomere length. Considering that HSC is susceptible to telomere attrition under hematopoietic stress, we investigated HSC function in G2 Dcaf11 KO mice after transplantation or 5-fluorouracil (5-FU) treatment, a cell cycle-dependent myelotoxic agent that kills proliferating cells, including cycling hematopoietic stem and progenitor cells (HSPCs). Given the function of Dcaf11 in the context of telomere length maintenance, we monitored how tolerant G2 Dcaf11 KO HSCs were of hematological stress by performing serial competitive repopulation assays with purified HSCs. Equal numbers of HSCs sorted from Dcaf11+/+ and G2 Dcaf11 KO mice were mixed with the same amount of competitor CD45.1 BM cells and transplanted into lethally irradiated CD45.1/2 recipients. In the primary transplantation, G2 Dcaf11 knockout donor HSCs did not show significant differences in repopulation capabilities compared to Dcaf11+/+ counterparts (Figure S5E). Four months after BMT, 1 × 106 BM MNCs from primary recipients were isolated and transplanted into lethally irradiated CD45.1/2 secondary recipients. However, we observed much lower relative PB and BM chimerism in the G2 Dcaf11 KO compared to the Dcaf11+/+ cell-transplanted group in the secondary transplantation than in the primary transplantation (Figure 5F). To gain further insight into Dcaf11 function during hematological stress, we evaluated telomere length in G2 Dcaf11 KO HSCs of primary and secondary transplantation mice. As shown, G2 Dcaf11 KO HSCs after the secondary transplantation had shortened telomere length compared with Dcaf11 KO HSCs in the primary transplantation (Figure S5F). Furthermore, to gain further insight into Dcaf11 function during chemotherapy hematological stress, we treated Dcaf11 KO and WT mice with 5-FU. Following a single dose of 5-FU, the absolute numbers of HSPCs (LSK) and LT-HSC (CD34–FLT3+LSK) populations were significantly decreased in G2 Dcaf11 KO bone marrow (Figure 5G). Taken together, our results indicate that Dcaf11 loss results in defects in stem cell capacity during stress hematopoiesis in vivo in a cell-autonomous manner. Next, we asked whether Zscan4 is the major downstream effector responsible for Dcaf11-induced telomere elongation. Forced expression of Zscan4 led to significantly elongated telomeres in Dcaf11 KO ESCs while knockdown of Zscan4 notably abolished telomere elongation effect caused by Dcaf11 OE (Figures S6A and S6B). Subsequently, we explored the molecular basis of Dcaf11 in reactivating Zscan4. DDB1 and CUL4-associated factors (DCAFs) act as E3 substrate receptors to recognize the substrate and recruit the ubiquitination machinery for subsequent degradation (Lee and Zhou, 2007Lee J. Zhou P. DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase.Mol. Cell. 2007; 26: 775-780Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The WD40 domain defines a critical signature of DCAFs (Lee and Zhou, 2007Lee J. Zhou P. DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase.Mol. Cell. 2007; 26: 775-780Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). To determine the domain function of Dcaf11, we overexpressed a series of truncated versions of Dcaf11 in ESCs (Figures 6A and 6B ). Only the full-length Dcaf11 could upregulate Zscan4 expression (Figure 6B). To gain insight int" @default.
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• W3113714347 date "2021-04-01" @default.
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• W3113714347 title "Dcaf11 activates Zscan4-mediated alternative telomere lengthening in early embryos and embryonic stem cells" @default.
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• W3113714347 doi "https://doi.org/10.1016/j.stem.2020.11.018" @default.
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