DCAF 13 promotes pluripotency by negatively regulating SUV 39H1 stability during early embryonic development

Article14 August 2018free access Source DataTransparent process DCAF13 promotes pluripotency by negatively regulating SUV39H1 stability during early embryonic development Yin-Li Zhang orcid.org/0000-0001-8353-9615 Life Sciences Institute, Zhejiang University, Hangzhou, China Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Long-Wen Zhao orcid.org/0000-0002-4964-9578 Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Jue Zhang orcid.org/0000-0001-5742-1277 Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Rongrong Le Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Shu-Yan Ji Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Chuan Chen Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Yawei Gao Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Dali Li Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Shaorong Gao Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Heng-Yu Fan Corresponding Author [email protected] orcid.org/0000-0003-4544-4724 Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yin-Li Zhang orcid.org/0000-0001-8353-9615 Life Sciences Institute, Zhejiang University, Hangzhou, China Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Long-Wen Zhao orcid.org/0000-0002-4964-9578 Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Jue Zhang orcid.org/0000-0001-5742-1277 Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Rongrong Le Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Shu-Yan Ji Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Chuan Chen Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Yawei Gao Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Dali Li Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Shaorong Gao Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China Search for more papers by this author Heng-Yu Fan Corresponding Author [email protected] orcid.org/0000-0003-4544-4724 Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Author Information Yin-Li Zhang1,2,‡, Long-Wen Zhao1,‡, Jue Zhang1,‡, Rongrong Le3, Shu-Yan Ji1, Chuan Chen3, Yawei Gao3, Dali Li4, Shaorong Gao3 and Heng-Yu Fan *,1 1Life Sciences Institute, Zhejiang University, Hangzhou, China 2Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China 3Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China 4Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86-571-88981370; E-mail: [email protected] EMBO J (2018)37:e98981https://doi.org/10.15252/embj.201898981 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mammalian oocytes and zygotes have the unique ability to reprogram a somatic cell nucleus into a totipotent state. SUV39H1/2-mediated histone H3 lysine-9 trimethylation (H3K9me3) is a major barrier to efficient reprogramming. How SUV39H1/2 activities are regulated in early embryos and during generation of induced pluripotent stem cells (iPSCs) remains unclear. Since expression of the CRL4 E3 ubiquitin ligase in oocytes is crucial for female fertility, we analyzed putative CRL4 adaptors (DCAFs) and identified DCAF13 as a novel CRL4 adaptor that is essential for preimplantation embryonic development. Dcaf13 is expressed from eight-cell to morula stages in both murine and human embryos, and Dcaf13 knockout in mice causes preimplantation-stage mortality. Dcaf13 knockout embryos are arrested at the eight- to sixteen-cell stage before compaction, and this arrest is accompanied by high levels of H3K9me3. Mechanistically, CRL4-DCAF13 targets SUV39H1 for polyubiquitination and proteasomal degradation and therefore facilitates H3K9me3 removal and zygotic gene expression. Taken together, CRL4-DCAF13-mediated SUV39H1 degradation is an essential step for progressive genome reprogramming during preimplantation embryonic development. Synopsis DCAF13 is a novel CRL4 E3 ubiquitin ligase adaptor that prevents histone modification by the SUV39H1 methyltransferase, thereby allowing preimplantation embryonic development. DCAF13 is a bona fideCRL4 E3 ligase adaptor highly expressed in preimplantation-stage embryos. DCAF13 mouse knockout causes embryonic lethality at the morula stage. CRL4-DCAF13 mediates SUV39H1 polyubiquitination and proteasomal degradation. DCAF13 deletion inhibits removal of histone H3-Lys9 trimethylation marks, thereby inhibiting zygotic gene expression. Introduction During early preimplantation development of mammalian embryos, genomes are highly asymmetric in epigenetic modifications of DNA and of the associated chromatin and undergo dramatic reorganization (Feil, 2009). These changes participate in the establishment of stable and heritable epigenetic modifications and may occur simultaneously during development and cell differentiation (Smith & Meissner, 2013; Hatanaka et al, 2015). Maternal factors involved in the regulation of zygotic genome reprogramming have been extensively studied (Ancelin et al, 2016; Zhang et al, 2016). Nonetheless, these maternal factors are presumably downregulated sharply after fertilization (Lee et al, 2014). Therefore, by continuing and substituting the function of maternal factors, products of early responsive genes after zygotic gene activation also play key roles in reprogramming of the genomes and in the support of early development of normal and cloned embryos. With the help of advances in single-cell sequencing techniques, gene expression profiles of murine and human early embryos have been extensively studied (Hou et al, 2013; Xue et al, 2013). On the other hand, the functions and biochemical properties of these early zygotic genes have yet to be fully identified. Mammalian oocytes and zygotes are different from somatic cells in their ability to reprogram a somatic cell nucleus into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT) (Lu & Zhang, 2015). Nonetheless, the majority of SCNT embryos fail to develop to term because of undefined reprogramming defects (Niemann, 2016). SUV39H1-mediated histone H3 lysine-9 trimethylation (H3K9me3) of the donor cell genome is a major barrier to efficient reprogramming by SCNT. Removal of this epigenetic mark either through ectopic expression of an H3K9me3-specific demethylase, KDM4D or KDM5B, in oocytes or through a knockdown of the H3K9 methyltransferases (SUV39H1/2) in donor cells not only attenuates the zygotic gene activation defect but also greatly improves the reprogramming efficiency of SCNT (Matoba et al, 2014; Liu et al, 2016). Nevertheless, it remains unclear how the SUV39H1/2 activities are disabled in early embryos derived from normal fertilization or in the process of induced pluripotent stem cell (iPSC) reprogramming. CRL4 ubiquitin E3 ligase in oocytes is crucial for female fertility. Oocyte-specific deletion of damaged DNA-binding protein 1 (DDB1)—the linker protein of the CRL4 complex—causes rapid primordial follicle loss and premature ovarian insufficiency (Yu et al, 2013, 2015b). CUL4, one of three founding cullins conserved from yeast to humans, uses DDB1 as a linker to interact with a subset of WD40-repeat-containing DDB1/CUL4-associated factors (DCAFs) that serve as substrate receptors, forming as many as 90 E3 complexes (Lee & Zhou, 2007). Among these, DCAF1 has been identified as a major maternal substrate adaptor of CRL4 that maintains oocyte survival (Yu et al, 2013). CRL4DCAF1 regulates expression of genes essential for oocyte survival and ovulation in primordial follicles and for paternal DNA demethylation upon fertilization, partially by modulating TET1/2/3 activities through monoubiquitination of a conserved lysine site to regulate DNA methylation levels in oocytes (Yu et al, 2013). Nonetheless, Ddb1-null embryos have more severe developmental defects than Tet3-null embryos (Gu et al, 2011), indicating that CRL4 supports early embryonic development by additional mechanisms. In this study, we report DCAF13 as a novel CRL4 adaptor that is essential for preimplantation embryonic development. Dcaf13 is an early zygotic gene in both murine and human embryos and is expressed mainly at eight-cell to morula stages. Dcaf13 knockout murine embryos are arrested at the eight- to sixteen-cell stage before compaction, and this situation causes preimplantation mortality. Mechanistically, CRL4DCFA13 directs SUV39H1/2 to polyubiquitination and proteasomal degradation and thereby triggers histone H3 lysine-9 demethylation and zygotic genome reprogramming. Results DCAF13 is a conserved DDB1/CUL4-associated factor that is specifically induced in early embryos By analyzing the expression profile of 14 DCAFs detected in human oocytes and early embryos by single-cell RNA sequencing (Hou et al, 2013; Yan et al, 2013), we noticed that DCAF13, a putative CRL4 substrate adaptor, was an early zygotic gene whose expression was specifically induced as early as the four- to eight-cell stage (Fig 1A). Quantitative RT–PCR and data from RNA-seq datasets (GSE70605) in mouse oocytes and early embryos both showed that murine Dcaf13 mRNA was also transiently expressed during four-cell to morula stages, in comparison with other DCAFs (Fig EV1A and B). Western blot and immunofluorescence results showed that DCAF13 was expressed as early as the four-cell stage and continued to accumulate in morulae and blastocysts (Fig 1B and C). Moreover, DCAF13 was mainly located in the nucleoli of blastomeres from four-cell to blastocyst stages, as indicated by its co-localization with well-established nucleolar marker B23 (also known as NPM1; Fig 1C). Furthermore, DCAF13 was uniformly distributed in trophectoderm cells and inner cells mass, which was indicated by co-staining of CDX2 and DCAF13 in mouse blastocyst embryos (Fig EV1C). Figure 1. Dcaf13 is a gene zygotically expressed early during human and murine embryonic development mRNA profiles of 14 DCAFs detected in human oocytes and early embryos by RNA-seq. The relative mRNA level in germinal vesicle (GV) stage oocytes was set to 1.0, and fold changes at different stages are shown. TE, trophectoderm; PE, primitive endoderm. Western blotting of DCAF13 and DDB1 in mouse oocytes and preimplantation embryos. ERK1/2 and α-tubulin were immunoblotted as loading controls. Total protein samples from 100 oocytes or embryos were loaded onto each lane. Confocal microscopic images of DCAF13 (green) and B23 (red) immunofluorescence in mouse oocytes and preimplantation embryos. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). At each stage, more than 30 oocytes or embryos were examined, with similar results. Scale bar = 10 μm. Source data are available online for this figure. Source Data for Figure 1 [embj201898981-sup-0003-SDataFig1.jpg] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Quantitative RT–PCR analysis showing mRNA level changes in indicated DCAFs in mouse preimplantation embryos at different stages The mRNA relative level of indicated DCAFs by qRT–PCR at each developmental stage is presented as mean ± SD (n = 30 embryos for each time point). The FPKM of detected DCAFs in RNA-seq data (GSE70605) at each developmental stage of mouse early embryos. Representative fluorescent images showing DCAF13 (green) and CDX2 (red) immunoreactivity in E4.5 mouse embryos (n = 25). The nuclei were stained with DAPI (blue). The arrow points to the inner cell mass (ICM) within white dotted circle. Scale bar = 10 μm. Download figure Download PowerPoint The Dcaf13 gene is conserved from yeast to mammals, but its function has not been identified in any species. We cloned the mouse Dcaf13 cDNA from a mouse ovary cDNA pool (Yu et al, 2016). It encodes a highly conserved protein of 445 amino acid residues with a molecular weight of 52 kDa; this protein contains seven WD40 repeats at the N terminus and a SOF1 domain at the C terminus (Appendix Fig S1). Co-immunoprecipitation results in HeLa cells indicated that DCAF13 interacts with CRL4 linker protein DDB1; the DCAF13–DDB1 interaction was abrogated by WD domain deletion but was strengthened by SOF1 domain deletion (Fig EV2A and B). Click here to expand this figure. Figure EV2. DCAF13 is a CRL4 E3 ligase adaptor and localized in the nucleolus A diagram of FLAG-tagged DCAF13, DCAF13ΔSOF1, and DCAF13ΔWD40 constructs. Co-IP results in HeLa cells showing DDB1 binding with full-length and truncated DCAF13. Immunofluorescence results of FLAG showing intracellular localization of full-length and truncated DCAF13 in HeLa cells. Nuclei were stained with DAPI, and nucleoli were labeled with B23. Scale bar = 10 μm. Immunofluorescence results showing intracellular localization of endogenous DCAF13 and B23 in mouse embryonic fibroblasts (MEFs). Scale bar = 10 μm. Source data are available online for this figure. Download figure Download PowerPoint We also examined the subcellular localization of DCAF13 in somatic cells. Both overexpressed FLAG-DCAF13 and endogenous DCAF13 were co-localized with B23 and were enriched in nucleoli of HeLa cells and MEFs (Fig EV2C and D). Deletion of the SOF1 domain, but not WD40 repeats, abrogated the nucleolar localization of DCAF13 (Fig EV2C). This result is in agreement with other reports, which show that the SOF1 domain mediates protein–RNA interactions in the yeast nucleolus (Jansen et al, 1993; Bax et al, 2006). DCAF13 is crucial for early embryonic development in mice To elucidate the function of DCAF13 in mice, we generated Dcaf13 knockout mouse strains using a TALEN-based gene targeting approach. Two independent strains containing frame shift mutations at an Alu I restriction site within Dcaf13 exon 2 were obtained and analyzed (Fig EV3A and B). For both strains, when Dcaf13+/− males and females were crossed, the ratio of newborn Dcaf13+/+, Dcaf13+/−, and Dcaf13−/− pups was close to 1:2:0. The 8-bp deletion Dcaf13 mutant strain was used in all of the following experiments. We examined genotypes of the embryos from day postcoitus (dpc) 8.5 to dpc 16.5 and did not find any Dcaf13−/− embryos (Fig 2A). At dpc 3–4, Dcaf13 knockout embryos were identified by DNA genotyping (Fig EV3C) and DCAF13 immunofluorescence (Fig 2B). These embryos were morphologically normal up to the eight-cell stage; however, they did not compact, failed to develop into blastocysts, and died at the morula stage. Notably, the nucleolar B23 signals were also weakened in Dcaf13−/− embryos (Fig 2B). These results indicate that the Dcaf13 knockout causes early embryonic death. Click here to expand this figure. Figure EV3. Generation of Dcaf13 knockout mouse strains Schematic representation of mouse Dcaf13 genes, TALEN target sequences, and the mutations obtained. Alu I site is highlighted in red. Alu I digestion result of PCR fragment amplified from WT mice, heterozygous mice, and homozygous mice embryos carrying the Dcaf13 8-bp deletion mutation (Dcaf13+/− and Dcaf13−/−). Dcaf13 genotyping results of blastocysts obtained from Dcaf13+/− mice. Different upstream primers (GT-W2 and GT-M2 in Appendix Table S1) and a common downstream primer (GT-R in Appendix Table S1) were designed to specifically amplified WT and mutated alleles of Dcaf13. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. DCAF13 is essential for murine preimplantation embryonic development Genotyping results on Dcaf13+/+, Dcaf13+/−, and Dcaf13−/− mouse embryos and offspring. Note that no Dcaf13−/− embryos or pups were obtained at or after 8.5 dpc. DCAF13 (green) and B23 (red) immunofluorescence in embryos at E2.5, E3.5, and E4.5 collected from Dcaf13+/− female mice mated with adult Dcaf13+/− male mice. DIC images were taken from the same batch of embryos before fixation and immunofluorescence. The embryos were genotyped after the DIC images were taken in order to assign them as either wild type or mutant. n = 4 mice at each time point. Scale bars = 10 μm. DCAF13 immunofluorescence in eight-cell WT embryos, which were microinjected with Dcaf13 siRNAs (siDcaf13) or control siRNAs (siControl). n = 25 embryos for each group. Scale bars = 10 μm. In vivo fertilized eggs were collected from oviducts, microinjected with siControl (n = 91) or siDcaf13 (n = 117), and then cultured for 72 h in vitro. The percentage of embryos that reached two-cell, four-cell, eight-cell, morula, and blastocyst stages at 48, 60, 72, 84, and 96 h post-hCG (human chorionic gonadotropin) injection were calculated and presented as developmental rate. Error bars indicate mean ± SD. Representative images showing the development of preimplantation embryos at E4.5, with or without the Dcaf13 knockdown. Scale bars = 100 μm. Download figure Download PowerPoint Because Dcaf13−/− embryos were obtained in small numbers and could not be identified for detailed analyses before processing for genotyping, we employed an RNA interference (RNAi) approach to assess DCAF13 function in preimplantation development, by injecting Dcaf13-targeting small interfering RNAs (siRNAs) into the cytoplasm of wild-type (WT) zygotes. An immunofluorescence assay revealed that DCAF13 was greatly downregulated in siRNA-injected embryos at the eight-cell stage (Fig 2C). Just as the Dcaf13−/− embryos, most Dcaf13 knockdown embryos failed to undergo compaction and showed developmental arrest at the morula stage (Fig 2D and E). CRL4DCAF13 regulates histone H3 lysine-9 methylation and heterochromatin by targeting SUV39H1 for polyubiquitination and degradation Because murine preimplantation embryos are inconvenient for extensive biochemical analyses, we studied biochemical functions of DCAF13 primarily in HeLa cells and MEFs and then verified the results in murine preimplantation embryos. The DCAF13 knockdown by RNAi inhibited proliferation and colony growth in HeLa cells (Fig 3A). Nonetheless, these cells tested negative for apoptotic marker cleaved caspase 3 or DNA damage marker pH2AX, indicating that DCAF13 depletion did not cause growth retardation by inducing programmed cell death (Fig EV4A–C). DCAF13-depleted cells were first identified by negative DCAF13 staining (Fig 3B). They changed their shape from flat to round and showed dense DNA staining as well as increased histone H3 trimethylation at lysine-9 (H3K9me3, a heterochromatin marker, Fig 3B and C, circled by dotted lines). Being related to the H3K9me3 upregulation, H3K9me2 levels were also slightly increased, but H3K9 acetylation (H3K9ac) was only mildly decreased after the DCAF13 knockdown (Fig EV4D–F). In contrast, other histone modifications, for example, H2A-K119 monoubiquitination, (H2A-K119ub1) were unaffected (Fig EV4G). Interestingly, H3K27me3 was slightly increased after the Dcaf13 knockdown (Fig EV4H and I). Figure 3. DCAF13 regulates H3K9me3 and SUV39H1 levels in HeLa cells and preimplantation embryos A. Images of colony formation and proliferation rates of HeLa cells with or without Dcaf13 knockdown. Proliferation rates were determined by a CCK8 proliferation assay (n = 5). Optical density at 450 nm (OD450) values was converted to cell numbers. B. DCAF13 immunofluorescence (red) in control and Dcaf13 knockdown HeLa cells co-stained with α-tubulin (green) and DAPI (blue). DCAF13-negative cells are circled by dotted lines. Scale bar = 10 μm. C. H3K9me3 immunofluorescence in control and Dcaf13 knockdown HeLa cells. DCAF13 knockdown cells are circled by dotted lines. The white box around the nucleus showing H3K9me3 signal was zoomed out on the right panel. Three independent experiments were conducted. Scale bar = 10 μm. D. Western blot analysis of the indicated proteins in control and Dcaf13 or Ddb1 knockdown cells. Histone H3 was immunoblotted as a loading control. Three independent experiments were performed in HeLa cells. The intensity of SUV39H1 and H3K9me3 relative to histone H3 quantified in ImageJ software was calculated and shown as mean ± SD. **P < 0.01; *P < 0.05, calculated by two-tailed Student's t-test. E, F. SUV39H1 (E) and H3K9me3 (F) immunofluorescence in eight- to sixteen-cell embryos, which were microinjected with control or Dcaf13 siRNA at the zygote stage. The nucleolar protein B23 was co-stained as an immunofluorescence marker. DNA was labeled by DAPI. At least 30 embryos in each group were examined and showed similar results. Scale bars = 10 μm. Source data are available online for this figure. Source Data for Figure 3 [embj201898981-sup-0004-SDataFig3.jpg] Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Effect of Dcaf13 depletion on DNA damage, apoptosis, and histone modifications A, B. Immunofluorescence images of rH2AX (A) and cleaved caspase 3 (B) in HeLa cells at 48 h after control siRNA or siDcaf13 transfection. Nuclei were labeled with DAPI (blue), and cell morphology was demonstrated by α-tubulin (green) immunostaining. Cells treated with 10 mM VP16 for 2 h before fixation were stained in parallel as a positive control. C. Western blotting results showing the levels of rH2AX and cleaved caspase 3 in HeLa cells at 48 h after transfection with control siRNA or siDcaf13. D–F. Immunofluorescence (D, E) and Western blotting (F) results showing levels of dimethylated histone H3 at lysine-9 (H3K9me2) and acetylated histone H3 at lysine-9 (H3K9Ac) in HeLa cells transfected with control and Dcaf13 siRNAs for 48 h. Scale bars = 10 μm. The dotted circles indicate Dcaf13-knockdown cells. G–I. Immunofluorescence (G, H) and Western blotting (I) results showing levels of ubiquitinated histone H2A at lysine-119 (H2A-K119ub) and trimethylated histone H3 at lysine-27 (H3K27me3) in HeLa cells transfected with control and Dcaf13 siRNAs for 48 h. Scale bars = 10 μm. Source data are available online for this figure. Download figure Download PowerPoint In addition to H3K9me3, both DCAF13- and DDB1-depleted cells showed increased levels of heterochromatin protein 1α (HP1α) and H3K9 methyltransferase SUV39H1 (Fig 3D). Going back to the in vivo system, we also found that H3K9me3 and SUV39H1 were remarkably upregulated and deposited clearly at the nuclear periphery and nuclear particles in DCAF13 knockdown eight- to sixteen-cell embryos (Fig 3E and F). Previous mass spectrometry results have shown that DCAF13 interacts with SUV39H1 (Yang et al, 2015). Therefore, we hypothesized that CRL4DCAF13 regulates histone H3K9 trimethylation and chromatin conformation changes by targeting SUV39H1. Co-immunoprecipitation results revealed that DCAF13 and DDB1 interacted with SUV39H1 (Fig 4A) and its homolog SUV39H2 (Appendix Fig S2A and B). Overexpression of DCAF13 or DDB1 significantly increased the polyubiquitination of SUV39H1 (Fig 4B) and SUV39H2 (Appendix Fig S2C). Conversely, when endogenous DCAF13 or DDB1 was depleted by siRNAs, SUV39H1 polyubiquitination was decreased (Fig 4C). We analyzed the degradation rates of SUV39H1 in cells cultured in the presence of cycloheximide (CHX), a protein synthesis inhibitor. The SUV39H1 protein was mostly degraded within 8 h after CHX treatment but was stabilized in DCAF13- or DDB1-depleted cells (Fig 4D and E). In HeLa cells transfected with a mCherry-tagged SUV39H1, immunofluorescence assay results suggested that mCherry-SUV39H1 and DCAF13 can be detected in the entire nucleus, but the signal in nucleolus is much stronger than that in the nucleoplasm (Fig 4F). Furthermore, we separated proteins in nucleoplasm and nucleoli and detected the presence of DCAF13 and SUV39H1. Fibrillarin and lamin A/C were blotted as marker proteins of nucleoli and nucleoplasm, respectively (Fig 4G). Endogenous DCAF13 and SUV39H1 were present in both fractions, but DCAF13 was more abundant in nucleoli than in nucleoplasm (Fig 4G). Endogenous SUV39H1 did not show an enrichment in nucleolus as the overexpressed mCherry-SUV39H1 detected in Fig 4F. This might be caused by the differences in intracellular SUV39H1 abundance. These results provide evidence that SUV39H1 is an accessible substrate of DCAF13. Figure 4. CRL4DCAF13 targets SUV39H1 for polyubiquitination and degradation A, B. Co-immunoprecipitation (Co-IP) experiments showing interactions of SUV39H1 with DDB1-DCAF13 (A) and SUV39H1 polyubiquitination (B). HeLa cells transiently transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with an anti-HA affinity gel. Input cell lysates and precipitates were immunoblotted with antibodies against FLAG, HA, and MYC. C. IP followed by Western blotting showing SUV39H1 polyubiquitination in control HeLa cells and those transfected with Dcaf13 or Ddb1 siRNAs. D, E. Cycloheximide (CHX)-chasing experiments showing SUV39H1 stability. HeLa cells transfected with control, Dcaf13 (D), or Ddb1 siRNAs (E) were treated with 100 μg/ml CHX to inhibit de novo protein synthesis. At the indicated time points after the CHX treatment, the cells were lysed for immunoblotting. The relatively stable proteins ERK1 and ERK2 were immunoblotted as an internal control. F. HeLa cells were transfected with expression plasmids encoding mCherry or mCherry-SUV39H1 and were fixed for immunofluorescent staining with nucleoli marker B23 (left panels) or DCAF13 (right panels) 24 h later. Nuclei were stained with DAPI. Scale bar = 10 μm. G. The nuclei, nucleoli, and nucleoplasm of HeLa cells were isolated and subjected to immunoblot with DCAF13, SUV39H1, fibrillarin, and lamin A/C. The result shows that both DCAF13 and SUV39H1 have nuclear and nucleolar localization. H. Representative embryo images at E4.5. Zygotes were microinjected with mRNAs encoding mCherry (as control), mCherry-SUV39H1, or mCherry-SUV39H1AD (inactive) and cultured for 4 days. Scale bar = 100 μm. I. Developmental rates of embryos microinjected with mRNAs encoding WT or mutated SUV39H1 as in (H). The numbers of eight-cell embryos, morulae, and blastocysts were counted at E2.5, E3, and E4.5, respectively. Error bars represent mean ± SD of three independent experiments. J. Immunofluorescence of H3K9me3 and HP1α in eight-cell embryos that were microinjected with mRNAs encoding mCherry or mCherry-SUV39H1 at the zygote stage. Scale bars = 10 μm. Source data are available online for this figure. Source Data for Figure 4 [embj201898981-sup-0005-SDataFig4.jpg] Download figure Download PowerPoint CRL4DCAF13-mediated SUV39H1 degradation is necessary for preimplantation embryonic development Next, we tested whether impaired SUV39H1 degradation contributed to the early embryonic death caused by Dcaf13 deletion. Microinjection of mRNAs encoding SUV39H1, but not a catalytic-activity-dead SUV39H1 mutant (NHSC residues in the catalytic domain were mutated to NLAA, SUV39H1AD; Rea et al, 2000), into mouse zygotes, significantly decreased their rates of development into blastocysts (Fig 4H and I). Overexpression of SUV39H1 also increased H3K9me3 and HP1α levels in blastomeres at the eight-cell stage (Fig 4J). These phenotypic features were similar to those caused by DCAF13 depletion in earl