UHRF1 phosphorylation by cyclin A2/cyclin-dependent kinase 2 is required for zebrafish embryogenesis

Abstract

Ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) is regulated at the transcriptional level during the cell cycle and in developing zebrafish embryos. We identify phosphorylation as a novel means of regulating UHRF1 and demonstrate that Uhrf1 phosphorylation is required for gastrulation in zebrafish. Human UHRF1 contains a conserved cyclin-dependent kinase 2 (CDK2) phosphorylation site at Ser-661 that is phosphorylated in vitro by CDK2 partnered with cyclin A2 (CCNA2), but not cyclin E. An antibody specific for phospho-Ser-661 recognizes UHRF1 in both mammalian cancer cells and in nontransformed zebrafish cells, but not in zebrafish bearing a mutation in ccna2. Depleting Uhrf1 from zebrafish embryos by morpholino injection causes arrest before gastrulation and early embryonic death. This phenotype is rescued by wild-type UHRF1, but not by UHRF1 in which the phospho-acceptor site is mutated, demonstrating that UHRF1 phosphorylation is essential for embryogenesis. UHRF1 was detected in the nucleus and cytoplasm, whereas nonphosphorylatable UHRF1 is unable to localize to the cytoplasm, suggesting the importance of localization in UHRF1 function. Together, these data point to an essential role for UHRF1 phosphorylation by CDK/CCNA2 during early vertebrate development.

FIGURE 1:

UHRF1 is phosphorylated by CCNA2/CDK2 at Ser-661. (A) Domain structure of UHRF1 and alignment of the conserved CDK2 phosphorylation site in vertebrate UHRF1. Canonical consensus is bolded (SPXY: X = any amino acid; Y = basic amino acid). The putative phosphorylation residue is underlined. (B) Left, Coomassie-stained gel shows equal loading of histone H1. Right, autoradiograph of the phosphorylated histone H1. (C) Top, Schematic representation of UHRF1 truncation mutations used as substrates for CDK2 kinase assays. Black box indicates the CDK2 phosphorylation site (SPRR). Left, Coomassie staining showing equal loading in all lanes. CCNA2/CDK2 phosphorylates a truncated UHRF1 that retains the SPRR motif (lanes 7 and 9) but a truncated mutant lacking SPRR (lane 11) is barely phosphorylated. CCNE1/CDK2 has almost no kinase activity against UHRF1.

FIGURE 2:

α-pS661 detects phospho-UHRF1 in vivo and phosphorylation is eliminated when Ser-661 is changed to glycine. (A) HCT116 cells transfected with empty plasmid (CONTROL), FLAG epitope–tagged UHRF1 (UHRF1), or UHRF1 in which Ser-661 is changed to an alanine (UHRF1^S661A). α-pS661 specifically recognizes a protein of appropriate size in FLAG-UHRF1 cells, but not in FLAG-UHRF1^S661A cells, whereas both FLAG-UHRF1 and FLAG-UHRF1^S661A are recognized by α-UHRF1 and α-FLAG. (B) Endogenous UHRF1 is phosphorylated on Ser-661. HCT116 cell lysates blotted with Preimmune (PI) or α-pS661 sera that was preincubated with either the phosphorylated or a cognate nonphosphorylated form of the immunizing peptide. Actin used as a loading control. (C) Lysates from HCT116 cells were immunoprecipitated with anti-UHRF1 antibody and then treated with or without λ-phosphatase. Immunoblotting (IB) with α-UHRF1 and with α-pS661 shows that phosphatase treatment reduces recognition of UHRF1 by α-pS661 but not by α-UHRF1.

FIGURE 3:

Phosphorylation of UHRF1 requires ccna2. (A) Experimental design. Embryos obtained from a cross of heterozygous ccna2^hi2696 mutants were injected (arrow) with a construct containing UHRF1-EGFP driven by the heat shock (hsp70l) promoter. Mutants (red) were separated from wild-type (tan) siblings based on phenotype at 3 dpf, heat-shocked at 4 dpf, and stained for the presence of phosphorylated UHRF1. (B) Immunofluorescence of 4-dpf wild-type (WT) and ccna2^hi2696 embryos shows that ccna2^hi2696 mutants do not have phosphorylation of UHRF1 despite overexpression of UHRF1, as seen by the GFP, due to mosaic expression from the Tg(hsp70I: UHRF1-EGFP) transgene. Mosaic expression is obtained by this method, so not all nuclei are labeled with GFP. Scale bar: 25 μm.

FIGURE 4:

uhrf1 is essential for zebrafish development. (A) Early embryonic development of uninjected, standard control morpholino-injected, and uhrf1 morpholino-injected embryos. uhrf1 morphants display a distinct developmental arrest phenotype leading to early embryonic death. Scale bar: 500 μm. (B) By 50% epiboly, uhrf1 morphants exhibit decreased survival to 61% compared with 99% of control. Total number of embryos and experiments are noted under each bar. *, p < 0.0001 by Fisher's exact test. (C) Left, uhrf1 morphants at 24 hpf are characterized by a small head, underdeveloped eye (arrow), and abnormal brain, typified by the depressed midbrain–hindbrain boundary (arrowhead) and dilated ventricle (caudal to arrowhead). Scale bar: 100 μm. Right, uhrf1 morphants at 5 dpf have a small liver as visualized in Tg(fabp10:dsred) zebrafish. All morphants have a small liver in three experiments. Scale bar: 500 μm. (D) uhrf1 morphants have a significant abnormal phenotype at 24 hpf. Total number of embryos and experiments are noted under each bar; *, p < 0.0001 by Fisher's exact test.

FIGURE 5:

UHRF1 levels are tightly regulated, and phosphorylation of UHRF1 at Ser-661 is essential for zebrafish embryogenesis. (A) Western blot with α-myc of 6, 12, and 24 hpf embryos injected with 400 pg of mRNA encoding full-length WT-UHRF1 and UHRF1^S661A. Tubulin is the loading control. (B) Both depletion and overexpression of UHRF1 are deleterious to embryonic survival. Only 57% of morphants and 61% of embryos injected with 6X-myc-UHRF1 survive to 50% epiboly. Survival improves to 81% when the morpholino is coinjected with the 6X-myc-UHRF1 mRNA (compare columns 2–4 and columns 3–4; for both, p < 0.0001 by Fisher's exact test). Coinjection of morpholino and mRNA encoding UHRF1^S661A mRNA results in no difference in mortality as compared with morpholino alone; p = 0.9146 by Fisher's exact test. mRNA = 400 pg. The total number of embryos and experiments are noted under each bar.

FIGURE 6:

Localization of UHRF1 changes with its phosphorylation. Cos7 cells transfected with UHRF1-GFP, UHRF1^S661G-GFP, or UHRF1^S661E-GFP show that phosphorylation of UHRF1 promotes cytoplasmic localization in these cells. Top panel, GFP signal in transfected cells. Second row, DAPI signal only. Third row, merge of top two panels. Bottom panel, maximum projection, merge. Magnification: 63×; scale bar: 10 μm.

FIGURE 7:

Phosphorylated endogenous Uhrf1 is cytoplasmic. (A) α-pS661 detects both overexpressed and endogenous phosphorylated UHRF1 in zebrafish. Top, GFP signal in hepatocytes of Tg(fabp10:UHRF1-GFP); middle, immunofluorescence with α-pS661; bottom, merge with DAPI. The gut and liver are labeled. Magnification: 20×; scale bar: 250 μm. (B) Sections of 5-dpf zebrafish expressing GFP-tagged UHRF1 under the control of the hepatocyte specific promoter (Tg(fabp10:UHRF1-EGFP)). Left, GFP expression in hepatocytes; right, immunofluorescence with α-pS661 (top) or preimmune (bottom). Confocal images taken with identical parameters. Scale bar: 50 μm. (C) Left, immunofluorescence of enterocytes with α-pS661 or preimmune. Right, merge with DAPI. Inset, 63× magnification of endogenous phosphorylated UHRF1 in the zebrafish gut with predominant nuclear localization. Confocal images taken with identical parameters. Scale bar: 50 μm. (D) Endogenous UHRF1 is phosphorylated in early development as seen with immunofluorescence on cryosections at 50% epiboly. Bottom, left, immunofluorescence on cryosections of embryos at 50% epiboly with preimmune sera; middle, DAPI alone; right, merge. Top, left, immunofluorescence with α-pS661; middle, DAPI alone; right, merge. Magnification: 63×; scale bar: 25 μm.