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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.

SUBMITTER: Chu J

PROVIDER: S-EPMC3248904 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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

Chu Jaime J Loughlin Elizabeth A EA Gaur Naseem A NA SenBanerjee Sucharita S Jacob Vinitha V Monson Christopher C Kent Brandon B Oranu Amanke A Ding Yuanying Y Ukomadu Chinweike C Sadler Kirsten C KC

Molecular biology of the cell 20111109 1

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 cyc ...[more]

PMID: 22072796

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Project description:NF-kappaB plays a central role in cytokine-inducible inflammatory gene expression. Previously we empirically determined the identity of 92 members of the genetic network under direct NF-kappaB/RelA control that show marked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigate this network further, we have applied a recently developed two-step chromatin immunoprecipitation assay that accurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducible RelA binding occurs with similar kinetics on all NF-kappaB-dependent genes, serine 276 (Ser(276))-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-beta, interleukin-8 (IL-8), and IkappaBalpha. Previous work has shown that TNF-inducible RelA Ser(276) phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. To further understand the role of phospho-Ser(276) RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser(276) formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser(276) RelA is required only for activation of IL-8 and Gro-beta, with IkappaBalpha being unaffected. These data were confirmed in experiments using RelA(-/-) murine embryonic fibroblasts reconstituted with a RelA Ser(276)Ala mutation. In addition, we observe that phospho-Ser(276) RelA binds the positive transcription elongation factor b (P-TEFb), a complex containing the cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short interfering RNA (siRNA)-mediated knockdown shows that the phospho-Ser(276) RelA-P-TEFb complex is required for IL-8 and Gro-beta gene activation but not for IkappaBalpha gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser(276) RelA formation and chromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domain phosphorylation on the IL-8 and Gro-beta genes. The second involves a phospho-Ser(276) RelA-independent activation of genes preloaded with Pol II, exemplified by the IkappaBalpha gene. Together, these data suggest that the binding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlled by a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.

Dataset Information

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

Publications

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

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