Cell cycle-regulated phosphorylation of p220 (NPAT) by cyclin E/Cdk2 in Cajal bodies promotes histone gene transcription

Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Genes & Development (Impact Factor: 10.8). 10/2000; 14(18):2298-313.
Source: PubMed


Cyclin E/Cdk2 acts at the G1/S-phase transition to promote the E2F transcriptional program and the initiation of DNA synthesis. To explore further how cyclin E/Cdk2 controls S-phase events, we examined the subcellular localization of the cyclin E/Cdk2 interacting protein p220(NPAT) and its regulation by phosphorylation. p220 is localized to discrete nuclear foci. Diploid fibroblasts in Go and G1 contain two p220 foci, whereas S- and G2-phase cells contain primarily four p220 foci. Cells in metaphase and telophase have no detectable focus. p220 foci contain cyclin E and are coincident with Cajal bodies (CBs), subnuclear organelles that associate with histone gene clusters on chromosomes 1 and 6. Interestingly, p220 foci associate with chromosome 6 throughout the cell cycle and with chromosome 1 during S phase. Five cyclin E/Cdk2 phosphorylation sites in p220 were identified. Phospho-specific antibodies against two of these sites react with p220 within CBs in a cell cycle-specific manner. The timing of p220 phosphorylation correlates with the appearance of cyclin E in CBs at the G1/S boundary, and this phosphorylation is maintained until prophase. Expression of p220 activates transcription of the histone H2B promoter. Importantly, mutation of Cdk2 phosphorylation sites to alanine abrogates the ability of p220 to activate the histone H2B promoter. Collectively, these results strongly suggest that p220(NPAT) links cyclical cyclin E/Cdk2 kinase activity to replication-dependent histone gene transcription.

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Available from: Tianlin Ma, Apr 28, 2014
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    • "Note that our the Drosophila histone gene locus that contained U7 snRNP (Liu et al., 2006), a factor essential for generating the unique histone mRNA 3′ end (Strub and Birnstiel, 1986; Mowry and Steitz, 1987). Similar factors necessary for histone transcription and pre-mRNA processing are found in both vertebrate and Drosophila HLBs, including human nuclear protein mapped to the mutated ataxia telangiectasia locus (NPAT), which was identified as a cyclin E/Cdk2 substrate essential for histone mRNA expression (Ma et al., 2000; Zhao et al., 2000; Wei et al., 2003; Miele et al., 2005). The multi sex combs (mxc) locus encodes the Drosophila orthologue of NPAT. "
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    ABSTRACT: Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi-Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel SIF (Self Interaction Facilitator) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or SIF domains severely impairs Mxc function in vivo resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Lastly, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis. © 2015 by The American Society for Cell Biology.
    Molecular Biology of the Cell 02/2015; 26(8). DOI:10.1091/mbc.E14-10-1445 · 4.47 Impact Factor
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    • "FLASH also plays a significant role in the transcriptional regulation of histone genes. The FLASH binding partner, NPAT (p220), is an activator of histone gene transcription [9], [13] under the control of cyclin E/Cdk2 kinase. Moreover, CHIP assays demonstrated that FLASH interacts with histone gene promoter sequences [8]. "
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    PLoS ONE 03/2012; 7(3):e32971. DOI:10.1371/journal.pone.0032971 · 3.23 Impact Factor
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    • "In human cells, cyclin E–CDK2 colocalizes with the NPAT proteins to histone gene clusters in subnuclear Cajal bodies (Ma et al. 2000; Zhao et al. 2000). Notably, phosphorylation of NPAT by cyclin E–CDK2 activates histone gene transcription at the G1/S transition (Zhao et al. 1998, 2000; Ma et al. 2000; Ye et al. 2003). However, the underlying molecular mechanisms remain to be identified. "
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    ABSTRACT: The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach-release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters.
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