The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.     

The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth

E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.     

Fukutin-related protein associates with the sarcolemmal dystrophin-glycoprotein complex

Mutations in fukutin-related protein (FKRP) give rise to mild and more severe forms of muscular dystrophy. FKRP patients have reduced glycosylation of the extracellular protein dystroglycan, and FKRP itself shows sequence similarity to glycosyltransferases, implicating FKRP in the processing of dystroglycan. However, FKRP localization is controversial, and no FKRP complexes are known, so any FKRP-dystroglycan link remains elusive. Here, we demonstrate a novel FKRP localization in vivo; in mouse, both endogenous and recombinant FKRP are present at the sarcolemma. Biochemical analyses revealed that mouse muscle FKRP and dystroglycan co-enrich and co-fractionate, indicating that FKRP coexists with dystroglycan in the native dystrophin-glycoprotein complex. Furthermore, FKRP sedimentation shifts with dystroglycan in disease models involving the dystrophin-glycoprotein complex, and sarcolemmal FKRP immunofluorescence mirrors that of dystroglycan in muscular dystrophy mice, suggesting that FKRP localization may be mediated by dystroglycan. These data offer the first evidence of an FKRP complex in muscle and suggest that FKRP may influence the glycosylation status of dystroglycan from within the sarcolemmal dystrophin-glycoprotein complex.     

The retinoblastoma protein physically associates with the human cdc2 kinase.

The protein product (pRB) of the retinoblastoma susceptibility gene functions as a negative regulator of cell proliferation, and its activity appears to be modulated by phosphorylation. Using a new panel of anti-human pRB monoclonal antibodies, we have investigated the biochemical properties of this protein. These antibodies have allowed us to detect a pRB-associated kinase that has been identified as the cell cycle-regulating kinase p34cdc2 or a closely related enzyme. Since this associated kinase phosphorylates pRB at most of the sites used in vivo, these results suggest that this kinase is one of the major regulators of pRB. The associated kinase activity follows the pattern of phosphorylation seen for pRB in vivo. The associated kinase activity is not seen in the G1 phase but appears in the S phase, and the levels continue to increase throughout the remainder of the cell cycle.     

Proliferating cell nuclear antigen associates with histone deacetylase activity, integrating DNA replication and chromatin modification

Faithful inheritance of the chromatin structure is essential for maintaining the gene expression integrity of a cell. Histone modification by acetylation and deacetylation is a critical control of chromatin structure. In this study, we test the hypothesis that histone deacetylase 1 (HDAC1) is physically associated with a basic component of the DNA replication machinery as a mechanism of coordinating histone deacetylation and DNA synthesis. Proliferating cell nuclear antigen (PCNA) is a sliding clamp that serves as a loading platform for many proteins involved in DNA replication and DNA repair. We show that PCNA interacts with HDAC1 in human cells and in vitro and that a considerable fraction of PCNA and HDAC1 colocalize in the cell nucleus. PCNA associates with histone deacetylase activity that is completely abolished in the presence of the HDAC inhibitor trichostatin A. Trichostatin A treatment arrests cells at the G(2)-M phase of the cell cycle, which is consistent with the hypothesis that the proper formation of the chromatin after DNA replication may be important in signaling the progression through the cell cycle. Our results strengthen the role of PCNA as a factor coordinating DNA replication and epigenetic inheritance.     

Structure of the yeast Hst2 protein deacetylase in ternary complex with 2'-O-acetyl ADP ribose and histone peptide

Sir2 proteins are NAD(+)-dependant protein deactylases that have been implicated in playing roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. To define the mechanism of Sir2 activity, we report the 1.5 A crystal structure of the yeast Hst2 (yHst2) Sir2 protein in ternary complex with 2'-O-acetyl ADP ribose and an acetylated histone H4 peptide. The structure captures both ligands meeting within an enclosed tunnel between the small and large domains of the catalytic protein core and permits the assignment of a detailed catalytic mechanism for the Sir2 proteins that is consistent with solution and enzymatic studies. Comparison of the ternary complex with the yHst2/NAD(+) complex, also reported here, and nascent yHst2 structure also reveals that NAD(+) binding accompanies intramolecular loop rearrangement for more stable NAD(+) and acetyl-lysine binding, and that acetyl-lysine peptide binding induces a trimer-monomer protein transition involving nonconserved Sir2 residues.     

Hairless contains a novel nuclear matrix targeting signal and associates with histone deacetylase 3 in nuclear speckles

Hair follicle cycling is a highly regulated and dynamic cellular process consisting of phases of growth, regression, and quiescence. The hairless (hr) gene encodes a nuclear factor that is highly expressed in the skin, where it appears to be an essential regulator during the regression in the catagen hair follicle. In hairless mice, as well as humans with congenital atrichia, the absence of hr protein initiates a premature and abnormal catagen due to defects in the signaling required for hair follicle remodeling. Here, we report that hr protein is a nuclear protein that is tightly associated with the nuclear matrix scaffold. Using a series of deletion constructs of the mouse hr gene, we monitored the sub-cellular localization of the recombinant protein by in situ immunolocalization and biochemical fractionation after nuclear matrix extraction of transiently transfected cells. We identified a novel nuclear matrix-targeting signal (NMTS) in the hr protein and mapped the domain to amino acid residues 111-186 of the mouse hr sequence. Furthermore, we provide evidence that this region not only mediates the interaction of hr with components of the nuclear architecture, but also specifies the sub-nuclear location of the hr protein to nuclear domains containing deacetylase activity. The N-terminal region directs hr to a speckled nuclear pattern that co-localizes with the histone deacetylase 3 (HDAC), but not with HDAC1 or HDAC7. Based on our findings, we propose that hr protein is part of a specific multi-protein repressor complex and that hr may be involved in chromatin remodeling.