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Supplementary Table 1, Figures 1-7, Methods from hPuf-A/KIAA0020 Modulates PARP-1 Cleavage upon Genotoxic Stress

Human hPuf-A/KIAA0020 was first identified as a new minor histocompatibility antigen in 2001. Its zebrafish orthologue contains six Pumilio-homology RNA-binding domains and has been shown to participate in the development of eyes and primordial germ cells, but the cellular function of hPuf-A remains unclear. In this report, we showed that hPuf-A predominantly localized in the nucleoli with minor punctate signals in the nucleoplasm. The nucleolar localization of hPuf-A would redistribute to the nucleoplasm after the treatment of RNA polymerase inhibitors (actinomycin D and 5,6-dichlorobenzimidazole riboside) and topoisomerase inhibitors [camptothecin (CPT) and etoposide]. Interestingly, knockdown of hPuf-A sensitized cells to CPT and UV treatment and cells constitutively overexpressing hPuf-A became more resistant to genotoxic exposure. Affinity gel pull-down coupled with mass spectrometric analysis identified PARP-1 as one of the hPuf-A interacting proteins. hPuf-A specifically interacts with the catalytic domain of PARP-1 and inhibits poly(ADP-ribosyl)ation of PARP-1 in vitro. Depletion of hPuf-A increased the cleaved PARP-1 and overexpression of hPuf-A lessened PARP-1 cleavage when cells were exposed to CPT and UV light. Collectively, hPuf-A may regulate cellular response to genotoxic stress by inhibiting PARP-1 activity and thus preventing PARP-1 degradation by caspase-3. Cancer Res; 71(3); 1126–34. ©2011 AACR.

PHRF1 (PHD and RING finger domain-containing protein 1) suppresses acute promyelocytic leukemia (APL) by promoting TGIF (TG-interacting factor) ubiquitination, while the PML-RARα protein interferes with PHRF1-mediated TGIF breakdown to facilitate APL. Beyond its role in APL tumorigenesis, PHRF1 contributes to non-homologous end-joining by linking H3K36 methylation and Nbs1 upon DNA damage insults. However, little is known regarding its function in tumor invasion. Here we highlight the unreported details of PHRF1 in the invasion of lung cancer cells by modulating the transcriptional level of ZEB1, a prominent regulator involved in epithelial-mesenchymal transition. PHRF1 associated with the phosphorylated C-terminal repeat domain of Rpb1, the large subunit of RNA polymerase II, through its C-terminal Set2 Rpb1 Interacting (SRI) domain. Chromatin immunoprecipitation revealed that PHRF1 bound to the proximal region adjacent to the transcription start site of ZEB1. SRI-deleted PHRF1 neither associated with Rpb1 nor increased ZEB1's expression. Collectively, PHRF1 might take the stage at migration and invasion by modulating the expression of ZEB1.

PHRF1 is involved in transforming growth factor β (TGF-β) signaling to constrain the formation of acute promyelocytic leukemia (APL) in mouse APL models. PHRF1 also participates in modulating non-homologous end-joining. However, the role of PHRF1 in mammalian dendrite architecture and synaptic plasticity is unclear. Here, we investigated the role of PHRF1 in dendritic formation in the murine hippocampus using Camk2a promoter driven-iCre recombinase to conduct a PHRF1 conditional knockout, namely PHRF1Δ/Δ, in the forebrain region. PHRF1Δ/Δ mice developed normally, but exhibited anxiety-like behaviors and displayed defective spatial memory. Alterations of dendritic complexity in apical and basal dendrites of pyramidal neurons were noticed in PHRF1Δ/Δ mutants. Furthermore, electrical stimulation in the hippocampal CA1 region after the TGF-β1 treatment showed a reduced synaptic plasticity in PHRF1Δ/Δ mice. Immunoblotting analysis indicated that PHRF1 ablation affected the TGF-β signaling. Collectively, our results demonstrate that PHRF1 is important for the dendritic architecture and required for spatial memory formation in the hippocampus.

Flavonoids are well-known antioxidants and have shown the ability to prevent tumor formation and recurrence. Especially in dietary flavonoids, they have provided convenience and consistence of intake for long-term prevention of tumor formation. Previous reports suggested that S100 calcium-binding protein A7 (S100A7) might activate epithelial&ndash, mesenchymal transition (EMT) signaling and promote the metastasis of tumor cells, however, the regulatory signaling was unclear. In this study, we found that S100A7 was highly expressed in cancer cells and could be reduced by luteolin (Lu) and quercetin (Qu) through Src/Stat3 signaling. We found that the protein levels of S100A7, phosphorylated Src (p-Src), and p-Stat3 were increased in A431-III cells. Flavonoids Lu and Qu reduce protein levels of p-Src, p-Stat3 and S100A7 in A431-III cells. Treatment of A431-III cells with Src inhibitor SU6656 and Stat3 inhibitor S3I-201 also reduced the protein levels of S100A7. Transactivation activity of 5&prime, upstream regions of S100A7 was activated by Stat3 but was reduced by treatment with Lu, Qu, SU6656 and S3I-201. The treatment also reduced the migratory and invasive abilities of A431-III cells. In a further analysis of EMT markers, the protein level of E-cad increased and that of Twist decreased after treatment with the inhibitors and flavonoids. Overexpression of S100A7 decreased the protein level of E-cad and increased the Twist level, whereas knockdown of S100A7 had the opposite effects. Treatment with S3I-201, Lu and Qu, compared to the control, were found to decrease metastasis of tumor cells in zebrafish larvae. These results suggest that Lu and Qu may inhibit Src/Stat3/S100A7 signaling to reduce tumorigenesis of cancer cells.

Human PUF-A/PUM3 is a RNA and DNA binding protein participating in the nucleolar processing of 7S to 5.8S rRNA. The nucleolar localization of PUF-A redistributes to the nucleoplasm upon the exposure to genotoxic agents in cells. However, little is known regarding the roles of PUF-A in tumor progression. Phosphoprotein database analysis revealed that Y259 phosphorylation of PUF-A is the most prevalent residue modified. Here, we reported the importance of PUF-A’s phosphorylation on Y259 in tumorigenesis. PUF-A gene was knocked out by the Crispr/Cas9 method in human cervix epithelial HeLa cells. Loss of PUF-A in HeLa cells resulted in reduced clonogenic and lower transwell invasion capacity. Introduction of PUF-AY259F to PUF-A deficient HeLa cells was unable to restore colony formation. In addition, the unphosphorylated mutant of PUF-A, PUF-AY259F, attenuated PUF-A protein stability. Our results suggest the important role of Y259 phosphorylation of PUF-A in cell proliferation.

Vilse/Arhgap39 is a Rho GTPase activating protein (RhoGAP) and utilizes its WW domain to regulate Rac/Cdc42-dependent morphogenesis in Drosophila and murine hippocampal neurons. However, the function of Vilse in mammalian dendrite architecture and synaptic plasticity remained unclear. In the present study, we aimed to explore the possible role of Vilse in dendritic structure and synaptic function in the brain. Homozygous knockout of Vilse resulted in premature embryonic lethality in mice. Changes in dendritic complexity and spine density were noticed in hippocampal neurons of Camk2a-Cre mediated forebrain-specific Vilse knockout (VilseΔ/Δ) mice. VilseΔ/Δ mice displayed impaired spatial memory in water maze and Y-maze tests. Electrical stimulation in hippocampal CA1 region revealed that the synaptic transmission and plasticity were defected in VilseΔ/Δ mice. Collectively, our results demonstrate that Vilse is essential for embryonic development and required for spatial memory.

The Tristetraprolin (TTP) protein family includes four mammalian members (TTP, TIS11b, TIS11d, and ZFP36L3), but only one in Drosophila melanogaster (DTIS11). These proteins bind target mRNAs with AU-rich elements (AREs) via two C3H zinc finger domains and destabilize the mRNAs. We found that overexpression of mouse TIS11b or DTIS11 in the Drosophila retina dramatically reduced eye size, similar to the phenotype of eyes absent (eya) mutants. The eya transcript is one of many ARE-containing mRNAs in Drosophila. We showed that TIS11b reduced levels of eya mRNA in vivo. In addition, overexpression of Eya rescued the TIS11b overexpression phenotype. RNA pull-down and luciferase reporter analyses demonstrated that the DTIS11 RNA-binding domain is required for DTIS11 to bind the eya 3' UTR and reduce levels of eya mRNA. Moreover, ectopic expression of DTIS11 in Drosophila S2 cells decreased levels of eya mRNA and reduced cell viability. Consistent with these results, TTP proteins overexpressed in MCF7 human breast cancer cells were associated with eya homologue 2 (EYA2) mRNA, and caused a decrease in EYA2 mRNA stability and cell viability. Our results suggest that eya mRNA is a target of TTP proteins, and that downregulation of EYA by TTP may lead to reduced cell viability in Drosophila and human cells.

Human p29 is a chromatin-associated protein and the silencing of p29 expression increases cell population in G(1) phase and decreases phosphorylation levels of Chk1 and Chk2 in response to UV treatment. To further characterize the function of p29, U2OS and Fanconi anemia complementation group G (FA-G) cells with constitutive p29 expression have been established. Analyses of these cells identified increased phosphorylation levels of Chk1 and Chk2, which were accompanied by elevated amounts of chromatin-associated Mre11-Rad50-Nbs1 complex and ATR-IP. Monoubiquitination of the FA ID complex was restored in p29 stably expressing FA-G cells. Moreover, lower tumor incidence was observed in mp29 transgenic mice after UV irradiation. These results suggest the involvement of p29 in the DNA damage responses and Fanconi anemia pathway.

DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.

Astrin is a microtubule-associated protein and localizes with mitotic spindles in the M-phase. We silenced the expression of astrin protein and tested the cell viability in response to paclitaxel treatment in paclitaxel-sensitive and paclitaxel-resistant cells. We found that the absence of astrin by siRNA resulted in the activation of a p53-dependent apoptosis, which elevated pro-apoptotic Bax expression and increased the activity of caspase-3 in astrin-depleted cells. The HPV18 E6 transcription was found to be inhibited along with the increase expression of p53. Intriguingly, the expression of astrin decreased in paclitaxel-sensitive HeLa cells but remained steady in paclitaxel-resistant cells in response to paclitaxel treatment. Furthermore, we identified that the depletion of astrin caused more cell death both in paclitaxel-sensitive and -resistant cells in combination with paclitaxel treatment. These findings suggest that the silencing of astrin induce a p53-dependent apoptosis and has an additive effect on paclitaxel treatment.

Study objectives To report the efficacy and findings of a large-scale preventive screening program for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) using amplification of the virus from a nasopharyngeal swab (NPS) obtained from the health-care workers (HCWs). Design A prospective observational study. Setting A medical center in Taiwan. Participants Two hundred thirty HCWs. Intervention NPS examination for the presence of SARS-CoV by two nested reverse transcription-polymerase chain reaction (RT-PCR) assays. Measurements and results During the outbreak of severe acute respiratory syndrome (SARS), NPS polymerase chain reaction screening of HCWs for SARS-CoV was performed. SARS-CoV was examined by two nested RT-PCRs and a quantitative RT-PCR. Serum-specific antibodies were assessed by enzyme immunoassay and indirect immunofluorescence. We monitored 230 HCWs, including 217 first-line HCWs and 13 non–first-line HCWs. One hundred ninety first-line HCWs and 13 non–first-line HCWs had negative results in both nested RT-PCR assays. Two first-line HCWs who were positive on both nested RT-PCR assays had SARS. They had 16,900 ± 7,920 copies (mean ± SD) of RNA per milliliter in the NPS and had detectable anti-SARS antibodies. The remaining 25 first-line HCWs were negative for the first nested RT-PCR but positive for the second nested RT-PCR. Their corresponding titers were 338 ± 227 copies of RNA per milliliter; antibodies developed in none of these 25 HCWs. The expression and function of angiotensin-converting enzyme-2 were not different among these HCWs. This study shows that colonization of SARS-CoV occurred in 25 of 217 well-protected first-line HCWs on a SARS-associated service, but they remained seronegative. Conclusion With the second RT-PCR assay more sensitive than the first RT-PCR assay, we are able to show that approximately 11.5% of well-protected HCWs exposed to SARS patients or specimens may have colonization without seroconversion. Only those with significant clinical symptoms or disease would have active immunity. Thus, regular NPS screening for nested RT-PCR assays in conjunction with a daily recording of body temperature in all first-line HCWs may provide an effective way of early detection.

Human p29 has been demonstrated in the yeast two-hybrid method and in vitro GST pull-down assay to associate with GCIP, a cyclin D interacting protein. In this study, we describe the cloning and genomic structure of the mouse homologue, mp29. The overall mouse mp29 amino acid sequence is highly identical (91%) to human p29. Polyclonal antibody against mp29 was raised and the subcellular localization of mp29 was identified to be in the nucleus. Genomic clones containing mp29 gene were isolated and this gene was divided into seven exons spanning 9 kb of genomic DNA. The transcription initiation site of mp29 gene was determined to be 94 bp upstream of the translation initiation codon and the first 140 bp proximal TATA-less promoter region is required to activate minimal transcription of mouse mp29 gene in mammalian cells.

We examined the inhibitory mechanism of byakangelicol, isolated from Angelica dahurica, on interleukin-1beta (IL-1beta)-induced cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human pulmonary epithelial cell line (A549). Byakangelicol (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 expression and PGE2 release. The selective COX-2 inhibitor, NS-398 (0.01-1 microM), and byakangelicol (10-50 microM) both concentration-dependently inhibited the activity of the COX-2 enzyme. Byakangelicol, at a concentration up to 200 microM, did not affect the activity and expression of COX-1 enzyme. IL-1beta-induced p44/42 mitogen-activated protein kinase (MAPK) activation was inhibited by the MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor, PD 98059 (30 microM), while byakangelicol (50 microM) had no effect. Treatment of cells with byakangelicol (50 microM) or pyrrolidine dithiocarbamate (PDTC; 50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol, translocation of p65 NF-kappaB from the cytosol to the nucleus and the NF-kappaB-specific DNA-protein complex formation. Taken together, we have demonstrated that byakangelicol inhibits IL-1beta-induced PGE2 release in A549 cells; this inhibition may be mediated by suppression of COX-2 expression and the activity of COX-2 enzyme. The inhibitory mechanism of byakangelicol on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. Therefore, byakangelicol may have therapeutic potential as an anti-inflammatory drug on airway inflammation.

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.

Seventeen kilobases of genomic DNA containing the promoter and the coding region of the round-spotted pufferfish JAK1 gene was isolated and completely sequenced. This gene consists of 25 exons and 24 introns spanning about 13.5 kb, compared to > 30kb in carp JAK1 gene. Primer extension analysis revealed one transcription initiation site which was 376 bp upstream of the translation initiation site. The sequence of the 2.9 kb region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including HNF-5, GCF, Sp1, CRE, AP2, GATA, GAGA, E2A, p53, and NF-IL6. When this region was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme three times more efficiently than could the common carp JAK1 promoter.

hPuf-A is a member of RNA-binding PUF family that regulates mRNA translation. Redistribution of hPuf-A from the nucleolus to the nucleoplasm upon genotoxic stress modulates the poly(ADP-ribosyl)ation activity of PARP-1. Here, we report a novel function of hPuf-A involved in promoting breast cancer progression. Immunohistochemical studies showed higher expression levels of hPuf-A in stage I, II, III, and IV breast cancer specimens in contrast with those of hPuf-A in ductal carcinoma in situ. The presence of hPuf-A is highly associated with colony formation capacities in breast cancer T47D and MDA-MB-231 cells. Xenograft growth of hPuf-A-silenced and hPuf-A overexpressing MDA-MB-231 cells in nude mice was substantially in concert with colony formation capacities. This promoting effect of hPuf-A in tumorigenesis might be correlated with the regulation of its associated mRNAs, such as RbAp48 and DDX3. Collectively, hPuf-A may have diagnostic values in breast cancer progression.

A 3.7-kb cDNA encodes the carp JAK1 kinase of 1,156 amino acid residues. The overall amino acid sequence identity between carp JAK1 and murine JAK1, JAK2, JAK3, and human TYK2 is 57%, 35.5%, 31.3%, and 42.4%, respectively. In addition, carp JAK1 shows higher sequence homology to mammalian JAK1 in both the kinase-like (JH2) and kinase (JH1) domains (approximately 70% identity). Therefore, carp JAK1 is a homolog of mammalian JAK1. To investigate the possible function of JH2 domain, full-length, and various truncated forms of carp JAK1 were produced in the baculovirus system. Our results demonstrate that c-JH1 and c-JH2 associate with each other and c-JH2 can be tyrosine-phosphorylated by c-JAK1 and by c-JH(1 + 2). The JAK1 gene was also isolated from a carp genomic library and characterized. This gene is divided into 24 exons spanning at least 31 kb of genomic DNA. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. The 2.5-kb DNA region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including NF-IL6, HNF-5, AP1, GHF-5, and E2A. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 16 times more efficiently than the promoterless pCAT-Basic. Deletion analysis defined a positive regulatory region between -1,023 and -528. A smaller region (-181 to +59) without any typical TATA-box sequences, G + C-rich sequences, or other binding sequences for known transcription factors still had promoter activity. Constructs without this region did not have detectable promoter activity. This suggests that this region of DNA may play an important role in the expression of carp JAK1 gene.

Lipopolysaccharide (LPS) treatment causes the marked changes of gene expression in macrophages. Tristetraprolin (TTP), which is an mRNA-destabilizing protein that negatively regulates the expression of pro-inflammatory mediators, is induced by LPS. To delineate the molecular mechanism of LPS-stimulated TTP expression, several inhibitors blocking different signaling pathways were used initially. We observed that inhibitors of the NF-κB signaling pathway could down-regulate the TTP expression during LPS-induction. Consistently, TTP expression was increased upon recombinant TNFα stimulation which activates NF-κB signaling. The 5′ regulatory region of zfp36 gene spanning from −2 k to +50 was isolated, which contained a putative NF-κB–binding site located in −1859 to −1850. Analysis of luciferase reporter activity driven by a serial 5′-deletion of TTP promoter showed that NF-κB inhibitor-mediated suppression of LPS or TNFα induced activity was through the predicted κB-binding sites. When the NF-κB-binding site was mutated, the TTP promoter decreased its response to the ectopic expression of NF-κB. Physical interaction analysis including oligonucleotides competition, gel shift and chromatin immunoprecipitation assays demonstrated that NF-κB activated by LPS or TNFα bound to the TTP promoter specifically. These results suggested that during LPS stimulation, NF-κB signaling were activated to regulate the transcription of TTP mRNA.

Human hPuf-A/KIAA0020 was first identified as a new minor histocompatibility antigen in 2001. Its zebrafish orthologue contains six Pumilio-homology RNA-binding domains and has been shown to participate in the development of eyes and primordial germ cells, but the cellular function of hPuf-A remains unclear. In this report, we showed that hPuf-A predominantly localized in the nucleoli with minor punctate signals in the nucleoplasm. The nucleolar localization of hPuf-A would redistribute to the nucleoplasm after the treatment of RNA polymerase inhibitors (actinomycin D and 5,6-dichlorobenzimidazole riboside) and topoisomerase inhibitors [camptothecin (CPT) and etoposide]. Interestingly, knockdown of hPuf-A sensitized cells to CPT and UV treatment and cells constitutively overexpressing hPuf-A became more resistant to genotoxic exposure. Affinity gel pull-down coupled with mass spectrometric analysis identified PARP-1 as one of the hPuf-A interacting proteins. hPuf-A specifically interacts with the catalytic domain of PARP-1 and inhibits poly(ADP-ribosyl)ation of PARP-1 in vitro. Depletion of hPuf-A increased the cleaved PARP-1 and overexpression of hPuf-A lessened PARP-1 cleavage when cells were exposed to CPT and UV light. Collectively, hPuf-A may regulate cellular response to genotoxic stress by inhibiting PARP-1 activity and thus preventing PARP-1 degradation by caspase-3. Cancer Res; 71(3); 1126–34. ©2011 AACR.

Emerging evidence shows that glycogen synthase kinase 3beta (GSK3beta) is involved in mitotic division and that inhibiting of GSK3beta kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3beta involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3beta interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3beta and is phosphorylated at Thr-111, Thr-937 ((S/T)P motif) and Ser-974/Thr-978 ((S/T)XXX(S/T)-p motif; p is a phosphorylatable residue). Inhibition of GSK3beta impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3beta. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3beta. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3beta-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3beta differentially increases GSK3beta-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3beta interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3beta-Astrin complex may also facilitate further physiological and pathological phosphorylation.

Human Ninein (hNinein) is implicated in centrosomal microtubule nucleation and microtubule anchoring in interphase cells and may act as a scaffold protein, but its direct interaction partners remain unexplored in the centrosome. In this report, we show clearly that a spindle-associated protein, Astrin, interacts and co-localizes with hNinein at the centrosome during the S and G2 phases, and this complex may dissociate in the M phase. We also demonstrate that the truncated forms of hNinein, which could interfere with gamma-tubulin and function as dominant-negative mutants, are able to affect Astrin localization to the centrosome. Moreover, siRNA-mediated knockdown of hNinein in HeLa cells causes Astrin to fail to target to the centrosome, whereas hNinein can localize at the centrosome in the absence of Astrin. In addition, reduction in hNinein protein levels causes mislocalization of Astrin with the spindle apparatus and results in the formation of an aberrant mitotic spindle. Collectively, these data suggest that hNinein is required for targeting Astrin to the centrosome during the S and G2 phases. We therefore propose a model wherein hNinein regulates the dynamic movement of Astrin throughout the cell cycle and this interaction, in turn, is required for maintenance of centrosome/spindle pole integrity.

Methylated histone readers are critical for chromatin dynamics, transcription, and DNA repair. Human PHRF1 contains a plant homeodomain (PHD) that recognizes methylated histones and a RING domain, which ubiquitinates substrates. A recent study reveals that PHRF1 is a tumor suppressor that promotes TGF-β cytostatic signaling through TGIF ubiquitination. Also, PHRF1 is a putative phosphorylation substrate of ataxia telangiectasia-mutated/ataxia telangiectasia and Rad3-related kinases; however, the role of PHRF1 in DNA damage response is unclear. Here we report a novel function of PHRF1 in modulating non-homologous end-joining (NHEJ). PHRF1 quickly localizes to DNA damage lesions upon genotoxic insults. Ablation of PHRF1 decreases the efficiency of plasmid-based end-joining, whereas PHRF1 overexpression leads to an elevated NHEJ in H1299 reporter cells. Immunoprecipitation and peptide pull-down assays verify that PHRF1 constitutively binds to di- and trimethylated histone H3 lysine 36 (H3K36) (H3K36me2 and H3K36me3) via its PHD domain. Substitution of S915DT917E to ADAE in PHRF1 decreases its affinity for NBS1. Both PHD domain and SDTE motif are required for its NHEJ-promoting activity. Furthermore, PHRF1 mediates PARP1 polyubiquitination for proteasomal degradation. These results suggest that PHRF1 may combine with H3K36 methylation and NBS1 to promote NHEJ and stabilize genomic integrity upon DNA damage insults.

Human p29 is a newly identified nuclear protein whose function is largely undetermined. We found that p29 associated with chromatin, interacted with MCM3, and localized with proliferating cell nuclear antigen foci in the S phase. Silencing of p29 using small interfering RNA duplexes reduced DNA synthesis and increased the expression of p107, a member of the RB family, and of cyclin-dependent kinase inhibitor p21, accompanied with a decreased expression of DNA polymerase α. Lethal events consisting of premature chromatin condensation with a reduced Chk1 phosphorylation were observed in p29-depleted cells in response to UV irradiation. Intriguingly, the phosphorylation of ataxia telangectasia-mutated kinases at S1981 was suppressed in p29-depleted HeLa cells with UV irradiation, but not in hydroxyurea- and ionizing radiation-treated cells. Taken together, these results reveal a novel function of p29 in the regulation of DNA replication checkpoint responses. (Cancer Res 2006; 66(17): 8484-91)

To understand the roles of heptad repeat 1(HR1) and HR2 of the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) in virus-cell interactions, the conserved Leu or Ile residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an α-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-weight S L form, while the wild type and all other mutants produced similar levels of both the S L form and the slower-migrating, higher-molecular-weight S H form. The wild-type S L form was processed to the S H form, whereas the S L form of the 913P mutant was inefficiently converted to the S H form after biosynthesis. None of these mutations affected cell surface expression or binding to its cognate ACE2 receptor. In a human immunodeficiency virus type 1/SARS S coexpression study, all mutants except the 913P mutant incorporated the S H form into the virions as effectively as did the wild-type S H form. The mutation at Ile-1151 did not affect membrane fusion or viral entry. The impaired viral entry of the 927P, 941P, 955P, and 1165P mutants was due to their inability to mediate membrane fusion, whereas the defect in viral entry of the 1179P mutant occurred not at the stage of membrane fusion but rather at a postfusion stage. Our study demonstrates the functional importance of HR1 and HR2 of the SARS-CoV spike protein in membrane fusion and viral entry.

From the methanolic extract of the leaves of Ficus ruficaulis Merr. var. antaoensis, 5-O-beta-D-glucopyranosyl-6-hydroxyangelicin (1), 6-O-beta-D-glucopyranosyl-5-hydroxyangelicin (2), 5,6-O-beta-D-diglucopyranosylangelicin (3), 8-O-beta-D-glucopyranosyl-5-hydroxypsoralen (4), 5-O-beta-D-glucopyranosyl-8-hydroxypsoralen (5), 3,4,5-trihydroxydehydro-alpha-ionol-9-O-beta-D-glucopyranoside (6), rutin (7), and isoquercitrin (8) were isolated. The structures of compounds 1-4 were elucidated by the analysis of their spectroscopic data. Their in vitro antiproliferation activities were also evaluated.

Early detection and identification of SARS-CoV-infected patients and actions to prevent transmission are absolutely critical to prevent another SARS outbreak. Antibodies that specifically recognize the SARS-CoV spike and nucleocapsid proteins may provide a rapid screening method to allow accurate identification and isolation of patients with the virus early in their infection. For this reason, we raised peptide-induced polyclonal antibodies against SARS-CoV spike protein and polyclonal antibodies against SARS-CoV nucleocapsid protein using 6x His nucleocapsid recombinant protein. Western blot analysis and immunofluorescent staining showed that these antibodies specifically recognized SARS-CoV.

Human astrin is a newly identified microtubule-associated protein, which is highly expressed in the testis. Silencing of astrin has resulted in growth arrest and apoptotic cell death. In this study, we describe the cloning and genomic structure of mastrin, the mouse counterpart to astrin. The overall mouse mastrin amino-acid sequence is 66% identical to human astrin. Mastrin protein was demonstrated to localize to mitotic spindles during mitosis. Genomic clones containing mastrin gene were isolated; the gene was found to have 24 exons spanning 24kb of genomic DNA. Deletion analysis of 5(')-flanking sequences demonstrated that the first 120bp proximal to the TATA-less promoter region is necessary for minimal transcription of the mouse mastrin gene.

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS84AP, corresponding to S136 in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish α-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.

We analyzed genomic aberrations in 20 cervical adenocarcinomas by comparative genomic hybridization (CGH). Most tissue samples (85%) showed DNA copy number changes; gains were more common than losses. The most consistent region of chromosomal gain was mapped to chromosome arm 3q, found in 70% of the cases, with a minimal common region of 3q28-ter. Other recurrent amplifications of genetic material were detected on 17q (45%), 1p (30%), 1q (25%), and 11q (20%). High-level copy number increases were found in chromosomal regions 3q27-ter and 9pter-13. DNA losses were seldom observed, occurring primarily in underrepresented regions of chromosome arms 4q, 13q, and 18q. The presence of high-risk human papilloma virus genomes in the cervical adenocarcinoma samples was detected in 90% of the cases. However, there was no correlation between human papilloma virus type and the pattern of genomic changes. This study is the first report of CGH analysis in human cervical adenocarcinoma. Among the major genomic alterations, our results demonstrate the importance of DNA copy increases of chromosome arm 3q in the development of cervical adenocarcinoma and identify other amplified chromosomal regions that are also associated with cervical carcinogenesis.

One novel gene product, hMAP126, was demonstrated to interact with p29 in the yeast two-hybrid assay. The full-length cDNA of hMAP126 has been obtained and encodes a protein of 1120 amino acids. Multiple tissue Northern blot analysis showed that hMAP126 was abundantly expressed in the testis. Polyclonal antiserum against hMAP126 was raised and affinity-purification of anti-hMAP126 antibodies was performed. The subcellular distribution of hMAP126 was localized to the mitotic spindle. Furthermore, hMAP126 was identified to be post-translationally modified and phosphorylated by p34 cdc2 kinase in vitro. Taken together, we have isolated a novel protein, hMAP126, which may be involved in the functional and dynamic regulation of mitotic spindles.

The rad17 gene of Schizosaccharomyces pombe plays an important role as a checkpoint protein following DNA damage and during DNA replication. The human homologue of S. pombe rad17, Hrad17, was recently identified, but its function has not yet been established. Using the yeast two-hybrid system, we determined that HRad17 can interact with a nucleolar protein, NHP2L1. This interaction was also demonstrated biochemically, in human cells. Immunofluorescence studies revealed that HRad17 and NHP2L1 colocalize to the nucleolus, and immunogold labeling further resolved the location of NHP2L1 to the dense fibrillar component of the nucleolus. Interestingly, the localization of HRad17 in the nucleolus was altered in response to UV irradiation. These results provide some insight into the DNA damage and replication checkpoint mechanisms of HRad17.

Human p29 is a putative component of spliceosomes, but its role in pre-mRNA is elusive. By siRNA knockdown and stable overexpression, we demonstrated that human p29 is involved in DNA damage response and Fanconi anemia pathway in cultured cells. In this study, we generated p29 knockout mice (mp29(GT/GT)) using the mp29 gene trap embryonic stem cells to study the role of mp29 in DNA damage response in vivo. Interruption of mp29 at both alleles resulted in embryonic lethality. Embryonic abnormality occurred as early as E6.5 in mp29(GT/GT) mice accompanied with decreased mRNA levels of α-tubulin and Chk1. The reduction of α-tubulin and Chk1 mRNAs is likely due to an impaired post-transcriptional event. An aberrant G2/M checkpoint was found in mp29 gene trap embryos when exposed to aphidicolin and UV light. This embryonic lethality was rescued by crossing with mp29 transgenic mice. Additionally, the knockdown of zfp29 in zebrafish resulted in embryonic death at 72 hours of development postfertilization (hpf). A lower level of acetylated α-tubulin was also observed in zfp29 morphants. Together, these results illustrate an indispensable role of mp29 in DNA checkpoint response during embryonic development.