Edward M Johnson

Publications of Edward M Johnson

• Addressing the Enigma of MCM8 in DNA Replication

  Authors: Dianne C. Daniel, Edward M. Johnson

  09/2011;

  ISBN: 978-953-307-259-3

• Regulation of PURA gene transcription by three promoters generating distinctly spliced 5-prime leaders: a novel means of fine control over tissue specificity and viral signals.

  Authors: Margaret J Wortman, Laura K Hanson, Luis Martínez-Sobrido, Ann E Campbell, Jonas A Nance, Adolfo García-Sastre, Edward M Johnson

  BMC molecular biology. 11/2010; 11:81.

  Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformationPurα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals. Here we report that human PURA (hPURA) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated hPURA 5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo. By co-transfecting hPURA reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate PURA transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of mPURA mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts. Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of PURA promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts.

• Role of Purα in the cellular response to ultraviolet-C radiation.

  Authors: Rafal Kaminski, Laurelle Cheeseboro, Shohreh Amini, Edward M Johnson, Martyn K White, Kamel Khalili, Armine Darbinyan

  Cell cycle (Georgetown, Tex.). 10/2010; 9(20):4164-73.

  Purα is a nucleic acid-binding protein with DNA-unwinding activity, which has recently been shown to have a role in the cellular response to DNA damage. We have investigated the function of Purα inPurα is a nucleic acid-binding protein with DNA-unwinding activity, which has recently been shown to have a role in the cellular response to DNA damage. We have investigated the function of Purα in Ultraviolet-C (UVC) radiation-induced DNA damage and nucleotide excision repair (NER). Mouse embryo fibroblasts from PURA(-/-) knockout mice, which lack Purα, showed enhanced sensitivity to UVC irradiation as assessed by assays for cell viability and clonogenicity compared to Purα positive control cultures. In reporter plasmid reactivation assays to measure the removal of DNA adducts induced in vitro by UVC, the Purα-negative cells were less efficient in DNA damage repair. Purα-negative cells were also more sensitive to UVC-induced DNA damage measured by Comet assay and showed a decreased ability to remove UVC-induced cyclobutane pyrimidine dimers. In wild-type mouse fibroblasts, expression of Purα is induced following S-phase checkpoint activation by UVC in a similar manner to the NER factor TFIIH. Moreover, co-immunoprecipitation experiments showed that Purα physically associates with TFIIH. Thus, Purα has a role in NER and the repair of UVC-induced DNA damage.

• Induction of bicalutamide sensitivity in prostate cancer cells by an epigenetic Puralpha-mediated decrease in androgen receptor levels.

  Authors: Xiaomei Liu, Alejandro Gomez-Pinillos, Xiaojun Liu, Edward M Johnson, Anna C Ferrari

  The Prostate. 09/2009;

  BACKGROUND: Increased androgen receptor (AR) levels support resistance to apoptosis and hormone therapy in advanced prostate cancer (PC). We recently linked the overexpression of AR inBACKGROUND: Increased androgen receptor (AR) levels support resistance to apoptosis and hormone therapy in advanced prostate cancer (PC). We recently linked the overexpression of AR in androgen-independent LNCaP cells (AI-cells) and tissues from castration-resistant patients to decreased nuclear levels of Pur-alpha (Puralpha) and loss from a protein complex bound to repressor sequences (ARS) in the 5'-UTR of AR. Strategies to regain control of increased AR transcription may overcome resistance of AI-cells and improve treatment outcomes. METHODS: MTT, real-time PCR, Western blot, ChIP, flow cytometry, and caspase 3/7 activation measured the effect on growth and targets of LBH589/bicalutamide treatment of AI-cells and androgen-dependent LNCaP cells (AD). RESULTS: Within 16 hr of treatment of AI-cells with low concentrations of the histone deacetylase inhibitor LBH589, a shift of cytoplasmic Puralpha restored the nuclear levels and the binding of Puralpha to the ARS. This was followed by a decline in AR-mRNA and protein reaching levels of parental AD-cells. The fraction of AI-cells in G1 increased and the cells in S phase decreased similar to AD-cells, and there was a modest caspase activation. Most notably, treatment of bicalutamide-resistant AI-cells with 10 nM LBH589 combined with 12.5 microM bicalutamide synergistically inhibited cell growth and induced a fivefold higher level of caspase 3/7 activation than observed in AD-cells. CONCLUSIONS: Low-dose LBH589 restores Puralpha binding to ARS and down-regulates AR transcription. Biologically, LBH589 reverses the resistance of AI-cells to bicalutamide and to apoptosis. The combination may restore the hormonal response of castration-resistant PC patients. Prostate (c) 2009 Wiley-Liss, Inc.

• SMAD Proteins of Oligodendroglial Cells Regulate Transcription of JC Viral Early and Late Genes Coordinately with the Tat Protein of Human Immunodeficiency Virus Type I.

  Authors: Michelle R. Stettner, Jonas A Nance, Clayton A. Wright, Yayoi Kinoshita, Woong-Ki Kim, Susan Morgello, Jay Rappaport, Kamel Khalili, Jennifer Gordon, Edward M Johnson

  The Journal of general virology. 05/2009;

  SummaryJC virus (JCV) is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the brain in AIDS. Although immunosuppression is involved inSummaryJC virus (JCV) is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the brain in AIDS. Although immunosuppression is involved in infection of the brain by JCV, a direct influence of human immunodeficiency virus type I (HIV-1) has also been established. The Tat protein of HIV-1 has been implicated in activation of the cytokine, TGF-beta, in HIV-1-infected cells and in stimulating JCV gene transcription and DNA replication in oligodendroglia, the primary CNS cell type infected by JCV in PML. Here we demonstrate that Tat can cooperate with SMAD proteins, the intracellular effectors of TGF-beta, at the JCV DNA control region (CR) to stimulate JCV gene transcription. Tat stimulates JCV early gene transcription in KG-1 oligodendroglial cells when either expressed via transfection or added exogenously. Using chromatin immunoprecipitation we show that exogenous Tat enhances binding of SMAD proteins 2, 3 and 4 and their binding partner Fast1 to the JCV CR in living cells. When SMADs 2, 3 and 4 are expressed together, Tat, expressed from plasmid pTat, stimulates transcription from both early and late gene promoters, the early promoter exhibiting stimulation of >100-fold. Tat, SMAD4 and polyoma JCV large T-antigen are all visualized in oligodendroglial cells at the border of an active PML lesion in the cerebral frontal lobe. These results reveal a positive reinforcement system in which the SMAD mediators of the TGF-beta system act cooperatively with Tat to stimulate JCV gene transcription.

• Androgen receptor overexpression in prostate cancer linked to Pur alpha loss from a novel repressor complex.

  Authors: Longgui G Wang, Edward M Johnson, Yayoi Kinoshita, James S Babb, Michael T Buckley, Leonard F Liebes, Jonathan Melamed, Xiao-Mei Liu, Ralf Kurek, Liliana Ossowski, Anna C Ferrari

  Cancer research. 05/2008; 68(8):2678-88.

  Increased androgen receptor (AR) expression and activity are pivotal for androgen-independent (AI) prostate cancer (PC) progression and resistance to androgen-deprivation therapy. We show that aIncreased androgen receptor (AR) expression and activity are pivotal for androgen-independent (AI) prostate cancer (PC) progression and resistance to androgen-deprivation therapy. We show that a novel transcriptional repressor complex that binds a specific sequence (repressor element) in the AR gene 5'-untranslated region contains Pur alpha and hnRNP-K. Pur alpha expression, its nuclear localization, and its AR promoter association, as determined by chromatin immunoprecipitation analysis, were found to be significantly diminished in AI-LNCaP cells and in hormone-refractory human PCs. Transfection of AI cells with a plasmid that restored Pur alpha expression reduced AR at the transcription and protein levels. Pur alpha knockdown in androgen-dependent cells yielded higher AR and reduced p21, a gene previously shown to be under negative control of AR. These changes were linked to increased proliferation in androgen-depleted conditions. Treatment of AI cells with histone deacetylase and DNA methylation inhibitors restored Pur alpha protein and binding to the AR repressor element. This correlated with decreased AR mRNA and protein levels and inhibition of cell growth. Pur alpha is therefore a key repressor of AR transcription and its loss from the transcriptional repressor complex is a determinant of AR overexpression and AI progression of PC. The success in restoring Pur alpha and the repressor complex function by pharmacologic intervention opens a promising new therapeutic approach for advanced PC.

• Colocalization of MCM8 and MCM7 with proteins involved in distinct aspects of DNA replication.

  Authors: Yayoi Kinoshita, Edward M Johnson, Ronald E Gordon, Heather Negri-Bell, Mark T Evans, Jennifer Coolbaugh, Yaseris Rosario-Peralta, Joseph Samet, Evelyn Slusser, Mark P Birkenbach, Dianne C Daniel

  Microscopy research and technique. 05/2008; 71(4):288-97.

  Minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. A subcomplex of the MCM2-7 family members, initially characterized in yeast, is thought to serve as aMinichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. A subcomplex of the MCM2-7 family members, initially characterized in yeast, is thought to serve as a eukaryotic DNA replicative helicase. MCM8 is a new family member, not present in yeast, which may function alone or with other family members in aspects of DNA metabolism, including replication initiation and elongation. Through the use of chromatin immunoprecipitation, we find that MCM8, like MCM7, colocalizes on a specific DNA segment of the c-MYC replication initiation zone (c-MYC replicator) with Cdc6, a protein potentially involved in loading MCM proteins onto DNA. The association between MCM8 and MCM7 peaks in mid G1, at the time of assembly of the prereplication complex. The association of both MCM proteins with Cdc6, however, continues even after DNA replication is complete. We also find that MCM8 colocalizes at the c-MYC replicator with chromatin-bound Cdk2. Our data indicate that any role MCM8 may play in elongation is likely to be discontinuous, in its association with DNA, from a potential role in initiation. Using immunogold electron microscopy we show that MCM8 and MCM7 differ in spatial relation to RPA70 during S phase. Our data strongly suggest that MCM8 functions with other known replication proteins in processes which accompany DNA replication, especially initiation, and which are specifically adapted to suit higher eukaryotes.

• Puralpha as a cellular co-factor of Rev/RRE-mediated expression of HIV-1 intron-containing mRNA.

  Authors: Rafal Kaminski, Nune Darbinian, Bassel E Sawaya, Dorota Slonina, Shohreh Amini, Edward M Johnson, Jay Rappaport, Kamel Khalili, Armine Darbinyan

  Journal of cellular biochemistry. 04/2008; 103(4):1231-45.

  To ensure successful replication, HIV-1 has developed a Rev-mediated RNA transport system that promotes the export of unspliced genomic RNA from nuclei to cytoplasm. This process requires the RevTo ensure successful replication, HIV-1 has developed a Rev-mediated RNA transport system that promotes the export of unspliced genomic RNA from nuclei to cytoplasm. This process requires the Rev responsive element (RRE) that is positioned in the viral transcript encoding Env protein, as well as in unspliced and singly spliced viral transcripts. We identified Puralpha, a single-stranded nucleic acid binding protein as a cellular partner for Rev that augments the appearance of unspliced viral RNAs in the cytoplasm. A decrease in the level of Puralpha expression by siRNA diminishes the level of Rev-dependent expression of viral RNA. Through its nucleic acid binding domain, Puralpha exhibits the ability to interact with the multimerization and RBD domains of Rev. Similar to Rev, Puralpha associates with RRE and in the presence of Rev forms a complex with slower electrophoretic mobility than those from Rev:RRE and Puralpha:RRE. The interaction of Puralpha with RRE occurs in the cytoplasm where enhanced association of Rev with RRE is observed. Our data indicate that the partnership of Puralpha with Rev is beneficial for Rev-mediated expression of the HIV-1 genome.

• Evidence for the involvement of Puralpha in response to DNA replication stress.

  Authors: Huichen Wang, Meijuan Wang, Krzysztof Reiss, Nune Darbinian-Sarkissian, Edward M Johnson, George Iliakis, Shohreh Amini, Kamel Khalili, Jay Rappaport

  Cancer biology & therapy. 05/2007; 6(4):596-602.

  Puralpha is a sequence-specific nucleic acid binding protein that is involved in multiple cellular functions including regulation of transcription, initiation of DNA replication, cell cyclePuralpha is a sequence-specific nucleic acid binding protein that is involved in multiple cellular functions including regulation of transcription, initiation of DNA replication, cell cycle progression, and neuronal cell differentiation. Its potential role in DNA repair has not been explored. We have now analyzed the role of Puralpha in the cellular response to replication-associated DNA repair of double-strand breaks (DSBs) using Puralpha knockout mouse embryo fibroblasts (MEFs). We found that Puralpha negative cells are hypersensitive to the DNA replication inhibitor, hydroxyurea (HU), and that HU induces excessive DSBs, which delayed the resumption of cell cycle progression after HU treatment. Reintroduction of Pura into Pura null cells reduced the accumulation of DSBs and enhanced DNA end joining. These results suggest a role for Puralpha as a caretaker protein that is involved in the repair of DSBs induced by stalled replication forks.

• Expression of p53 in virally infected tubular cells in renal transplant patients with polyomavirus nephropathy.

  Authors: David B Weinreb, Garrett T Desman, David E Burstein, Dae Un Kim, Steven H Dikman, Edward M Johnson

  Human pathology. 07/2006; 37(6):684-8.

  Polyomavirus (PV) infection is associated with ureteral stenosis, hemorrhagic cystitis, and interstitial nephritis in renal transplant patients. The 3 PVs detected in human beings-BK virus, JC virus,Polyomavirus (PV) infection is associated with ureteral stenosis, hemorrhagic cystitis, and interstitial nephritis in renal transplant patients. The 3 PVs detected in human beings-BK virus, JC virus, and simian virus 40-each encode highly homologous forms of a large T antigen, a transcriptional and replicational regulatory protein. We describe immunohistochemical findings in 5 renal transplant patients who developed PV nephropathy (PVN) and a sixth patient with both PVN and PV infection of the bladder mucosa. Polyomavirus infection was confirmed by immunohistochemical detection of T antigen in kidney and bladder biopsies. We report on the expression of p53 specific to virally infected cells in all biopsies positive for T antigen. Examination of posttransplant biopsies obtained from these 6 patients before they were diagnosed with PVN revealed no expression of T antigen or p53. Accumulation of p53 in PV-infected cells may occur in response to binding of p53 by T antigen, resulting in stabilization of p53. These results provide the first evidence for intracellular actions of PV T antigen in the context of nonneoplastic diseases.

• Role of Pur alpha in targeting mRNA to sites of translation in hippocampal neuronal dendrites.

  Authors: Edward M Johnson, Yayoi Kinoshita, David B Weinreb, Margaret J Wortman, Ruth Simon, Kamel Khalili, Bettina Winckler, Jennifer Gordon

  Journal of neuroscience research. 06/2006; 83(6):929-43.

  Using genetic inactivation in the mouse, PURA, encoding Pur alpha, is demonstrated to be essential for developmentally-timed dendrite formation in the cerebellum and hippocampus. Comparison of RNAUsing genetic inactivation in the mouse, PURA, encoding Pur alpha, is demonstrated to be essential for developmentally-timed dendrite formation in the cerebellum and hippocampus. Comparison of RNA species bound by Pur alpha prompts the hypothesis that Pur alpha functions with non-coding RNA in transport of certain mRNA molecules to sites of translation in dendrites. Pur alpha binds to human BC200 RNA, implicated in dendritic targeting, and this has homologies to 7SL RNA, implicated in compartmentalized translation. Results using hippocampal rat neurons in situ show that Pur alpha binds to BC1 RNA, implicated in dendritic targeting as a mouse counterpart of BC200, and to mRNA molecules translated in dendrites; Pur alpha is specifically located in dendrites, where it is colocalized with Map2, but not in axons, where it fails to colocalize with Ankyrin G. Pur alpha and Staufen are colocalized at dendritic sites of mRNA translation. Microtubule disruptors inhibit Pur alpha dendritic targeting and allow its mislocalization to axons. Using mouse brain, double-RNA immunoprecipitation places Pur alpha together with Staufen or FMRP on BC1 RNA and specific mRNA species in vivo. These results help define a mechanism by which Pur alpha targets specific mRNA molecules to sites of dendritic translation.

• Renal transplant patient with polyoma virus bladder infection and subsequent polyoma virus nephropathy.

  Authors: David B Weinreb, Garrett T Desman, David E Burstein, Steven H Dikman, Edward M Johnson

  International journal of urology : official journal of the Japanese Urological Association. 05/2006; 13(4):439-41.

  Polyoma virus nephropathy (PVN) is a significant cause of renal allograft dysfunction in transplant patients. A 58-year-old male received a cadaveric renal transplant and 12 weeks later presentedPolyoma virus nephropathy (PVN) is a significant cause of renal allograft dysfunction in transplant patients. A 58-year-old male received a cadaveric renal transplant and 12 weeks later presented with fever, diarrhea, and dysuria. He was diagnosed with a polyoma virus infection of the bladder by a transurethral bladder biopsy. One year post-transplant, he presented with renal allograft dysfunction and was diagnosed by biopsy with PVN of the non-native kidney. The diagnosis of a polyoma virus infection was confirmed by immunoreactivity to the polyoma T-antigen. We suggest that polyoma virus infection of the bladder be included in the differential diagnosis of urinary dysfunction in post-transplant patients, as such infections might be an under-recognized comorbidity in individuals with PVN.

• Polyoma virus infection is a prominent risk factor for bladder carcinoma in immunocompetent individuals.

  Authors: David B Weinreb, Garrett T Desman, May Jennifer M Amolat-Apiado, David E Burstein, James H Godbold, Edward M Johnson

  Diagnostic cytopathology. 03/2006; 34(3):201-3.

  Despite various reports of BK viral (BKV) DNA sequences or proteins in tumors of the urogenital tract, there has been no study statistically linking infection by this polyoma virus (PV) to tumorDespite various reports of BK viral (BKV) DNA sequences or proteins in tumors of the urogenital tract, there has been no study statistically linking infection by this polyoma virus (PV) to tumor development. All PV are potential transforming viruses, the large T-antigen of which interacts with tumor suppressor proteins. Here, we have performed a cross-sectional study of 3,782 patients having had urine cytologic analyses, comparing those diagnosed with PV infection with those not so diagnosed. In order to focus on immunocompetent individuals, renal transplant patients, for whom a diagnosis of PV infection followed immunosuppressive therapy, were excluded. Among the 133 immunocompetent patients diagnosed with PV infection, the most frequently occurring neoplasms were bladder carcinoma (15.8%) and prostate carcinoma (3.8%). The incidence of bladder carcinoma was sufficient to statistically establish temporality in a two-sided test, linking a prior diagnosis of PV infection to a subsequent diagnosis of bladder carcinoma (odds ratio = 3.419, P < 0.001).

• Mechanism of DNA binding and localized strand separation by Pur alpha and comparison with Pur family member, Pur beta.

  Authors: Margaret J Wortman, Edward M Johnson, Andrew D Bergemann

  Biochimica et biophysica acta. 04/2005; 1743(1-2):64-78.

  Pur alpha is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Pur alpha unwinds a double-strandedPur alpha is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Pur alpha unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Pur alpha are essential for both ss- and duplex DNA binding. Pur alpha binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Pur alpha since removal of Pur alpha in the gel eliminates the series and since Pur alpha binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Pur alpha binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Pur beta lacking the Pur alpha C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Pur alpha can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Pur alpha.

• Functional interaction of Puralpha with the Cdk2 moiety of cyclin A/Cdk2.

  Authors: Hong Liu, Sharon M Barr, Caryn Chu, D Stave Kohtz, Yayoi Kinoshita, Edward M Johnson

  Biochemical and biophysical research communications. 04/2005; 328(4):851-7.

  Puralpha is a sequence-specific single-stranded nucleic acid-binding protein and a member of the highly conserved Pur family. Puralpha has been shown to colocalize with cyclin A/Cdk2 and toPuralpha is a sequence-specific single-stranded nucleic acid-binding protein and a member of the highly conserved Pur family. Puralpha has been shown to colocalize with cyclin A/Cdk2 and to coimmunoprecipitate with cyclin A during S-phase. Here we show that this interaction is mediated by a specific affinity of Puralpha for Cdk2. In pull-down assays GST-Puralpha efficiently binds Cdk2 and Cdk1, binds Cdk4 less efficiently, and does not display binding to Cdk6. Puralpha stimulates several-fold the phosphorylation in vitro of histone H1 by cyclin A/Cdk2, produced from baculovirus constructs. Double chromatin immunoprecipitation using antibodies to Cdk2 and Puralpha reveals that both proteins colocalize in HeLa cells to DNA segments upstream of the c-MYC gene. Pur family member Purgamma colocalizes with Cdk2 to a specific DNA segment in this region.

• Internalization of exogenous human immunodeficiency virus-1 protein, Tat, by KG-1 oligodendroglioma cells followed by stimulation of DNA replication initiated at the JC virus origin.

  Authors: Dianne C Daniel, Yayoi Kinoshita, M Aamir Khan, Luis Del Valle, Kamel Khalili, Jay Rappaport, Edward M Johnson

  DNA and cell biology. 01/2005; 23(12):858-67.

  JC virus (JCV) is the etiological agent of an opportunistic brain infection, progressive multifocal leukoencephalopathy (PML), in AIDS. PML is fatal in approximately 4% of HIV-infected individuals,JC virus (JCV) is the etiological agent of an opportunistic brain infection, progressive multifocal leukoencephalopathy (PML), in AIDS. PML is fatal in approximately 4% of HIV-infected individuals, and although the overall incidence has fallen due to highly aggressive antiretroviral therapy (HAART), this percent has remained steady. It has been shown that the Tat protein of human immunodeficiency virus-1 (HIV-1) interacts in cells with cellular protein Puralpha. This interaction can stimulate transcription of both HIV-1 and JCV genes. HIV-1, however, infects primarily microglia and astrocytes in the brain, whereas JCV infects primarily oligodendrocytes. Although HIV-1 has been shown capable of infecting oligodendrocytes in vitro (Albright et al., 1996), no instance of viral coinfection of such cells with JCV has been reported. Tat is known to be secreted from cells in which it is made. Here we ask whether such exogenous Tat can influence JCV replication in oligodendrocytes. We find that glial cells infected with either HIV-1 or JCV are in proximity at the outer edge of PML lesions. Exogenous Tat is avidly incorporated into cultured KG-1 oligodendroglioma cells over a 72-h period and is colocalized with endogenous Puralpha both nuclear and juxtanuclear. At concentrations in the medium well below the pM range, Tat stimulates several-fold the replication in vivo of DNA initiated at the JCV origin. These results define a pathway by which a protein made by HIV-1 can directly affect the course of infection by another disease-causing virus.

• Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle.

  Authors: Yayoi Kinoshita, Edward M Johnson

  The Journal of biological chemistry. 09/2004; 279(34):35879-89.

  The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4,The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G(1) and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G(1). MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G(2), reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G(2), at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G(1) followed by a recruitment to multiple sites in chromatin subsequent to replication.

• Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse.

  Authors: Kamel Khalili, Luis Del Valle, Vandhana Muralidharan, William J Gault, Nune Darbinian, Jessica Otte, Ellen Meier, Edward M Johnson, Dianne C Daniel, Yayoi Kinoshita, Shohreh Amini, Jennifer Gordon

  Molecular and cellular biology. 11/2003; 23(19):6857-75.

  The single-stranded DNA- and RNA-binding protein, Puralpha, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, andThe single-stranded DNA- and RNA-binding protein, Puralpha, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA(-/-) mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA(-/-) mice. Levels of proliferation were also absent or low in several other tissues of the PURA(-/-) mice, including those of myeloid lineage, whereas those of PURA(+/-) mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Puralpha and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA(+/+) mice, but both were absent in dendrites of PURA(-/-) mice. Puralpha and Cdk5 can be coimmunoprecipitated from brain lysates of PURA(+/+) mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Puralpha in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

• A new member of the MCM protein family encoded by the human MCM8 gene, located contrapodal to GCD10 at chromosome band 20p12.3-13.

  Authors: Edward M Johnson, Yayoi Kinoshita, Dianne C Daniel

  Nucleic acids research. 07/2003; 31(11):2915-25.

  The MCM8 protein from HeLa cells, a new member of the MCM family, co-isolates through several steps with MCM6 and MCM7, and MCM8 co-immunoprecipitates with MCM4, MCM6 and MCM7, proteins reportedlyThe MCM8 protein from HeLa cells, a new member of the MCM family, co-isolates through several steps with MCM6 and MCM7, and MCM8 co-immunoprecipitates with MCM4, MCM6 and MCM7, proteins reportedly forming a helicase complex involved in initiation of DNA replication. MCM8 mRNA is expressed in placenta, lung and liver, but is also significantly expressed in adult heart, a tissue with a low percentage of proliferating cells. The MCM8 gene, consisting of 19 exons, is located contrapodal to a gene, consisting of 11 exons, encoding a homolog of the yeast GCD10 gene product. The region between these two transcription units, comprising as few as 62 bp, is TATA-less and highly GC-rich, containing multiple CpG units. MCM8 expression is altered in certain forms of neoplasia. In a case of choriocarcinoma MCM8 mRNA is aberrant, leading to expression of a protein lacking 16 amino acids. In several cases of colon adenocarcinoma MCM8 expression is greatly reduced relative to matched non-cancerous tissue. The potential helicase domain of MCM8 is different from those of other MCM proteins in that it is more homologous to canonical ATP-binding domains of other known helicases. Results suggest that MCM8 may interact with other MCM proteins to alter the function of the replicative MCM protein complex.

• Distinct proteins encoded by alternative transcripts of the PURG gene, located contrapodal to WRN on chromosome 8, determined by differential termination/polyadenylation.

  Authors: Hong Liu, Edward M Johnson

  Nucleic acids research. 07/2002; 30(11):2417-26.

  A gene encoding a new member of the Pur protein family, Purgamma, has been detected upstream of, and contrapodal to, the gene encoding the Werner syndrome helicase, Wrn, at human chromosome bandA gene encoding a new member of the Pur protein family, Purgamma, has been detected upstream of, and contrapodal to, the gene encoding the Werner syndrome helicase, Wrn, at human chromosome band 8p11-12. Both the PURG and WRN genes initiate transcription at multiple sites, the major clusters of which are approximately 90 bp apart. A segment containing this region strongly promotes transcription of a reporter gene in both directions. Both promoters are TATA-less and CAAT-less and both are positively regulated by Sp1 elements. While promoter elements for the two genes are interleaved, in the contrapodal direction, certain elements critical for each gene are distinct. Sequencing of cDNAs for Purgamma mRNA reveals that two alternative coding sequences are generated from a single gene, resulting in different Purgamma C-termini. PURG-A mRNA consists of a single intronless transcript of approximately 3 kb. PURG-B mRNA results from transcription through the PURG-A polyadenylation site and splicing out of an intron of >30 kb. In this unique example of a switch, splicing of a single intron either occurs or does not occur depending upon differential termination/polyadenylation. PURG-B is the primary PURG transcript detected in testis, but it is undetectable in all members of a normal adult tissue cDNA panel. PURG-A levels are low or undetectable in the normal tissue panel, but they are greatly elevated in all members of a tumor tissue panel. PURG-B is detected in several tumor panel members.