Our Science – Brady Website

John N. Brady, Ph.D.

Laboratory of Cellular Oncology Head, Virus Tumor Biology Section Senior Investigator Building 41 Room B201 Bethesda, MD 20892 Phone:   301-496-0986 Fax:   301-496-4951 E-Mail:   bradyj@mail.nih.gov

Biography

Dr. Brady joined the NCI in 1984, where he is presently chief of the Virus Tumor Biology Section, Laboratory of Cellular Oncology. Under the guidance of Dr. Richard Consigli, he obtained his Ph.D. from Kansas State University studying the molecular structure of polyoma virus. Dr. Brady later studied SV40 transcription regulation as a staff fellow with Dr. Norman Salzman, National Institute of Allergy and Infectious Diseases, and Dr. George Khoury, NCI. His current research interests center on eukaryotic gene regulation, tumor suppressor proteins, and viral transformation.

Research

MOLECULAR BIOLOGY OF HUMAN RETROVIRUSES

The laboratories research program is focused on the human retroviruses-human T cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus (HIV). HTLV-1 is closely associated with the human cancer adult T cell leukemia (ATL) and the neurologic disease HTLV-1-associated myelopathy (HAM/TSP). HIV is the etiologic agent for acquired immunodeficiency syndrome (AIDS). The laboratory is interested in interactions between the virus and cell that influence viral gene regulation, viral pathogenesis, and oncogenic transformation. Highlights from recent research projects are given below.


INHIBITION OF p53 TUMOR SUPPRESOR BY HTLV-1 TAX PROTEIN

p65/RelA Inhibits p53 Transcription Activity by Direct Interaction and Inhibition of Preinitiation Complex Formation. NF-kB activation plays a critical role in oncogenesis by HTLV-I, the etiologic agent of ATL, and is indispensable for maintenance of the malignant phenotype. In T-lymphocytes, Tax-mediated p53 inhibition is dependent on Tax activation of the NF-kB pathway and is linked to p53 phosphorylation. Recently we reported that blocking NF-kB transcriptional activation in HTLV-I-transformed cells restores p53 activity. Further, using MEF null cells and antisense oligonucleotides to inhibit expression of NF-kB family members, we demonstrated that the p65 subunit of NF-kB is uniquely involved in p53 inhibition. Coimmunoprecipitation assays showed an interaction between p65 and p53 in HTLV-I-transformed cells. In transient transfection assays, Tax induced the p53-p65 interaction. Phosphorylation of p53 at serines 15 and 392 was critical for complex formation. Importantly, Tax-mediated p53 inhibition correlated with p65 and p53 interaction. Using ChIP assays in HTLV-I-transformed cells, we found that p53 and p65 form a complex on the inactive, p53-responsive, MDM2 promoter. Consistent with reduced transcriptional activity, TFIID binding was not observed. These studies identify a unique mechanism for p53 regulation by the p65/RelA subunit of NF-kB. (Blood, 2004)

A novel NF-kB pathway involving IKKBeta and p65/RelA Ser-536 phosphorylation results in p53 inhibition in the absence of NF-kB transcription activity. Nuclear factor kappa B (NF-kB) plays an important role in regulating cellular transformation and apoptosis. Human T-cell lymphotrophic virus type I (HTLV-I) Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NF-kB. We have previously demonstrated that Tax inhibits p53 activity through the p65/RelA subunit of NF-kB. We now present evidence which suggest that upstream kinase IKKBeta plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblasts (MEFs) IKKBeta-/- cells did not support Tax-mediated p53 inhibition while MEFs lacking IKKalpha allowed Tax inhibition of p53. Second, transfection of IKKBeta wild type (WT), but not a kinase dead (KD) mutant, into IKKalpha-/- cells rescued p53 inhibition by Tax. Third, the IKKBeta specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA-/- cells transfected with p65/RelA WT or mutant (MT) plasmids. Moreover, Tax induces p65/RelA Ser-536 phosphorylation in WT or IKKalpha -/- cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKBeta-/- cells, suggesting a link between IKKβ and p65/RelA phosphorylation. Consistent with this observation, blocking IKKBeta kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in HTLV-I transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NF-κB transcription activation pathway. Our work, therefore, describes a novel Tax-NF-κB p65/RelA pathway that functions to inhibit p53, but does not require NF-κB transcription activity. (J. Biol. Chem., 2005)

Activated AKT regulates NF-kB activation, p53 inhibition and cell survival in HTLV-1-transformed cells. AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-κB activation, p53 inhibition and cell survival. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant resulted in increased Tax-mediated NF-kB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT SiRNA prevented NF-kB activation and inhibition of p53. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKb phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKKb phosphorylation of IkBa in vitro suggesting selective activity of AKT on the IKKb complex. Finally, AKT pro-survival function in HTLV-1-transformed cells is linked to expression of the Bcl-xL gene. We suggest that AKT plays a role in activation of pro-survival pathways in HTLV-1-transformed cells, possibly through NF-kB activation and inhibition of p53 transcription activity. (Oncogene, 2005)

PI3/AKT inhibition induces caspase-dependent apoptosis in HTLV-1 transformed cellsThe phosphatidylinositol-3-kinase (PI3K) and AKT (Protein Kinase B) signaling pathways play an important role in regulating cell cycle progression and cell survival. In previous studies, we demonstrated that AKT is activated in HTLV-1 transformed cells and that Tax activation of AKT is linked to p53 inhibition and cell survival. In the present study, we extend these observations to identify regulatory pathways affected by AKT in HTLV-1-transformed cells. We demonstrate that inhibition of AKT reduces the level of phosphorylated Bad, an important member of the pro-apoptotic family of proteins. Consistent with the decrease of phosphorylated Bad, cytochrome c is released from the mitochondria and caspase 9 is activated. Pre-treatment of the cells with caspase-9 specific inhibitor z-LEHD-FMK or pan caspase inhibitor Ac-DEVD-CHO prevented LY294002-induced apoptosis. Of interest, p53 siRNA prevents LY294002-induced apoptosis in HTLV-1-transformed cells, suggesting that p53 reactivation is linked to apoptosis. In conclusion, the AKT pathway is involved in targeting multiple proteins which regulate caspase- and p53-dependent apoptosis in HTLV-1-transformed cells. Since AKT inhibitors simultaneously inhibit NF-κB and activate p53, these drugs should be promising candidates for HTLV-1-associated cancer therapy. (Virology, In press, 2007)


HTLV-1 TRANSCRIPTION REGULATION

Tax relieves transcriptional repression by promoting HDAC1 release from the HTLV-I LTR. Expression of human T-cell leukemia virus type I (HTLV-I) is regulated by the viral transcriptional activator Tax. Tax activates viral transcription through interaction with the cellular transcription factor CREB and the coactivators CBP/p300. We have analyzed the role of histone deacetylase-1 (HDAC1) on HTLV-I gene expression in HTLV-I transformed cells and in a cell line that contains an integrated single copy of the HTLV-I LTR. TSA, a HDAC inhibitor, enhanced Tax expression in the HTLV-I transformed cells, whereas overexpression of HDAC1 repressed Tax transactivation in the cell line with the integrated template. ChIP analysis of the interaction of transcription factors, coactivators, and HDACs with the basal and activated HTLV-I promoter revealed that HDAC1 is associated with the inactive, but not the Tax transactivated HTLV-I promoter. Furthermore, in vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrated a direct physical association between Tax and HDAC1. Importantly, biotinylated chromatin pull-down assays indicated that Tax inhibits and/or dissociates the binding of HDAC1 to the HTLV-I promoter. Our results provide evidence that Tax interacts directly with HDAC1 and regulates the binding of the repressor to the HTLV-I promoter. (J. Virol., 2005)

Tax interacts with P-TEFb in a novel manner to stimulate HTLV-1 transcription
Human T-lymphotropic virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose function is essential for viral transcription and replication. Tax transactivates the viral LTR through a series of protein-protein interactions which facilitate CREB and CBP/p300 binding. In addition Tax dissociates transcription repressor HDAC1 interaction with the CREB-response element. The subsequent events through which Tax interacts and communicates with RNA polymerase II (RNAP II) and cyclin-dependent kinases (CDKs) are not clearly understood. Here we present evidence that Tax recruits P-TEFb (CDK9/cyclin T1) to the viral promoter. The recruitment likely involves protein-protein interactions since Tax associates with P-TEFb in vitro as demonstrated by GST fusion protein pull-down assays and in vivo as shown by co-immunoprecipitation assays. Functionally, SiRNA directed toward CDK9 inhibited Tax transactivation in transient assays. Consistent with these findings, depletion of CDK9 from nuclear extracts inhibited Tax transactivation in vitro. Reconstitution of the reaction with WT P-TEFb, but not a kinase-dead mutant, recovered HTLV-1 transcription. Moreover, the addition of the CDK9 inhibitor flavopiridol blocked Tax transactivation in vitro and in vivo. Interestingly, we found Tax regulates CDK9 kinase activity through a novel autophosphorylation pathway. (J. Virology, 2006)

Modulation of Brd4/P-TEFb interaction by the HTLV-1 Tax protein Positive transcription elongation factor (P-TEFb), which is composed of CDK9 and cyclinT1, plays an important role in cellular and viral gene expression. Our lab has recently demonstrated that P-TEFb is required for Tax transactivation of the viral LTR. P-TEFb is found in two major complexes, the inactive form which is associated with inhibitory subunits 7SK snRNA and HEXIM1 and the active form which is associated with, at least in part, Brd4. In the following study, we have analyzed the effect of Brd4 on HTLV-1 transcription. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose dependent manner. In vitro binding studies suggest that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclinT1. Tax interacts with cyclinT1 amino acids 426-533 which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates Serine 2 phosphorylation of the RNA Pol II CTD, suggesting Tax regulates the functionality of P-TEFb. Our results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation. (J. Virology, In press, 2007)

CARM1 enhances transcriptional activity of the HTLV-1 long terminal repeat through a direct interaction with Tax. In this study, we demonstrate that the coactivator associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro GST-Tax binding assays, in vivo coimmunoprecipitation and confocal microscopy experiments. Lastly, chromatin immunoprecipitation (ChIP) analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17 and R26). (J. Virology, 2006)

Coordination of RNAP II CTD phosphorylation and histone methylation by the P-TEFb kinase activity during HIV-1 transcription. The HIV-1 Tat protein recruits positive transcription elongation factor b (P-TEFb) to the TAR RNA structure to facilitate formation of processive transcription elongation complexes (TECs). In HIV-1 transcription elongation complexes, P-TEFb phosphorylates RNAP II CTD and the transcription elongation factors SPT5 and Tat-SF1 in a Tat/TAR-dependent manner. Using in vivo ChIP analysis to examine the role of the Tat/TAR-specific CDK9 kinase activity in the regulation of HIV-1 transcription elongation and histone methylation, we have revealed several unique properties of the HIV-1 transcription complexes. First, the RNAP II CTD is uniquely phosphorylated near the promoter and at downstream coding regions. Second, the stable association of SPT5 with the transcription elongation complexes is dependent upon P-TEFb kinase activity. Third, P-TEFb kinase activity is critical for the induction of methylation of histone H3 on HIV-1 genes. Our data therefore suggests that the potent anti-viral activity of flavopiridol, a potent P-TEFb kinase inhibitor that inhibits CTD phosphorylation, stable SPT5 binding and histone methylation, is due to its ability to inhibit several critical and unique steps in HIV-1 transcription elongation. (J. Virol. 78:13522-13533, 2004)

This page was last updated on 6/11/2008.