EBV nuclear antigen EBNALP dismisses transcription repressors NCoR and RBPJ from enhancers and EBNA2 increases NCoR-deficient RBPJ DNA binding

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) protein is a transcriptional regulator of viral and cellular genes that is essential for EBV-mediated immortalization of B lymphocytes in vitro. EBNA-3C can inhibit transcription through an association with the cellular DNA-binding protein Jκ, a function shared by EBNA-3A and EBNA-3B. Here, we report a mechanism by which EBNA-3C can activate transcription from the EBV latent membrane protein 1 (LMP-1) promoter in conjunction with EBNA-2. Jκ DNA-binding sites were not required for this activation, and a mutant EBNA-3C protein unable to bind Jκ activated transcription as efficiently as wild-type EBNA-3C, indicating that EBNA-3C can regulate transcription through a mechanism that is independent of Jκ. Furthermore, activation of the LMP-1 promoter is a unique function of EBNA-3C, not shared by EBNA-3A and EBNA-3B. The DNA element through which EBNA-3C activates the LMP-1 promoter includes a Spi-1/Spi-B binding site, previously characterized as an important EBNA-2 response element. Although this element has considerable homology to mouse immunoglobulin light chain promoter sequences to which the mouse homologue of Spi-1 binds with its dimerization partner IRF4, we demonstrate that the IRF4-like binding sites in the LMP-1 promoter do not play a role in EBNA-3C-mediated activation. Both EBNA-2 and EBNA-3C were required for transcription mediated through a 41-bp region of the LMP-1 promoter encompassing the Spi binding site. However, EBNA-3C had no effect on transcription mediated in conjunction with the EBNA-2 activation domain fused to the GAL4 DNA-binding domain, suggesting that it does not function as an adapter between EBNA-2 and the cellular transcriptional machinery. Like EBNA-2, EBNA-3C bound directly to both Spi-1 and Spi-B in vitro. This interaction was mediated by a region of EBNA-3C encompassing a likely basic leucine zipper (bZIP) domain and the ets domain of Spi-1 or Spi-B, reminiscent of interactions between bZIP and ets domains of other transcription factors that result in their targeting to DNA. There are many examples of regulation of the hematopoietic-specific Spi transcription factors through protein-protein interactions, and a similar regulation by EBNA-3C, in conjunction with EBNA-2, is likely to be an important and unique contribution of EBNA-3C to EBV-mediated immortalization.

Download Full-text

LIM Protein KyoT2 Negatively Regulates Transcription by Association with the RBP-J DNA-Binding Protein

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.644 ◽

1998 ◽

Vol 18 (1) ◽

pp. 644-654 ◽

Cited By ~ 128

Author(s):

Yoshihito Taniguchi ◽

Takahisa Furukawa ◽

Tin Tun ◽

Hua Han ◽

Tasuku Honjo

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Protein ◽

Dna Binding Protein ◽

Negative Regulator ◽

Nuclear Antigen ◽

Differential Splicing ◽

Barr Virus ◽

Lim Domains ◽

C Terminus

ABSTRACT The RBP-J/Su(H) DNA-binding protein plays a key role in transcriptional regulation by targeting Epstein-Barr virus nuclear antigen 2 (EBNA2) and the intracellular portions of Notch receptors to specific promoters. Using the yeast two-hybrid system, we isolated a LIM-only protein, KyoT, which physically interacts with RBP-J. Differential splicing gave rise to two transcripts of theKyoT gene, KyoT1 and KyoT2, that encoded proteins with four and two LIM domains, respectively. With differential splicing resulting in deletion of an exon, KyoT2 lacked two LIM domains from the C terminus and had a frameshift in the last exon, creating the RBP-J-binding region in the C terminus. KyoT1 had a negligible level of interaction with RBP-J. Strong expression of KyoT mRNAs was detected in skeletal muscle and lung, with a predominance of KyoT1 mRNA. When expressed in F9 embryonal carcinoma cells, KyoT1 and KyoT2 were localized in the cytoplasm and the nucleus, respectively. The binding site of KyoT2 on RBP-J overlaps those of EBNA2 and Notch1 but is distinct from that of Hairless, the negative regulator of RBP-J-mediated transcription in Drosophila. KyoT2 but not KyoT1 repressed the RBP-J-mediated transcriptional activation by EBNA2 and Notch1 by competing with them for binding to RBP-J and by dislocating RBP-J from DNA. KyoT2 is a novel negative regulatory molecule for RBP-J-mediated transcription in mammalian systems.

Download Full-text

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

Blood ◽

10.1182/blood.v70.4.1124.bloodjournal7041124 ◽

1987 ◽

Vol 70 (4) ◽

pp. 1124-1130

Author(s):

AL Epstein ◽

M Samoszuk ◽

E Stathopoulos ◽

GS Naeve ◽

CV Clevenger ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Dna Binding ◽

Cell Lines ◽

Solid Phase ◽

Specific Antigen ◽

Precursor Cells ◽

Myelogenous Leukemia ◽

Nuclear Antigen ◽

Cortex Of The Thymus

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Download Full-text

KSHV Episome Tethering Sites on Host Chromosomes: Regulation of Latency-Lytic Switch by the ChAHP complex

10.21203/rs.3.rs-136834/v2 ◽

2021 ◽

Author(s):

Ashish Kumar ◽

Yuanzhi Lyu ◽

Yuichi Yanagihashi ◽

Chanikarn Chantarasrivong ◽

Vladimir Majerciak ◽

...

Keyword(s):

Dna Binding ◽

Cell Nucleus ◽

Latent Infection ◽

Dna Binding Protein ◽

Lytic Replication ◽

Nuclear Antigen ◽

Host Chromosome ◽

Lncrna Expression ◽

Lytic Reactivation ◽

Genomic Regions

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with a CHD4 complex, ChAHP, at epigenetically active genomic regions and tethers frequently near centromeric regions of host chromosomes. The ChAHP complex also occupies the 5’-region of a highly-inducible lncRNAs and terminal repeats of KSHV genome with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation is accompanied by the detachment of KSHV episomes from host chromosome docking sites We propose a model in which elevated lncRNA expression determines the KSHV latency-lytic decision by regulating LANA/ChAHP DNA binding at inducible viral enhancers.

Download Full-text

Transcription factor NF-κB participates in regulation of epithelial cell turnover in the colon

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.6.g1282 ◽

2000 ◽

Vol 279 (6) ◽

pp. G1282-G1291 ◽

Cited By ~ 31

Author(s):

Mehmet Sait Inan ◽

Veronica Tolmacheva ◽

Qiang-Shu Wang ◽

Daniel W. Rosenberg ◽

Charles Giardina

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Transcription Factor ◽

Epithelial Cell ◽

Dna Binding ◽

Epithelial Cells ◽

Knockout Mice ◽

Nuclear Antigen ◽

Cell Turnover ◽

Tnf Α

The transcription factor nuclear factor (NF)-κB regulates the expression of genes that can influence cell proliferation and death. Here we analyze the contribution of NF-κB to the regulation of epithelial cell turnover in the colon. Immunohistochemical, immunoblot, and DNA binding analyses indicate that NF-κB complexes change as colonocytes mature: p65-p50 complexes predominate in proliferating epithelial cells of the colon, whereas the p50-p50 dimer is prevalent in mature epithelial cells. NF-κB1 (p50) knockout mice were used to study the role of NF-κB in regulating epithelial cell turnover. Knockout animals lacked detectable NF-κB DNA binding activity in isolated epithelial cells and had significantly longer crypts with a more extensive proliferative zone than their wild-type counterparts (as determined by proliferating cell nuclear antigen staining and in vivo bromodeoxyuridine labeling). Gene expression profiling reveals that the NF-κB1 knockout mice express the potentially growth-enhancing tumor necrosis factor (TNF)-α and nerve growth factor-α genes at elevated levels, with in situ hybridization localizing some of the TNF-α expression to epithelial cells. TNF-α is NF-κB regulated, and its upregulation in NF-κB1 knockouts may result from an alleviation of p50-p50 repression. NF-κB complexes may therefore influence cell proliferation in the colon through their ability to selectively activate and/or repress gene expression.

Download Full-text

DNA Binding and Modulation of Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.75.17.7882-7892.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 7882-7892 ◽

Cited By ~ 146

Author(s):

Alexander C. Garber ◽

Marla A. Shu ◽

Jianhong Hu ◽

Rolf Renne

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Viral Gene ◽

Mobility Shift ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is highly expressed in these malignancies and has been shown to play an important role in episomal maintenance, presumably by binding to a putative oriP. In addition, LANA modulates cellular and viral gene expression and interacts with the cellular tumor suppressors p53 and retinoblastoma suppressor protein. Many of these features are reminiscent of Epstein-Barr virus nuclear antigens (EBNAs), a family of six proteins expressed during latency. EBNA-1 is required for episome maintenance, binds to oriP, and strongly activates transcription from two promoters, including its own. We have previously shown that LANA can transactivate its own promoter and therefore asked whether LANA, like EBNA-1, activates transcription by direct binding to DNA. By using recombinant LANA expressed from vaccinia virus vectors for electrophoretic mobility shift assays, we found that LANA does not bind to its own promoter. In contrast, LANA binds specifically to sequences containing an imperfect 20-bp palindrome in the terminal repeat (TR) of KSHV. We further show that the C-terminal domain of LANA is sufficient for site-specific DNA binding. Unlike EBNA-1, which activates transcription through binding of oriP, we found that LANA inhibits transcription from a single TR binding site. A multimerized TR as found in the viral genome results in strong transcriptional suppression when linked to a heterologous promoter. These data suggest that LANA, although fulfilling functions similar to those of EBNA-1, does so by very different mechanisms.

Download Full-text

The Metastatic Suppressor Nm23-H1 Interacts with EBNA3C at Sequences Located between the Glutamine- and Proline-Rich Domains and Can Cooperate in Activation of Transcription

Journal of Virology ◽

10.1128/jvi.76.17.8702-8709.2002 ◽

2002 ◽

Vol 76 (17) ◽

pp. 8702-8709 ◽

Cited By ~ 40

Author(s):

Chitra Subramanian ◽

Erle S. Robertson

Keyword(s):

Dna Binding ◽

Nuclear Antigen ◽

Luciferase Reporter ◽

Luciferase Reporter Construct ◽

Barr Virus ◽

Activation Of Transcription ◽

Infected Cells ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. Recent studies have shown an in vitro and in vivo interaction between the EBV nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1, known to be downregulated in human invasive breast carcinoma. In this study, we have identified the domain of EBNA3C that specifically binds to Nm23-H1. This domain lies within the region comprising amino acids 637 to 675 of EBNA3C flanked by the proline- and glutamine-rich domains. Furthermore, we show that Nm23-H1 activates transcription when fused to the Gal4 DNA-binding domain and is coexpressed with a luciferase reporter construct containing the Gal4 binding sites upstream of a basal promoter. Gal4-Nm23-H1, when tethered to the promoter by binding to the Gal4 DNA binding sequences, consistently activated transcription. The level of activation increased when increasing amounts of Gal4-Nm23-H1 were introduced into the system. Moreover, EBNA3C when cotransfected with Gal4-Nm23-H1 enhanced the transcriptional activity. These results suggest that Nm23-H1 may have intrinsic transcription activities in EBV-infected cells and that this activity can be modulated in the presence of the essential latent antigen EBNA3C.

Download Full-text