- 现金
- 3592 元
- 精华
- 4
- 帖子
- 2070
- 注册时间
- 2002-11-20
- 最后登录
- 2014-10-10
|
1楼
发表于 2004-4-11 23:13
Journal of Virology, April 1999, p. 3197-3209, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transcriptional Repression of Human Hepatitis B Virus Genes by a bZIP Family Member, E4BP4
Chao-Kuen Lai and Ling-Pai Ting*
Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Shih-Pai, Taipei 11221, Taiwan, Republic of China
Received 22 September 1998/Accepted 17 December 1998
Box is an essential element of both the upstream regulatory sequence of the core promoter and the second enhancer, which positively regulate the transcription of human hepatitis B virus (HBV) genes. In this paper, we describe the cloning and characterization of a box binding protein, E4BP4. E4BP4 is a bZIP type of transcription factor. Overexpression of E4BP4 represses the stimulating activity of box in the upstream regulatory sequence of the core promoter and the second enhancer in differentiated human hepatoma cell lines. E4BP4 can also suppress the transcription of HBV genes and the production of HBV virions in a transient-transfection system that mimics the viral infection in vivo. Expression of an E4BP4 antisense transcript can, instead, elevate the transcription of the core promoter. A low abundance of E4BP4 protein and mRNA in differentiated human hepatoma cell lines is detected, and E4BP4 is not a major component of box binding proteins in untransfected differentiated human hepatoma cell lines. C/EBP and C/EBP, in contrast, are major components of the box binding activity present in nuclear extracts. E4BP4 has a stronger binding affinity towards box than the endogenous box binding activity present in nuclear extracts. Structure and function analysis of E4BP4 reveals that DNA binding activity is sufficient to confer the negative regulatory function of E4BP4. These results indicate that binding site occlusion is the mechanism whereby E4BP4 suppresses transcription in HBV.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Hepatitis B virus (HBV) is a small DNA virus with a partially double-stranded 3.2-kb genome. The genome organization of HBV is very compact, with four overlapping open reading frames coding for the surface, core, polymerase, and X proteins. The transcription of these open reading frames is under the control of four promoters: two for surface, one for core and polymerase, and one for X. Two enhancers, enhancer I and enhancer II, play important roles in the transcription regulation of these viral genes. The core promoter is composed of the basal core promoter and its upstream regulatory sequence (70). This promoter produces two 3.5-kb RNAs, i.e., the precore and pregenomic RNAs. Pregenomic RNA has dual functions: (i) it can be packaged into nucleocapsids (core particles) along with viral polymerase and serve as the template for reverse transcription, and (ii) it can serve as mRNA that encodes the core and polymerase proteins. Regulated expression of pregenomic RNA plays a pivotal role in the control of the viral replication cycle. A detailed understanding of the transcription control of viral genes may reveal new targets for therapeutic intervention.
The second enhancer of HBV has a unique bipartite structure in that the cooperation of two noncontiguous elements, box and box , is required for its enhancer function. It stimulates the transcriptional activity of the simian virus 40 (SV40) early promoter and the HBV surface and X promoters (67, 68). The second enhancer is colocalized with the core upstream regulatory sequence (CURS). Box , for example, is not only an essential component of the second enhancer but also a potent stimulatory element of the CURS. In other words, box can activate the basal core promoter from an upstream position in differentiated human hepatoma cell lines (HepG2 and HuH-7) (69, 70). A negative regulatory element, designated NRE, which represses the activities of enhancer II and the core promoter, was identified upstream of the CURS (44). The core promoter provides a valuable system to study the positive and negative transcription regulation of eukaryotic promoters.
Transcription regulation is governed by a constellation of trans-acting cellular factors that bind to specific cis-acting elements that act in either a positive or negative manner. Transcription initiation by RNA polymerase II involves a stepwise assembly of general transcription factors or a holoenzyme on a promoter template to form a preinitiation complex. Transcription activators may stimulate transcription by increasing the assembly of a preinitiation complex. Several distinct models have been proposed as the mechanism of transcription repression (20, 21, 24, 25, 29, 42, 52). In the competition model, repressors may bind directly at or near a transcription start site and compete with the formation of a preinitiation complex in the promoter. Alternatively, activators and repressors may compete for overlapping or closely linked binding sites. In the activator-sequestering model, repressors stoichiometrically bind to particular activators through protein-protein interactions, leading to the formation of complexes with reduced or no DNA binding activity. In the quenching model, repressors and activators may bind to adjacent, nonoverlapping DNA sequences, but the repressors neutralize the ability of the activators to transmit stimulatory signals to the basal transcription machinery. In the fourth model, direct repression, repressors may bind to any of the basal transcription factors, with RNA polymerase II itself, or with a corepressor that ultimately targets the basal machinery. Such interaction may interfere with the formation or the activity of the basal transcription preinitiation complex.
We have previously shown that C/EBP-like proteins can bind to box . C/EBPs (CCAAT/enhancer binding proteins) are a family of highly conserved, leucine zipper-type (bZIP) DNA binding proteins. Members identified so far are C/EBP, C/EBP (also known as NF-IL6, CRP2, LAP, and AGP/EBP), C/EBP (also known as NF-IL6 and CRP3), C/EBP, CRP1, Ig-C/EBP, and GADD153 (also known as CHOP) (1, 5, 6, 15, 31, 32, 36, 53, 54, 65). Different C/EBP family members are characterized by a high degree of sequence homology in the leucine zipper and basic regions. They have, however, much less conserved N-terminal regulatory and transactivation domains (5, 35). C/EBPs have the potential to form homo- and heterodimers with C/EBP family members or bZIP proteins or to interact with proteins that do not contain leucine zippers. Dimerization of C/EBPs is generally required for their DNA binding and transcription activation function (3, 10, 16, 18, 26, 33, 34, 37-41, 45, 46, 48, 58-65).
In this paper, we describe the cloning and characterization of a box binding protein, E4BP4. Overexpression of E4BP4 represses the stimulating activity of box in the CURS and the second enhancer. E4BP4 can also repress the transcription of HBV genes and the production of HBV virions in a transient-transfection system. Overexpression of an E4BP4 antisense transcript, on the other hand, can elevate the transcription of the core promoter. Though present in low abundance, E4BP4 can bind to the box sequence with higher affinity than the box binding activity present in nuclear extracts. Evidence that binding site occlusion is most likely the mechanism whereby E4BP4 suppresses transcription in HBV is presented.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Isolation of cDNA clones. A ZAPII cDNA library (prepared from Stratagene''''s ZAP cDNA synthesis kit) of human hepatoma HepG2 cells was screened with concatemerized double-stranded synthetic oligonucleotides of box by the method of Singh (57). The oligonucleotides contained the box sequence gatCCAAGGTCTTACATAAGAGGACTCTT and its complement, which corresponded to the box sequence extending from nucleotide (nt) 1644 to 1669 of HBV plus an MboI 5'''' overhang. The oligonucleotides were concatemerized to ~500 bp in size with T4 polynucleotide kinase and T4 DNA ligase and labeled with [-32P]dCTP by random prime labeling. All positive plaques were picked, replated, and clonally purified through secondary and tertiary screenings. cDNA inserts from positive clones were excised in the form of pBluescript plasmid (pSKP4) by coinfection with helper phage. DNA sequences were determined by dideoxy chain termination methods.
Plasmids. The HBV sequence used in the study is of the adw subtype. Numbering of the HBV sequence begins at the unique EcoRI site, which is nt 1. All reporter plasmids used in transfection experiments contain a head-to-tail trimeric tandem repeat, referred to as A3, of a 237-bp BclI-BamHI fragment from the SV40 polyadenylation signal. A3 is placed 5'''' of promoter sequences of interest and has been shown to stop transcription readthrough from spurious upstream initiation.
Plasmids pSV2CAT, p/BCP-CAT, pCURS/BCP-CAT, p(1613-1851)CAT, p(1687-1851)CAT, pSVpCAT/ENII, pSVpCAT/, and pHBV3.6 were described previously (44, 67, 68, 70).
The recombinant E4BP4 expression plasmid pXa-2-P4 was generated by cloning of the BamHI-KpnI fragment containing the E4BP4 open reading frame into the BamHI and KpnI sites of the PinPoint Xa-2 vector (Promega).
The plasmid pCMVP4 was generated by moving the cDNA inserts of pSKP4 into the BamHI and KpnI sites downstream of the cytomegalovirus (CMV) immediate-early promoter (CMVIE) in pCMVIE. To generate the FLAG-tagged E4BP4 construct, the BamHI-KpnI fragment containing the cDNA insert of pCMVP4 was cloned into the BglII and KpnI sites downstream of the CMVIE in the pFLAG-CMV2 expression vector (Kodak, New Haven, Conn.). The resulting plasmid, pf:E4BP4, was digested with ApaI and SalI and then recircularized to generate pf:E4BP4Apa and pf:E4BP4Sal, respectively. The plasmid pf:E4BP4Pvu was generated by cloning the BamHI-PvuII fragment from pf:E4BP4 into BglII- and SmaI-digested pFLAG-CMV2. To remove the repression domain of E4BP4, pCMVP4 was first digested with BstBI to derive a 5.6-kb BstBI fragment, which was subsequently digested with BamHI, followed by filling in of all 3''''-recessed ends with the Klenow fragment of Escherichia coli DNA polymerase. Two BamHI-BstBI fragments of 4,513 and 1087 bp, were generated. An 885-bp BamHI-HaeIII fragment was derived from the digestion of the 1,087-bp BamHI-BstBI fragment with HaeIII. The 4,513-bp BamHI-BstBI and 885-bp BamHI-HaeIII fragments were ligated together to generate pCMVP4Hae/BstB. The BamHI-KpnI fragment of pCMVP4Hae/BstB was cloned into the BglII and KpnI sites of the pFLAG-CMV2 expression vector to generate the plasmid pf:E4BP4Hae/BstB. All of these constructs were confirmed by DNA sequencing.
The E4BP4 antisense plasmid pCMV4Ns/s was generated by cloning of the 378-bp SalI-SspI fragment, which corresponds to the 5''''-end region of E4BP4 from nt 117 to 494, into the SalI-SmaI sites of the pCMVIE expression vector.
Bacterial fusion proteins. The E4BP4 expression construct pXa-2-P4 was used to express biotinylated fusion proteins in strain JM109. JM109 cells harboring E4BP4 vectors were grown to log phase and induced with 100 µM isopropyl--D-thiogalactopyranoside (IPTG) (Sigma). Six hours following induction, the bacteria were centrifuged and lysed in 1 mg of lysozyme per ml-0.1% Triton X-100-200 U of DNase. The lysates were then clarified by centrifugation at 10,000 × g for 15 min at 4°C and mixed with avidin resin (Promega). Following a 6-h incubation, the resin was washed three times in cold buffer (50 mM Tris-HCl, 4 mM dithiothreitol [DTT], 2 mM EDTA, 10% glycerol). If the fusion protein was to be eluted, the pelleted resin was washed with buffer containing 5 mM biotin. The eluent was aliquoted, quickly frozen under liquid nitrogen, and kept frozen at 70°C.
Preparation of anti-E4BP4 polyclonal antibody. Rabbits were immunized with a 16-amino-acid peptide corresponding to amino acids 446 to 461 of E4BP4. After three booster injections with conjugated peptide (the carrier protein was keyhole limpet hemocyanin or bovine serum albumin), rabbit sera were tested for reactivity with E4BP4 by immunoprecipitation and Western blotting.
Cell lines, transfection, and CAT assay. The culture and transfection of human hepatoma cell lines HepG2 and HuH-7 were performed as previously described (7). All plasmids used in one set of experiments were simultaneously prepared, checked for supercoiled forms, aliquoted in small amounts, and stored in 70% ethanol. Each set of experiments was performed with two different preparations of plasmids and repeated two to three times for each preparation. The chloramphenicol acetyltransferase (CAT) activity was normalized against the CAT activity exhibited by a control plasmid, pSV2CAT, which was taken as 100%. In pSV2CAT, the expression of the CAT gene is driven by the SV40 early promoter and 72-bp enhancer. When the CAT activity was high, assays were performed on serially diluted cell lysates to ensure that CAT activity fell in a linear range for all assays.
Preparation and heparin-Sepharose fractionation of nuclear extracts. Fractionated nuclear extracts from differentiated human hepatoma cell lines HepG2 and HuH-7 were prepared as previously described (8, 68). The crude and fractionated nuclear extracts were aliquoted, quickly frozen under liquid nitrogen, and kept frozen at 70°C.
Preparation of mini-nuclear extracts from transfected cells. Mini-nuclear extracts were prepared by the method of Schreiber et al. (56). HuH-7 cells were transiently transfected with pFLAG-CMV2, pf:E4BP4, and expression plasmids containing deletion mutants of f:E4BP4 by the calcium phosphate precipitation method. Forty-eight hours later, transfected HuH-7 cells were collected, washed with Tris-buffered saline (TBS) (10 mM Tris-HCl [pH 7.45] and 150 mM NaCl), and pelleted by centrifugation at 1,500 × g for 5 min. The cell pellet was resuspended in TBS, transferred into an Eppendorf tube, and pelleted again by being spun for 20 s in a microcentrifuge. TBS was removed, and the cell pellet was resuspended in cold buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride) by gentle pipetting. The cells were allowed to swell on ice for 15 min, after which a 10% solution of Nonidet P-40 was added and the tube was vigorously vortexed for 10 s. The homogenate was centrifuged for 30 s in a microcentrifuge. The nuclear pellet was resuspended in ice-cold buffer C (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride), and the tube was vigorously rocked at 4°C for 15 min on a shaking plate. The nuclear extract was centrifuged for 5 min in a microcentrifuge at 4°C, and the supernatant was aliquoted, quickly frozen under liquid nitrogen, and kept frozen at 70°C.
Gel shift analysis. The probe was prepared with annealed double-stranded oligonucleotide (100 ng) corresponding to the box sequence of HBV (Fig. 1A) and end labeled with [-32P]ATP and T4 polynucleotide kinase. Gel shifting and competition experiments were done as previously described (68) except that 5 instead of 10 µg of nuclear extract was used (68). Supershifts were generated with anti-E4BP4 antiserum, anti-FLAG M2 monoclonal antibody (Kodak), or anti-C/EBP polyclonal antibody for C/EBP, C/EBP, or C/EBP (Santa Cruz Biotechnology, Santa Cruz, Calif.).
[此贴子已经被作者于2004-4-11 10:17:27编辑过] |
|