Transcriptional Regulation of the TFIIH Transcription Repair Components XPB and XPD by the Hepatitis B Virus x Protein in Liver Cells and Transgenic Liver Tissue

Iris Jaitovich-Groisman,Naciba Benlimame,Betty L Slagle,Maite Hernandez Perez,Lesley Alpert,Daniel J Song,Nasser Fotouhi-Ardakani,Jacques Galipeau,Moulay A Alaoui-Jamali

doi:10.1074/jbc.m010852200

Iris Jaitovich-Groisman, Naciba Benlimame + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m010852200

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Abstract

Human hepatitis B virus is a risk factor for the development of hepatocellular carcinoma. The hepatitis B virus x protein (HBx) has been shown to inactivate the p53 tumor suppressor protein and impair DNA repair, cell cycle, and apoptosis mechanisms. Herein we report that HBx represses two components of the transcription-repair factor TFIIH, XPB (p89), and XPD (p80), both in p53-proficient and p53-deficient liver cells. This inhibition is observed while HBx maintains its transactivation function. Expression of HBx in liver cells results in down-regulation of endogenous XPB and XPD mRNAs and proteins; this inhibition is not observed with other TFIIH subunits, XPA or PCNA. In liver tissue from HBx transgenics, XPB and XPD proteins are down-regulated in comparison to matched normal liver tissue. HBx has been shown to interact with Sp1 transcription factor and affects its DNA binding activity. Sp1 is essential for the basal promoter activity of XPB in liver cells and Drosophila SL2 cells. In the Sp1-deficient SL2 cells, HBx-induced XPB and XPD inhibition is Sp1-dependent. In summary, our results provide evidence that HBx represses the expression of key TFIIH proteins at least in part through Sp1 elements; this repression may impair TFIIH function in DNA repair mechanisms.

Highlights

Chronic infection with human hepatitis B virus (HBV)1 is a risk factor for the development of hepatocellular carcinoma
Expression of hepatitis B virus x protein (HBx) resulted in ϳ65 and 37% reduction in pXPBCAT or pXPD-Chloramphenicol Acetyltransferase (CAT) activity, respectively, as compared with control cells transfected with pCMV (Fig. 1, A and B)
As compared with cells transfected with the control pCMV plasmid, expression of pCMV-HBx resulted in inhibition of CAT mRNA expression in both HepG2 and CCL13 cell lines co-transfected with either pXPD-CAT or pXPB-CAT plasmid (Fig. 2, A and B)

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructs—pXPB-CAT and pXPD-CAT vectors were constructed by ligating XPB or XPD promoters between the XbaI and the HindIII site of the pCAT basic vector (Promega, Madison, WI). Saos-2 is a human osteosarcoma cell line which lacks endogenous p53 These cells were grown in McCoy’s 5A medium supplemented with 15% FBS and penicillin-streptomycin (50 units/ml and 5 mg/ml, respectively). The appropriate cDNA volumes used were 0.1 and 1 ϫ 10Ϫ3 ␮l for each NER gene analyzed and ␤-actin, respectively These dilutions were determined to be in the linear range of each standard curve. Each sample was mixed with 0.25 mM dATP, dGTP, dTTP and 0.025 mM dCTP, 1 ϫ Taq polymerase buffer, 50 pmol of both forward and reverse NER gene specific primers or ␤-actin primers, 5 ␮Ci of [␣-32P]dCTP (Amersham Pharmacia Biotech), and 2.5 units of Taq polymerase in a final volume of 50 ␮l. The membranes were blocked with 10% low fat milk in PBS and incubated overnight with the corresponding antibody, mouse monoclonal anti-Sp1 (clone 1C6 from Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-XPB, or mouse monoclonal anti-

TABLE I Primer sequences used for NER genes

RESULTS

DISCUSSION