C/EBP-like proteins binding to the functional box-alpha and box-beta of the second enhancer of hepatitis B virus.

Abstract

The second enhancer (enhancer II) of hepatitis B virus is functionally liver specific. Located within an open reading frame of the virus and immediately upstream of the initiation sites of viral major transcripts, enhancer II furnishes a unique model for use in investigating the structure and function of an enhancer. In this study, two functional constituents, a 23-bp box-a and a 12-bp box-jI, are identified as being both necessary Control of gene transcription is in part regulated by the presence of cis-acting DNA elements that interact with specific nuclear transcription factors. Enhancers, which act in a position-and orientation-independent manner, are key regulatory elements in the transcriptional regulation of viral and cellular genes (18,26). The 72-bp repeat of simian virus 40 (SV40) is the best-characterized enhancer. The SV40 72-bp enhancer, which is composed of multiple regulatory cis-acting elements via the interaction with ubiquitous and cell-type-specific transcriptional factors, orchestrates the expression of viral genes in many cells (13,(28)(29)(30). We are interested in the study of gene regulation in hepatitis B virus (HBV), in particular its enhancer elements. HBV infection is one of the leading causes of acute and chronic hepatitis. The latter has been associated with liver cirrhosis and hepatocellular carcinoma (4). A better understanding of the molecular biology of HBV will provide deeper insight into its life cycle and the myriad disease processes in which it is involved. function in vivo (39). In this study, fine mapping of the enhancer region was analyzed with serial deletion mutants. We have identified a 23-bp sequence (designated box-cx) in the IT-A element and a 12-bp sequence (designated box-,) in the IT-B element, the interaction of which is both necessary and sufficient for enhancer II activity. Furthermore, proteins that display binding activities to box-a and box-, are found to be present in nuclear extracts of the differentiated human hepatoma cell line HepG2 by DNase I footprinting and gel shift analyses. It is likely that some of these binding proteins are C/EBP-like proteins, although they are not identical to C/EBP. MATERIALS AND METHODSPlasmid constructions. The plasmid with a 3' deletion of the enhancer II region was generated by BAL31 exonuclease digestion and confirmed by double-strand DNA sequencing. Fragments with serial 3' deletions were inserted downstream of the chloramphenicol acetyltransferase (CAT) gene of plasmid pA3SVpCAT (39) to generate a series of plasmids named pA3SVpCATENdl. To construct plasmids containing serial 3' deletions in IT-A but intact II-B elements, the fragment containing the IT-B sequence (nt 1704 to 1741) was added downstream of the enhancer II sequence in the pA3SVpCATENdl series of plasmids, which have lost II-B in its entirety, to make a series of plasmids designated pA3SVpCATAdlB. Plasmids containing the intact box-a (nt 1636 to 1668) but the sequential deletions from the 3' end of II-B were constructed by replacement of the TI-A sequen...

A study on sequence variations in pre‐S/surface, X and enhancer II/core promoter/precore regions of occult hepatitis B virus in non‐B, non‐C hepatocellular carcinoma patients in Taiwan

“…Moreover, the same sequence can also function as an upstream regulatory element activating the core promoter in a position-and orientation-dependent manner. [45][46][47] Although the precise mechanism is still unclear, our study suggested that G1721A was a useful viral marker for HCC in occult HBV carriers.…”

Wild-type levels of pregenomic RNA and replication but reduced pre-C RNA and e-antigen synthesis of hepatitis B virus with C(1653) --> T, A(1762) --> T and G(1764) --> A mutations in the core promoter.

“…2 A) and transcription factor binding sites (Lo! pez-Cabrera et al, 1990 ;Yuh & Ting, 1991 ;Chen et al, 1995 ;Raney et al, 1997) are shown.…”

“…The few mutations present in the upstream regulatory sequence (URS) of our promoter fragment are unlikely to exhibit a selective effect on one of the two core promoter transcripts because the URS regulates pre-C and C mRNA synthesis in a co-ordinate manner (Yuh et al, 1992). Of particular interest is the C-1653 T mutation in the URS as it is usually combined with the T-1762\A-1764 mutations, associated with fulminant hepatitis (Ogata et al, 1993 ;Hasegawa et al, 1994 ;Kaneko et al, 1995) and located in the alpha box, which is a strongly activating element of both enhancer II and core promoter (Yuh & Ting, 1991 ;Yuh et al, 1992). Interestingly, this mutation converts the alpha box binding site for C\EBP and related factors (Lo!…”

“…Nucleotide sequences of the mutant (Mut) and wild-type (Wt) (Galibert et al, 1979) Cp/EnII fragments functionally analysed. Regulatory elements of the Cp/EnII (Yuh & Ting, 1991 ;Yuh et al, 1992), the transcriptional start sites of the pre-C mRNA as determined by primer extension analysis (Fig. 2 A) and transcription factor binding sites (Lo!…”

“…Cells were harvested after 4 days. For primer extension analysis total RNA was purified using the guanidinium-lysis method (Chomczynski & Sacchi, 1987) and 20 µg thereof was denatured and hybridized to 0n2 pmol $#P-labelled primer P1 (1991( -1973. The primer was elongated with 5 U AMV reverse transcriptase (Promega) in the presence of 50 µg actinomycin D\ml and 1 U RNasin\µl (Promega).…”