Negative regulation of the human polyomavirus BK enhancer involves cell-specific interaction with a nuclear repressor.

Abstract

We have examined the cell type-specific regulation of the human BK virus (BKV) enhancer. This enhancer functions efficiently in cis to activate expression from the adenovirus major late promoter in the human kidney cell line, 293, and in a monkey kidney cell line, MK2, but not in the HeLa cell line. In gel retardation migration assays, specific BKV enhancer-protein complexes could be observed by using nuclear extracts prepared from each cell line. Moreover, a unique DNA-protein complex was observed by using the HeLa cell nuclear extracts. By DNase footprint analysis, four binding regions for HeLa cell nuclear proteins were defined within the BKV enhancer repeat region. Two of the protected regions encompassed nuclear factor 1 or CCAAT transcription factor binding sites. These nuclear factor 1 sites also were protected by nuclear proteins from the 293 and MK2 cell lines. The other two protected sites encompassed a region of symmetry which included a sequence similar to the simian virus 40 TC enhancer motif and to a conserved sequence present upstream or within the introns of several cellular genes. These two sites were not protected by either the 293 or MK2 nuclear proteins. Competition studies in transfected cells indicated that the reduced activity of the BKV enhancer in the HeLa cell line was due to negative regulation. Further, we have demonstrated that binding of a nuclear factor(s) to the HeLa cell-specific site is involved in the repression of enhancer activity.The control of transcription in eucaryotic cells is a complex process involving the interaction of cellular protein factors with specific gene sequences. A number of trans-and cis-acting elements involved in the regulation of transcription initiation have been described. For example, the cisacting enhancer sequence, first described as a repeat sequence in the regulatory region of simian virus 40 (SV40), can stimulate transcription from homologous and heterologous promoters, independent of position and orientation (for reviews, see references 18 and 32). The activity of enhancer sequences can be modulated by factors in trans as demonstrated by the ability of viral early proteins such as the adenovirus ElA proteins to repress (5,25,49) or stimulate (6, 21, 29) enhancer activity. In addition to viral trans-acting factors, a number of investigators have identified binding sites for nuclear proteins within enhancer sequences (2,3,15,30,37,40,41,47), and there is evidence that the binding of trans-acting proteins regulates enhancer activity (1,4,17,34,35,45,48,53).For both viral and cellular enhancer sequences, host cell specificity has been observed (21, 38), and recent studies have described host cell-specific binding of nuclear proteins to enhancer sequences (2,11,34,35,51). Host cell specificity of enhancer activity could be controlled by the presence or absence of either positively or negatively acting factors. For example, Atchison and Perry (1) suggest the kappa gene is regulated in development by the presence or absence of a positively acting fact...

Expression of the vasopressin and oxytocin genes in rats occurs in mutually exclusive sets of hypothalamic neurons

“…[16] and estrogen-responsive elements (ERE) [17] upstream of the transcription start site, suggesting that this gene is under steroid control (table 1). In this region the oxytocin gene also shares homology with a negative regulatory sequence motif (AGTGCATGACTGGGCAGC-CAGCCAGTGGCAG) of the human polyomavirus BK enhancer which binds a HeLa cell-specific potential repressor [18]. This sequence element overlaps partially with an ERE of the oxytocin gene -91 to -72) suggesting that positively and negatively acting protein factors may be involved in the expression of this gene.…”

E1A-induced enhancer activity of the poly(dG-dT).poly(dA-dC) element (GT element) and interactions with a GT-specific nuclear factor.

“…With increasing concentrations of 293 cell nuclear extract (lanes 1 to 3), we observed a protected region corresponding to the GT element. Approximately fourfold less nuclear extract was needed to obtain an equivalent level of protection of the BK virus enhancer sites I and III (11), suggesting that the activity or concentration of the GT binding factor is less than that of the BK virus enhancer-binding factor (data not shown). In gel retardation experiments using a 32P-labeled synthetic poly(GT) element (Fig.…”

“…The low-level activity of the BK virus enhancer in HeLa cells was shown previously to be due to repression by a specific nuclear factor (11). The ElA gene products have been shown to activate (3,10,22) as well as to repress (2, 19, 36) the activity of enhancer sequences, and we previously reported that the BK virus enhancer, which is stimulated by ElA in kidney cells (10), is repressed by the ElA proteins in the HeLa cell line (11). In the presence of the ElA proteins, the level of CAT from pBL2-CAT was reduced approximately 3.5-fold and that from pBL(GT)-CAT was reduced approximately 11-fold.…”

“…Experiments were performed to determine whether a specific nuclear factor(s) interacted with the GT element and, if so, whether or not the ElA protein(s) affected the interaction(s). Nuclear extracts were prepared, and specific binding of protein factors to the poly(GT)21 element was determined by DNase footprint and gel migration retardation analyses by using conditions described previously (11). To assure that nuclear extracts from each cell line were as equivalent as possible, extracts were prepared at the same time and under identical conditions and were shown to be transcriptionally active, as measured by runoff transcription from the MLP (12).…”

Subpopulations of non-coding control region variants within a cell culture-passaged stock of BK virus: sequence comparisons and biological characteristics

“…The biological importance of BKV NCCR variation probably ties with the resultant divergence in number, types and interdistance of cis-acting binding motifs for trans-acting factors (reviewed in Markowitz et al, 1990). Previous studies (Nowock et al, 1985;Grinnell et al, 1988;Markowitz & Dynan, 1988;Chakraborty & Das, 1991 ;Moens et al, 1994) have proven functional binding of the transcription factors Spl (Qa~-39), AP-I (P-P junction), NF-1 (P24-38, Ps-s/2r 3s in the P-block with internal 18 bp deletion, at the PQ junction, R32 46, R52_63 , $31_~ and 862_~gnogene) , the oestrogen receptor, the progesterone receptor and the glucocorticoid receptor (distal part of S-block). We deduced the number of binding sites for these proven transcription factors in the NCCR variants discussed here.…”