Repression of enhancer activity by the adenovirus E1A tumor protein(s) is mediated through a novel HeLa cell nuclear factor

A.E Reifel-Miller,D.T Berg,B.W Grinnell

doi:10.1016/s0021-9258(18)92783-3

Abstract

We have examined the mechanism for the host cell-dependent repression of enhancer activity by the adenovirus early region 1A (E1A) proteins. The enhancer used in this study, from the human BK virus P2, 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. In addition, enhancer activity can be stimulated by the E1A gene products in these cells. However, cis-enhancer activity is repressed in the HeLa cell line, and we demonstrate here that further repression can be induced by the E1A proteins. We show that the binding site for the negative regulatory factor involved in cis-repression, designated BK virus enhancer factor 1 (BEF-1), is also required for E1A-induced repression. Using gel mobility retardation assays, we demonstrated a 4-fold increase in active BEF-1 in nuclear extracts containing the E1A proteins. However, the E1A proteins did not change the binding pattern or the strength of binding of BEF-1 to its target sequence. BEF-1 was identified as a 98-kDa nuclear factor, and phosphorylation was shown to be important for DNA binding. Three potential nuclear factor 1 (NF-1) sites are present in the BEF-1-binding site. Using a known NF-1 site as competitor DNA in a gel mobility retardation assay, we provide evidence that BEF-1 may be a newly identified NF-1 family member. In addition, the sequence TGA present in the repressor-binding site was shown to be essential for high affinity binding of BEF-1. Overall, our data demonstrate that an enhancer can be repressed by the trans-activation of a negative regulatory factor.

Highlights

Site for the negative regulatory factor involvedciisn- The host cell specificity of enhancer activity could be conrepression,designated BK virusenhancerfactor 1 trolled by thepresence or absence of either positively or (BEF-l),is required forE 1A-induced repression. negatively acting factors
In the present study,we have examined induced repression the ligated oligonucleotides were blunted with the Klenow fragment at the enhancerof the human BK virus P2 (BKV-P2) (49) by of DNA polymerase I
We have shown that the activity of after transfection,the cells were trypsinized and washed, and thecell the BKV-P2 enhancer is host cell-dependent, being under lysates were analyzed for CHCls extraction and ethanol icol acetyltransferase (CAT) activity by the method of Gorman et negative regulation in HeLa cells, and thata 25-bp sequence spanning the junction between the 68- and 32-bp repeats is responsible for binding of a repressor protein (51)

Summary

RESULTS

T o determine the effect of the E1A proteins on the activity of the BKV-P2 enhancer in HeLa cells, where cis activity is repressed, the CAT gene expression plasmids shown in Fig. 1A were introduced into HeLacells withand without an E1Aexpressing vector. The level of CAT activity from pLP-CAT was 2-fold greater following cotransfection with pSVElA (lane 2) as compared to cotransfection with pSV2HNXB (lane 1) This result is in agreement with other investigators who have shown that theE1A proteins activatethe MLPapproximately 2-fold in HeLa cells (73, 74). Repression by ElA Is Mediated Through BEF-1-As indicated above, the BKV-P2 enhancer can be negatively regulated in HeLa cells through binding of a nuclear factor or factor complex (51) referred to asBEF-1. In these experiments, a relatively high level of pBL2-CAT was used, which gave approximatelya 15-fold increase in enhancer-dependent CAT activity relative to pLP-CAT. The amount of probe bound was much higher with the Ad5-

Ad5 dl312

B Phosphatase treated pg protein

DISCUSSION