Cooperativity between an Upstream TATA-like Sequence and a CAA Repeated Element Mediates E1A-dependent Negative Repression of the H-2K Class I Gene

Xiaoren Tang,Hai-Ou Li,Osamu Sakatsume,Tomohiro Ohta,Hatsumi Tsutsui,Arian F.A Smit,Masami Horikoshi,Phillipe Kourilsky,Alain Israël,Gabriel Gachelin,Kazushige Yokoyama

doi:10.1074/jbc.270.5.2327

Abstract

In primary rodent cells transformed by the E1A region of the highly oncogenic adenovirus type 12, repression of transcription mediated by the far upstream TATA-like element was observed only in conjunction with either possible juxtaposition of a CAA repeated element in the presence of E1A and was dependent upon the relative arrangement of both the TATA-like and CAA repeated motifs in both homologous and heterologous promoter constructs. A gel shift competition study demonstrated that the TATA-binding protein (TBP) or a TBP-like protein can bind to both the upstream TATA-like sequence and the regular TATA box on the H-2Kb basal promoter. Moreover, employing immunoselection and cyclic amplification and selection of targets (CASTing) methods with nuclear extracts derived from Ad12-E1A transformants, we have identified a high affinity binding site in the H-2Kb class I promoter for E1A-associated DNA-binding proteins. The sequences of the binding sites were identified and were found to contain both the upstream TATA-like motif and the CAA repeated motifs. Our results suggest that the TATA-like sequence in the far upstream region of the H-2Kb gene is one of the elements that is required for Ad12-E1A-mediated negative repression.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM I EMBL Data Bank with accession number(s) X151 04
A distal 5'-end element between -1,521 and -1,837 that contributes to negative regulation mediated by the ElA gene products was previously defined within the H-2K!' gene [9]
The results obtained with substitution mutations of the TATA-like sequence in this region showed that this negative regulation was caused by the TATA-like sequence

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM I EMBL Data Bank with accession number(s) X151 04. The level of binding to the R1 element has been shown to be higher in the case of extracts from AdS-transformed cells than in the case of extracts from Ad12-transformed cells (2729). We showed previously that a distal 5'-element (-1,521 to -1,837 relative to the start site of transcription) that contributes to E1A-mediated negative regulation is included in the promoter region of the H-2K!> gene [9]. The CAA repeated sequences in regions -1,736 to -1,689 and -1,616 to -1,535 are both necessary for full negative regulation of the H-2K!> gene by E1A. We report here that an additional element, a TATA-like sequence, in the far upstream region of the 5'flanking sequence (-1,773 to -1,767) is a key element in the negative regulation of expression of the MHC class I H-2K!> gene by E1A in conjunction with either upstream or downstream CAA repeats. Using an immunoselection and cyclic amplification and selection of targets (CASTing) method, we obtained direct evidence that Ad12-E1A can associate with the

Methods

Results

Conclusion