Nucleosomal Location of the STE6 TATA Box and Matα2p-Mediated Repression

Abstract

It has been proposed that yeast MATa cell-specific genes are repressed in MATα cells by the Matα2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the MATa-specific gene close to the nucleosomal pseudodyad. In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker. These plasmids contain different lengths of synthetic random DNA between the Matα2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid. We show that in MATα cells, a nucleosome is retained in an identical translational frame relative to the Matα2p operator in all the constructs investigated, irrespective of the sequence of the DNA wrapped onto the histone octamer. This result shows that the nucleosomal organization of the STE6 promoter in MATα cells is not conferred by the sequence of the promoter itself. No expression of the lacZ reporter gene was detectable in MATα cells in any of the constructs, even with the TATA box located in a short internucleosomal linker. These data indicate that repression of MATa-specific genes in MATα cells does not require the precise translational placement of the TATA box close to the nucleosomal pseudodyad; the gene remains repressed when the TATA box is located within the investigated 250-bp region in the organized chromatin domain abutting the Matα2p operator in MATα cells and may remain repressed with the TATA box located anywhere within this organized repression domain.