Functional requirements of histone deacetylases in Tup1-Ssn6 repression

The corepressor complex Tup1-Ssn6 regulates many classes of genes in yeast including cell type specific, glucose repressible, and DNA damage inducible. Tup1 and Ssn6 are recruited to target promoters through their interactions with specific DNA binding proteins such as α2, Mig1, and Crt1. Most promoters that are repressed by this corepressor complex exhibit a high degree of nucleosomal organization. This chromatin domain occludes transcription factor access to the promoter element resulting in gene repression. Previous work indicated that Tup1 interacts with underacetylated isoforms of H3 and H4, and that mutation of these histones synergistically compromises repression. These studies predict that Tup1-hypoacetyalted histone interaction is important to the repression mechanism, and in vivo hyperacetylation might compromise the corepressors ability to repress target genes. One way to alter histone acetylation levels in vivo is to alter the balance between histone acetyltransferases and histone deacetylases. To date five histone deacetylases (HDACs) have been identified in yeast Rpd3, Hos1, Hos2, Hos3 and Hda1. Deletion of single or double HDAC genes had little to no effect on Tup1-Ssn6 repression, but simultaneous deletion of three specific activities Rpd3, Hos1, and Hos2 abolished repression in vivo. Promoter regions of Tup1-Ssn6 target genes in these triple deacetylase mutant cells are dramatically hyperacetylated in both H3 and H4. Examination of bulk histone acetylation levels showed that this specific HDAC triple mutant combination (rpd3 hos1 hos2) caused a dramatic and concomitant hyperacetylation of both H3 and H4. The loss of repression in the rpd3 hos1 hos2 cells, but not in other mutants, is consistent with previous observations, which indicate that histones provide redundant functions in the repression mechanism and that high levels of acetylation are required to prevent Tup1 binding. Investigation into a potential direct interaction between the Tup1-Ssn6 corepressor complex and one or more HDAC activities showed that both Rpd3 and Hos2 interact with the corepressor complex in vivo. These findings indicate that Tup1-Ssn6 repression involves the recruitment of histone deacetylase activities to target promoters, where they locally deacetylate histone residues promoting Tup1-histone tail interaction to initiate and/or maintain the repressed state