Regulation of the HAP1 gene involves positive actions of histone deacetylases

Abstract

The yeast transcriptional regulator Hap1 promotes both transcriptional activation and repression. Previous studies have shown that Hap1 binds to the promoter of its own gene and represses its transcription. In this report, we identified the DNA site that allows Hap1-binding with high affinity. This Hap1-binding site contains only one CGG triplet and is distinct from the typical Hap1-binding upstream activation sequences (UASs) mediating transcriptional activation. Furthermore, at the HAP1 promoter, Ssa is bound to DNA with Hap1, whereas Hsp90 is not bound. Intriguingly, we found that histone deacetylases, including Rpd3, Hda1, Sin3 and Hos1, are not required for the repression of the HAP1 gene by Hap1. Rather, they are required for transcriptional activation of the HAP1 promoter, and this requirement is dependent on the HAP1 basal promoter. These results reveal a complex mechanism of transcriptional regulation at the HAP1 promoter, involving multiple DNA elements and regulatory proteins.

Fig. 1

The DNA-proteins complexes formed on various regions of the HAP1 promoter. Note that the “−” sign in the nucleotide number is omitted in the Figure. Extracts were prepared from cells bearing a Hap1 expression vector (+ Hap1) or an empty vector (− Hap1). Extracts were incubated with radiolabeled DNA fragments containing various regions of the HAP1 promoter.

Fig. 2

The effects of unlabeled competitor Hap1-binding site on the formation of DNA-protein complexes formed on various regions of the HAP1 promoter. Extracts were prepared from cells bearing a Hap1 expression vector (+ Hap1) or an empty vector (− Hap1). In lanes 4, 8, 12, 16 and 20, no extract (marked as F) was included. In lanes 3, 7, 10, 15 and 19 (marked as C), unlabeled DNA containing a high affinity Hap1-binding UAS site was included as competitor DNA.

Fig. 3

The effects of Hap1 antibodies on the formation of the DNA-protein complexes formed on various regions of the HAP1 promoter. Extracts were prepared from cells bearing a Hap1 expression vector (+ Hap1) or an empty vector (− Hap1). Extracts were incubated with radiolabeled DNA fragments containing various regions of the HAP1 promoter. In lanes 3, 7, 10, 15, 19 and 23 (+ Ab), antiserum against Hap1 was included in the reaction mixtures; in lanes 4, 8, 12, 16, 20 and 24, pre-immune serum was included in the reactions mixtures.

Fig. 4

(A) The levels of Hap1 proteins pulled down by DNA containing various HAP1 promoter regions. Extracts prepared from cells expressing Hap1 were used to perform DNA pull-downs with DNA containing various regions of the HAP1 promoter or a mutant Hap1-binding site (Mut, CGGACTCATCCG). (B) Sequence comparison of typical UAS sites mediating Hap1 transcriptional activation and the identified Hap1-binding site in the HAP1 promoter. Shown here are the UASs of the CYC1, CYT1, CTT1, and CYC7 promoters, and the −341 to −380 sequence of the HAP1 promoter. (C) Ssa, but not Hsp90, is associated with Hap1 on the HAP1 promoter. DNA pull-downs were performed with DNA containing the −341 to −380 or −345 to −373 sequence of the HAP1 promoter or a mutant Hap1-binding site (Mut, CGGACTCATCCG). In lanes 2, 4 and 6 (+ heme), 2 μg/ml heme was included in the reaction mixtures.