A Sequence within PHR1 Promoter Acts as a Gate for Cross-Talks between Damage-Signaling Pathway and Multi-Stress Response

Rph1 and Gisl are damage-responsive repressors involved in PHR1 expression. They have two $C_{2}$H/ sub 2/ zinc finger motifs as putative DNA binding domains and N-terminal conserved domain with unknown function. They are also found in the human retinoblastoma binding protein 2 and the mouse jumonji- encoded protein. The repressors are able to bind to A $G_{4}$ sequence within a 39-bp sequence called upstream repressing sequence of PHR1 promoter (UR $S_{PHR1}$) responsible for the damage-response of PHR1. We report here that Rph1 is predominantly localized in the nucleus as examined by fluorescence microscopic analysis with GFP-Rph1 fusion protein. On the basis of the fact that the A $G_{4}$ sequence that is recognized by Rph1 and Gisl is also recognized by Msn2 and Msn4 in a process of stress response, we a1so tried to examine the in vivo function of A $G_{4}$ and the role of Msn2 and Msn4 in PHR1 expression. Our results demonstrate that Msn2 and Msn4 are actually required for the basal transcription of PHR1 expression but not for its damage induction. When A $G_{4}$ sequence was inserted into the minimal promoter of the cyc1-LacZ reporter, the increased LacZ expression was observed indicating its involvement in transcriptional activation. The data suggest that the A $G_{4}$ is primarily required for basal transcriptional activation of PHR1 or CYC1 promoter through the possible involvement of Msn2 and Msn4. However, since the A $G_{4}$ is also involved in the repression of PHR1 via Rphl and Gisl, it is proposed that A $G_{4}$ functions as either URS or upstream activating sequence (UAS) depending on the promoter context.t.