Rme1p functions both as a repressor and an activator of transcription. The three zinc fingers in the Rme1p C-terminal region are required for DNA binding (Figure ) (Covitz and Mitchell, 1993
; Shimizu et al., 1997a
; Shimizu et al., 2001
). We have shown here that the Rme1p N-terminal region is necessary and sufficient for both repression and activation (Figures and ). Thus, the role of Rme1p as an effector of transcription is not simply to displace proteins that bind to overlapping DNA sites.
Figure 7 Structure-functional summary of Rme1p. The C-terminal part of Rme1p contains three zinc fingers (ZF boxes) and C-terminal segment (CTR) with properties of α-helix. This part of the protein is important for DNA- binding (Shimizu et al., 2001 ). (more ...)
The minimal regions required for repression and activation overlap completely and lie between amino acids 61 and 148 (Figure ). It is unusual for a protein to have a single effector region that directs both repression and activation. For example, Ume6p has a central repression domain that interacts with Sin3p and Rdp3p (Kadosh and Struhl, 1997
) and an N-terminal activation domain that interacts with Rim11p and Ime1p (Bowdish et al., 1995
; Rubin-Bejerano et al., 1996
; Malathi et al., 1997
). Rap1p has neighboring but separable repression and activation domains (Sussel and Shore, 1991
). It is possible that the single Rme1p transcriptional effector domain interacts with a single target protein whose activity—repression or activation—is dictated by neighboring proteins or the chromatin environment. A second possibility is that the N-terminal region of Rme1p can interact with two different proteins or complexes that individually yield exclusively repression or activation.
The Rme1p N-terminal effector domain shows no extensive homology to known activation or repression domains, and has no significant primary sequence identity to other proteins in current databases. However, the effector domains, does contain residues and possible secondary structures that are implicated in activation or repression by other transcription factors. For example, it contains stretches of bulky hydrophobic amino acids and charged residues (Figure ). Two regions (85–98 and 116–122) are predicted to form α-helices, which may facilitate protein–protein interactions. These patches of similarity to other repressors and activators are consistent with the possibility that Rme1p has interdigitated residues that contribute only to repression or activation. This model predicts that mutational alteration of specific Rme1p N-terminal residues may impair only repression or activation, in contrast to the broad effects of the deletions studied here. However, we note that random mutagenesis of RME1 has yielded numerous mutations that impair DNA binding, but none that specifically impair repression. It is possible that the individual Rme1p effector segments function redundantly, so that multiple point mutations would be necessary to inactivate a specific effector function. Our deletion analysis here is consistent with such a model, in that regions flanking the effector domain can augment repression and activation (Figure ).
The observation that Rme1p activation and repression domains overlap brings to the foreground the question of whether identical protein complexes form at Rme1p-repressed and Rme1p-activated promoters. One simple possibility is that the Mediator or a smaller Rgr1p-Sin4p complex is the Rme1p-interacting target, because these proteins act as both positive and negative regulators of transcription (Stillman et al., 1994
). However, we have shown clearly that Sin4p and Rgr1p are not required for Rme1p-mediated activation. Thus, if RNA polymerase II holoenzyme subcomplexes are direct Rme1p targets, there must be distinct subcomplexes that are brought to the Rme1p-repressed and Rme1p-activated promoters (Myers et al., 1999
). Perhaps recruitment of a Mediator subcomplex lacking Sin4p and Rgr1p prompts RNA polymerase II to activate transcription, as occurs when the Rme1p-binding site is situated in place of a UAS.
We have favored the model that Rgr1p-Sin4p is recruited by Rme1p at repressed promoters because it explains genetic relationships simply. However, we have recently observed that lexA-Rgr1p does not repress the lexO-IME1
test gene (Blumental-Perry, 2001
), whereas lexA-Rme1p derivatives are effective repressors. In addition, the fact that Rme1p repression excludes nearby transcriptional activators from DNA (Shimizu et al., 1997b
) is not an expected consequence of direct interaction between Rme1p and the Mediator. Thus, more complex biochemical relationships must be considered. One possibility, discussed previously (Shimizu et al., 1997b
), is that Rme1p repression depends on a nucleosome structure or density that is unachievable in rgr1
mutants. This model predicts that other mutations with similar effects on nucleosome structure will also impair Rme1p repression specifically. A second possibility is that a gene specifying the hypothetical Rme1p corepressor is not expressed in rgr1
mutants. Candidate corepressor genes may then be identified through genome-wide expression surveys. A direct approach to this question is to identify proteins that interact with the Rme1p effector domain. Overlap between Rme1p repression and activation regions precludes the use of conventional two-hybrid cloning, but we expect that biochemical identification of Rme1p effector region–interacting proteins, combined with chromatin immunoprecipitation of the RC, will provide a direct route to address these mechanistic questions.