By examining the molecular mechanisms of a transcriptional switching event that underlies a change in yeast mating type, we have observed that asg are rapidly derepressed upon loss of the α2 repressor. Strikingly, the accumulation of asg mRNAs is coincidental with the removal of α2 from its target promoters. This rapid derepression of cell-type-specific genes does not result from the dissociation of the Ssn6-Tup1 corepressor complex, which remains associated with asg promoters long after removal of α2. Rather, the asg appear to be primed for rapid derepression by Ssn6-Tup1 while still in the actively repressed state, poised to begin transcription as soon as the removal of α2 from asg promoters is initiated.
How does the cell maintain the cell-type-specific promoters in a stable and strongly repressed state but allow these same regulatory elements to transition to an active state on such a rapid timescale? Two ideas have been proposed previously for such a process. In one scenario, transcriptional initiation occurs in both the repressed and active states, but in the former, RNA polymerase II is stalled during the elongation phase, effectively blocking transcript production (18
). The best-characterized example of such a regulatory mechanism is the transcriptional control of the hsp70
locus in Drosophila
), where a preloaded polymerase allows for a rapid and robust induction of hsp70
transcription in response to environmental stress (63
). While removal of an elongation block allows for fast transcriptional responses because the enzymatic machinery is already engaged, the potential for spurious activation is probably high since only a single barrier to transcript production needs to be overcome. Leaky expression of hsp70
is not detrimental, but the inappropriate activation of other genes, such as proto-oncogenes and lineage-specifying developmental regulators, could be disastrous to the biology of the cell. Nevertheless, a paused polymerase has been mapped on an appreciable fraction of genes by ChIP (41
), although the biological significance of this localization for the expression of most of these genes is not yet clear. Observations from genome-wide studies that have mapped the posttranslational modifications of chromatin from embryonic stem (ES) cells have led to another hypothesis that could explain rapid switching between alternate transcriptional states. The promoters of genes encoding developmental regulators and key signaling factors, which are inactive in ES cells but induced rapidly upon cell differentiation, display so-called bivalent chromatin marks that have characteristics of both repressed and active transcriptional states at once (1
). Such hybrid configurations have been likened to a neutral or ambiguous state that can quickly transition to either extreme in response to external stimuli or intrinsic developmental cues; thus, the bivalent chromatin state may poise these inactive genes for subsequent activation. Compared to the single barrier blocking active transcription with an engaged but stalled polymerase, a mechanism involving bivalent marks could still receive regulatory inputs at the multiple steps along the pathway leading to the initiation of transcription. Thus, regulatory systems based on bivalent chromatin states may provide for transcriptional switches that transition rapidly given the appropriate signal but are still faithful and robust when confronted with spurious, weak, or transient signals.
Do such hybrid states represent the initiation of a transcriptional switch, or do the bivalent marks arise through a mechanism that is distinct from the active state? In the case of a
sg, the potent coactivator Gcn5 appears to be recruited to active promoters since its associated histone acetyltransferase (HAT) activity targets histone H3 in the resident nucleosomes. As expected, H3 is acetylated on K9, K14, K18, and K23 at a
sg promoters in the active state, but surprisingly, full acetylation of H3K9 under the same conditions is dependent upon the corepressor Tup1 (Fig. ). Since α2 recruits Ssn6-Tup1 through direct protein-protein interactions (33
) and is thought to be essential for the interaction of Ssn6-Tup1 with its a
sg targets, the finding that Tup1 has a functional role in the absence of α2 was not anticipated. However, Ssn6-Tup1 remains associated with a
sg promoters long after the loss of α2 (Fig. ) and has a positive role in STE6
expression (Fig. ). Nevertheless, Tup1 does not strongly occupy a
sg in a
cells, so a mechanism must exist to reduce the amount of Ssn6-Tup1 associated with promoters as cells transition to the fully active transcriptional state. A plausible model for such a mechanism is suggested by the preferential interaction of Ssn6-Tup1 with underacetylated histones (11
). After the loss of α2 from a
sg promoters, Gcn5-mediated acetylation of histone H3 likely increases the acetylation density of the H3 N-terminal tails by producing multiply acetylated isoforms. This enhanced density of H3 acetylation would impair the interaction of Ssn6-Tup1 with promoter chromatin, thereby leading to lower levels of the complex on its a
sg targets. Indeed, increasing histone acetylation by abrogating histone deacetylase activity strongly decreases the association of Tup1 with its target promoters (9
There are precedents for the persistent association of Ssn6-Tup1 with its regulatory targets after derepression (39
). For a subset of these genes (the glucose- and osmotic-shock-repressed genes), the DNA-binding repressors that recruit the Ssn6-Tup1 complex (Mig1 and Sko1, respectively) also remain bound under activating conditions. In these transcriptional switches, Ssn6-Tup1 transitions to a coactivator role after a signal-induced phosphorylation event on Mig1 or Sko1 disrupts a physical interaction between the DNA-binding proteins and Ssn6-Tup1 (44
). For a
sg, the functional transition in Ssn6-Tup1 occurs only after the loss of α2 from target promoters, suggesting that the effects of α2 on Ssn6-Tup1 activity serve an important role in repression. Thus, α2 may operate as the switching mechanism to transition the Ssn6-Tup1 complex from its function as a coactivator (where it serves to recruit the Gcn5 HAT [45
]) to that of a corepressor (recruiting, among other repressive factors, histone deacetylases [HDACs] [64
]). In this scenario, the Ssn6-Tup1 complex serves as a hub, coordinating distinct and antagonistic chromatin-modifying activities and perhaps functioning in a manner analogous to that of a simple automobile transmission or gearbox, shifting the histone acetylation reaction into forward (Gcn5 catalyzed) or reverse (HDAC catalyzed).
Strikingly, the control of Ssn6-Tup1 function by α2 is not absolute. In the repressed state where α2 is present, Gcn5 is still recruited to a
sg promoters, but under these conditions, the activity of Gcn5 is conspicuously constrained to a subset of its potential acetylation sites on histone H3 (H3K9 and H3K23) (Fig. ). Such a scenario may arise by the steric occlusion of some target lysine residues on the H3 amino-terminal tails by their interactions with the Ssn6-Tup1 corepressors (11
). Alternately, Gcn5 may have equal access to all sites, but HDACs play a role in establishing the acetylation pattern. In either case, Gcn5 is required for the rapid derepression of the a
upon loss of α2 (Fig. ) and the presence of partial H3 acetylation on a
sg promoters in α cells strongly correlates with the rapid derepression kinetics. Similar roles for Gcn5 and the SAGA complex in promoting the rapid kinetics of gene activation have also been observed for the inducible PHO5
). Interestingly, SAGA is recruited to the PHO5
promoter only under inducing conditions (2
), whereas Gcn5 is found at a
sg promoters in both the active and repressed states (Fig. ). These findings suggest that the Gcn5-containing complexes may regulate these different promoters in mechanistically distinct ways.
The partial acetylation of H3 at repressed promoters may generally be part of a mechanism to evoke rapid transcriptional induction. Of the known Ssn6-Tup1-targeted promoters, a
sg derepression occurs on the shortest timescale. For example, glucose-repressible and hypoxia-induced genes typically do not reach full activation until ~2 h after being induced (17
), and osmotic-shock genes display a 15-min lag before activation begins (50
). Although differences in the timing of perceiving and propagating the induction signal likely also contribute to these varied reactivation kinetics, the lag in the transcription of osmotic-stress genes is not the result of slow signal transduction or salt uptake since changes at these osmotic-regulated promoters are observed within 3 min of the addition of salt (49
). Despite this wide range of response times, our observations suggest a direct correlation between reactivation kinetics and H3K9 acetylation, with the most rapidly activated promoters containing marks of partial activation (Fig. ). In addition, mot3
Δ cells derepress a
sg very slowly and contain poorly acetylated nucleosomes in the repressed state (Fig. ). Thus, our work suggests that in addition to providing fidelity to transcriptional switches, such hybrid chromatin states may also actively contribute to the kinetics of such transitions.
Are hybrid states the result of partial activation or incomplete repression? At a
sg promoters, α2 does not saturate its target sites in the repressed state (65
), leading to the possibility that the activity of Ssn6-Tup1 cycles between corepressor and coactivator at individual promoters. In this scenario, the presence of α2 switches Ssn6-Tup1 to its repressor function, but the DNA-binding dynamics of α2 also create a state that is poised for rapid derepression by allowing the deposition of at least some active chromatin acetylation marks. Our studies suggest that the biologically stable repressed and active transcriptional states of a
sg are actually metastable forms connected by a continuum of partially active/partially repressed states. Master regulators like α2 play key roles in such dynamic configurations by allowing both the stable endpoints and the rapid transitions between the intermediate states. Such a mechanism is likely to be a widespread regulatory strategy, given the strong biological parallels between the phenotypic transitions that occur during yeast mating-type switching and those that take place in differentiation events in higher eukaryotes.