Yeast cells exposed to environmental stress respond through conserved MAPK pathways resulting in rapid transcriptional induction of stress-responsive genes. The regulation of gene transcription in eukaryotic cells involves a dynamic balance between packaging of regulatory sequences into chromatin and access of transcriptional regulators to these sequences (Cairns, 2009
). Here we describe a novel connection between MAPK signaling and chromatin remodeling. The combination of gene expression analyses, ChIP, and chromatin-remodeling assays allowed us to establish a model for the sequence of events during transcriptional initiation upon cell wall stress (). The MAPK Slt2 mediates, through Rlm1, nucleosome rearrangements at CWI-responsive genes by selective targeting of the SWI/SNF remodeling complex. This process is essential for assembly of the transcription preinitiation complex and proper orchestration of transcriptional adaptive responses.
FIGURE 8: Model for transcriptional activation of CWI-responsive genes. In MLP1, those exposed Rlm1-binding sites would allow transcription factor access to the promoter, whereas occluded binding sites within nucleosomes would not be accessible. Under cell wall (more ...)
Nucleosome positioning can influence the accessibility of the DNA-binding sites in the promoters to specific transcription factors, and consequently occupancy of such binding sites can be regulated by chromatin-remodeling factors (Li et al., 2007
; Jansen and Verstrepen, 2011
). The nucleosome-positioning pattern characterized here for MLP1
correlates well with data from multiple high-resolution genome-wide maps of in vivo nucleosomes across the S. cerevisiae
genome (Jiang and Pugh, 2009
). The MLP1
promoter presents two DNA-binding sites for Rlm1: BOX1 (−359/−350) and BOX2 (−510/−501). The former seems to be exposed in the linker DNA between nucleosomes −3 and −2, or partially exposed at the −2 nucleosome edge, whereas the latter would be completely occluded within nucleosome −3. The exposed site would allow the transcription factor Rlm1 access to the promoter in a first step, but chromatin remodeling would be required under stress to expose the additional nucleosomal site in a two-step model for activation (). Such a requirement has also been established at the yeast PHO5
promoter (Fascher et al., 1990
; Cairns, 2009
). In our ChIP assays, Rlm1 is bound to the promoter of the cell wall–responsive genes in the absence of stress. This basal binding of Rlm1 is independent of Slt2 kinase activity and phosphorylation of the MAPK by Mkk1/Mkk2, suggesting that Rlm1 preloaded into CWI-responsive genes could be inactive. However, this binding seems to be also partially dependent on the presence of Slt2. Because there is some Slt2 MAPK activity under vegetative growth conditions, a small amount of active Rlm1 could be bound to the CWI gene promoters in the absence of stress. In agreement, a very small amount of SWI/SNF also resides on the MLP1
promoter under nonstress conditions, which is dependent on Slt2 and Rlm1.
On cell wall stress, Slt2 interacts with and phosphorylates Rlm1, regulating its transcriptional activation function (Watanabe et al., 1997
; Jung et al., 2002
). We show here that recruitment of Rlm1 to cell wall stress genes is highly induced by stress and it requires phosphorylation by Slt2 and the activity of SWI/SNF. Because SWI/SNF is targeted to the promoters of the cell stress genes and this recruitment leads to clear alterations of nucleosome architecture, our data favor a model in which chromatin remodeling by the SWI/SNF complex would facilitate the binding of the transcription factor and then stimulate transcriptional initiation by Pol II (). Thus massive recruitment of Rlm1 to the MLP1
promoter under cell wall stress requires preliminary action by the SWI/SNF complex. In support of this model, it has been shown that the SWI/SNF complex can facilitate Gal4 binding to occluded sites to promote transcription in vivo by removing nucleosomes that occupy the Gal4-binding site (Burns and Peterson, 1997
The absence of Rlm1 also abolished the binding of SWI/SNF to the cell wall stress gene promoters. This interdependence between Rlm1 and SWI/SNF indicates a close functional connection between both elements for promoter recruitment. How is the remodeling complex recruited to specific CWI gene promoters? The existence of a direct physical interaction shown here, both in vitro and in vivo, between Rlm1 and SWI/SNF, and the fact that recruitment of SWI/SNF depends on MAPK and Rlm1 favor a model in which the remodeling complex is targeted to the promoters via interaction with the transcription factor Rlm1 (). Physical interaction with the specific transcription factor has been shown to be one of the main determinants for the recruitment of the SWI/SNF remodeling complex to different gene promoters (Cosma et al., 1999
; Neely et al., 1999
; Yudkovsky et al., 1999
). The interaction of activated Rlm1 and the remodeling complex could take place in solution, and then those Rlm1 DNA-binding sites that are not occupied by the transcription factor under nonactivating conditions would direct the remodeling complex to the chromatin (). Alternatively, Rlm1 prebound to the accessible DNA-binding sites within the promoter could be activated by Slt2, leading to the recruitment of SWI/SNF. The two alternatives do not exclude one another.
The mechanisms of regulation of gene expression proposed here are not restricted to MLP1 but they seem to represent a general mechanism for transcriptional activation of the CWI responsive–genes regulated by Rlm1. Supporting this, we show that 1) the global yeast gene expression profile in response to cell wall stress is severely affected in swi3Δ mutants, 2) the SWI/SNF complex is also recruited to other promoters of CWI-responsive genes, like SRL3 and YLR194C, in a Slt2- and Rlm1-dependent manner, 3) Rlm1 recruitment to these promoters is also dependent on SWI/SNF, and 4) deletion of SWI/SNF elements renders cells hypersensitive to cell wall stress.
As a conclusion, the results presented in this work characterize the SWI/SNF complex as a novel element of the CWI MAPK pathway. Recruitment of this complex to cell wall stress–responsive genes mediates the chromatin remodeling necessary for developing adequate transcriptional responses upon cell wall stress. In addition, several elements of the SAGA histone-modifying complex were also uncovered in the screening developed here. Future work is necessary to define the nature of possible cooperation between the two complexes in the regulation of these processes.