Deshaies-2005-01-00987B

NIH Support- This work was supported in part by an NIH Research Project Grant (GM065997) to R.J.D.

Supplementary Discussion for Table S2

Based on our findings with Gcn4, we propose a class of regulatory factors whose activity is required in a reaction, but whose subsequent turnover or removal promotes completion of the reaction or subsequent reaction cycles. Such ‘activation by destruction’ appears to play a role in numerous cellular processes, especially those involving DNA or RNA-mediated reactions. . For example, VIP1 and VirE2 enable uptake of the agrobacterium genome into the host nucleus. Subsequently, VirF, an F-box protein, promotes the degradation of VirE2 and VIP1, exposing the genome so that it can integrate 1. Given the diversity of other examples listed in Table S2, it is plausible that ‘activation by destruction’ may represent a regulatory mechanism for a large, underappreciated class of factors and may be an important determinant of infection and disease. The challenge remains for most of these factors to understand the mechanism by which turnover contributes to activator function.

Supplementary Discussion for Figure S2

Inspection of the microarray data from the Velcade analysis indicates that proteasome inhibition significantly (>2x) depressed the transcription of ~80 Gcn4 targets 2. Dozens of additional Gcn4 targets were down-regulated 1-2-fold.

Supplementary Discussion for Figure S2

ChIP analysis of Gal4 was performed with a strain in which the chromosomally-encoded Gal4 was fused to a tandem affinity purification (TAP) tag 3. To facilitate isolation, this tag contains the Z -domain from Protein A that recognizes immunoglobulin G (IgG). To confirm that Gal4-TAP was functional and regulated normally we repeated the proteasome inhibition analysis with MG132 and the pdr5, GAL4-TAP strain. As shown in figure S2, GAL1 transcripts were produced normally in this strain and their induction was inhibited by MG132 treatment.

Using the Gal4-TAP strain, we demonstrated that, unlike Gcn4, Gal4 occupancy at the GAL1 promoter did not change upon proteasome inhibition (Fig. 2c) This is presumably because the relative concentration of Gal4 allows it to constitutively occupy all of its several dozen target sites 4. By contrast Gcn4 does not fully occupy all of its sites, which may number in the thousands, in the growth conditions used 5.

Supplementary Discussion for Figure S3

One hypothesis to explain the effects of proteasome inhibition on Gcn4 and Gal4-mediated transcription proposes that MG132 treatment disrupts cellular signaling upstream of the activators 6. This explanation appears unlikely to apply to Gcn4, as the end-product of signaling in the Gcn4 pathway is the accumulation of the activator on cognate promoters 7, and proteasome inhibition promoted such accumulation (Fig. 2b). In contrast, Gal4 levels at cognate promoters is not regulated by signaling, but promoter association of the transcriptional repressor, Gal80, is diminished upon galactose induction 8. Therefore, we tested a strain in which the chromosomal GAL80 locus was TAP-tagged to monitor the behavior of Gal80 upon galactose induction in the presence of MG132. Proteasome inhibition had no effect on the promoter-association dynamics of Gal80-TAP, as judged by ChIP (Fig. S3). These findings imply that the impact of proteolysis on Gcn4 or Gal4-dependent transcription is not due to the role of the proteasome in cellular signaling.

Supplementary Table 1

STRAINRelevant genotypeReference

RJD 796204, ura3, leu2, trp1, cdc34-29

RJD 1114S288c, MATa, ura3, leu2, his3, pre1-1 pre4-110

RJD 1115S288c, MATa, ura3, leu2, his310

RJD 1174S288c, Mat a, his3 200, leu2-3,112, ura3-52 9

RJD 1721 (BY4741)MATa, his3-1, leu2-0, met15-0, ura3-0 Research Genetics

RJD 2125RJD 1174, GCN4 (Myc9)::HIS39

RJD 2141RJD 2125, cdc34-29

RJD 2152RJD 2125, cdc4-19

RJD 2159RJD 1174, gcn4 (Myc9)::URA3

RJD 2185RJD 1174, gcn4-3T2S (Myc9)::HIS39

RJD 2262RJD 1174, HIS4-LacZ-LEU2 This study

RJD 2264RJD 2262, srb10::hisG This study

RJD 2266RJD 2262, pho85::URA3 This study

RJD 2268RJD 2262, srb10::hisG, pho85::URA3 This study

RJD 2291RJD 2185, HIS3P-LacZ::LEU2 This study

RJD 2292RJD 2125, HIS3P-LacZ::LEU2 This study

RJD 2296gcn4 (HA3), HIS3P-LacZ::LEU2 This study

RJD 2308RJD 2296, cdc34-2 This study

RJD 2313RJD 2291, cdc34-2 This study

RJD 2314RJD 2292, cdc34-2 This study

RJD 2505W303, can1-100, leu2-3,-112, his3-11,-15, trp1-1, This study

ura3-1, ade2-1, pep4::LEU2, pdr5::KANR

RJD 3035 (RG 2409)RJD 1721, pdr5::KANR Research Genetics

RJD 3047 (SUB313)MATa, lys2-801, leu2-3,112, ura3-52, his3-A200,11

trpl-1, ubil::TRP1, ubi2-2:ura3, ubi3-ub2,

ubi4-2::LEU2, [pGALP-UBI][pCUP1P-UBI]

RJD 3048 (SUB314) as RJD 3047, except [pCUP1P-ubi]11

RJD 3049 (SUB315)as RJD 3047, except [pCUP1p-ubi-K48R]11

RJD 3037RJD 3035, GCN4 (Myc9)::HIS3 This study

RJD 3039RJD 3035, gcn4-3T2S (Myc9)::HIS3 This study

RJD 3042RJD 3035, GAL4 (TAP):: KANR This study

RJD 3045RJD 3035, GAL80 (TAP):: KANR This study

RJD 3041BY4741, GAL4 (TAP):: KANR Research Genetics

RJD 3044BY4741, GAL80 (TAP):: KANR Research Genetics

RJD 3137 (RH1796)W303, ade2-101, leu2-3, 112, suc2-9, trp1-901,12

GCRE-LacZ::URA3, gcn4::LEU2

RJD 3138 RJD 3137 + pME1092 (GCN4P-GCN4, TRP1)12

RJD 3139RJD 3138, pdr5::KANR (from cross w/ RJD 2505) This study

RJD 3140RJD 3138, CDC34 (from cross w/ RJD 796) This study

RJD 3141RJD 3138, cdc34-2 (from cross w/ RJD 796) This study

RJD 3142RJD 2262, cdc34-2 This study

RJD 3143RJD 2264, cdc34-2 This study

RJD 3144RJD 2266, cdc34-2 This study

RJD 3145RJD 2268, cdc34-2 This study

SUPPLEMENTARY TABLE 2

Target

/

Turnover factor

/

Biological Process

/

ref

Gcn4
VIP1, VirE2
Laforin
p21, p27
Securin
MuA / SCF(Cdc4)
VirF
Malin
SCF
APC
ClpX(P?) / Turnover of Gcn4 stimulates transcription of target genes.
See accompanying supplementary text.
Malin targets laforin, a protein phosphatase, for turnover. Recessive mutations in either give rise to accumulation of glycogen particles, resulting in Lafora disease.
p21 or p27 promotes cyclin-CDK nuclear accumulation, but also inhibits CDK. SCF-dependent turnover of p21 and p27 activates CDK to promote cell cycle progression.
Securin promotes proper localization of separase. APC targets securin for destruction to activate separase and chromosome segregation.
ClpX removes a protomer of MuA transposase from the complex intermediate to complete phage Mu transposition. ClpP role is unknown. / 1
13
14
15
16

Supplementary Methods

Media

Many of our analyses were performed with minimal drop-in medium, which here consists of yeast nitrogen base (YNB- Difco) and glucose plus only those amino acids and bases (typically leucine, histidine, and uracil) that the cell cannot synthesize due to auxotrophy. Under these conditions, the cell must synthesize most of its amino acids (except those for which it is auxotrophic), and to do so it relies on amino acid biosynthetic enzymes, whose production depends on Gcn4. Consequently, gcn4∆ cells are severely compromised for growth in this medium 17 (doubling time ~6 hours) and for transcription of many target genes (see Figs. 1e, 3e, and S4). The expression of target genes in GCN4 cells in this minimal medium is approximately half the maximal induction that is seen when cells are shifted from rich or replete medium to starvation medium (data not shown), allowing for the assessment of positive or negative effects on Gcn4-dependent transcription. To induce amino acid starvation cells were cultured in minimal medium to mid-log phase then harvested and resuspended in a similar medium that lacked leucine 7. In the cases where the cells were prototrophic for leucine, starvation was induced by the histidine analog, 3-aminotriazole (3-AT), which was added to 100mM in medium lacking histidine 7.

Strains used for GAL1 analysis were grown in YP-raffinose (3%) to mid-log (OD 0.5-1). Galactose was then added to 3% for 0.5 hr. For INO1 analysis, strains were grown in synthetic complete medium (SC) plus inositol to mid-log, then washed and cultured in SC lacking inositol for 0.5 hrs.

In all experiments involving MG132, cultures were grown to mid-log, then treated with 50 uM MG132 (dissolved in DMSO at 1000X) or DMSO for 0.5- 1 hour. Inductions were carried out after 0.5 hour incubation with MG132 or DMSO.

Ubiquitin Derivative Analysis

Strains 11 were used that lacked all chromosomal copies of ubiquitin and that harbored a plasmid expressing ubiquitin from the GAL1 promoter. In addition the strains carried plasmids that expressed from the CUP1 promoter either wild type ubiquitin (RJD 4001), no coding sequence (i.e. empty vector; RJD 4002), or the K48R version of ubiquitin (RJD 4003) . These strains were grown in minimal medium with galactose, then switched to glucose containing medium for 6 hrs to deplete WT ubiquitin and exclusively express the ubiquitin driven by the CUP1 promoter. Samples were prepared for RT-PCR as described in the text.

SCF Mutant Analysis

Thermosensitive mutant versions of CDC34 and CDC4 were analyzed at semi-permissive temperature, 30oC because Gcn4-dependent transcription is down-regulated at the restrictive temperature, 37oC, and defects associated with cell-cycle arrest are avoided at 30oC. Despite having little growth defect at 30oC, cdc34-2 mutants are clearly defective, as evidenced by synthetic lethality between cdc34-2 and other mutations even at low temperatures 18.

Supplementary Figure Legends

Figure S1- Proteasome inhibition represses the transcription of many Gcn4 targets and of INO1, a target of the transcriptional activators, Ino2 and Ino4. a, GCRE6-LacZ, pdr5cells were cultured in minimal medium, MG132 or DMSO was added for 0.5 hrs, and cells were processed for RT-PCR analysis of ADH5, ARG1, and ASN1 transcripts. b, pdr5cells were cultured in SC+inositol to mid-log phase, MG132 or DMSO were added for 0.5 hours, and then the cells were resuspended in SC lacking inositol for 0.5 hours. Cells were then processed for RT-PCR analysis of INO1 and ACT1 transcripts.

Figure S2- Gal4-TAP functions similarly to wild-type Gal4. pdr5GAL4-TAP strains were treated as in Fig. 1b.

Figure S3- Proteasome inhibition does not affect the dynamic association of Gal80 with a Gal4 target. A pdr5,GAL80-TAP strain was prepared for ChIP analysis as in Fig. 2c, except half of the culture was maintained in raffinose medium and half had galactose added. PCR was performed with primers to the promoter region of GAL1.

Figure S4- Gcn4 is required for the association of RNA polymerase II (polII) with HIS4. ChIP analysis of polII was performed as in Fig. 2b with GCN4-Myc9 and gcn4 strains grown in minimal medium.

Supplementary References

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