Britta Knight, Slawomir Kubik, Bhaswar Ghosh, et al.
nucleosomes control ribosomal protein gene transcription Two distinct promoter architectures centered on dynamic
Genes Dev. October , 2014 28: 2188 ribosomal protein gene transcription
Two distinct promoter architectures centered on dynamic nucleosomes control
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Supplemental Information Inventory (Knight et al., Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription)
Supplemental Data
Figure S1. RPG promoter characterization. Associated with Figure 1.
Figure S2. Rap1, Fhl1 and Hmo1-binding motifs. Associated with Figure 1.
Figure S3. Relationship between transcription factor binding and the Rap1-Hmo1 ChIP-seq peak distance. Associated with Figure 1.
Figure S4. Changes in RPG expression in response to different types of stress. Associated with Figure 1.
Figure S5. Mutation of two Rap1 binding sites in the RPL30 promoter. Associated with Figure 2.
Figure S6. Hmo1 binding at the rDNA locus and RPG promoters. Associated with Figure 4.
Figure S7. Mutation of Fhl1 binding sites in four RPG promoters. Associated with Figure 5.
Figure S8. MNase digested DNA fragments in divergent versus non-divergent promoters.
Associated with Figure 6.
Figure S9. The location of Rap1 in relation to fragile nucleosomes. Associated with Figure 6.
Table S1. Genotypes of yeast strains used in this study. Associated with all figures.
Table S2. ChIP-seq peak measurements and positions. Associated with Figure 1.
Table S3. RPG promoter YFP reporter library fluorescence. Associated with Figure 1.
Supplemental Materials and Methods
Supplemental References
RPL3 RPL4A RPL4B RPL15B RPS21A RPS28A RPS28B RPP1A RPP2B MRPL32 MRPS18
A B
Rap1 Fhl1 Ifh1 Hmo1classes of
classes of Rap1 sites4 5 0
Figure S1. RPG promoter characterization. (A) Abf1-FLAG occupancy (qPCR ChIP) on the
indicated promoters. Data are represented as mean ± SEM. (B) Cross-correlations between the differ-ent ChIP-seq profiles. (C) The classification of Rap1-bound RPG promoters based on the number and orientation of Rap1 binding sites as defined by the ChIP-seq derived weight matrix in Figure S2 with a threshold of 9.6 bits. There is a significant association between promoter category and Rap1 class (p=0.025; Fisher’s exact test), with class 1 having more category I and less category II promoters than expected (adjusted residuals = ±2) and class 5 having more category II and less category I promoters than expected (adjusted residuals = ±2.6). (D) Box plots showing the Rap1 ChIP-seq signal for each of the classes of Rap1-bound RPG promoters. Horizontal lines in the boxes represent the medians, the boxes represent the 25 and 75 percentiles, the whiskers represent the 2.5 and 97.5 percentiles, and the outliers are represented by dots.
Rap1 motifs
Figure S2. Rap1, Fhl1 and Hmo1-binding motifs. Sequence motifs of Rap1, Fhl1 and Hmo1 derived from MEME analysis of ChIP-seq data are shown together with motifs derived from in vitro MITOMI measurements and from previously published phylogenetic conservation analyses of different yeast species ( Siddharthan et al. 2005; Lavoie et al. 2010). (B) (B1) The Rap1 motif for class 1 (2 forward Rap1 sites). The numbers above the arrows indicate the maximum and minimum distance between the two sites for the 54 promoters. (B2) Rap1 motif for class 2 (1 forward and 1 reverse Rap1 site) (14 promoters). (B3) Rap1 motif for class 5 (1 forward Rap1 site) (43 promot-ers). (B4) Rap1 motif for non-RPG promoters (37 promotpromot-ers).
1 1
1 2
B
RPG promoters 2 forward Rap1 sites (54 promoters)RPG promoters 1 forward 1 reverse Rap1 sites (14 promoters)
0-24 bp
RPG promoters 1 forward Rap1 site
non-RPG promoters 1 forward Rap1 site B1
A B
C D
100 150 200 250 300
0.30.40.50.60.70.80.91.0
distance between Rap1 and Hmo1 peaks normalized Log transformed Rap1 ChIP-seq signal
100 150 200 250 300
0.30.40.50.60.70.80.91.0
distance bet ween Rap1 and Hmo1 peaks normalized Log transformed predicted Rap1 occupancy
100 150 200 250 300
0.800.850.900.95
distance bet ween Rap1 and Hmo1 peaks normalized Log transformed Fhl1 ChIP-seq signal
R=0.46 R=0.002
R=-0.32
δG = δGR Rap1 Rap1
Hmo1
Ifh1 δG = δGR + δGF + δGH-F + δGR-F >0 Rap1 Rap1
δG = 0
Fhl1
Hmo1
Ifh1 δG = δGF + δGH-F Fhl1
Figure S3. Relationship between transcription factor binding and the Rap1-Hmo1 ChIP-seq peak distance. (A) The relationship between the Rap1-Hmo1 ChIP-seq peak distance and the Rap1 ChIP-seq signals for the category I promoters. (B) The relationship between the Rap1-Hmo1 ChIP-seq peak distance and the Rap1 occupancy for the category I promoters. (C) The relationship between the Rap1-Hmo1 ChIP-seq peak distance and the Fhl1 ChIP-seq signal for the category I promoters. (D) The ratio of ChIP-seq signals for Rap1 and Fhl1 is related to the interaction ener-gies and the DNA binding affinities of Rap1, Fhl1 and Hmo1. For the category I promoters, the free energy of each configuration is shown above that particular configuration.
Knight_Fig. S3
category I
glucose limitation osmotic stress oxidative stress
category II -4
-3 -2 -1
fold change in mRNA levels fold change in mRNA levels fold change in mRNA levels
0
Rap1 Fhl1 Hmo1 TSS-Rap1 TSS-Fhl1 TSS-Hmo1 Rap1-Fhl1 Rap1-Hmo1 Fhl1-Hmo1 nucleosome full
Rap1 Fhl1 Hmo1 TSS-Rap1 TSS-Fhl1 TSS-Hmo1 Rap1-Fhl1 Rap1-Hmo1 Fhl1-Hmo1 nucleosome full 0.0
0.1
heat shock entry into stationary rapamycin treatment
-0.5 0.0 0.5 1.0
correlation coefficients (R)fold change in mRNA levels fold change in mRNA levels fold change in mRNA levels
category I
Figure S4. Changes in RPG expression in response to different types of stress. (A) Box plots showing the promoter activity of the three categories of promoters as defined in Figure 1A. Horizontal lines in the boxes represent the medians, the boxes represent the 25 and 75 percentiles, the whiskers represent the 2.5 and 97.5 percentiles, and the outliers are represented by dots. (B) The correlation coefficients between the different variables for category I and category II promoters. Full corresponds to a linear regression model incorporating the ten different explanatory variables, namely Fhl1, Rap1, and Hmo1 (log transformed signals), Fhl1, TSS-Rap1, and TSS-Hmo1 (distance of the peaks from TSS), nucleosome (average in vivo nucleosome occupancy within -200 bp of the TSS (Kaplan et al., 2009), Fhl1-Hmo1, Rap1-Hmo1, and Rap1-Fhl1 (distance between the indicated peaks). For category II promoters, Hmo1, TSS-Hmo1, Fhl1-Hmo1 and Rap1-Hmo1 are excluded from the full model. (C) The fraction of variance of the promoter activities explained by different linear models. The asterisk indicates a p-value of less than 0.05 based on an F-test. (D-I) Box plots as defined in (A). (D) Fold changes in mRNA expression (log ratio) at T=39°C relative to T=30°C. (E) Fold changes in mRNA expression (log ratio) at the stationary phase relative to the log phase. (F) Fold changes in mRNA expression (log ratio) when cells are treated with rapamycin. (G) Fold changes in mRNA expression under glucose dilution (log ratio) relative to normal condition. (H) Fold changes in mRNA expression exposed to NaCl (log ratio) relative to unstressed condition. (I) Fold changes in mRNA expression exposed to H2O2 (log ratio) relative to unstressed condition. The p-value for panels D through I indicates the probability of having unequal mean expression changes between category I and II genes based on a Welch two sample t-test.
TF ChIP TF-TSS distance TF-TF distance category I
Knight_Fig. S4
0.00 0.01 0.02 0.03 0.04
RNA Pol II (S5-P) ChIP in Rap1-AID WT
surface bound DNA (RFU) surface bound protein (RFU) surface bound DNA (RFU) surface bound protein (RFU)
B A
Figure S5. Mutation of two Rap1 binding sites in the RPL30 promoter. (A) The mutations that were made in the two Rap1 binding sites (Site 1 and Site 2) in the RPL30 promoter. (B) The in vitro binding affinity of the mutated Rap1 sites as measured by MITOMI. (C) RNA polymerase II (RNAPII) binding following Rap1-AID depletion as measured by ChIP, using an antibody directed against the Ser5 phosphorylated form of the CTD. RPS13, RPL30, and RPS30B promoters contain Rap1 binding sites, whereas the two category III promoters examined (RPS28A and RPP2B) do not. Data are represented as mean ± SEM.
Knight_Fig. S5
RDN37-1 RDN5-1
RPL30 RPS18B RPL28RPS30BRPL15ARPS13 relative fold enrichment (auxin/vehicle)
Figure S6. Hmo1 binding at the rDNA locus and RPG promoters. (A) The sequence including 200 bp around the Hmo1 peak was analyzed in category I promoters and the top 100 Hmo1 peaks in non-RPG promoters to determine the number of Hmo1 motifs present using a threshold of above 8 total bits. These values were plotted versus the Hmo1 ChIP-seq peak tag count for the corresponding peak. (B) Hmo1 binding on the indicated Cy5-labeled DNA templates was performed as described in Figure 4 and Materials and Methods. This is an independent replicate of the experi-ment shown in Figure 4. (C) Forward and reverse potential Hmo1 binding sites on the rDNA locus, as defined by the ChIP-seq derived weight matrix in Figure S2, plotted with Hmo1 ChIP-seq signal.
(D) Fhl1 promoter occupancy at the indicated times following auxin-induced depletion of AID-tagged Hmo1. Data are plotted as auxin relative to vehicle treatment, and normalized to t=0. Data are represented as mean ± SEM. (E) Hmo1 promoter occupancy at the indicated times on several category I RPG promoters after auxin-induced depletion of AID-tagged Fhl1. Data are plotted as auxin relative to vehicle treatment, and normalized to t=0. Data are represented as mean ± SEM.
Knight_Fig. S6
1 mutation –
3 mutations –