1. Volume 38, Issue 4, Pages 614-620 (May 2010)
The Telomere-Binding Protein Tbf1 Demarcates snoRNA Gene Promoters in Saccharomyces cerevisiae 
Milena Preti, Cyril Ribeyre, Chiara Pascali, Maria Cristina Bosio, Barbara Cortelazzi, Jacques Rougemont, Enrico Guarnera, Felix Naef, David Shore, Giorgio Dieci 
Molecular Cell 
Volume 38, Issue 4, Pages (May 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
Conserved Sequence Elements in Saccharomyces snoRNA Gene Promoters
(A) General snoRNA promoter organization. The most frequent sequences for the Reb1p-binding site and the RRPE are indicated below the corresponding boxes. Above the boxes are the absolute and (in parentheses) the percentage motif occurrence within the promoter set. The observed range of distances (in base pairs, bp) between the various promoter elements is indicated on the top. Horizontal arrows indicate the sequence element orientations. Dotted arrows indicate the different positions of the RRPE.
(B) Alignment of aRCCCTaa-containing snoRNA upstream sequences. Identical and conserved nucleotides are shaded in black and gray, respectively. Numbers on the right indicate the position with respect to either the TATA element or (when absent) to the start of the mature snoRNA coding sequence (in square brackets). The alignment does not include the promoter regions of SNR82, SNR86, and SNR17B (Tbf1 site not invariant in at least four of the analyzed genomes); of SNR80 (inverted site); and of NME1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Association of Tbf1p to snoRNA Gene Promoter Regions
Association of Tbf1-TAP with the indicated snoRNA gene promoter regions was assessed by PCR of both input and immunoprecipitated (IP) DNA samples with both target-specific and control intergenic primers. A quantitative estimate of target enrichment in the immunoprecipitated DNA is reported below each IP lane. Tel XII, subtelomeric junction region of chromosome XII-L (Fourel et al., 1999).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 ChIP-Seq Data Analysis
(A) Map of all 197 ChiP-seq sites on the 16 yeast chromosomes (y axis). Telomeric (green), snoRNA (red), and other (black) sites are indicated with their strength (tag counts) in logarithmic scale (gray line indicates 103 counts).
(B) Strength of sites varies across the three groups.
(C) Main sequence motifs detected de novo in windows of ±200 bp around the snoRNA sites and other sites. Telomeric sites are not investigated here because of the repetitive nature of telomeric sequences.
(D) Nucleosome occupancies at snoRNA Chip-seq sites. The 0 position corresponds to that of the highest scoring Tbf1-binding site in the expected orientation. Two independent nucleosome data sets are shown in the left (Kaplan et al., 2009) and right (Lee et al., 2007) panels.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Tbf1 Role and Interactions at Promoters
(A) Episomal SNR64 constructs with WT or mutated promoter region (illustrated at the top) were transformed into an snr64 null strain (mut-T, Tbf1 site mutant; mut-A, poly[dA:dT] mutant; mut-AT, double mutant). Total RNA from two (WT, mut-T, mut-A) or four (mut-AT) different transformants was subjected to northern analysis both with an snR64-specific probe and a U3 snoRNA internal control probe. The normalized levels of mature snR64 snoRNA (expressed as percentages of the WT levels) are reported on the right. Error bars, standard error in the analysis of two to four independent transformants.
(B) Strains carrying a Tbf1 site mutation at either SNR32 (snr32 mut-T) or SNR64 (snr64 mut-T) promoters and a 13xMyc-tagged allele of TBF1 were analyzed for Tbf1 binding to and snoRNA expression from the mutated SNR promoters (upper panels, black bars, WT; gray bars, Tbf1 site mutant; error bars, standard error in three independent experiments). Profiles of nucleosome occupancy at promoter regions are represented in the lower panels (left, WT and mutated SNR32 promoter; right, WT and mutated SNR64 promoter; black and gray lines refer to WT and mutated gene loci, respectively). The series of overlapping DNA amplicons used for upstream region scanning are shown under the plots.
(C) The extent of association of Vid22-13xMyc and Ygr071c-13xMyc proteins with snoRNA and non-snoRNA promoter regions was assessed by ChIP followed by qPCR and is expressed as relative −fold enrichment with respect to the enrichment of MEI4 (whose promoter was not bound by Tbf1), which was arbitrarily set to 1.
Molecular Cell  , DOI: ( /j.molcel )
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