Journal: PLoS Genetics
Article Title: Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II
doi: 10.1371/journal.pgen.1009828
Figure Lengend Snippet: (A) Most sumoylated proteins are associated with chromatin. Fractionation experiments were carried out in normally growing, unmodified yeast and human HeLa cells, then whole cell extracts (WCE) and indicated fractions were analyzed by immunoblot (IB) with indicated antibodies, including antibodies for the yeast Smt3 peptide (“SUMO”), human SUMO1 or SUMO2/3 isoforms, and histone H3. (B) Independent duplicate SUMO ChIP-seq experiments were performed in yeast, and sample alignments are shown from Replicate 1, using the Integrative Genomics Viewer (IGV) genomic alignment tool, with reads from the SUMO IP and corresponding inputs over selected short segments of Chromosomes XII and IV. Values correspond to maximum data range (read numbers) for the view shown and blue bars along the bottom represent gene positions, including for two non-RPGs, CCW12 and LYS20 , whose ORF orientations are indicated with arrows. See for a list of yeast genes associated with the 603 SUMO peaks. (C) Pie chart showing fractions of the 603 SUMO ChIP-seq peak set associated with different gene types. Non-RPG refers to protein-coding genes that are not ribosomal protein genes (RPGs). See for detailed description of peak classifications. (D ) Distribution of read counts for the 603 SUMO ChIP-seq peaks, separated by gene type, then ranked by normalized read counts. Normalized read counts were determined using DiffBind tool with two independent ChIP-seq replicates. (E) Sample SUMO peak alignments from Replicate 1 are shown for two RPGs and four non-RPGs. Peak alignments for TBP and Rap1 are also shown, using published ChIP-seq and ChIP-exo datasets, respectively, for comparison with SUMO peak positions (NCBI GEO database accession numbers GSM2870615 and GSE93662, respectively). Values refer to maximum data range (read numbers) for the view shown, and unnormalized alignments were generated using IGV.
Article Snippet: Antibodies used for immunoblots are: 1:500 yeast SUMO/Smt3 (y-84; Santa Cruz, sc-28649); 1:3000 GAPDH (Sigma, G9545); histone H3 (1:3000 for yeast immunoblot, 1:20,000 for HeLa immunoblot; Abcam, ab1791); 1:500 SUMO1 (Developmental Studies Hybridoma Bank, 21C7); 1:50 SUMO2/3 (Developmental Studies Hybridoma Bank, 8A2); 1:0000 α-tubulin (Cell Signaling Technology, 2144); 1:1000 HA (Novus, NB600-363); 1:1000 Rpb1 (8WG16; Abcam, ab817); and 1:1000 Rpb3 (Abcam; ab202893).
Techniques: Fractionation, Western Blot, ChIP-sequencing, Generated