The Ulp2 SUMO Protease Promotes Transcription Elongation through Regulation of Histone Sumoylation (2019)
Figure 1

Figure 1. Ulp2 is recruited to actively transcribed genes.

A Transcriptome re-analysis of ulp2Δ strains compared with WT (MHY1379) from previous RNA-seq experiments (Ryu et al, 2018). Two-dimensional agglomerative hierarchical clustering shows 2,903 significantly up- or down-regulated genes (P < 0.05) in triplicate RNA samples. Red and blue indicate up and down regulation of genes, respectively, and their intensity represents the relative gene expression changes.

B Summery of transcriptome data in (A). White and gray colors indicate up- and down-regulated genes, respectively, in ulp2Δ cells. The pie graph shows the percentages of significantly changed genes, except for 130 ribosomal protein genes (RPs), and are classified by FPKM (Fragments Per Kilobase of Million reads mapped) values in the bar graph.

C qRT-PCR analysis of the highly transcribed PMA1, ADH1 and PYK1 genes in ulp2Δ cells. Expression was measured relative to WT cells and data were normalized to SPT15 expression. Error bars indicate the standard deviations (SDs) from three independent RNA preparations. FPKMs of each gene in WT are shown in the bottom graph.

D Schematic diagram of PMA1, ADH1, PYK1 and CUP1 genes. The TATA/promoter (Pro) and open reading frame (ORF) are represented by black and white boxes, respectively. Bars with numbers below the genes show the relative positions of the PCR products used in the ChIP and qRT-PCR analyses and are used for identification in all later figures.

E ChIP analysis using IgG-Sepharose or anti-Flag-agarose beads in strains expressing TAP-tagged Paf1, Ubc9, or Ulp1 or Flag-tagged Ulp2. An untagged strain (MHY500) was used as a negative control for immunoprecipitation of Ulp2-Flag (Fig EV1A). The qPCR signals of the indicated genes were quantitated and normalized to an internal background control and the input DNA. The primer pairs used are indicated in (D). Quantification (described in Materials and Methods) presented as fold over background; a value of 1 indicates no signal detected above background signal at a nontranscribed locus, as marked with the horizontal line. Error bars indicate SDs calculated from three independent chromatin preparations.

F Occupancy of Rpb3 and Ulp2 at CUP1 gene was determined by ChIP in a strain expressing Flag-tagged Ulp2 using anti-Rpb3 antibody and anti-Flag agarose beads as shown in (E). 'Mock' indicates use of Protein G beads without added antibody. For CUP1 induction, cells were harvested at the indicated time points after adding CuSO4. "Pro" and "ORF" represent the positions of PCR fragments 1 and 2 from the CUP1 gene as described in (D). Black bar indicates a value of 1, the background signal. Error bars, SD from four independent experiments.

G A percentage graph of association of Ulp2-Flag with genes classified by FPKM in the file Dataset EV1. RPs and ND indicate ribosomal protein genes and genes not detected genes in our earlier RNA-seq experiments, respectively. The numbers above the graph indicate the total number of yeast genes in each expression category.

H Average plot of Ulp2-Flag occupancy at transcribed genes. The values of the y-axis indicate rescaled fold-enrichment, setting the maximum occupancy to 1 and the minimum occupancy to 0, and the x-axis indicates normalized distance from transcription start sites (TSS) and transcription end sites (TES). The dotted lines indicate the TSS and TES. The ChIP-seq data were obtained from duplicate experiments.

Data information: Asterisks indicate statistically significant differences of Paf1-TAP and Ubc9-TAP with Ulp1-TAP and Ulp2-Flag with No tag (Fig EV1A) in (E) and significant differences between uninduced and induced cells in (F) using a two-tailed Student's t test (*, P <0.05; **, P <0.01). See Dataset EV2 for qPCR raw data.