Fig 2: Depletion of Paf1C subunits leads to genome-wide changes in pA site usage.(A) Schematic depicting different types of APA sites and method to detect total and full-length transcript abundance. Solid boxes are exons, and open ones are introns. 3’UTR-APA sites are those located in the 3’UTR. CDS-APA sites are those located in introns or exons upstream of the 3’-most exon. (B) Left, normalized number of genes with regulated 3’UTR-APA in each sample as examined by Global Analysis of Alternative Polyadenylation (GAAP, see Materials and Methods for detail). Normalized number is based on (observed value—expected value). Red and blue bars represent genes with shortened 3’UTRs (Sh, proximal pA isoform up-regulated relative to distal pA) and lengthened 3’UTRs (Le, distal pA isoform up-regulated relative to proximal pA), respectively. FDR = 0.05 (Significance Analysis of Alternative Polyadenylation, SAAP, see Materials and Methods for detail) was used to select genes with significant 3’UTR-APA regulation. Only the top two most abundant APA isoforms (based on the number of PASS reads) of each gene were used for this analysis. Error bars show standard deviation based on 20 iterations of bootstrapping. Log2(Sh#/Le#) is log2(ratio) of the number of Sh genes to the number of Le genes. Right, genome browser tracks showing 3’READS data for a selected gene, Zfp260. The maximum value of y-axis for each track is indicated. APA sites are indicated by arrows. (C) Left, normalized number of genes with regulated CDS-APA as examined by GAAP. Red and blue bars represent genes with up-regulated CDS-APA isoforms (UP) and down-regulated isoforms (DN), respectively. All CDS-APA isoforms were combined and compared to all 3’-most exon isoforms combined by SAAP. FDR = 0.05 (SAAP) was used to select genes with significant CDS-APA regulation. Error bars are as described in panel B. Samples were sorted by the total number of genes with CDS-APA changes. Log2(UP#/DN#) is log2(ratio) of the number of UP genes to the number of DN genes. Right, genome browser tracks showing 3’READS data for a selected gene, Pcbp2. The maximum value of y-axis for each track is indicated. APA sites are indicated by arrows. (D) Left, normalized number of genes with regulated uaRNA expression. Red and blue bars represent genes with up-regulated (UP) and down-regulated (DN) uaRNA expression, respectively. Log2(UP#/DN#) is log2(ratio) of the number of UP genes to the number of DN genes. All uaRNAs were combined and compared to all sense strand transcripts whose pAs were beyond 2 kb from the TSS by SAAP. Q-value < 0.05 (SAAP) was used to select genes with a significant uaRNA expression difference. Error bars are as described in panels B and C. Right, metagene plots of uaRNA and sense strand RNA expression in siPaf1, siCdc73, siSki8 and siCtrl samples. Expression is represented by reads per million (RPM, pA site-supporting reads only) at pA positions. (E) RT-qPCR validation of impact on APA after depletion of Paf1, Cdc73, or Ski8. p-value indicates significant difference between Paf1- and Ski8-depleted cells. Negative control genes are indicated. (F) Heatmap showing common and distinct target genes with changes in CDS-APA after depletion of Paf1, Cdc73 or Ski8. Genes were sorted using the siPaf1 data. Samples were clustered using Pearson correlation. (G) Top, Venn diagrams depicting overlap of genes among Paf1C targets as detected by ChIP-seq, genes showing altered levels of expression in RNA-seq (fold-change >1.4, p-value<0.01 (Fisher's exact test)) and genes with changes in APA after depletion of Paf1C subunits (SAAP FDR = 0.05; see Materials and Methods). Right, data are shown in percentage of overlap.

Fig 3: Chromatin occupancy of Paf1C correlates with APA regulation after depletion of Paf1C subunits.(A) The average enrichment profiles of Paf1, Cdc73, and Ski8 were plotted on genes that showed regulated, low-expressed, or unaffected CDS-APA after depletion of Paf1, Cdc73 or Ski8. A region from 3 kb upstream of TSS to 3 kb downstream of TES was analyzed. (B) The average enrichment profiles of Paf1, Cdc73, and Ski8 were plotted over genes that showed shortening or unaffected length of 3’UTR after depletion of Paf1, Cdc73, or Ski8. A region from 2 kb up-or downstream of the two most regulated 3’UTR pA sites was analyzed (A distance between the proximal and distal pAs greater than 1 kb was required).

Fig 4: Parafibromin activates Wnt and Notch signals in a cooperative manner.(a,b) Wnt and Notch signals mutually potentiate parafibromin-dependent signal activation. Luciferase reporter assays using the indicated reporter plasmids in HEK293T cells. Total cell lysates (TCLs) were subjected to immunoblotting (n=3, mean±s.d. **P<0.01; ANOVA with Bonferroni's post hoc test). (c) ß-catenin stabilizes parafibromin/NICD interaction. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) and immunoblotted with the indicated antibodies. (d) Endogenous interaction between parafibromin, NICD and ß-catenin was increased on endogenous Wnt signal activation. HEK293T cells treated with or without 25 mM LiCl were lysed and sequentially immunoprecipitated (IP) with an anti-parafibromin antibody and immunoblotted. (e) Parafibromin forms a trimeric complex with ß-catenin and NICD. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies and immunoblotted. (f) A schematic view of Flag-tagged wild-type parafibromin and its derivative, parafibromin-357X. The cdc73 core homology domain (aa 357–531) and ß-catenin-binding site (aa 218–263) are indicated. (g) The C-terminal domain of parafibromin is required for NICD binding. HEK293T cells were transfected with the indicated vectors. TCLs were sequentially immunoprecipitated (IP) with the indicated antibodies.

Fig 5: Depletion of Paf1, Cdc73 but not Ski8 leads to prevalent activation of TSS-proximal intronic APA sites.(A) Up-regulation of intronic APA sites is biased toward the TSS after depletion of Paf1 or Cdc73, but not Ski8. Regions between TSS and the 3'-most splice site were divided into 5 equally-sized bins. “siCtrl" indicates the background distribution of intronic pAs whose usage was detected in control cells. The distribution of up-regulated intronic pAs in knock-down samples were compared to background. The p-values (Fisher’s exact test) for the percentage of pAs in Bin1, the 5’-most bin, for each depletion and control pair are 4x10-28, 4x10-19, and 0.02 for siPaf1, siCdc73, and siSki8, respectively. (B) TSS-proximal intronic pAs are more strongly activated as compared to downstream intronic pAs. +1, +2, middle, -2 and -1 introns are the first, second, middle (not first two or last two), second to the last, and the last introns, respectively. The error bar represents standard error of mean for all analyzed pAs in a given intron group. Expression changes are log2(ratio) of PASS reads in knock-down sample vs. control sample. (C) Top, Venn diagrams depicting overlap of genes with regulated intronic pAs between Paf1- or Cdc73-depletion and U1 inhibition. Bottom, data are shown in percentage of overlap.