Hill, et al. 1 Hill Supplemental Materials Included in this document are the following: -Supplemental Figure and Table legends for Figures S1-S8 and Tables S1-S6 -Supplemental Materials and Methods, these are more detailed methods and lists of all the primer and siRNA sequences -Supplemental References to accompany the supplemental Materials and Methods -Supplemental Figures S1-S8, these Figures provide controls for the main Figures in the paper Not included in this document, provided as a single Excel file with 6 sheets -Supplemental Tables S1-S6, these tables provide the full datasets from the complementary screens and information about each hit based on various analyses Hill, et al. 2 Supplemental Figure and Table Legends Hill Figure S1. Analysis of Y2H screen hits, Related to Figure 1: A) Based on the smallest fragment interaction detected for each bait, some of the interactions detected in the yeast two hybrid (Y2H) screen were mapped to specific functional domains of BRCA1. The interactors from our screen mapping to each domain are listed below the domains in black (RING, Exon 11, Coiled Coil Domain (CCD), and BRCTs). The interactors mapped to the CCD and BRCTs most certainly interact with the C-terminal domain of BRCA1, but due to the overlapping nature of the fragments it is possible that the CCD and BRCT estimates could overlap with each other. We have currently mapped proteins to one fragment or the other based on the smallest interacting domain, but further mapping will need to be done in the future to confirm where the C-terminal domain binding partners interact (CCD vs. BRCTs). In the bottom left part of this panel is a map of full length SETX, with which BRCA1 did interact by Y2H, and a C-terminal SETX fragment with which BRCA1 did not interact by Y2H. B) Map demonstrating the results of an analysis performed on each BRCA1 interacting partner to determine whether it was associated with a specific “GO annotation” of interest (transcription, replication, DNA damage), whether it had been identified in any completed “systematic screens” searching for cancer genes (Sleeping Beauty, GWAS, SCNA, Somatic Mutations, tumor virus protein interactors), whether it had been identified in any previous “BRCA1 protein-protein interaction (PPI)” screens, or whether it is a “known cancer gene” in the Sanger Census or other relevant lists. Hits that fit any of these criteria align with one or more black boxes, each representing one of the above-noted criteria. The hits that were validated through genetic analysis are represented in red. *See also Table S5. Hill, et al. 3 Hill Figure S2. Overexpression co-IPs of select screen hits with BRCA1 isoforms (related to Figure 1) and assessment of siRNAs used in various experiments (Related to Figures 2-4 for all siRNAs used): A) 293FT cells were transiently transfected with an HA-FLAG tagged gene of interest (TONSL, a TAP-MS identified interactor, and BARD1 in this case) and a myc-tagged BRCA1 isoform (Full Length BRCA1 p220 which we have referred to as BRCA1 throughout the text or the exon 11 missing isoform 11b). Lysates from these cells were immunoprecipitated with FLAG, electrophoresed, blotted with a myc antibody, and in some cases the blots stripped and then reacted with FLAG antibody. Arrows denote the migration of proteins of interest. B) HA-FLAG tagged TONSL was tandem affinity purified from the soluble nuclear and chromatin fractions of 293FT cells stably overexpressing this protein. A silver stain showing the results of these purifications along with those performed on extracts of cells transduced with empty vector can be seen in the left panel. A western blot of 10% of the purified fraction stained for BRCA1 is shown in the right panel. Endogenous p220/BRCA1 and 11b are marked by arrows. C) Co- transfections, IPs, and blots were performed as in panel A, but here with HA-FLAG tagged PPFIA1 (from the Y2H screen) or BARD1 as a control. Arrows indicate the location of p220/BRCA1 and 11b on this myc blot. D) Co-transfections, IPs, and blots were performed as noted for panel A, but here with HA-FLAG tagged MAP3K14 (from the Y2H screen) or BARD1 as a control. Arrows indicate the location of p220/BRCA1 and 11b in this myc blot. E) HA- FLAG-tagged MAP3K14 was immunoprecipitated from lysates of cells stably expressing it, and these IPs were blotted with a BRCA1 antibody (left panel) and a FLAG antibody (right panel). Arrows demonstrate the proteins of interest. F) Co-transfections, IPs, and blots were performed as in panel A but with HA-FLAG tagged CLK2 or CWF19L2 (both interactors from the Y2H screen). Arrows indicate the location of p220/BRCA1 and 11b on the myc blot shown in the Hill, et al. 4 left panel. This blot was stripped and re-probed with FLAG antibody, as shown in the right panel where arrows indicate the migration of CLK2 and CWF19L2. G) Co-transfections, IPs, and blots were performed as in panel A but with HA-FLAG tagged PRKAA2 (from the Y2H screen) or HA-FLAG-MLLT6 (a false positive from the Y2H screen). Arrows indicate the migration of p220/BRCA1 and 11b on this myc blot. H) Co-transfections, IPs, and blots were performed as in panel A this time with HA-FLAG tagged MCRS1 (from the Y2H screen) or BARD1 as a control. Arrows indicate the migration of p220/BRCA1 and 11b on this myc blot. I) IP-Western for BRCA1 in the WT/WT (WT), BRCA1+/- and TONSL+/- lines transfected with each BRCA1 siRNA noted in Figure 3D. The exon 13, exon 11, and 3’UTR targeting siRNAs were also used in multiple other experiments in WT/WT cells throughout the manuscript. J) IP-Western for TONSL in the WT/WT (WT), BRCA1+/-, and TONSL+/- lines with each TONSL siRNA noted in Figure 3E. These two TONSL siRNAs were also used in multiple other experiments in WT/WT cells throughout the manuscript. K) Western blot of extracts of the cells in Figure 4B that were transfected with each SSRP1 siRNA. The left panel is blotted with SSRP1, and the right panel is the same blot stripped and re-probed with a tubulin antibody to demonstrate equal loading. These two SSRP1 siRNAs were also used in multiple other experiments in WT/WT cells throughout the manuscript. L) IP-Western of WT/WT and BRCA1+/- cells transfected with the SUPT16H siRNAs noted in Figure 4C. These two SUPT16H siRNAs were also used in multiple other experiments in WT/WT cells throughout the manuscript. M) IP-Western of WT/WT (WT) and BRCA1+/- lines transfected with SETX siRNAs noted in Figure 3A. The IP was carried out with an antibody targeting the C-terminus of SETX and the blot with an antibody targeting the N-terminus. N) qRT-PCR on cDNAs generated from whole cell RNA harvested from cells studied in the TCEANC experiment in Hill, et al. 5 Figure 3B. The bars represent the average relative mRNA number (2-CT) of 5 replicates for the oligo pair/cell line, and the error bars represent the deviation between the replicates. O) qRT- PCR on cDNAs generated from whole cell RNA harvested from cells studied in the TCEA2 experiment in Figure 3C. The bars represent the average relative mRNA number (2-CT) of 5 replicates for the oligo pair/cell line, and the error bars represent the deviation between the replicates. Hill Figure S3. Dose curves for DRB, alpha-Amanitin, and UV sensitivity of various cell lines transfected with different siRNAs, Related to Figures 2 and 3: A) DRB dose curves for U2OS cells transfected with siGL2 or multiple BRCA1 specific siRNAs. U2OS cells were transfected with various siRNAs on days 1 and 2, and plated at a suitable density for colony formation on day 3. Four hours after plating, the media was changed on the cells to media containing various doses of DRB. 24 hours later, the DRB media was removed, the cells were washed with PBS, and fresh non-drug containing media was added. The cells were allowed to grow until suitably sized colonies had formed. The colonies were stained with crystal violet solution and counted. This experiment was repeated a minimum of three times for each siRNA. The percentage of cells surviving at each dose compared to the 0uM control was plotted in GraphPad Prism, and a non-linear regression curve was fit to these values from each experiment. The IC50 for each experiment was calculated based on the non-linear regression analysis. Shown here is the average curve from all of the experiments for each individual siRNA. The error bars at each data point represent the standard error of the mean (SEM) between values from the multiple experiments for that dose as calculated by GraphPad Prism. B) alpha-Amanitin dose curves for U2OS cells transfected with siGL2 or multiple BRCA1 specific siRNAs. U2OS Hill, et al. 6 cells were transfected with various siRNAs on days 1 and 2, and plated at a suitable density for colony formation on day 3. Four hours after plating, the media was changed on the cells to media containing various doses of alpha-Amanitin. 24 hours later, the alpha-Amanitin media was removed, the cells were washed with PBS, and fresh non-drug containing media was added. The cells were allowed to grow until suitably sized colonies had formed. The colonies were stained with crystal violet solution and counted. This experiment was repeated a minimum of three times for each siRNA. The percentage of cells surviving at each dose compared to the 0uM control was plotted in GraphPad Prism, and a non-linear regression curve was fit to these values from each experiment.