1 00:00:05,560 --> 00:00:10,200 >>WELCOME TO THE DNA REPAIR 2 00:00:10,200 --> 00:00:14,360 INTEREST GROUP MONTHLY LECTURES. 3 00:00:14,360 --> 00:00:17,800 I WILL BE THE HOST TODAY, EVEN 4 00:00:17,800 --> 00:00:19,240 THOUGH KEN KRAMER AND I ARE NO 5 00:00:19,240 --> 00:00:22,960 LONGER THE LEADERS OF THE 6 00:00:22,960 --> 00:00:25,400 INTEREST GROUP, THAT IS CHUN 7 00:00:25,400 --> 00:00:27,640 YANG AND KAREN USTEN, NOT ABLE 8 00:00:27,640 --> 00:00:29,280 TO BE PRESENT, ALTHOUGH CHUN IS 9 00:00:29,280 --> 00:00:33,080 HERE AND WILL HELP ME WITH 10 00:00:33,080 --> 00:00:34,440 LEADING THE QUESTIONS. 11 00:00:34,440 --> 00:00:39,120 I WANT TO MENTION THAT THE NEXT 12 00:00:39,120 --> 00:00:41,720 MEETING APRIL 11 IS CHRIS LORD, 13 00:00:41,720 --> 00:00:43,600 OPTIMIZING USE OF PARP 14 00:00:43,600 --> 00:00:46,560 INHIBITORS TO TREAT CANCER, AND 15 00:00:46,560 --> 00:00:52,160 ON TUESDAY MAY 9, PETER CHOIKA 16 00:00:52,160 --> 00:00:53,600 FROM SWITZERLAND, WE HAVE A 17 00:00:53,600 --> 00:00:57,280 REALLY GOOD PROGRAM THIS YEAR. 18 00:00:57,280 --> 00:01:00,640 AND WE'RE VERY PLEASED TO HAVE 19 00:01:00,640 --> 00:01:03,840 JESSICA DOWNS SPEAK TO US HERE 20 00:01:03,840 --> 00:01:09,680 TODAY NOW, AND HERE IS A PICTURE 21 00:01:09,680 --> 00:01:10,960 OF HER. 22 00:01:10,960 --> 00:01:12,400 AND I'M SHARING MY SCREEN HERE, 23 00:01:12,400 --> 00:01:14,320 I HOPE THAT WORKS. 24 00:01:14,320 --> 00:01:19,080 AND YOU CAN SEE JESSICA FROM 25 00:01:19,080 --> 00:01:22,240 THE -- PROFESSOR OF EPIGENETICS 26 00:01:22,240 --> 00:01:23,440 AND GENOME STABILITY, DEPUTY 27 00:01:23,440 --> 00:01:24,840 HEAD DIVISION OF CANCER BIOLOGY 28 00:01:24,840 --> 00:01:28,360 AT THE INSTITUTE OF CANCER 29 00:01:28,360 --> 00:01:32,320 RESEARCH DOWNTOWN LONDON. 30 00:01:32,320 --> 00:01:37,360 AND SHE HAS DONE A LOT OF VERY 31 00:01:37,360 --> 00:01:41,400 INTERESTING WORK RELATING TO 32 00:01:41,400 --> 00:01:43,160 EPIGENETICS CHROMATIN STRUCTURE 33 00:01:43,160 --> 00:01:44,880 AND DNA REPAIR. 34 00:01:44,880 --> 00:01:47,520 SHE RECEIVED HER Ph.D. FROM 35 00:01:47,520 --> 00:01:49,720 DEPARTMENT OF ZOOLOGY, 36 00:01:49,720 --> 00:01:51,680 UNIVERSITY OF CAMBRIDGE, AND 37 00:01:51,680 --> 00:01:55,760 WORKED THERE WITH STEVE JACKSON, 38 00:01:55,760 --> 00:01:57,240 AND THEN SHE CONTINUED IN 39 00:01:57,240 --> 00:01:59,120 JACKSON'S LAB AT A POSTDOC FOR 40 00:01:59,120 --> 00:02:01,200 SOME YEARS, AND THEN SHE WENT TO 41 00:02:01,200 --> 00:02:11,720 UNIVERSITY OF CAMBRIDGE AND WAS 42 00:02:12,000 --> 00:02:13,560 A GROUP LEADER THERE, LATER 43 00:02:13,560 --> 00:02:16,400 JOINED THE UNIVERSITY OF SUSSEX, 44 00:02:16,400 --> 00:02:17,400 PROFESSOR OF GENOME STABILITY, 45 00:02:17,400 --> 00:02:23,640 WE HAVE MANY FRIENDS THERE AND 46 00:02:23,640 --> 00:02:24,080 COLLEAGUES. 47 00:02:24,080 --> 00:02:27,480 LATER MOVED TO LONDON, DEPUTY 48 00:02:27,480 --> 00:02:29,200 HEAD OF DIVISION OF CANCER 49 00:02:29,200 --> 00:02:31,960 BIOLOGY AT INSTITUTE OF CANCER 50 00:02:31,960 --> 00:02:36,120 RESEARCH, SINCE 2019. 51 00:02:36,120 --> 00:02:37,320 AS I MENTIONED PUBLISHED MANY 52 00:02:37,320 --> 00:02:38,880 IMPORTANT PAPERS IN THE FIELD OF 53 00:02:38,880 --> 00:02:39,640 DNA REPAIR. 54 00:02:39,640 --> 00:02:46,880 SHE'S ALSO PART OF THE EDITORIAL 55 00:02:46,880 --> 00:02:48,400 LEADERSHIP OF DNA REPAIR, THE 56 00:02:48,400 --> 00:02:53,480 JOURNAL, AND MANY OTHER 57 00:02:53,480 --> 00:02:56,720 JOURNALS. 58 00:02:56,720 --> 00:03:02,280 SHE WILL TALK ABOUT THE SWI/SNF 59 00:03:02,280 --> 00:03:05,360 CHROMATIN REMODELING COMPLEX 60 00:03:05,360 --> 00:03:06,600 MAINTAINS GENOME STABILITY AT 61 00:03:06,600 --> 00:03:06,960 CENTROMERES. 62 00:03:06,960 --> 00:03:08,960 JESSICA, PLEASE TAKE IT OVER 63 00:03:08,960 --> 00:03:09,720 FROM HERE. 64 00:03:09,720 --> 00:03:10,680 >>THANK YOU VERY MUCH 65 00:03:10,680 --> 00:03:12,000 FOR THE INVITATION 66 00:03:12,000 --> 00:03:14,120 HERE, IT'S A GREAT PLEASURE. 67 00:03:14,120 --> 00:03:15,960 I'D LIKE TO TELL YOU ABOUT THE 68 00:03:15,960 --> 00:03:18,800 RECENT WORK ON THIS CHROMATIN 69 00:03:18,800 --> 00:03:19,800 REMODELING COMPLEX PBAF AND 70 00:03:19,800 --> 00:03:23,280 SOMEHOW WE FOUND A ROLE FOR IT 71 00:03:23,280 --> 00:03:25,720 AS CENTROMERES THAT'S IMPORTANT 72 00:03:25,720 --> 00:03:28,520 FOR MAINTAINING GENOME STABILITY 73 00:03:28,520 --> 00:03:29,160 AT CENTROMERIC SEQUENCES. 74 00:03:29,160 --> 00:03:32,800 THIS IS A SET OF RECENT WORKS 75 00:03:32,800 --> 00:03:35,120 UNPUBLISHED WORKS I'M KEEN TO 76 00:03:35,120 --> 00:03:37,840 GET PEOPLE'S FEEDBACK ON THIS. 77 00:03:37,840 --> 00:03:38,840 OKAY. 78 00:03:38,840 --> 00:03:40,800 SO, TO BEGIN, MY LAB, WE'VE 79 00:03:40,800 --> 00:03:43,320 ALWAYS BEEN INTERESTED IN 80 00:03:43,320 --> 00:03:44,800 UNDERSTANDING HOW CHROMATIN 81 00:03:44,800 --> 00:03:46,640 IMPACTS ON GENE OWN INTEGRITY, 82 00:03:46,640 --> 00:03:48,720 AND OBVIOUSLY YOU CAN SEE FROM 83 00:03:48,720 --> 00:03:50,560 THIS SLIDE IT'S TRYING TO 84 00:03:50,560 --> 00:03:51,960 REPRESENT HOW AS YOU ALL KNOW 85 00:03:51,960 --> 00:03:53,720 THAT THERE ARE MULTIPLE 86 00:03:53,720 --> 00:03:57,120 DIFFERENT WAYS THAT CHROMATIN 87 00:03:57,120 --> 00:03:59,000 BIOLOGY CAN INFLUENCE GENOME 88 00:03:59,000 --> 00:03:59,280 INTEGRITY. 89 00:03:59,280 --> 00:03:59,960 IT'S DYNAMICALLY INVOLVED IN 90 00:03:59,960 --> 00:04:02,960 ANYTHING TO DO WITH DNA 91 00:04:02,960 --> 00:04:10,160 METABOLISM SO WE CAN HAVE 92 00:04:10,160 --> 00:04:10,800 TRANSCRIPTIONAL REGULATION, DNA 93 00:04:10,800 --> 00:04:11,840 REPLICATION, WHAT I REALLY -- 94 00:04:11,840 --> 00:04:14,080 THE MAIN FOCUS OF THE LAB IS ON 95 00:04:14,080 --> 00:04:16,520 DOUBLE STRAND BREAK REPAIR AND 96 00:04:16,520 --> 00:04:17,640 HOW CHROMATIN INFLUENCES THE 97 00:04:17,640 --> 00:04:20,120 REPAIR OF THOSE BREAKS AND 98 00:04:20,120 --> 00:04:20,760 SIGNALING AROUND THEM. 99 00:04:20,760 --> 00:04:22,320 BUT THE STORY I'M GOING TO TELL 100 00:04:22,320 --> 00:04:26,440 YOU ABOUT TODAY KIND OF STEMS 101 00:04:26,440 --> 00:04:30,080 FROM WORK A NUMBER OF YEARS 102 00:04:30,080 --> 00:04:33,800 LOOKING AT RELATIONSHIP BETWEEN 103 00:04:33,800 --> 00:04:38,320 PBAF AND CHROMATIN ADHESION, AN 104 00:04:38,320 --> 00:04:41,320 INFLUENCE ON CENTROMERE 105 00:04:41,320 --> 00:04:44,200 STRUCTURE WHICH IS IMPORTANT FOR 106 00:04:44,200 --> 00:04:45,840 MAINTAINING GENOME INTEGRITY AT 107 00:04:45,840 --> 00:04:52,560 THOSE REGIONS, ALSO IN TERMS OF 108 00:04:52,560 --> 00:04:54,280 FIDELITY OF CHROMOSOME 109 00:04:54,280 --> 00:04:54,600 SEGREGATION. 110 00:04:54,600 --> 00:04:59,960 WE'RE INTERESTED IN REMODELING 111 00:04:59,960 --> 00:05:02,160 COMPLEX, MULTI-SUBUNIT COMPLEXES 112 00:05:02,160 --> 00:05:05,360 THAT USE ENERGY DIRECT FROM ATP 113 00:05:05,360 --> 00:05:06,240 HYDROLYSIS TO ORGANIZE THE 114 00:05:06,240 --> 00:05:07,640 RELATIONSHIP BETWEEN DNA AND 115 00:05:07,640 --> 00:05:08,400 HISTONES, OBVIOUSLY THAT CAN 116 00:05:08,400 --> 00:05:12,760 LEAD TO ALL KINDS OF DOWNSTREAM 117 00:05:12,760 --> 00:05:15,200 ACTIVITIES THAT ARE IMPORTANT 118 00:05:15,200 --> 00:05:19,880 FOR DNA METABOLISM BASED EVENTS. 119 00:05:19,880 --> 00:05:21,640 IN PARTICULAR, WE'RE FOCUSED ON 120 00:05:21,640 --> 00:05:24,040 THIS MAMMALIAN GROUP OF 121 00:05:24,040 --> 00:05:24,800 CHROMATIN REMODELING COMPLEXES. 122 00:05:24,800 --> 00:05:33,120 AS YOU CAN SEE YOU CAN DIVIDE 123 00:05:33,120 --> 00:05:36,520 SWI/SNF REMODELES INTO THREE 124 00:05:36,520 --> 00:05:42,320 BASED ON SUBUNIT COMPLEXES, BAF, 125 00:05:42,320 --> 00:05:45,160 PBAF OR NCBAF, SHARING A NUMBER 126 00:05:45,160 --> 00:05:54,240 OF CORE SUBUNITS IN GRAY, WITH A 127 00:05:54,240 --> 00:05:56,320 CATALYTIC SUBUNIT IN GREEN, I'LL 128 00:05:56,320 --> 00:05:57,080 MENTION THIS SUBUNIT LATER IN 129 00:05:57,080 --> 00:05:58,000 THE TALK. 130 00:05:58,000 --> 00:05:59,640 WE'VE SPENT A LOT OF TIME 131 00:05:59,640 --> 00:06:03,000 FOCUSING ON THIS PARTICULAR 132 00:06:03,000 --> 00:06:06,080 SUBUNIT OF PBAF, I'M GOING TO 133 00:06:06,080 --> 00:06:12,480 REFER TO AS INTERCHANGEABLY AS 134 00:06:12,480 --> 00:06:13,720 PBRM 1 OR BAF180. 135 00:06:13,720 --> 00:06:16,040 ONE THING THAT'S STRIKING ABOUT 136 00:06:16,040 --> 00:06:17,760 THIS FAMILY OF REMODELERS HOW 137 00:06:17,760 --> 00:06:18,960 FREQUENTLY THEY ARE MISREGULATED 138 00:06:18,960 --> 00:06:19,400 IN CANCER. 139 00:06:19,400 --> 00:06:21,720 YOU CAN SEE FROM THE HEAT MAP 140 00:06:21,720 --> 00:06:23,720 WHERE WE'VE GOT THE DIFFERENT 141 00:06:23,720 --> 00:06:25,800 SUBUNITS ACROSS THE BOTTOM OF 142 00:06:25,800 --> 00:06:28,200 THE HEAT MAP, AND THE -- LOTS OF 143 00:06:28,200 --> 00:06:29,480 CANCER TYPES ALONG THE SIDE OF 144 00:06:29,480 --> 00:06:31,240 THE HEAT MAP. 145 00:06:31,240 --> 00:06:32,760 THIS IS A PROJECT, IN FACT A 146 00:06:32,760 --> 00:06:34,320 LOCKDOWN PROJECT THAT WE DID A 147 00:06:34,320 --> 00:06:36,280 COUPLE YEARS AGO WHERE WE JUST 148 00:06:36,280 --> 00:06:39,400 ANALYZED EVERYTHING THAT WAS 149 00:06:39,400 --> 00:06:41,720 AVAILABLE IN BIOPORTAL AT THAT 150 00:06:41,720 --> 00:06:41,920 TIME. 151 00:06:41,920 --> 00:06:44,280 WHAT WAS PLOTTED IS FREQUENCE OF 152 00:06:44,280 --> 00:06:47,600 DELETERIOUS CHANGES TO THESE 153 00:06:47,600 --> 00:06:49,520 SUBUNITS OF SWI/SNF. 154 00:06:49,520 --> 00:06:53,680 ACROSS A GREAT CHANGE OF CANCER 155 00:06:53,680 --> 00:07:00,680 TYPES YOU SEE FREQUENT MUTATIONS 156 00:07:00,680 --> 00:07:02,560 AND MISREGULATION OF SWI/SNF. 157 00:07:02,560 --> 00:07:05,080 IF YOU ADD TOGETHER, YOU SEE 158 00:07:05,080 --> 00:07:07,160 THAT AROUND 30% OF HUMAN CANCERS 159 00:07:07,160 --> 00:07:11,200 HAVE MISREGULATION OF AT LEAST 160 00:07:11,200 --> 00:07:16,760 ONE SUBUNIT OF SWI/SNF, 161 00:07:16,760 --> 00:07:18,520 PROFOUNDLY MISREGULATED IN 162 00:07:18,520 --> 00:07:19,720 CANCER BIOLOGY. 163 00:07:19,720 --> 00:07:21,240 THERE'S ELEGANT WORK FROM OTHER 164 00:07:21,240 --> 00:07:23,280 LABS SHOWING THIS IS A BONA FIDE 165 00:07:23,280 --> 00:07:24,160 TUMOR SUPPRESSOR ACTIVITY. 166 00:07:24,160 --> 00:07:25,960 ONCE YOU LOSE IT, YOU OPEN THE 167 00:07:25,960 --> 00:07:30,280 DOOR TO THAT PROGRESSION TOWARDS 168 00:07:30,280 --> 00:07:30,600 TUMORIGENESIS. 169 00:07:30,600 --> 00:07:34,080 OUR LAB IS INTERESTED IN 170 00:07:34,080 --> 00:07:37,680 UNDERSTANDING HOW CHROMATIN 171 00:07:37,680 --> 00:07:39,080 BIOLOGY AND SWI/SNF IN 172 00:07:39,080 --> 00:07:44,040 PARTICULAR CONTRIBUTES TO GENE 173 00:07:44,040 --> 00:07:45,480 OWN INSTABILITY, MULTIPLE 174 00:07:45,480 --> 00:07:48,760 CELLULAR ACTIVITIES COULD 175 00:07:48,760 --> 00:07:50,840 POTENTIALLY BE IMPORTANT FOR 176 00:07:50,840 --> 00:07:56,120 TUMORIGENESIS, I'VE LISTED THOSE 177 00:07:56,120 --> 00:07:57,080 HERE, GENE EXPRESSION CHANGES, 178 00:07:57,080 --> 00:08:01,440 NOT THE LEAST AMONG THEM. 179 00:08:01,440 --> 00:08:04,320 AND THERE'S NO DOUBT CELL TYPE 180 00:08:04,320 --> 00:08:05,600 AND CONTEXT-SPECIFIC INFLUENCES 181 00:08:05,600 --> 00:08:08,680 ON WHICH OR ANY OF THESE ARE 182 00:08:08,680 --> 00:08:09,560 ACTUALLY IMPORTANT FOR 183 00:08:09,560 --> 00:08:10,560 TUMORIGENESIS. 184 00:08:10,560 --> 00:08:12,040 AND AGAIN JUST TO REMIND YOU 185 00:08:12,040 --> 00:08:13,640 THIS PARTICULAR STORY I WANT TO 186 00:08:13,640 --> 00:08:15,040 TELL YOU ABOUT CAME OUT OF SOME 187 00:08:15,040 --> 00:08:20,720 WORK WE DID A FEW YEARS BACK 188 00:08:20,720 --> 00:08:23,080 LOOKING AT HOW PBAF INFLUENCES 189 00:08:23,080 --> 00:08:23,600 SISTER ADHESION. 190 00:08:23,600 --> 00:08:25,720 I'LL SHOW ONE OF THE KEY 191 00:08:25,720 --> 00:08:28,720 FINDINGS FROM THAT PROJECT THAT 192 00:08:28,720 --> 00:08:29,240 WE HAD. 193 00:08:29,240 --> 00:08:34,160 SO, THIS IS SOME WORK WE DID 194 00:08:34,160 --> 00:08:36,680 LOOKING AT SISTER CHROMATID AT 195 00:08:36,680 --> 00:08:39,600 HE'S IN ABSENCE OF THE SUBUNIT 196 00:08:39,600 --> 00:08:46,200 OF PBAF. 197 00:08:46,200 --> 00:08:48,680 WE'RE USE FISH PROBES, TO 198 00:08:48,680 --> 00:08:50,320 CHROMOSOME ARMS OR TELOMERES, 199 00:08:50,320 --> 00:08:52,080 WHAT WE FOUND WHEN WE COMPARED 200 00:08:52,080 --> 00:08:56,040 THE CONTROL CELLS WHICH ARE IN 201 00:08:56,040 --> 00:09:02,480 BLACK HERE TO THOSE WHERE WE 202 00:09:02,480 --> 00:09:06,480 DEPLETED PBRM 1 OR BAF 180, YOU 203 00:09:06,480 --> 00:09:07,920 LOOK AT THE READOUT. 204 00:09:07,920 --> 00:09:11,000 YOU CAN SEE IN THE ABSENCE OR 205 00:09:11,000 --> 00:09:13,320 WHEN PBRM 1 IS DEPLETED THE 206 00:09:13,320 --> 00:09:14,840 DISTANCE INCREASES, OR THE 207 00:09:14,840 --> 00:09:17,600 DISTRIBUTION INCREASES IN THIS 208 00:09:17,600 --> 00:09:19,040 TOP PANEL WHEN WE'RE LOOKING 209 00:09:19,040 --> 00:09:21,520 AFTER THE CELLS USING A 210 00:09:21,520 --> 00:09:22,680 CENTROMERE-SPECIFIC PROBE. 211 00:09:22,680 --> 00:09:25,320 BUT INTERESTINGLY WE DIDN'T SEE 212 00:09:25,320 --> 00:09:26,480 THAT SAME DIFFERENCE WITH PROBES 213 00:09:26,480 --> 00:09:35,960 TO CHROMOSOME ARMS OR TELOMERES. 214 00:09:35,960 --> 00:09:38,160 THAT'S SOMETHING WE WANTED TO 215 00:09:38,160 --> 00:09:40,760 DELVE INTO TO UNDERSTAND MORE 216 00:09:40,760 --> 00:09:41,240 FULLY. 217 00:09:41,240 --> 00:09:42,760 ONE OF THE OTHER THINGS WE'VE 218 00:09:42,760 --> 00:09:44,320 BEEN DOING IN THE LAB IS TO 219 00:09:44,320 --> 00:09:48,920 CREATE A WIDE PANEL OF ISOGENIC 220 00:09:48,920 --> 00:09:54,400 CELL SIGNS, DISRUPTING WITH 221 00:09:54,400 --> 00:09:55,760 CRISPR/CAS9. 222 00:09:55,760 --> 00:09:59,280 WE'VE CREATED BETWEEN 3 AND 8 223 00:09:59,280 --> 00:10:00,000 CLONALLY DERIVED PBRM1 DEFICIENT 224 00:10:00,000 --> 00:10:02,880 LINES IN A NUMBER OF CELL LINE 225 00:10:02,880 --> 00:10:03,320 BACKGROUNDS. 226 00:10:03,320 --> 00:10:07,280 WE'VE GOT A FEW NON-CANCER, JUST 227 00:10:07,280 --> 00:10:09,920 IMMORTALIZED CELL LINES SUCH AS 228 00:10:09,920 --> 00:10:12,800 BECOMING WIDELY USED EPITHELIAL 229 00:10:12,800 --> 00:10:14,520 CELL LINE AND A FEW 230 00:10:14,520 --> 00:10:22,760 CANCER-DIRECT CELL LINES AS 231 00:10:22,760 --> 00:10:23,680 WELL. 232 00:10:23,680 --> 00:10:28,600 CREATED CELL LINES WAS TO DO 233 00:10:28,600 --> 00:10:29,480 WHOLE PROTEOME ANALYSIS. 234 00:10:29,480 --> 00:10:30,800 ONE THING WE WANTED TO ASK IS 235 00:10:30,800 --> 00:10:33,560 WHETHER OR NOT WE COULD IDENTIFY 236 00:10:33,560 --> 00:10:36,800 WHAT WE CALL CORE PATHWAYS THAT 237 00:10:36,800 --> 00:10:44,760 ARE REGULATED BY SWI/SNF AND BY 238 00:10:44,760 --> 00:10:46,040 PBRM1. 239 00:10:46,040 --> 00:10:48,000 THERE WOULD BE PATHWAYS WHERE 240 00:10:48,000 --> 00:10:48,760 MISREGULATION WAS APPARENT IN 241 00:10:48,760 --> 00:10:51,400 ONE OR MAYBE A COUPLE CELL LINES 242 00:10:51,400 --> 00:10:52,480 BECAUSE IT WAS VERY SPECIFIC 243 00:10:52,480 --> 00:10:59,720 EITHER TO THAT TISSUE TYPE OR TO 244 00:10:59,720 --> 00:11:02,120 THAT GENETIC CONTEXT BUT WE 245 00:11:02,120 --> 00:11:04,880 MIGHT SEE PATHWAYS DISRUPTED OR 246 00:11:04,880 --> 00:11:06,360 DYSREGULATED ACROSS THESE CELL 247 00:11:06,360 --> 00:11:07,480 LINES GIVING INDICATION OF WHAT 248 00:11:07,480 --> 00:11:15,040 SORT OF THINGS ARE SORT OF 249 00:11:15,040 --> 00:11:16,560 BROADLY MISREGULATED WHEN PBRM1 250 00:11:16,560 --> 00:11:17,880 IS DEFICIENT, TO THINK ABOUT 251 00:11:17,880 --> 00:11:19,400 THIS FROM THE POINT OF VIEW OF 252 00:11:19,400 --> 00:11:21,360 HOW WIDELY THIS IS MISREGULATED 253 00:11:21,360 --> 00:11:23,600 IN CANCER, AND IF WE HAVE A 254 00:11:23,600 --> 00:11:25,760 CANCER THAT HAS A MUTATION, LOSS 255 00:11:25,760 --> 00:11:29,480 OF FUNCTION MUTATION IN PBRM1 256 00:11:29,480 --> 00:11:32,640 ARE THERE PREDICTIONS WE COULD 257 00:11:32,640 --> 00:11:34,120 MAKE ABOUT MISREGULATED PATHWAYS 258 00:11:34,120 --> 00:11:36,040 OR PHENOTYPES ASSOCIATED WITH 259 00:11:36,040 --> 00:11:36,360 THAT. 260 00:11:36,360 --> 00:11:37,120 WE ANALYZED THESE DATA. 261 00:11:37,120 --> 00:11:38,560 ONE OF THE THINGS WE WERE KEEN 262 00:11:38,560 --> 00:11:42,160 TO LOOK AT BASED ON PREVIOUS 263 00:11:42,160 --> 00:11:46,120 OBSERVATIONS AROUND SISTER 264 00:11:46,120 --> 00:11:48,520 CHROMATID ADHESION AT 265 00:11:48,520 --> 00:11:49,160 CENTROMERES WAS CENTROMERE 266 00:11:49,160 --> 00:11:50,400 ASSOCIATED PROTEINS AND 267 00:11:50,400 --> 00:11:50,880 PATHWAYS. 268 00:11:50,880 --> 00:11:53,160 WE FOUND THAT FELL INTO ONE OF 269 00:11:53,160 --> 00:11:57,960 THESE TYPE OF CATEGORIES, 270 00:11:57,960 --> 00:11:58,920 BROADLY ASSOCIATED WITH PBRM1 271 00:11:58,920 --> 00:12:01,320 LOSS, ALL OF THE CLONES IN ALL 272 00:12:01,320 --> 00:12:02,520 CELL LINE BACKGROUNDS ARE THE 273 00:12:02,520 --> 00:12:04,680 SAME CHANGES, AND THOSE ARE 274 00:12:04,680 --> 00:12:05,240 REPRESENTED HERE. 275 00:12:05,240 --> 00:12:07,640 SO, THIS IS -- THESE ARE DATA 276 00:12:07,640 --> 00:12:10,600 PLOTTED FROM THE RPE1 CELL LINE, 277 00:12:10,600 --> 00:12:14,000 I'M GOING TO PRIMARILY SHOW THAT 278 00:12:14,000 --> 00:12:18,800 CELL LINE-BASED DATA UNLESS I 279 00:12:18,800 --> 00:12:21,200 SPECIFY OTHERWISE. 280 00:12:21,200 --> 00:12:23,560 WE'VE GOT LOG TWO FOLD CHANGE IN 281 00:12:23,560 --> 00:12:26,680 KNOCKOUT COMPARED TO PARENTAL. 282 00:12:26,680 --> 00:12:27,920 WE'VE DIVIDED THESE PROTEINS 283 00:12:27,920 --> 00:12:29,840 BASED ON THEIR ORGANIZATION SORT 284 00:12:29,840 --> 00:12:31,520 OF ROUGHLY ACCORDING TO THIS 285 00:12:31,520 --> 00:12:33,240 SCHEMATIC IN THE BOTTOM, SO 286 00:12:33,240 --> 00:12:38,640 COMING FROM THE MOST CLOSELY 287 00:12:38,640 --> 00:12:42,440 ASSOCIATED MOVING OUTWARD TO 288 00:12:42,440 --> 00:12:43,880 CONNECTING WITH MICROTUBE ALLS, 289 00:12:43,880 --> 00:12:47,880 YOU CAN SEE OUTER CORE, INNER 290 00:12:47,880 --> 00:12:49,800 CORE, OTHER 291 00:12:49,800 --> 00:12:50,440 CENTROMERE-ASSOCIATED PROTEINS. 292 00:12:50,440 --> 00:12:52,440 ACROSS THE BOARD WE DON'T HAVE 293 00:12:52,440 --> 00:12:53,920 DRAMATIC DECREASE OF PROTEIN 294 00:12:53,920 --> 00:12:54,920 LEVELS OF INDIVIDUAL PROTEINS 295 00:12:54,920 --> 00:12:58,120 BUT THEY ARE ALL SIGNIFICANTLY 296 00:12:58,120 --> 00:12:59,560 AND REPRODUCIBLY DOWN IN THE 297 00:12:59,560 --> 00:12:59,960 KNOCKOUTS. 298 00:12:59,960 --> 00:13:02,920 AS I SAY, THIS PATHWAY ON 299 00:13:02,920 --> 00:13:04,760 ALTHOUGH IT DOESN'T LOOK LIKE 300 00:13:04,760 --> 00:13:07,400 THE SAME PATTERN, THEY HAVE 301 00:13:07,400 --> 00:13:08,840 DOWNREGULATION ACROSS THIS PANEL 302 00:13:08,840 --> 00:13:10,120 OF PROTEINS. 303 00:13:10,120 --> 00:13:12,120 SO, WE THOUGHT THIS WAS 304 00:13:12,120 --> 00:13:14,760 EXTREMELY INTERESTING GIVEN WHAT 305 00:13:14,760 --> 00:13:16,680 WE'VE SEEN PREVIOUSLY AND 306 00:13:16,680 --> 00:13:17,560 MECHANISTICALLY THE FIRST THING 307 00:13:17,560 --> 00:13:19,760 WE THOUGHT WAS MAYBE THE GENES 308 00:13:19,760 --> 00:13:21,640 ARE ALL REGULATED BY PBAF, WHEN 309 00:13:21,640 --> 00:13:24,040 WE KNOCK OUT ONE OF THE SUBUNITS 310 00:13:24,040 --> 00:13:29,160 OF PBA, IF WE SEE DOWNREGULATION 311 00:13:29,160 --> 00:13:30,200 OF PROTEINS. 312 00:13:30,200 --> 00:13:31,040 SO, WE LOOKED TO SEE WHETHER OR 313 00:13:31,040 --> 00:13:33,240 NOT WE HAD A SORT OF BROAD 314 00:13:33,240 --> 00:13:35,520 REGULATION OF THE GENES THAT 315 00:13:35,520 --> 00:13:36,840 ENCODE THESE DIFFERENT PROTEINS, 316 00:13:36,840 --> 00:13:38,600 AND WE DID THIS AGAIN ACROSS THE 317 00:13:38,600 --> 00:13:40,200 DIFFERENT CELL LINES AND IN A 318 00:13:40,200 --> 00:13:41,200 COUPLE DIFFERENT WAYS BUT WHAT 319 00:13:41,200 --> 00:13:45,040 I'LL SHOW HERE IS THE RNAseq 320 00:13:45,040 --> 00:13:46,200 DATA FROM RPE1. 321 00:13:46,200 --> 00:13:47,920 AGAIN, PLOTTED ON THE TOP THIS 322 00:13:47,920 --> 00:13:48,880 IS THE PROTEOME DATA. 323 00:13:48,880 --> 00:13:51,640 AND ON THE BOTTOM IS THE 324 00:13:51,640 --> 00:13:53,080 RNAseq DATA, PLOTTED IN THE 325 00:13:53,080 --> 00:13:54,960 SAME WAY, LOG TWO-FOLD CHANGE, 326 00:13:54,960 --> 00:13:56,000 KNOCKOUT RELATIVE TO PARENTAL. 327 00:13:56,000 --> 00:13:57,520 WHAT YOU CAN SKI HERE AGAIN 328 00:13:57,520 --> 00:13:59,520 THESE ARE LINED UP IN THE SAME 329 00:13:59,520 --> 00:14:01,800 ORDER, THERE'S A LITTLE BIT OF 330 00:14:01,800 --> 00:14:02,360 DIFFERENCE BECAUSE OBVIOUSLY 331 00:14:02,360 --> 00:14:03,120 THERE'S SOME THINGS WE COULD 332 00:14:03,120 --> 00:14:05,040 PICK UP IN THE RNAseq WE 333 00:14:05,040 --> 00:14:06,960 DIDN'T PICK UP IN THE PROTEOME 334 00:14:06,960 --> 00:14:08,280 BUT GENERALLY SPEAKING YOU CAN 335 00:14:08,280 --> 00:14:10,880 SEE THAT THERE IS NOT A GREAT 336 00:14:10,880 --> 00:14:13,000 CORRELATION BETWEEN THE PROTEIN 337 00:14:13,000 --> 00:14:14,080 LEVELS AND TRANSCRIPT LEVELS, 338 00:14:14,080 --> 00:14:16,920 AND INDEED IF YOU LOOK AT SOME 339 00:14:16,920 --> 00:14:20,520 OF THE TRANSCRIPT LEVELS 340 00:14:20,520 --> 00:14:23,160 DECREASED IN THE PBRM1 KNOCKOUT 341 00:14:23,160 --> 00:14:25,280 SUCH AS THIS HERE WE DON'T SEE A 342 00:14:25,280 --> 00:14:26,320 GREAT CORRELATION WITH WHAT'S 343 00:14:26,320 --> 00:14:36,160 HAPPENING AT THE PROTEIN LEVEL 344 00:14:36,160 --> 00:14:38,760 WITH CENFAS. 345 00:14:38,760 --> 00:14:42,960 WE DID A CUT AND RUN MAPPING OF 346 00:14:42,960 --> 00:14:46,120 THE CATALYTIC SUBUNIT OF PBAF TO 347 00:14:46,120 --> 00:14:47,040 SEE GLOBALLY WHERE THIS IS 348 00:14:47,040 --> 00:14:47,400 BOUND. 349 00:14:47,400 --> 00:14:50,520 AND OF COURSE WE CAN MAP THAT TO 350 00:14:50,520 --> 00:14:51,400 GENE PROMOTERS AND ENHANCERS AND 351 00:14:51,400 --> 00:15:01,840 WHAT WE FIND IS THERE'S A 352 00:15:03,040 --> 00:15:04,160 LIMITED ASSOCIATION OF SMARCA4. 353 00:15:04,160 --> 00:15:06,480 WE DON'T SEE STRONG EVIDENCE 354 00:15:06,480 --> 00:15:08,320 DOWNREGULATION IS DOWN TO 355 00:15:08,320 --> 00:15:11,200 MISREGULATION OF THE GENES OF 356 00:15:11,200 --> 00:15:12,520 TRANSCRIPTION OF THOSE GENES. 357 00:15:12,520 --> 00:15:14,360 SO WERE DO WE SEE 358 00:15:14,360 --> 00:15:14,760 DOWNREGULATION? 359 00:15:14,760 --> 00:15:16,040 WE STARTED THINKING ABOUT IT THE 360 00:15:16,040 --> 00:15:17,600 OTHER WAY AROUND, IT WAS A 361 00:15:17,600 --> 00:15:21,120 CONSEQUENCE OF A FAILURE TO 362 00:15:21,120 --> 00:15:25,240 ASSEMBLE INTO THE PROTEIN 363 00:15:25,240 --> 00:15:25,920 COMPLEX. 364 00:15:25,920 --> 00:15:31,680 SO EVERYONE WILL BE FAMILIAR, I 365 00:15:31,680 --> 00:15:34,080 PUT PBAF HERE, WHEN WE MAKE A 366 00:15:34,080 --> 00:15:40,400 KNOCK OUTCELL LINE OR DEPLETE BY 367 00:15:40,400 --> 00:15:45,760 S RNA WE USE STABILITY. 368 00:15:45,760 --> 00:15:52,640 LEVEL OF PROTEINS DOWN ACROSS 369 00:15:52,640 --> 00:15:54,200 THIS ASSOCIATED WITH CENTROMERE 370 00:15:54,200 --> 00:15:57,600 MIGHT BE A READOUT OF FAILURE TO 371 00:15:57,600 --> 00:15:59,520 ASSEMBLE INTO THE COMPLEXES 372 00:15:59,520 --> 00:16:02,520 WHICH THEN WOULD STABILIZE THE 373 00:16:02,520 --> 00:16:04,040 PROTEIN LEVELS GLOBALLY. 374 00:16:04,040 --> 00:16:05,160 AND THAT'S ACTUALLY NOT 375 00:16:05,160 --> 00:16:06,400 UNREASONABLE THOUGHT WHEN YOU 376 00:16:06,400 --> 00:16:07,760 THINK THAT THE CHROMATIN 377 00:16:07,760 --> 00:16:11,160 STRUCTURE AT CENTROMERES IS 378 00:16:11,160 --> 00:16:11,960 OBVIOUSLY VERY COMPLICATED AND 379 00:16:11,960 --> 00:16:15,720 WHAT YOU NEED TO DO IS 380 00:16:15,720 --> 00:16:18,480 REORGANIZE FIRST THAT AT THIS 381 00:16:18,480 --> 00:16:19,560 INDIVIDUAL LEVEL REORGANIZE 382 00:16:19,560 --> 00:16:25,600 NUCLEOSOMES TO PREVENT ASSEMBLY 383 00:16:25,600 --> 00:16:26,920 OF VARIOUS ASSOCIATED PROTEINS 384 00:16:26,920 --> 00:16:31,400 AND THEN BUILD THIS COMPLEX 385 00:16:31,400 --> 00:16:32,840 OUTWARD TOWARD MICROTUBULES AND 386 00:16:32,840 --> 00:16:34,880 WE HAD A DEFECT IN SISTER 387 00:16:34,880 --> 00:16:36,200 CHROMATID ADHESION AND WE KNOW 388 00:16:36,200 --> 00:16:38,200 FROM A LOT OF PEOPLE'S WORK 389 00:16:38,200 --> 00:16:41,080 THERE ARE THREE DIMENSIONAL 390 00:16:41,080 --> 00:16:47,680 ORGANIZATION OF CENTROMERES AND 391 00:16:47,680 --> 00:16:48,520 PERICENTROMERES THAT INVOLVES 392 00:16:48,520 --> 00:16:49,160 LOOPING. 393 00:16:49,160 --> 00:16:51,680 A FAILURE OF CHROMATIN REMODELER 394 00:16:51,680 --> 00:16:53,840 TO DOUGH TO CENTROMERES AND 395 00:16:53,840 --> 00:16:55,880 ORGANIZE THIS CHROMATIN COULD 396 00:16:55,880 --> 00:16:57,600 POTENTIALLY LEAD TO A SLIGHT 397 00:16:57,600 --> 00:16:59,360 DECREASE IN THE EFFICIENCY OR 398 00:16:59,360 --> 00:17:01,120 ABILITY OF THESE PROTEIN 399 00:17:01,120 --> 00:17:02,960 COMPLEXES TO ASSEMBLE IN THE 400 00:17:02,960 --> 00:17:04,440 APPROPRIATE WAY. 401 00:17:04,440 --> 00:17:05,280 SO, WE WANTED TO SEE WHETHER OR 402 00:17:05,280 --> 00:17:08,320 NOT THERE WAS ANY EVIDENCE THAT 403 00:17:08,320 --> 00:17:11,080 EITHER THE STRUCTURE OR 404 00:17:11,080 --> 00:17:12,160 INTEGRITY OF THESE SEQUENCES WAS 405 00:17:12,160 --> 00:17:14,600 IMPAIRED IN ANY WAY IN THE 406 00:17:14,600 --> 00:17:15,680 ABSENCE OF PBRM1. 407 00:17:15,680 --> 00:17:18,960 I'LL SHOW YOU A FEW EXAMPLES OF 408 00:17:18,960 --> 00:17:19,720 THOSE DATA. 409 00:17:19,720 --> 00:17:21,800 IN THIS EXPERIMENT WHAT WE DID 410 00:17:21,800 --> 00:17:26,960 WAS JUST TO SIMPLY USE FISH 411 00:17:26,960 --> 00:17:31,800 PROBES AGAINST CENTROMERIC 412 00:17:31,800 --> 00:17:34,200 SEQUENCES IN CHROMOSOME 2 OR 10. 413 00:17:34,200 --> 00:17:36,960 AND VISUALIZE THOSE 414 00:17:36,960 --> 00:17:37,320 MICROSCOPICALLY. 415 00:17:37,320 --> 00:17:38,800 HOPEFULLY YOU CAN SEE 416 00:17:38,800 --> 00:17:41,080 SUBJECTIVELY IN THE IMAGES IS 417 00:17:41,080 --> 00:17:42,440 THAT THE INTENSITY AND SIZE OF 418 00:17:42,440 --> 00:17:44,480 THE SIGNALS THAT WE SEE IN THE 419 00:17:44,480 --> 00:17:48,400 KNOCKOUT CELLS IN THE BOTTOM TWO 420 00:17:48,400 --> 00:17:50,080 ROWS IS GREATER THAN THAT WE SEE 421 00:17:50,080 --> 00:17:51,400 IN THE PARENTAL. 422 00:17:51,400 --> 00:17:55,240 THIS IS QUANTIFIED ON THE OTHER 423 00:17:55,240 --> 00:17:56,520 SIDE OF THE SLIDE. 424 00:17:56,520 --> 00:17:58,040 AND I PUT IN HERE THESE CHANGES 425 00:17:58,040 --> 00:18:00,440 ARE OBSERVED IN ALL OF THE CELL 426 00:18:00,440 --> 00:18:03,200 LINES WE'VE TESTED SO FAR, 427 00:18:03,200 --> 00:18:04,040 SUGGESTING THAT THESE 428 00:18:04,040 --> 00:18:09,360 CENTROMERES ARE NOT CREATING 429 00:18:09,360 --> 00:18:10,080 THIS THREE DIMENSIONAL STRUCTURE 430 00:18:10,080 --> 00:18:12,400 IN THE SAME WAY IN THE KNOCKOUTS 431 00:18:12,400 --> 00:18:13,720 AS IN PARENTAL, AT LEAST THAT'S 432 00:18:13,720 --> 00:18:17,520 ONE INTERPRETATION OF THESE 433 00:18:17,520 --> 00:18:17,720 DATA. 434 00:18:17,720 --> 00:18:22,000 IT'S WORTH NOTING THIS IS A 435 00:18:22,000 --> 00:18:24,880 FEATURE OF PBRM1 KNOCKOUT IN THE 436 00:18:24,880 --> 00:18:25,360 ABSENCE OF PERTURBATION. 437 00:18:25,360 --> 00:18:28,240 THIS IS SIMPLY HOW THEY LOOK 438 00:18:28,240 --> 00:18:29,880 UNDER NORMAL GROWTH CONDITIONS. 439 00:18:29,880 --> 00:18:31,760 THIS IS THE CELL LINES WE'VE 440 00:18:31,760 --> 00:18:34,920 LOOKED AT SO FAR INCLUDING 441 00:18:34,920 --> 00:18:36,360 CANCER DIRECT AND NONCANCER 442 00:18:36,360 --> 00:18:36,680 DIRECT. 443 00:18:36,680 --> 00:18:40,080 WHAT ABOUT THE INTEGRITY OF 444 00:18:40,080 --> 00:18:41,280 THESE CENTROMERIC REPEATS? 445 00:18:41,280 --> 00:18:43,720 WE WANTED TO LOOK AT THAT AND WE 446 00:18:43,720 --> 00:18:48,680 DECIDED TO USE AN ASSAY THAT WAS 447 00:18:48,680 --> 00:18:50,680 DEVELOPED WHERE THEY WERE 448 00:18:50,680 --> 00:18:56,120 LOOKING AT THE PROTECTIVE 449 00:18:56,120 --> 00:19:00,200 ABILITY OF THE CENP-A, AS WELL 450 00:19:00,200 --> 00:19:04,480 AS ASSOCIATED PROTEIN CENP-C AND 451 00:19:04,480 --> 00:19:04,960 CENP-T. 452 00:19:04,960 --> 00:19:09,800 AND WHAT THEY DID HERE WAS TO 453 00:19:09,800 --> 00:19:12,800 USE BRD, BRDC LABELING TO 454 00:19:12,800 --> 00:19:14,960 INCORPORATE INTO THE DNA, AND 455 00:19:14,960 --> 00:19:18,600 THEN THEY COULD PROCESS THAT 456 00:19:18,600 --> 00:19:19,880 WITH NUCLEASE DIGESTION IN ORDER 457 00:19:19,880 --> 00:19:23,640 TO GIVE YOU A STRAND-SPECIFIC 458 00:19:23,640 --> 00:19:29,080 LABELING, USING -- YOU CAN SEE 459 00:19:29,080 --> 00:19:32,920 FLUORESCENTLY LABELED PROBES 460 00:19:32,920 --> 00:19:33,720 AGAINST THE CENP DNA. 461 00:19:33,720 --> 00:19:35,880 IF YOU DO THAT IN NORMAL CELLS, 462 00:19:35,880 --> 00:19:39,080 WHAT YOU GET IS THIS VERY LOVELY 463 00:19:39,080 --> 00:19:39,960 SIGNAL WHERE ONE SISTER 464 00:19:39,960 --> 00:19:44,000 CHROMATIN LIGHTS UP WITH RED, 465 00:19:44,000 --> 00:19:45,120 ONE WITH GREEN. 466 00:19:45,120 --> 00:19:49,320 BUT IF YOU'VE GOT INAPPROPRIATE 467 00:19:49,320 --> 00:19:50,640 RECOMBINATION OR REARRANGEMENT 468 00:19:50,640 --> 00:19:51,640 BETWEEN THESE REPETITIVE 469 00:19:51,640 --> 00:19:52,960 SEQUENCES IN THE CENTROMERES YOU 470 00:19:52,960 --> 00:19:54,920 START TO SEE THESE ABERRANT 471 00:19:54,920 --> 00:19:56,240 SIGNALS WHERE YOU GET DIFFERENT 472 00:19:56,240 --> 00:20:00,280 PATTERNS OF RED AND GREEN 473 00:20:00,280 --> 00:20:01,880 ASSOCIATED WITH SISTER 474 00:20:01,880 --> 00:20:03,120 CHROMATIDS, AND IMAGES SHOWN ON 475 00:20:03,120 --> 00:20:05,200 THE BOTTOM, YOU CAN SEE IN THE 476 00:20:05,200 --> 00:20:06,520 CONTROL CELL LINE YOU'VE GOT 477 00:20:06,520 --> 00:20:08,280 REALLY LOVELY GREEN AND RED 478 00:20:08,280 --> 00:20:12,440 SIGNALS, AND THEN WHEN THEY 479 00:20:12,440 --> 00:20:13,760 DEPLETE CENP-A, SIGNALS BECOME 480 00:20:13,760 --> 00:20:19,760 NO LONGER SORT OF BEAUTIFULLY 481 00:20:19,760 --> 00:20:20,840 PATTERNED ACCORDING TO SISTER 482 00:20:20,840 --> 00:20:21,840 CHROME SIDS. 483 00:20:21,840 --> 00:20:24,600 WE SET THIS UP TO SEE WHETHER 484 00:20:24,600 --> 00:20:28,080 LOSS OF PBRM 1 WOULD HAVE IMPACT 485 00:20:28,080 --> 00:20:29,840 ON INTEGRITY OF CENTROMERIC 486 00:20:29,840 --> 00:20:30,240 SEQUENCES. 487 00:20:30,240 --> 00:20:32,480 DATA THAT WE GOT ARE SHOWN HERE. 488 00:20:32,480 --> 00:20:34,880 SO THIS IS -- YOU CAN SEE THIS 489 00:20:34,880 --> 00:20:37,920 IS A SET OF CHROMOSOMES LABELED 490 00:20:37,920 --> 00:20:39,920 IN THE WAY DESCRIBED PREVIOUSLY. 491 00:20:39,920 --> 00:20:45,160 YOU GET REALLY NICE ONE RED 492 00:20:45,160 --> 00:20:50,320 SIGNAL, ONE GREEN WITH SISTER 493 00:20:50,320 --> 00:20:51,400 CHROMATIDS. 494 00:20:51,400 --> 00:20:52,520 POSITIVE CONTROL, DELETED 495 00:20:52,520 --> 00:20:54,680 CENP-A, THAT LEADS TO A DECREASE 496 00:20:54,680 --> 00:20:57,840 IN THE NUMBER OF NORMAL SIGNALS 497 00:20:57,840 --> 00:20:59,520 ASSOCIATED WITH SISTER 498 00:20:59,520 --> 00:20:59,920 CHROMATIDS. 499 00:20:59,920 --> 00:21:01,680 AND THESE ARE THE DATA WITH THE 500 00:21:01,680 --> 00:21:02,040 CONTROL. 501 00:21:02,040 --> 00:21:04,920 SO THIS IS WHAT WE FOUND IN -- 502 00:21:04,920 --> 00:21:11,280 WHEN WE QUANTITATED NUMBER OF 503 00:21:11,280 --> 00:21:13,000 ABNORMAL MITOSIS, THIS IS WHAT 504 00:21:13,000 --> 00:21:16,720 WE SAW. 505 00:21:16,720 --> 00:21:19,000 WHEN KEY DEPLETE CENP-A THERE'S 506 00:21:19,000 --> 00:21:20,960 INCREASE IN NORMAL MITOSIS, AS 507 00:21:20,960 --> 00:21:22,160 WE WOULD EXPECT. 508 00:21:22,160 --> 00:21:24,040 WHEN WE LOOKED AT PARENTAL CELL 509 00:21:24,040 --> 00:21:25,680 LINE AND KNOCKOUTS WE SAW 510 00:21:25,680 --> 00:21:26,200 SOMETHING SIMILAR. 511 00:21:26,200 --> 00:21:29,440 THIS AGAIN IS THE PARENTAL, 512 00:21:29,440 --> 00:21:30,640 WHICH LOOKS LIKE NON-TARGETING 513 00:21:30,640 --> 00:21:32,040 CONTROL WITH S RNA, AND THEN 514 00:21:32,040 --> 00:21:37,200 THESE ARE THE TWO PBRM1 CLONES, 515 00:21:37,200 --> 00:21:39,040 THE RPE CELL LINE. 516 00:21:39,040 --> 00:21:41,320 WE SAW SOMETHING THAT WAS FAIRLY 517 00:21:41,320 --> 00:21:44,920 SIMILAR TO WHAT THEY WERE SEEING 518 00:21:44,920 --> 00:21:46,320 WITH CENP-A DEPLETION, LOSING 519 00:21:46,320 --> 00:21:49,720 ABILITY OR LOSING PROTECTION OF 520 00:21:49,720 --> 00:21:52,600 THESE CENTROMERIC SEQUENCE WHEN 521 00:21:52,600 --> 00:21:56,640 PBRM1 IS MISSING. 522 00:21:56,640 --> 00:21:58,400 SO, I MENTIONED THAT WE FOUND 523 00:21:58,400 --> 00:22:01,360 THIS BY SORT OF LOOKING AT THE 524 00:22:01,360 --> 00:22:02,600 PROTEOME ACROSS OUR DIFFERENT 525 00:22:02,600 --> 00:22:03,920 CELL LINE MODELS WE GENERATED IN 526 00:22:03,920 --> 00:22:12,880 THE LAB, WHICH ARE GREAT BECAUSE 527 00:22:12,880 --> 00:22:13,920 THEY ARE ISOGENIC, BUT THE 528 00:22:13,920 --> 00:22:14,920 ORIGINAL HERE WAS THIS IS 529 00:22:14,920 --> 00:22:17,360 SOMETHING THAT MIGHT BE 530 00:22:17,360 --> 00:22:21,080 GENERALLY TRUE, IN LOTS OF 531 00:22:21,080 --> 00:22:22,320 DIFFERENT CELLS LACKING PBRM1, 532 00:22:22,320 --> 00:22:24,160 MORE BROADLY THINK FROM A CANCER 533 00:22:24,160 --> 00:22:26,680 POINT OF VIEW. 534 00:22:26,680 --> 00:22:29,800 SO, ONE OF THE THINGS WE DID WAS 535 00:22:29,800 --> 00:22:34,440 TO DOWNLOAD SOME DATA FROM THE 536 00:22:34,440 --> 00:22:37,280 STUDIES WHERE THEY DID THE 537 00:22:37,280 --> 00:22:38,920 CANCER CELL LINE ENCYCLOPEDIA, 538 00:22:38,920 --> 00:22:43,440 AND IF YOU REMEMBER WE CAN'T SEE 539 00:22:43,440 --> 00:22:45,720 THIS CHANGE IN CENTROMERE AT THE 540 00:22:45,720 --> 00:22:47,480 TRANSCRIPT LEVEL, SO WE 541 00:22:47,480 --> 00:22:50,160 DOWNLOADED THE DATA WHERE THERE 542 00:22:50,160 --> 00:22:55,360 WERE FULL PROTEOMES, NOT LOOKING 543 00:22:55,360 --> 00:22:59,720 AT ISOGENIC PAIRS, WHAT WE DID 544 00:22:59,720 --> 00:23:01,720 WAS ORDER BASED ON PBRM1 545 00:23:01,720 --> 00:23:03,200 EXPRESSION FROM PROTEOMIC DATA, 546 00:23:03,200 --> 00:23:04,760 SHOWN HERE. 547 00:23:04,760 --> 00:23:08,920 WE'VE GOT EACH COLUMN HERE 548 00:23:08,920 --> 00:23:12,320 REPRESENTS A CELL LINE WITH THE 549 00:23:12,320 --> 00:23:14,080 ENCYCLOPEDIA WITH PROTEOMIC DATA 550 00:23:14,080 --> 00:23:14,360 AVAILABLE. 551 00:23:14,360 --> 00:23:17,360 BELOW IT IN THE HEAT MAP SHOWING 552 00:23:17,360 --> 00:23:23,480 RELATIVE SCALED ABUNDANCE OF THE 553 00:23:23,480 --> 00:23:24,320 CENTROMERE-ASSOCIATED PROTEINS 554 00:23:24,320 --> 00:23:27,080 IN PROTEOMES IN THE 555 00:23:27,080 --> 00:23:27,720 CENTROMERE-ASSOCIATED PROTEINS, 556 00:23:27,720 --> 00:23:29,440 IN THE DATA. 557 00:23:29,440 --> 00:23:31,640 AND WHAT YOU CAN SEE RED IS 558 00:23:31,640 --> 00:23:35,920 HIGH, BLUE IS LOW, AS YOU GO 559 00:23:35,920 --> 00:23:39,680 FROM HIGH TO LOW LEVELS YOU SEE 560 00:23:39,680 --> 00:23:44,080 A GENERAL CHANGE IN THE 561 00:23:44,080 --> 00:23:45,200 CORRELATION AND ABUNDANCE OF 562 00:23:45,200 --> 00:23:47,360 CENTROMERE-ASSOCIATED PROTEINS, 563 00:23:47,360 --> 00:23:51,280 SOMETHING MORE BROADLY TRUE WHEN 564 00:23:51,280 --> 00:23:54,800 YOU LOSE PBRM1 YOU GET DECREASE 565 00:23:54,800 --> 00:23:57,000 IN CENTROMERE-SPECIFIC PROTEINS. 566 00:23:57,000 --> 00:24:00,240 WHAT ABOUT IMPACT ON FRAGILITY 567 00:24:00,240 --> 00:24:00,840 OR INSTABILITY? 568 00:24:00,840 --> 00:24:03,480 EVEN IN THE ABSENCE OF 569 00:24:03,480 --> 00:24:05,080 PERTURBATION WE WERE SEE 570 00:24:05,080 --> 00:24:05,880 REARRANGEMENTS OF CENTROMERES IN 571 00:24:05,880 --> 00:24:06,360 THE SYSTEM. 572 00:24:06,360 --> 00:24:08,720 PREVIOUSLY WHEN WE FOUND THIS 573 00:24:08,720 --> 00:24:10,800 DEFECT IN SISTER CHROMATID 574 00:24:10,800 --> 00:24:15,400 COHESION WE LOOKED AT 575 00:24:15,400 --> 00:24:18,040 ANEUPLOIDY, THERE WAS AN 576 00:24:18,040 --> 00:24:21,760 INCREASED LEVEL OF ANEUPLOIDY IN 577 00:24:21,760 --> 00:24:22,520 WHOLE CHROMOSOME INSTABILITY 578 00:24:22,520 --> 00:24:24,120 WITH LOSS OF PBRM1. 579 00:24:24,120 --> 00:24:27,080 IS THIS TRUE IN CANCER SAMPLES? 580 00:24:27,080 --> 00:24:30,600 DO YOU SEE EITHER WHOLE ARM OR 581 00:24:30,600 --> 00:24:31,720 WHOLE CHROMOSOME INSTABILITY 582 00:24:31,720 --> 00:24:33,160 ASSOCIATED WITH PBRM1 LOSS? 583 00:24:33,160 --> 00:24:35,000 FORTUNATELY FOR US A BEAUTIFUL 584 00:24:35,000 --> 00:24:38,600 STUDY CAME OUT FROM FLORIAN 585 00:24:38,600 --> 00:24:41,200 MARKOWITZ'S LAB LAST YEAR 586 00:24:41,200 --> 00:24:42,760 LOOKING AT CHROMOSOME 587 00:24:42,760 --> 00:24:47,240 INSTABILITY BROADLY, USING -- 588 00:24:47,240 --> 00:24:49,760 LOOKING PAN-CANCER, TRYING TO 589 00:24:49,760 --> 00:24:51,640 UNDERSTAND SIGNATURES, THAT WERE 590 00:24:51,640 --> 00:24:53,160 APPARENT IN CANCER SAMPLES. 591 00:24:53,160 --> 00:24:57,480 AND THEY DID THAT, AND THEY 592 00:24:57,480 --> 00:24:59,520 IDENTIFIED 17 SIGNATURES 593 00:24:59,520 --> 00:25:03,360 ASSOCIATED WITH CHROMOSOME 594 00:25:03,360 --> 00:25:07,520 INSTABILITY BASED ON DIFFERENT 595 00:25:07,520 --> 00:25:11,680 FEATURES AND LOOKED TO SEE 596 00:25:11,680 --> 00:25:13,160 WHETHER THERE WERE GENETIC 597 00:25:13,160 --> 00:25:20,080 DETERMINANTS, THIS IS ONE OF THE 598 00:25:20,080 --> 00:25:22,320 PAPERS SHOWING THAT, YOU CAN SEE 599 00:25:22,320 --> 00:25:29,840 THE 17 17 SIGNATURE SIGNATURES, 600 00:25:29,840 --> 00:25:30,920 LOSS WAS ASSOCIATED WITH 601 00:25:30,920 --> 00:25:32,480 SIGNATURE 1. 602 00:25:32,480 --> 00:25:35,080 SIGNATURE 1 WHEN THEY WERE 603 00:25:35,080 --> 00:25:37,160 DESCRIBING THESE SIGNATURES AND 604 00:25:37,160 --> 00:25:37,880 BASED ON MOLECULAR 605 00:25:37,880 --> 00:25:39,360 CHARACTERISTICS OF WHAT THEY 606 00:25:39,360 --> 00:25:43,520 WERE SEEING IS ASSOCIATED WITH 607 00:25:43,520 --> 00:25:44,600 DEFECTS IN MITOSIS, AND 608 00:25:44,600 --> 00:25:47,240 ASSOCIATED WITH EITHER WHOLE ARM 609 00:25:47,240 --> 00:25:48,560 OR WHOLE CHROMOSOME CHANGES. 610 00:25:48,560 --> 00:25:49,560 THIS BEAUTIFULLY FIT WITH WHAT 611 00:25:49,560 --> 00:25:52,040 WE WERE FINDING IN OUR CELL LINE 612 00:25:52,040 --> 00:25:54,800 MODELS, SUGGESTING THIS IS 613 00:25:54,800 --> 00:25:56,800 ACTUALLY PERHAPS MORE BROADLY 614 00:25:56,800 --> 00:25:58,320 ASSOCIATED WITH CANCER SAMPLES 615 00:25:58,320 --> 00:26:01,120 AND SOMETHING THAT'S RELEVANT IN 616 00:26:01,120 --> 00:26:02,120 CANCER BIOLOGY. 617 00:26:02,120 --> 00:26:03,920 SO, WHAT WE WANTED TO DO WAS 618 00:26:03,920 --> 00:26:04,960 PUSH THIS SYSTEM. 619 00:26:04,960 --> 00:26:07,400 SO FAR WE'VE JUST BEEN MAPPING 620 00:26:07,400 --> 00:26:08,880 AND CHARACTERIZING WHAT'S 621 00:26:08,880 --> 00:26:13,960 HAPPENING IN THE ABSENCE OFPBRM1 622 00:26:13,960 --> 00:26:16,240 UNDER NORMAL CONDITIONS IN THE 623 00:26:16,240 --> 00:26:16,960 CELLS. 624 00:26:16,960 --> 00:26:19,840 HERE WE'VE SEEN WHETHER OR NOT 625 00:26:19,840 --> 00:26:21,320 THEY SHOW HYPERSENSITIVITY TO 626 00:26:21,320 --> 00:26:22,160 MITOTIC PERTURBATION. 627 00:26:22,160 --> 00:26:23,800 WHAT I'M GOING TO SHOW IS AN 628 00:26:23,800 --> 00:26:25,720 EXPERIMENT THAT WE DID WHERE WE 629 00:26:25,720 --> 00:26:29,800 JUST TREATED THE CELLS WITH CDK 630 00:26:29,800 --> 00:26:31,920 1 INHIBITOR FOR 24 HOURS, 631 00:26:31,920 --> 00:26:36,840 REPORTED IN LITERATURE, CELLS 632 00:26:36,840 --> 00:26:38,160 WITH PROBLEMS AT CENTROMERES ARE 633 00:26:38,160 --> 00:26:39,040 PARTICULARLY SENSITIVE TO. 634 00:26:39,040 --> 00:26:42,960 YOU CAN SEE THE SCHEME WE USED, 635 00:26:42,960 --> 00:26:45,280 SHOWN HERE, TREATED WITH CDK1 636 00:26:45,280 --> 00:26:46,720 INHIBITOR FOR 24 HOURS, WASHED 637 00:26:46,720 --> 00:26:49,680 IT OFF AND ALLOWED CELLS TO 638 00:26:49,680 --> 00:26:52,000 REENTER CELL CYCLE AND MONITORED 639 00:26:52,000 --> 00:26:55,040 AT 24 AND 48 HOURS POST I GUESS 640 00:26:55,040 --> 00:26:56,760 RELEASE FROMMED DRUG. 641 00:26:56,760 --> 00:26:59,120 AND WE JUST MONITORED THEM 642 00:26:59,120 --> 00:26:59,440 VISUALLY. 643 00:26:59,440 --> 00:27:01,520 WE WERE LOOKING AT THEIR NUCLEI 644 00:27:01,520 --> 00:27:02,600 AND SCORING THEM AS TO WHETHER 645 00:27:02,600 --> 00:27:04,840 OR NOT THEY LOOKED NORMAL OR 646 00:27:04,840 --> 00:27:05,280 ABNORMAL. 647 00:27:05,280 --> 00:27:07,120 OF COURSE, WE REALIZE THE ONES 648 00:27:07,120 --> 00:27:09,720 WE'RE SCORING AS NORMAL COULD 649 00:27:09,720 --> 00:27:11,840 STILL HAVE GONE THROUGH AN 650 00:27:11,840 --> 00:27:14,440 ABNORMAL MITOSIS BUT WE'RE JUST 651 00:27:14,440 --> 00:27:18,400 LOOKING AT THIS MORE GROSS 652 00:27:18,400 --> 00:27:20,120 OUTCOME OF MITOTIC PERTURBATION. 653 00:27:20,120 --> 00:27:22,120 AND PUT THEM INTO THESE VARIOUS 654 00:27:22,120 --> 00:27:24,520 CATEGORIES FROM FAIRLY MILD TO 655 00:27:24,520 --> 00:27:26,600 WHAT IS QUITE CLEARLY NOT A 656 00:27:26,600 --> 00:27:28,120 SUCCESSFUL MITOSIS EVENT AT THE 657 00:27:28,120 --> 00:27:28,680 END HERE. 658 00:27:28,680 --> 00:27:31,080 AND WHAT YOU CAN SEE IN THE 659 00:27:31,080 --> 00:27:33,240 PARENTAL CELL LINE IS THAT IN 660 00:27:33,240 --> 00:27:34,640 UNTREATED CONDITIONS, AS YOU 661 00:27:34,640 --> 00:27:36,200 EXPECT, VAST MAJORITY OF THE 662 00:27:36,200 --> 00:27:37,200 CELLS LOOK NORMAL. 663 00:27:37,200 --> 00:27:41,120 IF YOU TREAT THEM FOR 24 HOURS 664 00:27:41,120 --> 00:27:42,520 WITH CDK1 INHIBITOR AND RELEASE 665 00:27:42,520 --> 00:27:44,520 YOU CAN SEE INCREASE IN THINGS 666 00:27:44,520 --> 00:27:46,840 THAT HAVE GONE AWRY IN THESE 667 00:27:46,840 --> 00:27:47,280 CELLS. 668 00:27:47,280 --> 00:27:48,480 THEY ARE STARTING TO RECOVER A 669 00:27:48,480 --> 00:27:49,880 LITTLE BIT BY 48 HOURS BUT 670 00:27:49,880 --> 00:27:52,080 THERE'S STILL SOME THINGS THAT 671 00:27:52,080 --> 00:27:53,200 HAVE GONE WRONG THERE. 672 00:27:53,200 --> 00:27:56,040 WHEN WE DO THE SAME THING IN THE 673 00:27:56,040 --> 00:27:58,120 KNOCKOUT CLONES IN THE CELL LINE 674 00:27:58,120 --> 00:27:59,160 BACKGROUND, IN THE UNTREATED 675 00:27:59,160 --> 00:28:01,320 CELLS AGAIN WE'RE NOT SEEING A 676 00:28:01,320 --> 00:28:03,760 MASSIVE IMPACT AT THIS STAGE, SO 677 00:28:03,760 --> 00:28:06,880 NORMALLY DURING MITOSIS THEY ARE 678 00:28:06,880 --> 00:28:07,600 PROGRESSING APPROPRIATELY. 679 00:28:07,600 --> 00:28:09,160 EVEN THOUGH AS WE KNOW THEY HAVE 680 00:28:09,160 --> 00:28:11,440 ACTUALLY GOT SORT OF BIGGER 681 00:28:11,440 --> 00:28:13,280 CENTROMERES AND THEY ARE 682 00:28:13,280 --> 00:28:15,680 RECOMBINING MORE THAN THE 683 00:28:15,680 --> 00:28:16,240 PARENTAL. 684 00:28:16,240 --> 00:28:20,480 BUT WHEN WE CREED WITH CDK1 685 00:28:20,480 --> 00:28:21,920 INHIBITOR TO PUSH THINGS WE SEE 686 00:28:21,920 --> 00:28:25,160 FOR CELLS WITH PROBLEMS AS THEY 687 00:28:25,160 --> 00:28:27,000 GO THROUGH MITOSIS COMPARED TO 688 00:28:27,000 --> 00:28:28,640 PARENTAL, CONSISTENT WITH IMPACT 689 00:28:28,640 --> 00:28:31,160 ON CENTROMERE BIOLOGY. 690 00:28:31,160 --> 00:28:32,600 WE DID ANOTHER EXPERIMENT, IN A 691 00:28:32,600 --> 00:28:34,360 LOT OF DIFFERENT CELL LINES BUT 692 00:28:34,360 --> 00:28:37,120 I'M SHOWING THIS BECAUSE HERE 693 00:28:37,120 --> 00:28:38,800 WE'RE NOT USING ISOGENIC CELL 694 00:28:38,800 --> 00:28:39,560 LINE MODELS. 695 00:28:39,560 --> 00:28:43,640 WE LOOKED AT A PANEL OF RENAL 696 00:28:43,640 --> 00:28:44,720 CANCER-DERIVED CELL LINES, AND 697 00:28:44,720 --> 00:28:48,520 THIS IS WHERE YOU SEE VERY 698 00:28:48,520 --> 00:28:50,080 FREQUENT PBRM1 MUTATION, LOSS OF 699 00:28:50,080 --> 00:28:50,880 FUNCTION MUTATIONS THAT RESULT 700 00:28:50,880 --> 00:28:52,880 IN LOSS OF PROTEIN EXPRESSION. 701 00:28:52,880 --> 00:28:55,360 AND YOU CAN SEE, AGAIN, WE'VE 702 00:28:55,360 --> 00:29:03,240 GOT THREE THAT HAVEPBRM1 703 00:29:03,240 --> 00:29:05,400 EXPRESSION, NOW DEPLETING TO 704 00:29:05,400 --> 00:29:08,800 PROMOTE MITOTIC TRANSITION, 705 00:29:08,800 --> 00:29:10,760 USING TWO siRNAs AGAINST 706 00:29:10,760 --> 00:29:13,960 CYCLIN B1 THOSE LACKING BPRM 1 707 00:29:13,960 --> 00:29:15,920 ARE MORE SENSITIVE TO DEPLETION 708 00:29:15,920 --> 00:29:18,160 THAN THOSE THAT DON'T LACK 709 00:29:18,160 --> 00:29:18,360 PBRM1. 710 00:29:18,360 --> 00:29:19,320 THIS SUGGESTED WE MIGHT HAVE 711 00:29:19,320 --> 00:29:22,160 SOMETHING THAT WE COULD ACTUALLY 712 00:29:22,160 --> 00:29:28,080 MAKE USE OF IN TERMS OF CLINICAL 713 00:29:28,080 --> 00:29:28,720 POTENTIAL. 714 00:29:28,720 --> 00:29:30,080 SO, WHAT WE NOTED THERE WAS VERY 715 00:29:30,080 --> 00:29:35,520 LITTLE, AS I SHOWED WITH THE CDK 716 00:29:35,520 --> 00:29:37,680 1 INHIBITOR DATA WHEN WE DON'T 717 00:29:37,680 --> 00:29:39,800 PROCURE THINGS THERE ARE CHANGES 718 00:29:39,800 --> 00:29:42,080 WE CAN DETECT BUT OVERALL 719 00:29:42,080 --> 00:29:44,160 FITNESS OF CELLS LOOKS OKAY AND 720 00:29:44,160 --> 00:29:47,400 VAST MAJORITY GO THROUGH MITOSIS 721 00:29:47,400 --> 00:29:48,360 NOR OR LESS NORMALLY. 722 00:29:48,360 --> 00:29:50,200 PERHAPS THE REASON IS BECAUSE 723 00:29:50,200 --> 00:29:52,200 THEY HAVE GOT INTACT SPINDLE 724 00:29:52,200 --> 00:29:53,240 ASSEMBLY CHECKPOINT, AND THAT 725 00:29:53,240 --> 00:29:55,040 THEY MIGHT BE RELIANT ON THAT. 726 00:29:55,040 --> 00:29:58,160 AND THIS OF COURSE IS A USEFUL 727 00:29:58,160 --> 00:29:59,840 THING TO UNDERSTAND BECAUSE 728 00:29:59,840 --> 00:30:02,160 INHIBITORS OF THE SPINDLE 729 00:30:02,160 --> 00:30:02,920 ASSEMBLY CHECKPOINTS IN THE 730 00:30:02,920 --> 00:30:08,720 CLINIC COULD BE A GOOD WAY OF 731 00:30:08,720 --> 00:30:09,440 TREATING PBRM1 DEFICIENT CANCERS 732 00:30:09,440 --> 00:30:14,360 IS HE WE WANTED TO ASK WHETHER 733 00:30:14,360 --> 00:30:16,360 CELLS WITH SENSITIVE TO NPS 734 00:30:16,360 --> 00:30:19,840 INHIBITORS, THIS IS SHOWING 735 00:30:19,840 --> 00:30:21,520 RPE1-BASED SYSTEM, RED LINES ARE 736 00:30:21,520 --> 00:30:23,600 THE TWO CLONES, BLACK LINE IS 737 00:30:23,600 --> 00:30:26,200 THE PARENTAL, WE'RE USING THREE 738 00:30:26,200 --> 00:30:29,600 DRUGS HERE. 739 00:30:29,600 --> 00:30:33,520 SO WE'VE GOT AN ASTRAZENECA DRUG 740 00:30:33,520 --> 00:30:36,040 INHIBITOR, AND ONE THAT WAS 741 00:30:36,040 --> 00:30:40,760 GENERATED IN THE ICR, THIS BOS 742 00:30:40,760 --> 00:30:42,840 NPS1 INHIBITOR, YOU CAN SEE TO 743 00:30:42,840 --> 00:30:44,000 VARYING DEGREES KNOCKOUT LINES 744 00:30:44,000 --> 00:30:46,560 ARE MORE SENSITIVE TO NPS1 745 00:30:46,560 --> 00:30:47,840 INHIBITORS THAN PARENTAL. 746 00:30:47,840 --> 00:30:49,800 SO THIS SEEMED SORT OF 747 00:30:49,800 --> 00:30:52,440 ENCOURAGING THING TO LOOK AT IN 748 00:30:52,440 --> 00:30:54,080 MORE DETAIL, WHAT WE WANTED TO 749 00:30:54,080 --> 00:30:56,320 DO WAS GO TO A MOUSE MODEL AND 750 00:30:56,320 --> 00:31:01,840 SEE WHETHER OR NOT TREATING 751 00:31:01,840 --> 00:31:03,400 CELLS WITH NPS 1 INHIBITORS IN 752 00:31:03,400 --> 00:31:07,040 MICE COULD HAVE IMPACT ON 753 00:31:07,040 --> 00:31:08,200 DIFFERENTIAL SENSITIVITY. 754 00:31:08,200 --> 00:31:09,120 WE GENERATED KNOCKOUTS IN A 755 00:31:09,120 --> 00:31:10,720 MOUSE CELL LINE. 756 00:31:10,720 --> 00:31:14,480 THIS IS THE MOUSE B16 CELL LINE, 757 00:31:14,480 --> 00:31:18,920 A MELANOMA LINE, USEFUL, GOOD 758 00:31:18,920 --> 00:31:21,160 FOR DOING MOUSE XENOGRAFT WORK, 759 00:31:21,160 --> 00:31:22,640 AND BY DOING IT IN MOUSE CELL 760 00:31:22,640 --> 00:31:27,800 LINE AT ALLOWED TO GO INTO 761 00:31:27,800 --> 00:31:28,520 IMMUNOCOMPETENT MOUSE MODEL, 762 00:31:28,520 --> 00:31:29,440 RELATED TO ANOTHER PROJECT IN 763 00:31:29,440 --> 00:31:32,480 THE LAB WHERE WE SEE CHANGES IN 764 00:31:32,480 --> 00:31:35,080 INNATE IMMUNE SIGNALING WHEN WE 765 00:31:35,080 --> 00:31:39,400 LOSE PBRM1 1 AND WANTED TO KILL 766 00:31:39,400 --> 00:31:40,600 TWO BIRDS WITH ONE STONE. 767 00:31:40,600 --> 00:31:42,560 WE NEEDED TO SEE WHETHER OR NOT 768 00:31:42,560 --> 00:31:46,040 WE GOT THE SAME IMPACT ON 769 00:31:46,040 --> 00:31:47,360 CENTROMERES, AS WE DID IN THE 770 00:31:47,360 --> 00:31:49,120 MOUSE CELL LINE, AS WE DID IN 771 00:31:49,120 --> 00:31:50,520 THE HUMAN CELL LINES. 772 00:31:50,520 --> 00:31:54,120 THE FIRST THING DID WAS TO LOOK 773 00:31:54,120 --> 00:31:55,800 AT PROTEOME ANALYSIS, AND AGAIN 774 00:31:55,800 --> 00:31:58,800 I'M PLOTTING THE SAME DATA, SORT 775 00:31:58,800 --> 00:32:01,760 OF LOG TWO-FOLD CHANGE VERSUS 776 00:32:01,760 --> 00:32:02,600 PARENTAL, VERY SATISFYINGLY AS 777 00:32:02,600 --> 00:32:06,080 YOU CAN SEE ACROSS THESE VARIOUS 778 00:32:06,080 --> 00:32:07,000 DIFFERENT COMPLEXES ASSOCIATED 779 00:32:07,000 --> 00:32:10,400 WITH CENTROMERES WE CAN SEE 780 00:32:10,400 --> 00:32:12,920 REDUCTION ACROSS THE BOARD OF 781 00:32:12,920 --> 00:32:14,680 THESE PROTEIN LEVELS. 782 00:32:14,680 --> 00:32:17,840 ARE THE CELLS SENSITIVE TO NPS1 783 00:32:17,840 --> 00:32:18,160 INHIBITORS? 784 00:32:18,160 --> 00:32:20,480 AGAIN THE ANSWER IS YES. 785 00:32:20,480 --> 00:32:23,800 WE'VE GOT THE ICR DIRECT 786 00:32:23,800 --> 00:32:30,760 COMPOUND AND ASTRAZENECA 787 00:32:30,760 --> 00:32:31,640 GENERATED COMPOUNDS, WE SEE 788 00:32:31,640 --> 00:32:32,280 SENSITIVITY TO BOTH. 789 00:32:32,280 --> 00:32:34,040 TWO THINGS ABOUT THE MOUSE CELL 790 00:32:34,040 --> 00:32:36,680 WORK THAT WAS PERHAPS SLIGHTLY 791 00:32:36,680 --> 00:32:38,640 SUBOPTIMAL, ONE IS THAT OUR 792 00:32:38,640 --> 00:32:43,880 COLLABORATORS WERE RESTRICTED TO 793 00:32:43,880 --> 00:32:45,440 USING THIS ICR-GENERATED MPS1 794 00:32:45,440 --> 00:32:47,960 INHIBITOR, WE SEE BIGGER EFFECTS 795 00:32:47,960 --> 00:32:48,800 WITH ASTRAZENECA ONE, SOMETHING 796 00:32:48,800 --> 00:32:51,880 WE WANTED TO COME BACK TO, TO 797 00:32:51,880 --> 00:32:53,520 UNDERSTAND BETTER. 798 00:32:53,520 --> 00:33:00,840 SECOND THING, THIS IS THE 799 00:33:00,840 --> 00:33:06,600 SCHEMATIC OF THE APPROACH. 800 00:33:06,600 --> 00:33:08,720 THE THE STUDY ENDED, THE MOUSE 801 00:33:08,720 --> 00:33:12,760 HOUSE WAS BEING CLOSED FOR 802 00:33:12,760 --> 00:33:14,760 REFURBISHMENT SO WE HAD TO END 803 00:33:14,760 --> 00:33:16,440 THE EXPERIMENT. 804 00:33:16,440 --> 00:33:20,120 WHAT WE DID WAS EITHER TAKE THE 805 00:33:20,120 --> 00:33:22,960 PARENTAL B16 LINE OR TWO 806 00:33:22,960 --> 00:33:24,040 KNOCKOUTS OF PBRM1, PUT THEM 807 00:33:24,040 --> 00:33:25,920 INTO GROUPS OF MICE WHERE THEY 808 00:33:25,920 --> 00:33:32,760 WERE TREATED WITH THE VEHICLE OR 809 00:33:32,760 --> 00:33:35,120 WITH THE MPS1 INHIBITOR AND LOOK 810 00:33:35,120 --> 00:33:36,960 AT SURVIVAL AND TUMOR GROWTH. 811 00:33:36,960 --> 00:33:40,280 THIS IS THE SURVIVAL CURVE ON 812 00:33:40,280 --> 00:33:44,000 THIS SIDE, THE PARENTAL. 813 00:33:44,000 --> 00:33:50,040 YOU CAN SEE MPS1 INHIBITOR IS 814 00:33:50,040 --> 00:33:51,680 LIMITED AND WASN'T STATISTICALLY 815 00:33:51,680 --> 00:33:51,960 SIGNIFICANT. 816 00:33:51,960 --> 00:33:58,760 THE OTHER TWO PANELS ARE 817 00:33:58,760 --> 00:33:59,720 KNOCKOUT LINES, IMPACT ON 818 00:33:59,720 --> 00:34:04,000 SURVIVAL, IN THIS CASE WITH THE 819 00:34:04,000 --> 00:34:05,680 B25 WAS SIGNIFICANT INCREASE IN 820 00:34:05,680 --> 00:34:06,640 SURVIVAL, DIDN'T REACH 821 00:34:06,640 --> 00:34:07,880 SIGNIFICANCE WITH THE OTHER 822 00:34:07,880 --> 00:34:09,840 CLONE BUT WHAT YOU'LL ALSO NOTE 823 00:34:09,840 --> 00:34:11,920 IS THAT EVERYTHING ENDS ABRUPTLY 824 00:34:11,920 --> 00:34:12,600 AT DAY 42. 825 00:34:12,600 --> 00:34:14,200 IF YOU LOOK AT THE NUMBER OF 826 00:34:14,200 --> 00:34:16,840 MICE THAT WERE STILL ALIVE AT 827 00:34:16,840 --> 00:34:19,360 DAY 42 YOU SEE THERE ARE A 828 00:34:19,360 --> 00:34:20,440 REASONABLE PERCENTAGE IN MOUSE 829 00:34:20,440 --> 00:34:22,640 GROUPS FROM THE KNOCKOUT THAT 830 00:34:22,640 --> 00:34:25,040 HAVE BEEN TREATED WITH MPS1 831 00:34:25,040 --> 00:34:26,600 INHIBITOR, SOMEBODY WE WANTED TO 832 00:34:26,600 --> 00:34:29,000 DIG INTO AND LOOK AT A BIT MORE 833 00:34:29,000 --> 00:34:29,560 BECAUSE WE THINK 834 00:34:29,560 --> 00:34:33,560 PRELIMINARYARIAL AT LEAST THIS 835 00:34:33,560 --> 00:34:36,920 IS GIVING A GOOD INDICATION THAT 836 00:34:36,920 --> 00:34:40,920 COULD BE SOMETHING YOU CONSIDER 837 00:34:40,920 --> 00:34:42,760 USING WITH PBRM1 CANCERS IN THE 838 00:34:42,760 --> 00:34:43,960 CLINIC. 839 00:34:43,960 --> 00:34:47,480 GETTING BACK TO MECHANISMS, SO 840 00:34:47,480 --> 00:34:51,200 OUR HYPOTHESIS IS THAT PBRM1 AND 841 00:34:51,200 --> 00:34:53,200 PBAF REMODELING COMPLEX IS 842 00:34:53,200 --> 00:34:55,920 WORKING DIRECTLY AT CENTROMERES 843 00:34:55,920 --> 00:34:58,040 IN ORDER TO CREATE A SUBSTRATE 844 00:34:58,040 --> 00:34:59,840 WHICH IS ACTUALLY A GOOD 845 00:34:59,840 --> 00:35:02,280 PLATFORM FOR ASSEMBLY FOR THESE 846 00:35:02,280 --> 00:35:06,520 PROTEINS AND THEY CAN ASSEMBLE 847 00:35:06,520 --> 00:35:10,360 IN THE ABSENCE OF PBRM1 BUT NOT 848 00:35:10,360 --> 00:35:14,760 AS EFFICIENTLY IN THE PRESENCE. 849 00:35:14,760 --> 00:35:16,560 LOSS OF PROTEINS LEADS TO 850 00:35:16,560 --> 00:35:19,800 PROBLEMS AND VULNERABLE TO 851 00:35:19,800 --> 00:35:20,800 MITOTIC STRESS. 852 00:35:20,800 --> 00:35:22,160 SO, PREDICTION THEREFORE IS THAT 853 00:35:22,160 --> 00:35:26,640 WE SHOULD BE ABLE TO SEE PBRM1 854 00:35:26,640 --> 00:35:32,160 AND PBAF COMPLEX OR CENTROMERIC 855 00:35:32,160 --> 00:35:34,360 OR PERICENTROMERIC CHROMATIN. 856 00:35:34,360 --> 00:35:36,040 THIS IS REPETITIVE ELEMENTS 857 00:35:36,040 --> 00:35:37,880 ASSOCIATED WITH CENTROMERES FROM 858 00:35:37,880 --> 00:35:43,200 THE LATEST TELOMERE-TO-TELOMERE 859 00:35:43,200 --> 00:35:45,040 ANALYSIS OF HUMAN CHROMOSOMES 860 00:35:45,040 --> 00:35:47,000 LOOKING AT REPETITIVE DNA IN 861 00:35:47,000 --> 00:35:55,200 PRETTY AMAZING DETAIL. 862 00:35:55,200 --> 00:35:58,040 WE USED THIS T-TO-T GENOME GENES 863 00:35:58,040 --> 00:36:00,680 TO ALIGN MAPPING DATA, USING CUT 864 00:36:00,680 --> 00:36:05,440 AND RUN TO MAP SMARC4 TO THE 865 00:36:05,440 --> 00:36:14,920 GENOME TO SEE WHERE IT WAS 866 00:36:14,920 --> 00:36:17,200 ALIGNING, AND WE CENP TO LOOK AT 867 00:36:17,200 --> 00:36:21,280 THINGS BINDING TO REPETITIVE 868 00:36:21,280 --> 00:36:23,120 SEQUENCES, TO SEE IF SMARC4 IS 869 00:36:23,120 --> 00:36:23,360 BINDING. 870 00:36:23,360 --> 00:36:25,960 WE CHOSE THAT BECAUSE WE COULD 871 00:36:25,960 --> 00:36:31,360 THEN LOOK IN THEPBRM1 KNOCKOUT 872 00:36:31,360 --> 00:36:35,840 TO DESEE -- TO SEE IF IT'S 873 00:36:35,840 --> 00:36:41,080 ASSOCIATING ON CENTROMERIC 874 00:36:41,080 --> 00:36:41,600 ALLIANCES. 875 00:36:41,600 --> 00:36:43,680 THIS IS A SCREEN SHOT OF THE OF 876 00:36:43,680 --> 00:36:49,640 ASSOCIATION OF THESE PROTEINS 877 00:36:49,640 --> 00:36:50,720 WITH CENTROMERES. 878 00:36:50,720 --> 00:36:55,440 I WANTED TO SHOW THE PATTERN 879 00:36:55,440 --> 00:36:59,880 ACROSS THE CENTROMERIC REGION. 880 00:36:59,880 --> 00:37:02,840 WE'VE ANNOTATED IN BLACK, 881 00:37:02,840 --> 00:37:04,280 REPEATS WHICH CONTAIN ALPHA 882 00:37:04,280 --> 00:37:06,800 SATELLITE SEQUENCES IN RED, 883 00:37:06,800 --> 00:37:09,640 WE'VE GOT PERIPHERALLY TO THAT 884 00:37:09,640 --> 00:37:12,400 MORE DIVERGENT ALPHA SATELLITE 885 00:37:12,400 --> 00:37:16,080 SEQUENCES AND OTHER REPETITIVE 886 00:37:16,080 --> 00:37:18,440 SEQUENCES SUCH AS HSAT 2 AND 3, 887 00:37:18,440 --> 00:37:21,600 AND IN GRAY TRANSITION ARMS 888 00:37:21,600 --> 00:37:23,000 WHICH HAVE THESE, AGAIN, 889 00:37:23,000 --> 00:37:25,960 REPETITIVE SEQUENCES BUT NOW 890 00:37:25,960 --> 00:37:28,720 WE'RE LOOKING AT SOME 891 00:37:28,720 --> 00:37:31,720 INTERSPERSED ACTIVE GENES AND SO 892 00:37:31,720 --> 00:37:32,000 FORTH. 893 00:37:32,000 --> 00:37:40,120 SO WHERE ARE THINGS BINDING? 894 00:37:40,120 --> 00:37:42,640 WE DID CENP-B, YOU CAN SEE 895 00:37:42,640 --> 00:37:42,960 ENRICH. 896 00:37:42,960 --> 00:37:45,200 ACROSS HIGHER ORDER OF REPEATS, 897 00:37:45,200 --> 00:37:46,480 WHERE YOU EXPECT TO SEE B 898 00:37:46,480 --> 00:37:51,200 ASSOCIATION, THAT ENRICHMENT, 899 00:37:51,200 --> 00:37:56,480 COMPARED TO IgG NEGATIVE 900 00:37:56,480 --> 00:37:56,880 CONTROL. 901 00:37:56,880 --> 00:37:59,840 WE IS SMARCA4 WITH A KNOCKOUT 902 00:37:59,840 --> 00:38:01,320 CELL LINE, INSTEADING USING 903 00:38:01,320 --> 00:38:02,720 IgG TO USE THE ANTIBODY 904 00:38:02,720 --> 00:38:08,840 AGAINST SMARC4 IN CELLS THAT 905 00:38:08,840 --> 00:38:10,480 DON'T CONTAIN SMARCA4. 906 00:38:10,480 --> 00:38:13,000 KNOCKOUT NEGATIVE CONTROL IN 907 00:38:13,000 --> 00:38:16,640 GRAY, AND ASSOCIATION OF SMARCA4 908 00:38:16,640 --> 00:38:18,400 WITH CHROMATIN IN THE PARENTAL 909 00:38:18,400 --> 00:38:19,760 CELLS IS IN GREEN. 910 00:38:19,760 --> 00:38:25,360 WHAT YOU CAN SEE WE GET A NICE 911 00:38:25,360 --> 00:38:26,280 ENRICHMENT OF SMARCA4 ACROSS THE 912 00:38:26,280 --> 00:38:34,560 SAME REGION OF THE CENTROMERE WE 913 00:38:34,560 --> 00:38:36,640 SEE CENP-B ENRICHMENT AND ACROSS 914 00:38:36,640 --> 00:38:37,280 THE CENTROMERES. 915 00:38:37,280 --> 00:38:39,040 AS WE WERE ANALYZING THIS WE 916 00:38:39,040 --> 00:38:40,800 WANTED TO GET A FULLER PICTURE, 917 00:38:40,800 --> 00:38:44,080 FROM THIS IT WAS REASONABLE TO 918 00:38:44,080 --> 00:38:45,400 CONCLUDE WE HAVE ENRICHMENT OF 919 00:38:45,400 --> 00:38:52,640 SMARCA4 AT CENTROMERES BUT WE'RE 920 00:38:52,640 --> 00:38:54,360 RUNNING INTO A COUPLE ISSUES TO 921 00:38:54,360 --> 00:38:57,960 SEE WHERE IT IS BINDING, ONE YOU 922 00:38:57,960 --> 00:38:59,520 CAN SEE FROM TWO NEGATIVE 923 00:38:59,520 --> 00:39:03,200 CONTROLS YOU GET SOME BACKGROUND 924 00:39:03,200 --> 00:39:03,960 READS FROM REPETITIVE SEQUENCES 925 00:39:03,960 --> 00:39:06,080 THAT ARE A LITTLE BIT HIGHER 926 00:39:06,080 --> 00:39:07,040 THAN FROM NON-REPETITIVE 927 00:39:07,040 --> 00:39:07,800 SEQUENCES. 928 00:39:07,800 --> 00:39:10,760 THAT'S ACTUALLY BEEN REPORTED IN 929 00:39:10,760 --> 00:39:11,360 THE LITERATURE. 930 00:39:11,360 --> 00:39:15,280 WE CAN LOOK AT WHAT'S ENRICHED 931 00:39:15,280 --> 00:39:16,000 ABOVE THAT. 932 00:39:16,000 --> 00:39:20,960 THE OTHER CHALLENGING ISSUE IS 933 00:39:20,960 --> 00:39:22,160 MAPPING READS, INTERESTED TO 934 00:39:22,160 --> 00:39:25,680 UNDERSTAND WHERE THINGS COULD 935 00:39:25,680 --> 00:39:26,680 MAP MULTIPLE PLACES LOSING 936 00:39:26,680 --> 00:39:28,280 ENRICHMENT TO TELL US IT WAS 937 00:39:28,280 --> 00:39:29,880 BOUND AT A SICK SEQUENCE. 938 00:39:29,880 --> 00:39:35,120 IN ORDER TO GET A MORE -- I 939 00:39:35,120 --> 00:39:39,240 GUESS A FULLER PICTURE EXACTLY 940 00:39:39,240 --> 00:39:44,080 WHERE SMARCA4 IS BINDING, WE 941 00:39:44,080 --> 00:39:50,000 DECIDED TO USE THIS ANALYSIS, A 942 00:39:50,000 --> 00:39:53,800 SEQUENCE OF K AS A SUBSEQUENCE 943 00:39:53,800 --> 00:39:57,040 OF SOME SORT OF LARGER READ THAT 944 00:39:57,040 --> 00:40:01,320 YOU'RE LOOKING AT. 945 00:40:01,320 --> 00:40:04,640 LOOKING AT 50 K-MERS, AND THIS 946 00:40:04,640 --> 00:40:07,600 WE SORT OF FOLLOWED THE APPROACH 947 00:40:07,600 --> 00:40:13,800 THAT WAS TAKEN BY THE STRAIGHT 948 00:40:13,800 --> 00:40:17,760 LAB, USING MAPPING OF CENP-A TO 949 00:40:17,760 --> 00:40:21,480 IDENTIFY SEQUENCES ENRICHED, 950 00:40:21,480 --> 00:40:22,920 K-MERS ENRICHED IN THE CENP-A 951 00:40:22,920 --> 00:40:26,280 READS TO GET A SENSE OF 952 00:40:26,280 --> 00:40:27,680 CENTROMERIC SEQUENCES. 953 00:40:27,680 --> 00:40:29,840 WE FOLLOWED THAT APPROACH TO ASK 954 00:40:29,840 --> 00:40:31,400 THE QUESTION OF PATTERN OF 955 00:40:31,400 --> 00:40:32,880 SEQUENCES BOUND BY SMARCA4? 956 00:40:32,880 --> 00:40:34,440 I WILL BRIEFLY GO THROUGH THIS 957 00:40:34,440 --> 00:40:36,400 FOR THE SAKE OF TIME BUT HAPPY 958 00:40:36,400 --> 00:40:37,880 TO SPEAK WITH PEOPLE LATER ABOUT 959 00:40:37,880 --> 00:40:40,320 IT IF THEY ARE INTERESTED, 960 00:40:40,320 --> 00:40:41,960 ESSENTIALLY WE TOOK THOSE CUT 961 00:40:41,960 --> 00:40:45,160 AND RUN READS ASSOCIATED WITH 962 00:40:45,160 --> 00:40:48,120 THE CENTROMERIC REGION INCLUDING 963 00:40:48,120 --> 00:40:49,640 TRANSICS A LITTLER, NORMALIZE 964 00:40:49,640 --> 00:40:53,600 K-MER COUNT AND THE KEY THING 965 00:40:53,600 --> 00:40:56,120 CALCULATING THEM, THE FOLD 966 00:40:56,120 --> 00:40:57,640 CHANGES, ADJUSTED P-VALUES OF 967 00:40:57,640 --> 00:41:00,040 K-MERS BETWEEN OUR POSITIVE 968 00:41:00,040 --> 00:41:06,840 CONTROL OR NOT POSITIVE 969 00:41:06,840 --> 00:41:08,160 CONTROLS, THE CENP-B AND 970 00:41:08,160 --> 00:41:09,560 NEGATIVE CONTROL. 971 00:41:09,560 --> 00:41:12,720 YOU'RE LOOKING AT ENRICHED K 972 00:41:12,720 --> 00:41:23,200 MERS ASSOCIATED WITH CENP-B, 973 00:41:28,200 --> 00:41:30,200 WHAT TYPE OF CENTROMERE SEQUENCE 974 00:41:30,200 --> 00:41:31,520 ARE THESE ASSOCIATED WITH RATHER 975 00:41:31,520 --> 00:41:34,920 THAN WHAT POSITION ALONG HIGHER 976 00:41:34,920 --> 00:41:38,760 ORDER REPEAT DOES IT BIND TO. 977 00:41:38,760 --> 00:41:40,080 AND AS A NEGATIVE -- EXTRA 978 00:41:40,080 --> 00:41:43,480 NEGATIVE CONTROL WE TOOK THE 979 00:41:43,480 --> 00:41:53,640 ENRICHED K-MERS, GOT A LOT FOR 980 00:41:53,640 --> 00:41:53,960 CENP-B 981 00:41:53,960 --> 00:41:56,360 FOR SMARC4s, IF YOU HAD THEM 982 00:41:56,360 --> 00:41:57,480 RANDOMLY ASSOCIATED WITH GENOME 983 00:41:57,480 --> 00:41:59,280 JUST TO SORT OF GIVE OURSELVES 984 00:41:59,280 --> 00:42:00,640 EXTRA CONFIDENCE THAT ANY 985 00:42:00,640 --> 00:42:01,960 PATTERN WE WERE SEEING WAS REAL 986 00:42:01,960 --> 00:42:05,360 AND NOT A CONSEQUENCE OF 987 00:42:05,360 --> 00:42:07,040 DIFFERENT PROPORTIONS OF DNA 988 00:42:07,040 --> 00:42:10,760 THAT WAS OCCUPIED BY THE VARIOUS 989 00:42:10,760 --> 00:42:12,280 TYPES OF CENTROMERE REPEATS. 990 00:42:12,280 --> 00:42:13,560 I HOPE THAT MAKES SENSE. 991 00:42:13,560 --> 00:42:13,760 OKAY. 992 00:42:13,760 --> 00:42:14,440 WE DID THAT. 993 00:42:14,440 --> 00:42:18,080 AS I SAY WE DID THIS SHUFFLED 994 00:42:18,080 --> 00:42:20,760 AND AS WE ANTICIPATED, AS WE 995 00:42:20,760 --> 00:42:24,240 HOPED, WHICH IS TO SAY IT WAS A 996 00:42:24,240 --> 00:42:25,800 VERY FLAT ASSOCIATION, SO THE 997 00:42:25,800 --> 00:42:28,120 NUMBER OF K-MERS WHEN YOU 998 00:42:28,120 --> 00:42:29,960 SHUFFLE THE LOCATIONS THAT ARE 999 00:42:29,960 --> 00:42:32,560 RANDOMLY ASSOCIATED WITH THESE 1000 00:42:32,560 --> 00:42:34,560 DIFFERENT TYPES OF REPEATS 1001 00:42:34,560 --> 00:42:35,600 PRETTY MUCH CORRESPONDS TO HOW 1002 00:42:35,600 --> 00:42:38,640 MUCH DNA SEQUENCE THAT OCCUPIES 1003 00:42:38,640 --> 00:42:45,440 IN ANY GIVEN CENTROMERE. 1004 00:42:45,440 --> 00:42:48,080 WE DID IT WITH CENP-B, MAPPING 1005 00:42:48,080 --> 00:42:51,160 BACK PRIMARILY TO THE HIGHER 1006 00:42:51,160 --> 00:42:53,280 ORDER REPEATS, SO THE ACTIVE 1007 00:42:53,280 --> 00:42:55,560 HIGHER ORDER REPEATS ASSOCIATED 1008 00:42:55,560 --> 00:42:58,160 WITH CENTROMERES, WHAT YOU WOULD 1009 00:42:58,160 --> 00:43:00,480 PREDICT AND DID THIS WITH SMARC4 1010 00:43:00,480 --> 00:43:05,920 AND GOT A PATTERN LIKELY, 1011 00:43:05,920 --> 00:43:09,000 BIGGEST ENRICHMENT IN HIGHEST 1012 00:43:09,000 --> 00:43:13,800 ORDER REPEATS FOLLOWED BY HSAT 1 1013 00:43:13,800 --> 00:43:18,200 AND 2, AND LOW LEVEL WITH 1014 00:43:18,200 --> 00:43:19,440 TRANSITION ARMS, WE STILL NEED 1015 00:43:19,440 --> 00:43:22,880 TO DO MORE WORK BUT ALMOST A 1016 00:43:22,880 --> 00:43:24,080 REASONABLE INTERPRETATION, VERY 1017 00:43:24,080 --> 00:43:25,320 SPECIFIC POINTS ASSOCIATED WITH 1018 00:43:25,320 --> 00:43:26,720 GENES IN THOSE TRANSITION ARMS. 1019 00:43:26,720 --> 00:43:29,080 BUT REALLY WHAT WE'RE GETTING IS 1020 00:43:29,080 --> 00:43:31,000 A BIG ENRICHMENT OF REPETITIVE 1021 00:43:31,000 --> 00:43:32,560 SEQUENCES ASSOCIATED WITH HIGHER 1022 00:43:32,560 --> 00:43:33,000 ORDER REPEATS. 1023 00:43:33,000 --> 00:43:37,960 SO THIS IS GREAT. 1024 00:43:37,960 --> 00:43:40,400 BASICALLY FROM BOTH WE CAN SAY 1025 00:43:40,400 --> 00:43:43,200 WITH CONFIDENCE SMARCA4 IS 1026 00:43:43,200 --> 00:43:45,880 ASSOCIATED WITH CENTROMERES AND 1027 00:43:45,880 --> 00:43:48,320 PARTICULARLY WITH HIGHER ORDER 1028 00:43:48,320 --> 00:43:49,480 REPEATS IN CENTROMERES. 1029 00:43:49,480 --> 00:43:53,960 WHAT HAPPENS WITH THE SAME THING 1030 00:43:53,960 --> 00:43:56,880 IN THE PBRM1 KNOCKOUT? 1031 00:43:56,880 --> 00:43:58,240 SHUFFLE LOOKS THE SAME. 1032 00:43:58,240 --> 00:44:01,640 CENP-B LOOKS THE SAME. 1033 00:44:01,640 --> 00:44:04,320 WE CAN CONCLUDE CENP-B 1034 00:44:04,320 --> 00:44:05,880 ASSOCIATION TO PARTICULAR 1035 00:44:05,880 --> 00:44:07,640 SEQUENCES, THE ABILITY TO FIND 1036 00:44:07,640 --> 00:44:09,160 THE SEQUENCES AND PATTERN OF 1037 00:44:09,160 --> 00:44:11,680 BINDING IS UNAFFECTED BY THE 1038 00:44:11,680 --> 00:44:16,040 ABSENCE OF PBRM1. 1039 00:44:16,040 --> 00:44:17,200 WHAT HAPPEN WAS SMARCA4? 1040 00:44:17,200 --> 00:44:19,880 IF WE LAYER THE DATA, THERE WERE 1041 00:44:19,880 --> 00:44:20,640 NONE. 1042 00:44:20,640 --> 00:44:24,160 WE FOUND NO K-MERS ENRICHED IN 1043 00:44:24,160 --> 00:44:26,680 THE SMARCA4 CUT AND RUN DATASET 1044 00:44:26,680 --> 00:44:29,680 IN THE ABSENCE OF PBRM1 COMPARED 1045 00:44:29,680 --> 00:44:30,880 TO NEGATIVE CONTROLS. 1046 00:44:30,880 --> 00:44:33,440 SO THE ABILITY OF SMARCA4 TO GET 1047 00:44:33,440 --> 00:44:35,520 TO THOSE PARTICULAR SITES IN 1048 00:44:35,520 --> 00:44:39,800 CENTROMERES IS DEPENDENT ON 1049 00:44:39,800 --> 00:44:41,280 PBRM1. 1050 00:44:41,280 --> 00:44:41,600 SOAK. 1051 00:44:41,600 --> 00:44:44,320 WHAT IS PBAF DOING AT THESE 1052 00:44:44,320 --> 00:44:46,400 REGIONS IN THE CENTROMERE IN 1053 00:44:46,400 --> 00:44:50,000 ORDER TO PROMOTE ASSEMBLY OF THE 1054 00:44:50,000 --> 00:44:50,800 CENTROMERE-ASSOCIATED PROTEINS 1055 00:44:50,800 --> 00:44:56,560 AND OF COURSE PREVENT 1056 00:44:56,560 --> 00:44:57,560 INAPPROPRIATE RECOMBINATION OF 1057 00:44:57,560 --> 00:44:58,160 SO FORTH? 1058 00:44:58,160 --> 00:45:01,680 AN OBVIOUS THING TO LOOK AT, 1059 00:45:01,680 --> 00:45:02,120 TRANSCRIPTION. 1060 00:45:02,120 --> 00:45:04,280 WE'VE GOT A CHROMATIN REMODELER 1061 00:45:04,280 --> 00:45:06,080 WHICH WE KNOW ASSOCIATES WITH 1062 00:45:06,080 --> 00:45:09,240 HIGHER ORDER REPEATS, IMPORTANT 1063 00:45:09,240 --> 00:45:12,000 FOR CENTROMERE STRUCTURE AND 1064 00:45:12,000 --> 00:45:13,280 FUNCTION, AND WORK FROM LABS 1065 00:45:13,280 --> 00:45:19,000 SHOWS THERE'S A LOW LEVEL OF 1066 00:45:19,000 --> 00:45:19,600 CENTROMERE-DERIVED TRANSCRIPTS 1067 00:45:19,600 --> 00:45:22,720 IMPORTANT FOR ASSEMBLY OF THESE 1068 00:45:22,720 --> 00:45:24,800 PROTEINS AT CENTROMERES AND FOR 1069 00:45:24,800 --> 00:45:25,600 KINETIC ATTACHMENT. 1070 00:45:25,600 --> 00:45:27,320 THIS SEEMED LIKE A REASONABLE 1071 00:45:27,320 --> 00:45:28,840 HYPOTHESIS THAT WE'VE GOT 1072 00:45:28,840 --> 00:45:31,560 SOMETHING THAT'S ACTUALLY BEING 1073 00:45:31,560 --> 00:45:32,760 TRANSCRIPTIONALLY MISREGULATED 1074 00:45:32,760 --> 00:45:34,520 IN ABSENCE OF PBAF, AND WHAT 1075 00:45:34,520 --> 00:45:35,400 PEOPLE HAVE SHOWN, THIS IS 1076 00:45:35,400 --> 00:45:38,280 IMPORTANT FOR THE NEXT SLIDE, IS 1077 00:45:38,280 --> 00:45:42,880 THAT EITHER TOO MUCH OR TOO 1078 00:45:42,880 --> 00:45:45,240 LITTLE TRANSCRIPTION FROM THESE 1079 00:45:45,240 --> 00:45:48,560 CENTROMERIC SEQUENCES IS ENOUGH 1080 00:45:48,560 --> 00:45:51,040 TO IMPAIR CENTROMERE ASSEMBLY, 1081 00:45:51,040 --> 00:45:53,480 CENTROMERE ASSEMBLY AND CORE 1082 00:45:53,480 --> 00:45:54,440 ATTACHMENTS IN CENTROMERE 1083 00:45:54,440 --> 00:45:55,200 FUNCTION. 1084 00:45:55,200 --> 00:45:56,120 WE LOOKED AT TRANSCRIPTION, WE 1085 00:45:56,120 --> 00:46:00,240 DID THIS A COUPLE WAYS. 1086 00:46:00,240 --> 00:46:02,440 HERE IS DATA USING PRIMER PAIRS 1087 00:46:02,440 --> 00:46:03,440 AGAINST ALPHA SATELLITE 1088 00:46:03,440 --> 00:46:07,480 SEQUENCES PRIMARILY BUT WE ALSO 1089 00:46:07,480 --> 00:46:10,480 GOT AN HSAT 2 SET OF SEQUENCES. 1090 00:46:10,480 --> 00:46:13,640 THE PARENTAL IS SHOWN IN BLACK. 1091 00:46:13,640 --> 00:46:15,480 TWO PBRM1 KNOCKOUTS IN BLUE, IN 1092 00:46:15,480 --> 00:46:17,440 TWO CELL LINES, AND WE GET 1093 00:46:17,440 --> 00:46:18,320 MISREGULATION OF TRANSCRIPTION 1094 00:46:18,320 --> 00:46:20,160 BUT IN FACT IT'S UPREGULATED IN 1095 00:46:20,160 --> 00:46:21,240 THE KNOCKOUTS AND WE SEE THE 1096 00:46:21,240 --> 00:46:28,360 SAME THING WHEN WE LOOK AT 1097 00:46:28,360 --> 00:46:28,960 RNAseq DATASETS, LOOKING INTO 1098 00:46:28,960 --> 00:46:32,840 THIS TO SEE IF WE CAN RELATE TO 1099 00:46:32,840 --> 00:46:35,600 K-MERS AND POSITION OF PBAF 1100 00:46:35,600 --> 00:46:35,960 BINDING. 1101 00:46:35,960 --> 00:46:38,760 TO PULL INTO A MODEL, TO FINISH, 1102 00:46:38,760 --> 00:46:43,120 WHAT WE KNOW NOW IS THAT PBAF IS 1103 00:46:43,120 --> 00:46:48,880 RECRUITED -- IS ASSOCIATED WITH 1104 00:46:48,880 --> 00:46:49,560 CENTROMERES, WITH OTHER 1105 00:46:49,560 --> 00:46:54,560 REPETITIVE ELEMENTS, IN THE 1106 00:46:54,560 --> 00:46:57,480 ABSENCE PBAF FAILS TO GET TO 1107 00:46:57,480 --> 00:47:00,840 CHROMATIN, AS A CONSEQUENCE WE 1108 00:47:00,840 --> 00:47:04,400 THINK THAT THAT -- THAT LEADS TO 1109 00:47:04,400 --> 00:47:09,400 INAPPROPRIATE REGULATION OF THE 1110 00:47:09,400 --> 00:47:09,840 CENTROMERE-SPECIFIC 1111 00:47:09,840 --> 00:47:11,280 TRANSCRIPTIONS AND THAT IS SHOWN 1112 00:47:11,280 --> 00:47:12,800 TO BE IMPORTANT FOR COMPLEX 1113 00:47:12,800 --> 00:47:13,080 ASSEMBLY. 1114 00:47:13,080 --> 00:47:15,720 IF YOU DON'T HAVE SAY 1115 00:47:15,720 --> 00:47:16,640 APPROPRIATE COMPLEX ASSEMBLY 1116 00:47:16,640 --> 00:47:19,240 THAT LINKS US INTO ALL THESE 1117 00:47:19,240 --> 00:47:22,160 PHENOTYPES WE'RE SEEING, 1118 00:47:22,160 --> 00:47:23,840 DECREASE IN 1119 00:47:23,840 --> 00:47:25,040 CENTROMERE-ASSOCIATED COHESION, 1120 00:47:25,040 --> 00:47:26,480 INCREASED CENTROMERE FRAGILITY, 1121 00:47:26,480 --> 00:47:34,000 WHOLE ARM AND WHOLE CHROMATIN 1122 00:47:34,000 --> 00:47:34,360 INSTABILITY. 1123 00:47:34,360 --> 00:47:36,440 AND WITH THAT I'LL BRIEFLY 1124 00:47:36,440 --> 00:47:37,520 MENTION ALL OF THE WONDERFUL 1125 00:47:37,520 --> 00:47:39,400 PEOPLE WHO ARE INVOLVED. 1126 00:47:39,400 --> 00:47:41,800 IN MY LAB, KAREN LANE IS A 1127 00:47:41,800 --> 00:47:44,440 FANTASTIC POSTDOC WHO HAS DRIVEN 1128 00:47:44,440 --> 00:47:46,600 THIS PROJECT AND DONE CELL 1129 00:47:46,600 --> 00:47:49,240 MOLECULAR BIOLOGY, WITH A LOT OF 1130 00:47:49,240 --> 00:47:51,560 INPUT FROM JEFFREY FUNG, WHO HAS 1131 00:47:51,560 --> 00:47:54,040 RECENTLY LEFT TO DO POSTDOC AT 1132 00:47:54,040 --> 00:47:54,640 THE CRICK. 1133 00:47:54,640 --> 00:48:02,800 LILLIAN WU DID THE CUT AND RUN 1134 00:48:02,800 --> 00:48:04,760 ASSAYS WITH SMARCA4 AND ALLISON 1135 00:48:04,760 --> 00:48:12,840 DID A FORMAL JOB WITH -- 1136 00:48:12,840 --> 00:48:14,840 PHENOMENAL JOB WITH K-MER 1137 00:48:14,840 --> 00:48:15,200 ANALYSIS. 1138 00:48:15,200 --> 00:48:23,360 PROTEOMEIC ANALYSIS IS DONE WITH 1139 00:48:23,360 --> 00:48:25,000 GREAT COLLABORATORS WITH JYOTI. 1140 00:48:25,000 --> 00:48:27,320 I'LL STOP AND TAKE QUESTIONS. 1141 00:48:27,320 --> 00:48:27,840 THANK YOU. 1142 00:48:27,840 --> 00:48:30,280 >>THANK YOU VERY MUCH FOR AN 1143 00:48:30,280 --> 00:48:31,160 EXCELLENT TALK. 1144 00:48:31,160 --> 00:48:33,400 REALLY BEAUTIFUL DATA AND VERY 1145 00:48:33,400 --> 00:48:37,160 INTERESTING AND CHALLENGING. 1146 00:48:37,160 --> 00:48:38,960 I'M WAITING FOR SOME CHAT 1147 00:48:38,960 --> 00:48:41,200 MESSAGE QUESTIONS TO COME HERE. 1148 00:48:41,200 --> 00:48:45,400 AND I GUESS I WOULD LIKE TO ASK 1149 00:48:45,400 --> 00:48:48,240 ABOUT THE RELATION TO DNA DAMAGE 1150 00:48:48,240 --> 00:48:50,840 AND REPAIR MAYBE HERE BECAUSE 1151 00:48:50,840 --> 00:48:57,280 YOU SAY THAT DISRUPTING 1152 00:48:57,280 --> 00:48:59,400 CENTROMERES WITH PBAF AND PBRM1 1153 00:48:59,400 --> 00:49:01,520 DEPLETION CAUSES CHANGES IN 1154 00:49:01,520 --> 00:49:02,320 SURVIVAL, IN TRANSCRIPTIONAL 1155 00:49:02,320 --> 00:49:05,200 THINGS AND STATUS. 1156 00:49:05,200 --> 00:49:07,360 DOES IT CHANGE DNA DAMAGE IN 1157 00:49:07,360 --> 00:49:10,760 RELATION, DOES IT CHANGE DNA 1158 00:49:10,760 --> 00:49:11,000 REPAIR? 1159 00:49:11,000 --> 00:49:15,280 >>YEAH, THAT IS AN EXCELLENT 1160 00:49:15,280 --> 00:49:16,360 QUESTION. 1161 00:49:16,360 --> 00:49:18,440 SO, THERE WAS A RECENT -- WELL, 1162 00:49:18,440 --> 00:49:21,720 A NUMBER OF PAPERS AROUND DNA 1163 00:49:21,720 --> 00:49:24,920 DAMAGE RESPONSES IN CENTROMERES 1164 00:49:24,920 --> 00:49:26,880 BUT RECENT PAPER SHOWED THERE 1165 00:49:26,880 --> 00:49:28,840 ARE DOUBLE-STRAND BREAKS 1166 00:49:28,840 --> 00:49:31,680 NORMALLY ASSOCIATED WITH THESE 1167 00:49:31,680 --> 00:49:34,720 CENTROMERIC SEQUENCES, AND SO 1168 00:49:34,720 --> 00:49:37,040 ONE REALLY ATTRACTIVE 1169 00:49:37,040 --> 00:49:40,120 POSSIBILITY IS THAT BECAUSE WE 1170 00:49:40,120 --> 00:49:44,120 KNOW THAT PBAF PLAYS A ROLE AT 1171 00:49:44,120 --> 00:49:46,000 DOUBLE-STRAND BREAKS, AND SO ONE 1172 00:49:46,000 --> 00:49:46,600 ATTRACTIVE POSSIBILITY WE'RE 1173 00:49:46,600 --> 00:49:49,640 LOOK BOOK IS IS THERE'S A 1174 00:49:49,640 --> 00:49:50,960 RELATIONSHIP BETWEEN PBAF 1175 00:49:50,960 --> 00:49:51,680 TRANSCRIPTION AND THE 1176 00:49:51,680 --> 00:49:53,120 DOUBLE-STRAND BREAKS, AND OF 1177 00:49:53,120 --> 00:49:59,040 COURSE WHICH WAY AROUND IT GOES 1178 00:49:59,040 --> 00:50:00,160 IS NOT ENTIRELY CLEAR BUT ONE 1179 00:50:00,160 --> 00:50:05,080 WAY YOU CAN IMAGINE IS THAT WE 1180 00:50:05,080 --> 00:50:07,920 KNOW PBAF ASSOCIATES IS 1181 00:50:07,920 --> 00:50:08,560 SEQUENCES TO RESTRAIN 1182 00:50:08,560 --> 00:50:09,720 TRANSCRIPTION, IT COULD BE TOO 1183 00:50:09,720 --> 00:50:12,840 MUCH TRANSCRIPTION BRINGS IN THE 1184 00:50:12,840 --> 00:50:15,240 SORT OF ACTIVITIES LIKE 1185 00:50:15,240 --> 00:50:16,360 TOPOISOMERASES WHICH CREATE 1186 00:50:16,360 --> 00:50:17,960 BREAKS THAT LEAD TO FRAGILITY 1187 00:50:17,960 --> 00:50:19,000 WE'RE SEEING. 1188 00:50:19,000 --> 00:50:23,360 THERE MIGHT BE A REALLY STRONG 1189 00:50:23,360 --> 00:50:24,200 ASSOCIATION, THAT'S SOMETHING WE 1190 00:50:24,200 --> 00:50:25,200 WANT TO LOOK INTO TO SEE WHETHER 1191 00:50:25,200 --> 00:50:28,080 OR NOT NUMBER AND LOCATION OF 1192 00:50:28,080 --> 00:50:28,920 DOUBLE-STRAND BREAKS IS 1193 00:50:28,920 --> 00:50:37,800 DIFFERENT IN PRESENCE OR ABSENCE 1194 00:50:37,800 --> 00:50:39,440 OF PBRM1. 1195 00:50:39,440 --> 00:50:40,120 >>RELATING TO ACCESSIBILITY 1196 00:50:40,120 --> 00:50:44,440 ISSUE, AND I GUESS THE SURVIVAL 1197 00:50:44,440 --> 00:50:46,640 DEFICIENCY THAT YOU SEE, IS THAT 1198 00:50:46,640 --> 00:50:47,920 THEN POSSIBLY RELATED TO 1199 00:50:47,920 --> 00:50:48,400 DOUBLE-STRAND BREAKS? 1200 00:50:48,400 --> 00:50:51,600 >>YEAH, I THINK IT COULD BE. 1201 00:50:51,600 --> 00:50:54,200 WE SEE -- IT'S REALLY DIFFICULT 1202 00:50:54,200 --> 00:50:55,840 TO DISENTANGLE THESE THINGS 1203 00:50:55,840 --> 00:50:57,600 BECAUSE WE ALSO SEE A DECREASE 1204 00:50:57,600 --> 00:51:00,840 IN SURVIVAL WHEN WE USE 1205 00:51:00,840 --> 00:51:02,200 REPLICATION STRESS. 1206 00:51:02,200 --> 00:51:04,480 AND SO, YOU KNOW, WHAT WE WANT 1207 00:51:04,480 --> 00:51:06,320 TO TRY TO GET AT IS WHETHER OR 1208 00:51:06,320 --> 00:51:07,760 NOT WE CAN LOOK AT THE FATE OF 1209 00:51:07,760 --> 00:51:10,840 THE CELLS AFTER WE TREAT THEM 1210 00:51:10,840 --> 00:51:14,520 WITH, SAY, REPLICATION STRESS 1211 00:51:14,520 --> 00:51:15,960 VERSUS NPS1 INHIBITOR TO ASK 1212 00:51:15,960 --> 00:51:20,120 EXACTLY HOW THEY DIED, WAS IT 1213 00:51:20,120 --> 00:51:23,520 DOWN TO THE DNA DAMAGE AND 1214 00:51:23,520 --> 00:51:24,600 FAILURE OR INAPPROPRIATE 1215 00:51:24,600 --> 00:51:26,160 RECOMBINATION OF FAILURE TO DEAL 1216 00:51:26,160 --> 00:51:33,480 WITH THINGS AT CENTROMERES, YOU 1217 00:51:33,480 --> 00:51:37,920 KNOW, AS A CONSEQUENCE OF TAKING 1218 00:51:37,920 --> 00:51:39,040 AWAY THE CHECKPOINT ACTIVITY. 1219 00:51:39,040 --> 00:51:46,160 >>A QUESTION, GREAT TALK, GREAT 1220 00:51:46,160 --> 00:51:47,040 ANALYSIS. 1221 00:51:47,040 --> 00:51:50,440 ONE QUESTION, IS THE CENP 1222 00:51:50,440 --> 00:51:54,800 COHESION PHENOTYPE DEPENDENT ON 1223 00:51:54,800 --> 00:51:55,920 ACTIVITY OF PBAF? 1224 00:51:55,920 --> 00:51:56,240 >>FANTASTIC. 1225 00:51:56,240 --> 00:51:58,520 HELLO AND THANK YOU VERY MUCH. 1226 00:51:58,520 --> 00:52:00,760 YEAH, IT'S A GREAT QUESTION. 1227 00:52:00,760 --> 00:52:05,000 YES, WE DID THE EXPERIMENT A 1228 00:52:05,000 --> 00:52:06,840 COUPLE WAYS, WE USED ATPase 1229 00:52:06,840 --> 00:52:08,720 MUTANTS OF SMARCA4 BUT NOW 1230 00:52:08,720 --> 00:52:13,760 WHAT'S WONDERFUL FOR US THERE 1231 00:52:13,760 --> 00:52:15,840 ARE BOTH ATPase INHIBITORS, 1232 00:52:15,840 --> 00:52:17,360 AND WE REPEATED A LOT OF 1233 00:52:17,360 --> 00:52:20,520 EXPERIMENTS AND LOOKED AT THE 1234 00:52:20,520 --> 00:52:27,080 RESPONSES TO THE PROTACs AND 1235 00:52:27,080 --> 00:52:29,600 ATPase INHIBITOR, YOU NEED 1236 00:52:29,600 --> 00:52:31,160 REMODELING TO GET ADHESION AND 1237 00:52:31,160 --> 00:52:36,000 PREVENT THESE SORTS OF ISSUES. 1238 00:52:36,000 --> 00:52:38,800 >>DO YOU WANT TO READ THE NEXT 1239 00:52:38,800 --> 00:52:39,040 QUESTION? 1240 00:52:39,040 --> 00:52:40,320 >>YES. 1241 00:52:40,320 --> 00:52:43,040 SO WE HAVE A QUESTION FROM 1242 00:52:43,040 --> 00:52:43,320 ALEXANDER. 1243 00:52:43,320 --> 00:52:45,080 HAVE YOU LOOKED AT THE COHESION 1244 00:52:45,080 --> 00:52:50,680 LEVEL BY CUT AND RUN IN THE 1245 00:52:50,680 --> 00:52:51,600 ABSENCE OF PBMR1? 1246 00:52:51,600 --> 00:52:55,120 DO YOU THINK THIS IS BECAUSE OF 1247 00:52:55,120 --> 00:52:59,720 TRANSCRIPTION EFFECTS OR 1248 00:52:59,720 --> 00:53:00,520 PROBLEMS WITH COHESION LOADING? 1249 00:53:00,520 --> 00:53:04,400 >>THAT'S A GREAT QUESTION. 1250 00:53:04,400 --> 00:53:06,520 IN FACT, INTERESTINGLY, THE 1251 00:53:06,520 --> 00:53:07,720 PHENOTYPES FROM YEAST WERE WHAT 1252 00:53:07,720 --> 00:53:10,480 MADE US LOOK AT SISTER CHROMATID 1253 00:53:10,480 --> 00:53:13,080 COHESION IN THE MAMMALIAN SYSTEM 1254 00:53:13,080 --> 00:53:15,280 TO SEE WHETHER THAT WAS 1255 00:53:15,280 --> 00:53:16,360 CONSERVED, SO, YEAH. 1256 00:53:16,360 --> 00:53:21,280 WHAT WE FIND IS THERE'S NO 1257 00:53:21,280 --> 00:53:23,360 DEFECT IN TRANSCRIPTION OF ANY 1258 00:53:23,360 --> 00:53:24,280 COHESION-ASSOCIATED -- THE 1259 00:53:24,280 --> 00:53:26,560 PATHWAY, TRANSCRIPT IS NORMAL IN 1260 00:53:26,560 --> 00:53:27,560 ABSENCE OF PBRM1. 1261 00:53:27,560 --> 00:53:30,840 BACK WHEN WE FOUND THE DEFECT WE 1262 00:53:30,840 --> 00:53:32,240 ACTUALLY TRIED LOOKING AT 1263 00:53:32,240 --> 00:53:33,680 PROTEIN LEVELS GLOBALLY AND WE 1264 00:53:33,680 --> 00:53:35,960 TRIED LOOKING AT PROTEIN LEVELS 1265 00:53:35,960 --> 00:53:37,040 ASSOCIATED WITH CHROMATIN, AND 1266 00:53:37,040 --> 00:53:43,120 WE NEVER FOUND ANYTHING THAT WE 1267 00:53:43,120 --> 00:53:44,520 THOUGHT WAS CONVINCINGLY 1268 00:53:44,520 --> 00:53:45,200 DEFECTIVE. 1269 00:53:45,200 --> 00:53:47,800 SO, WE WOULD HAVE SAID IT LOOKS 1270 00:53:47,800 --> 00:53:49,440 LIKE COHESION ASSOCIATION AND 1271 00:53:49,440 --> 00:53:53,280 LOADING ARE UNAFFECTED BY THE 1272 00:53:53,280 --> 00:53:54,480 ABSENCE OF PBRM1. 1273 00:53:54,480 --> 00:53:55,880 HAVING SAID THAT I THINK WE'VE 1274 00:53:55,880 --> 00:53:57,640 GOT BETTER TOOLS NOW THAT WE CAN 1275 00:53:57,640 --> 00:54:02,920 USE, AS I JUST MENTIONED WITH 1276 00:54:02,920 --> 00:54:04,320 INHIBITORS AND PROTACs, AND 1277 00:54:04,320 --> 00:54:06,400 THESE KNOCKOUT LINES INSTEAD OF 1278 00:54:06,400 --> 00:54:08,080 USING DEPLETION, AND SO WE'VE 1279 00:54:08,080 --> 00:54:09,960 JUST STARTED TO REVISIT THAT 1280 00:54:09,960 --> 00:54:10,360 QUESTION. 1281 00:54:10,360 --> 00:54:13,000 SO I STILL THINK THAT'S ON THE 1282 00:54:13,000 --> 00:54:14,800 TABLE AS A POSSIBILITY THAT 1283 00:54:14,800 --> 00:54:18,480 YOU'RE FAILING TO LOAD COHESION 1284 00:54:18,480 --> 00:54:21,640 TO APPROPRIATE PLACES IN AND 1285 00:54:21,640 --> 00:54:22,240 AROUND THE CENTROMERE. 1286 00:54:22,240 --> 00:54:24,120 >>GREAT. 1287 00:54:24,120 --> 00:54:28,080 THERE'S A QUESTION FROM IRENE, 1288 00:54:28,080 --> 00:54:29,840 WONDERFUL WORK, WHAT DO YOU 1289 00:54:29,840 --> 00:54:33,680 THINK PBAF IS DOING IN REPEATED 1290 00:54:33,680 --> 00:54:35,840 REGIONS SURROUNDING THE 1291 00:54:35,840 --> 00:54:36,120 CENTROMERE? 1292 00:54:36,120 --> 00:54:38,240 >>OH, YEAH, THANKS, IRENE. 1293 00:54:38,240 --> 00:54:39,120 GREAT QUESTION. 1294 00:54:39,120 --> 00:54:41,560 AND I HAVE NO IDEA. 1295 00:54:41,560 --> 00:54:43,520 SO, THIS IS PART OF THE REASON 1296 00:54:43,520 --> 00:54:47,120 WE WANTED TO DO THAT K-MER 1297 00:54:47,120 --> 00:54:48,520 ANALYSIS TO GET AT THE QUESTION 1298 00:54:48,520 --> 00:54:51,200 WHAT IS BINDING AND WHEN. 1299 00:54:51,200 --> 00:54:54,840 SO, ONE OF THE THINGS THAT WE 1300 00:54:54,840 --> 00:54:56,040 FOUND WHICH WE WANT TO SORT OF 1301 00:54:56,040 --> 00:54:58,920 DIG INTO A LITTLE BIT MORE, IS 1302 00:54:58,920 --> 00:55:00,800 THAT THERE'S AN ASSOCIATION 1303 00:55:00,800 --> 00:55:03,800 BETWEEN PBAF BINDING AND 1304 00:55:03,800 --> 00:55:05,040 STRUCTURED DNAs. 1305 00:55:05,040 --> 00:55:07,040 AND SO ONE OF THE QUESTIONS IS 1306 00:55:07,040 --> 00:55:08,880 WHETHER THIS IS ACTUALLY AROUND 1307 00:55:08,880 --> 00:55:10,920 THE SEQUENCES PER SE, OR WHETHER 1308 00:55:10,920 --> 00:55:14,200 IT'S THEIR PROPENSITY TO FORM 1309 00:55:14,200 --> 00:55:14,960 DIFFERENT DNA STRUCTURES. 1310 00:55:14,960 --> 00:55:17,760 AND WHETHER OR NOT THAT'S 1311 00:55:17,760 --> 00:55:19,360 ACTUALLY DIFFERENT BETWEEN THE 1312 00:55:19,360 --> 00:55:20,280 DIFFERENT REPEATS BECAUSE 1313 00:55:20,280 --> 00:55:22,800 OBVIOUSLY THEY ALL HAVE 1314 00:55:22,800 --> 00:55:23,880 DIFFERENT PROPERTIES. 1315 00:55:23,880 --> 00:55:24,960 SO, YEAH, IT'S EARLY DAYS. 1316 00:55:24,960 --> 00:55:27,240 I DON'T KNOW THE ANSWER TO THAT 1317 00:55:27,240 --> 00:55:28,240 QUESTION BUT IT'S DEFINITELY 1318 00:55:28,240 --> 00:55:31,000 SOMETHING WE WANT TO GET VASTLY 1319 00:55:31,000 --> 00:55:32,840 MORE INTO AND I WOULD LOVE TO 1320 00:55:32,840 --> 00:55:37,200 HEAR YOUR VIEWS ON IT. 1321 00:55:37,200 --> 00:55:45,760 1322 00:55:45,760 --> 00:55:50,480 >>AND WALT SAYS THANK YOU. 1323 00:55:50,480 --> 00:55:52,520 SO, I WANTED TO MAYBE JUST ASK 1324 00:55:52,520 --> 00:55:54,720 ABOUT THE -- YOU STARTED WITH 1325 00:55:54,720 --> 00:55:56,200 TALKING ABOUT DIFFERENT CANCERS, 1326 00:55:56,200 --> 00:55:58,000 ROLE OF DIFFERENT CANCERS, HOW 1327 00:55:58,000 --> 00:55:59,320 DO YOU SEE THAT? 1328 00:55:59,320 --> 00:56:00,880 CAN YOU EXPLORE THIS A LITTLE 1329 00:56:00,880 --> 00:56:01,120 MORE? 1330 00:56:01,120 --> 00:56:03,280 HOW DO WE CONNECT THIS WHOLE 1331 00:56:03,280 --> 00:56:04,280 THING AND WHAT CANCERS IN 1332 00:56:04,280 --> 00:56:06,240 PARTICULAR SHOULD BE OF 1333 00:56:06,240 --> 00:56:06,480 INTEREST? 1334 00:56:06,480 --> 00:56:08,000 >>YEAH, YEAH, THAT'S A REALLY 1335 00:56:08,000 --> 00:56:09,000 GOOD QUESTION. 1336 00:56:09,000 --> 00:56:10,280 AND SOMETHING THAT WE'VE BEEN 1337 00:56:10,280 --> 00:56:14,720 THINKING A LOT ABOUT RECENTLY. 1338 00:56:14,720 --> 00:56:17,560 SO, PBRM1 IS LOST IN ABOUT 40% 1339 00:56:17,560 --> 00:56:18,520 OF RENAL CANCERS, WHICH IS PART 1340 00:56:18,520 --> 00:56:21,360 OF THE REASON WE USED THAT PANEL 1341 00:56:21,360 --> 00:56:24,440 OF RENAL CANCER CELL LINES. 1342 00:56:24,440 --> 00:56:26,200 HAVING SAID THAT, WE ACTUALLY 1343 00:56:26,200 --> 00:56:30,600 DON'T SEE THAT THIS IS A RENAL 1344 00:56:30,600 --> 00:56:31,320 CANCER-SPECIFIC PHENOTYPE. 1345 00:56:31,320 --> 00:56:32,000 >>IT'S VERY BROAD. 1346 00:56:32,000 --> 00:56:32,880 >>YEAH. 1347 00:56:32,880 --> 00:56:36,160 SEEMS MORE BROAD. 1348 00:56:36,160 --> 00:56:37,600 SO, BUT YOU PROBABLY WILL HAVE 1349 00:56:37,600 --> 00:56:41,440 ALSO NOTICED THE DEGREE OF 1350 00:56:41,440 --> 00:56:47,720 SENSITIVITY THAT WE'RE SEEING TO 1351 00:56:47,720 --> 00:56:51,400 EITHER CDK-IS, BETTER IS THE NPS 1352 00:56:51,400 --> 00:56:52,360 INHIBITORS, IT'S NOT HUGELY 1353 00:56:52,360 --> 00:56:53,080 DRAMATIC. 1354 00:56:53,080 --> 00:56:57,600 SO I FEEL LIKE IN TERMS OF 1355 00:56:57,600 --> 00:57:00,800 THINKING ABOUT THE SORT OF 1356 00:57:00,800 --> 00:57:01,680 IMPACTING CANCER, IT WOULD BE 1357 00:57:01,680 --> 00:57:05,080 GREAT IF WE COULD COME UP WITH 1358 00:57:05,080 --> 00:57:05,680 SOMETHING THAT WAS 1359 00:57:05,680 --> 00:57:06,240 THERAPEUTICALLY, YOU KNOW, 1360 00:57:06,240 --> 00:57:08,480 SOMETHING THAT WOULD GO AND GIVE 1361 00:57:08,480 --> 00:57:09,920 US A HUGE THERAPEUTIC INDEX BUT 1362 00:57:09,920 --> 00:57:11,320 I THINK WHAT THIS MIGHT BE 1363 00:57:11,320 --> 00:57:14,640 TELLING US MORE IS EVOLUTION OF 1364 00:57:14,640 --> 00:57:16,680 CANCERS, IT'S ALMOST LIKE THE 1365 00:57:16,680 --> 00:57:21,720 RECOMBINATION AT THE CENTROMERES 1366 00:57:21,720 --> 00:57:22,600 HAPPENING EVEN UNDER NORMAL 1367 00:57:22,600 --> 00:57:24,120 CONDITIONS, SEEING THEM GROW 1368 00:57:24,120 --> 00:57:25,640 UNDER NON-STRESSED CONDITIONS, 1369 00:57:25,640 --> 00:57:28,320 YOU'RE GETTING REARRANGEMENTS 1370 00:57:28,320 --> 00:57:30,560 AND YOU'RE GETTING 1371 00:57:30,560 --> 00:57:31,240 RECOMBINATION. 1372 00:57:31,240 --> 00:57:33,760 SO, YOU KNOW, THE GENOMES ARE -- 1373 00:57:33,760 --> 00:57:34,520 IT'S VERY PLASTIC. 1374 00:57:34,520 --> 00:57:35,960 AND SO YOU COULD IMAGINE 1375 00:57:35,960 --> 00:57:37,240 ACTUALLY THIS IS ONE OF THE 1376 00:57:37,240 --> 00:57:39,640 REASONS WHY THESE THINGS ARE 1377 00:57:39,640 --> 00:57:41,080 ACTUALLY SO INSIDIOUS AS TUMOR 1378 00:57:41,080 --> 00:57:43,400 SUPPRESSORS, THAT ONCE YOU LOSE 1379 00:57:43,400 --> 00:57:52,920 THEM, THEY GO THROUGH MITOSIS, 1380 00:57:52,920 --> 00:57:54,360 BUT THINGS ARE REARRANGING IN A 1381 00:57:54,360 --> 00:57:56,840 WAY THAT'S NOT APPROPRIATE. 1382 00:57:56,840 --> 00:58:00,480 IN ANOTHER STUDY WE FOUND LOSS 1383 00:58:00,480 --> 00:58:02,320 OF SWI/SNF UPREGULATES PATHWAYS 1384 00:58:02,320 --> 00:58:04,720 THAT ALLOWS YOU TO TOLERATE 1385 00:58:04,720 --> 00:58:09,200 ANEUPLOIDY BETTER SO IT'S A TERM 1386 00:58:09,200 --> 00:58:09,480 COMBINATION. 1387 00:58:09,480 --> 00:58:12,840 THEY SAME TIME YOU'RE MISSING 1388 00:58:12,840 --> 00:58:14,360 WITH FIDELITY OF CHROMOSOME 1389 00:58:14,360 --> 00:58:15,840 SEGREGATION YOU'RE ALSO TAKING 1390 00:58:15,840 --> 00:58:17,720 AWAY SOME OF THE BREAKS THAT THE 1391 00:58:17,720 --> 00:58:20,360 CELL HAS THAT WOULD NORMALLY 1392 00:58:20,360 --> 00:58:23,720 ACTUALLY MAKE THAT A LESS-FIT 1393 00:58:23,720 --> 00:58:24,000 CELL. 1394 00:58:24,000 --> 00:58:27,080 SO, I MEAN IT'S EXACTLY THE 1395 00:58:27,080 --> 00:58:31,200 RIGHT COMBINATION FOR A CANCER 1396 00:58:31,200 --> 00:58:32,600 CELL, A DANGEROUS COMBINATION BY 1397 00:58:32,600 --> 00:58:38,120 LOSING THE ONE PROTEIN COMPLEX? 1398 00:58:38,120 --> 00:58:42,480 >>THERE'S A LOT OF DNA REPAIR 1399 00:58:42,480 --> 00:58:42,960 CONNECTIONS TO RENAL. 1400 00:58:42,960 --> 00:58:46,080 >>YEAH, I THINK THAT RENAL 1401 00:58:46,080 --> 00:58:48,440 CANCER IN A WAY IS -- IT'S 1402 00:58:48,440 --> 00:58:53,040 NOT -- THERE'S NOT A SORT OF 1403 00:58:53,040 --> 00:59:02,920 BIOLOGICAL REASON IN TERMS 1404 00:59:02,920 --> 00:59:03,120 OFPBRM1. 1405 00:59:03,120 --> 00:59:09,560 RENAL CELLS LOSE ONE COPE OF 1406 00:59:09,560 --> 00:59:16,360 CHROMOSOME 3P, WELCOME 1407 00:59:16,360 --> 00:59:18,400 HAPLOINSUFFICIENT, WHERE PBRM1 1408 00:59:18,400 --> 00:59:23,120 SITS, ALL ON 3P, NOW 1409 00:59:23,120 --> 00:59:24,000 HAPLOINSUFFICIENT, AND SOME 1410 00:59:24,000 --> 00:59:27,120 REALLY NICE WORK FROM A COUPLE 1411 00:59:27,120 --> 00:59:28,120 LABS DOING MOUSE STUDIES SHOWED 1412 00:59:28,120 --> 00:59:32,080 YOU COULD JUST GET RID OF VHL 1413 00:59:32,080 --> 00:59:36,360 AND PBRM1, ENOUGH TO DRIVE RENAL 1414 00:59:36,360 --> 00:59:38,320 CANCER. 1415 00:59:38,320 --> 00:59:39,200 SO IT'S -- THE OTHER GENES 1416 00:59:39,200 --> 00:59:40,600 OBVIOUSLY COULD PLAY A ROLE SO 1417 00:59:40,600 --> 00:59:43,040 IT'S KIND OF JUST BAD LUCK IN 1418 00:59:43,040 --> 00:59:44,960 RENAL CANCER THAT YOU ARE 1419 00:59:44,960 --> 00:59:46,520 ALREADY HAPLOINSUFFICIENT, IT 1420 00:59:46,520 --> 00:59:50,280 JUST TAKES THAT ONE HIT. 1421 00:59:50,280 --> 00:59:52,880 YOU SEE LOSS AT LOWER LEVELS IN 1422 00:59:52,880 --> 00:59:54,800 OTHER CANCERS, I SUSPECT A LOT 1423 00:59:54,800 --> 00:59:56,040 OF PHENOTYPES ARE ALSO IMPORTANT 1424 00:59:56,040 --> 00:59:56,480 THERE. 1425 00:59:56,480 --> 00:59:58,600 IT'S JUST THE FREQUENCY IS SO 1426 00:59:58,600 --> 01:00:01,800 MUCH LOWER BECAUSE YOU NEED THE 1427 01:00:01,800 --> 01:00:06,040 TWO HITS, THAN WHAT YOU SEE IN 1428 01:00:06,040 --> 01:00:06,440 RENAL CANCER. 1429 01:00:06,440 --> 01:00:10,040 >>INTERESTING. 1430 01:00:10,040 --> 01:00:15,560 CHUN, DO YOU HAVE ANY QUESTIONS? 1431 01:00:15,560 --> 01:00:18,560 >>NO. 1432 01:00:18,560 --> 01:00:18,880 THANK YOU. 1433 01:00:18,880 --> 01:00:23,440 >> IRENE SAYS GREAT, I'LL FOLLW 1434 01:00:23,440 --> 01:00:24,000 UP. 1435 01:00:24,000 --> 01:00:24,520 >>GREAT. 1436 01:00:24,520 --> 01:00:24,840 THANK YOU. 1437 01:00:24,840 --> 01:00:26,840 >>WE'RE OUT OF TIME. 1438 01:00:26,840 --> 01:00:31,440 THANK YOU SO MUCH FOR A REALLY 1439 01:00:31,440 --> 01:00:32,640 ELEGANT LECTURE, GREAT QUESTIONS 1440 01:00:32,640 --> 01:00:33,240 ANSWERED. 1441 01:00:33,240 --> 01:00:37,480 AND WE WILL BE IN TOUCH WITH YOU 1442 01:00:37,480 --> 01:00:38,880 ABOUT POTENTIALLY POSTING THE 1443 01:00:38,880 --> 01:00:41,120 TALK AT OUR WEBSITE, IF THAT'S 1444 01:00:41,120 --> 01:00:41,480 OKAY WITH YOU. 1445 01:00:41,480 --> 01:00:43,680 >>ABSOLUTELY. 1446 01:00:43,680 --> 01:00:44,200 THANK YOU VERY MUCH. 1447 01:00:44,200 --> 01:00:45,920 >>THANK YOU TO THE ATTENDEES 1448 01:00:45,920 --> 01:00:47,040 FOR LISTENING. 1449 01:00:47,040 --> 01:00:49,240 SEE YOU IN A MONTH AT THE NEXT 1450 01:00:49,240 --> 01:00:50,520 DNA REPAIR INTEREST GROUP 1451 01:00:50,520 --> 01:00:52,960 SEMINAR. 1452 01:00:52,960 --> 00:00:00,000 >>THANK YOU.