1 00:00:06,314 --> 00:00:08,149 >> GOOD AFTERNOON EVERYONE. 2 00:00:08,149 --> 00:00:09,951 WELCOME TO THE WEDNESDAY 3 00:00:09,951 --> 00:00:12,887 AFTERNOON LECTURE SERIES. I AM 4 00:00:12,887 --> 00:00:16,257 -- CHIEF OF THE FUNCTIONAL-- 5 00:00:16,257 --> 00:00:25,834 SECTION IN THE NAIAB 6 00:00:25,834 --> 00:00:26,334 LABORATORY, PLEASED TO 7 00:00:26,334 --> 00:00:29,637 INTRODUCE OUR GUEST SPEAKER, 8 00:00:29,637 --> 00:00:30,238 WHO IS CONDUCTING SOME OF THE 9 00:00:30,238 --> 00:00:34,776 MOST EXCITING WORK TODAY ON 10 00:00:34,776 --> 00:00:37,245 HIGH MASS SPECTROMETRY, AND 11 00:00:37,245 --> 00:00:37,879 SOME CHROMATIN REGULATION FIRST 12 00:00:37,879 --> 00:00:42,584 I HAVE FEW ANNOUNCEMENTS. 13 00:00:42,584 --> 00:00:43,151 THE FIRST CONCERNS CONTINUING 14 00:00:43,151 --> 00:00:46,054 MEDICAL EDUCATION CREDITS. THE 15 00:00:46,054 --> 00:00:49,757 CME CODE FOR TODAY IS 50129. I 16 00:00:49,757 --> 00:00:57,932 REPEAT THAT, 50129. 17 00:00:57,932 --> 00:01:00,802 ALSO FOR THOSE OF YOU WATCHING 18 00:01:00,802 --> 00:01:01,336 REMOTELY, YOU CAN SUBMIT 19 00:01:01,336 --> 00:01:03,638 QUESTIONS AT ANY TIME, BY 20 00:01:03,638 --> 00:01:05,540 CLICKING ON THE BUTTON JUST 21 00:01:05,540 --> 00:01:06,774 BELOW YOUR VIDEOCAST WINDOW 22 00:01:06,774 --> 00:01:15,683 THAT SAYS "SEND LIVE FEEDBACK." 23 00:01:15,683 --> 00:01:16,284 FOR THOSE PRESENT HERE THERE 24 00:01:16,284 --> 00:01:20,955 ARE TWO MICS IN THE AISLES THAT 25 00:01:20,955 --> 00:01:21,589 YOU WILL BE ABLE TO USE AT THE 26 00:01:21,589 --> 00:01:24,192 END OF THE LECTURE. 27 00:01:24,192 --> 00:01:26,995 PLEASE NOTE ALSO THAT TODAY'S 28 00:01:26,995 --> 00:01:29,797 LECTURE IS THE FINAL IN A 29 00:01:29,797 --> 00:01:30,298 SERIES. WE WILL TAKE A 30 00:01:30,298 --> 00:01:31,432 TWO-MONTH BREAK AND BEGIN A NEW 31 00:01:31,432 --> 00:01:33,201 SEASON ON SEPTEMBER 11, WITH A 32 00:01:33,201 --> 00:01:37,372 LECTURE BY DR. EVE MARDER 33 00:01:37,372 --> 00:01:38,106 (SOUNDS LIKE) A BRANDEIS 34 00:01:38,106 --> 00:01:40,542 UNIVERSITY. 35 00:01:40,542 --> 00:01:42,410 OUR SPEAKER TODAY IS DR. 36 00:01:42,410 --> 00:01:46,781 BENJAMIN GARCIA. THE RAYMOND H. 37 00:01:46,781 --> 00:01:50,051 WALKOFF DISTINCT PROFESSOR OF 38 00:01:50,051 --> 00:01:52,120 DIALOGICAL CHEMISTRY, PROFESSOR 39 00:01:52,120 --> 00:01:52,720 OF BIOCHEMISTRY AND MOLECULAR 40 00:01:52,720 --> 00:01:54,455 BIOPHYSICS AND HEAD OF THE 41 00:01:54,455 --> 00:01:56,624 DEPARTMENT OF BIOCHEMISTRY AND 42 00:01:56,624 --> 00:01:57,125 MOLECULAR BIOPHYSICS AT 43 00:01:57,125 --> 00:01:57,759 WASHINGTON UNIVERSITY SCHOOL OF 44 00:01:57,759 --> 00:02:00,361 MEDICINE. 45 00:02:00,361 --> 00:02:03,364 DR. GARCIA RECEIVED HIS PHD IN 46 00:02:03,364 --> 00:02:06,367 ANALYTICAL CHEMISTRY FROM THE 47 00:02:06,367 --> 00:02:06,868 UNIVERSITY OF VIRGINIA, 48 00:02:06,868 --> 00:02:07,502 CHARLOTTESVILLE. HE THEN SPENT 49 00:02:07,502 --> 00:02:11,372 HIS POSTDOCTORAL YEAR AT THE 50 00:02:11,372 --> 00:02:11,873 UNIVERSITY OF ILLINOIS, 51 00:02:11,873 --> 00:02:14,776 URBANA-CHAMPAIGN, AND UNDER THE 52 00:02:14,776 --> 00:02:17,345 MENTORSHIP OF NEIL-- FROM 2008 53 00:02:17,345 --> 00:02:20,481 - 2012 HE WORKED AT PRINCETON 54 00:02:20,481 --> 00:02:21,816 UNIVERSITY WHERE HE WAS AT THE 55 00:02:21,816 --> 00:02:22,417 PROFESSOR. AND HE JOINED THE 56 00:02:22,417 --> 00:02:25,587 UNIVERSITY OF PENNSYLVANIA 57 00:02:25,587 --> 00:02:26,221 SCHOOL OF MEDICINE IS DIRECTOR 58 00:02:26,221 --> 00:02:30,758 OF QUANTITATIVE PROTEOMICS. AND 59 00:02:30,758 --> 00:02:39,334 IN 2019 HE BECAME THE-- 60 00:02:39,334 --> 00:02:39,934 PRESIDENTIAL PROFESSOR AND IN 61 00:02:39,934 --> 00:02:40,535 2021 MOVED TO THE WASHINGTON 62 00:02:40,535 --> 00:02:44,839 UNIVERSITY SCHOOL OF MEDICINE 63 00:02:44,839 --> 00:02:45,473 IN ST. LOUIS WHERE HE ATTAINED 64 00:02:45,473 --> 00:02:45,940 HIS CURRENT TITLE AS 65 00:02:45,940 --> 00:02:49,344 DISTINGUISHED PROFESSOR. 66 00:02:49,344 --> 00:02:49,844 HE IS VERY ACTIVE IN THE 67 00:02:49,844 --> 00:02:53,848 PROTEOMICS COMMUNITY. HE IS 68 00:02:53,848 --> 00:02:54,349 PART OF THE NOMINATING 69 00:02:54,349 --> 00:02:54,849 COMMUNITY FOR THE HUMAN 70 00:02:54,849 --> 00:02:55,516 PROTEOMIC ORGANIZATION. HEAD OF 71 00:02:55,516 --> 00:02:57,819 THE DIVERSITY, EQUITY AND 72 00:02:57,819 --> 00:03:00,221 INCLUSION COMMITTEE FOR THE 73 00:03:00,221 --> 00:03:00,822 AMERICAN CHEMICAL SOCIETY AND 74 00:03:00,822 --> 00:03:08,796 THE MENTOR FOR THE ASB, 75 00:03:08,796 --> 00:03:09,330 NIH-FUNDED PROGRAM CALLED 76 00:03:09,330 --> 00:03:09,864 MAXIMIZE OPPORTUNITY FOR 77 00:03:09,864 --> 00:03:12,667 SCIENTIFIC ACADEMIC STUDY. 78 00:03:12,667 --> 00:03:13,268 HE IS ALSO ASSOCIATE EDITOR OF 79 00:03:13,268 --> 00:03:17,605 AMERICAN CHEMISTRY, AND THE 80 00:03:17,605 --> 00:03:18,139 EDITORIAL BOARD MEMBER OF 81 00:03:18,139 --> 00:03:19,140 NUMEROUS JOURNALS IN OUR FIELD 82 00:03:19,140 --> 00:03:20,408 AND HAS RECEIVED SEVERAL HONORS 83 00:03:20,408 --> 00:03:21,009 ALONG THE WAY AS WELL AS YOU 84 00:03:21,009 --> 00:03:22,710 MIGHT IMAGINE, HE WAS NAMED A 85 00:03:22,710 --> 00:03:30,118 HIGHLY CITED RESEARCHER IN 86 00:03:30,118 --> 00:03:30,752 2022. HE HAS RECEIVED THE HUMAN 87 00:03:30,752 --> 00:03:31,252 PROTEOMIC ORGANIZATION 88 00:03:31,252 --> 00:03:32,420 DISCOVERY IN PROTEOMIC SCIENCE 89 00:03:32,420 --> 00:03:33,054 AWARD. AND WAS ELECTED A FELLOW 90 00:03:33,054 --> 00:03:34,322 OF THE ROYAL SOCIETY OF 91 00:03:34,322 --> 00:03:39,927 CHEMISTRY. 92 00:03:39,927 --> 00:03:40,461 BENJAMIN'S PRIMARY RESEARCH 93 00:03:40,461 --> 00:03:41,896 FOCUS HAS BEEN TO CATEGORIZE 94 00:03:41,896 --> 00:03:44,399 THE ROLE OF POST-TRANSLATIONAL 95 00:03:44,399 --> 00:03:47,568 MODIFICATIONS OR PTMS ON 96 00:03:47,568 --> 00:03:49,704 PROTEINS INVOLVED IN HUMAN 97 00:03:49,704 --> 00:03:51,105 BIOLOGY, ESPECIALLY INTERESTED 98 00:03:51,105 --> 00:03:58,680 IN UNDERSTANDING HOW PROTEINS 99 00:03:58,680 --> 00:03:59,247 AND NUCLEIC ACID IS MEDIATE 100 00:03:59,247 --> 00:04:02,717 CANONICAL FUNCTIONS. AND THAT 101 00:04:02,717 --> 00:04:03,351 HE HAS BROUGHT EXPECTATION MANY 102 00:04:03,351 --> 00:04:13,828 AREAS INCLUDING CHEMISTRY. 103 00:04:17,532 --> 00:04:18,099 PARTICULARLY IN THE AREA OF MAS 104 00:04:18,099 --> 00:04:24,072 SPECTROMETRY BASED CHEMISTRY, 105 00:04:24,072 --> 00:04:24,672 AND HIS JOKE IS TO BRING MASS 106 00:04:24,672 --> 00:04:29,977 SPECTROMETRY TO THE MASSES. AN 107 00:04:29,977 --> 00:04:32,113 ANALYSIS OF HISTONE PTNS AND 108 00:04:32,113 --> 00:04:34,282 CHROMATIN EXTRACTORS. 109 00:04:34,282 --> 00:04:38,086 HE ALSO EXEMPLIFIES THAT BY HIS 110 00:04:38,086 --> 00:04:39,087 RELENTLESS ADVOCACY DURING THE 111 00:04:39,087 --> 00:04:48,830 YEARLY -- WHERE SEVERAL VERY, 112 00:04:48,830 --> 00:04:49,430 VERY GOOD METHODS COMPETE TO 113 00:04:49,430 --> 00:04:58,506 BECOME MIDDLE OF THE YEAR. AND 114 00:04:58,506 --> 00:04:59,040 ALL OF THE YEARS AT LEAST 115 00:04:59,040 --> 00:05:01,409 SEVERAL YEARS IN A ROW MASS 116 00:05:01,409 --> 00:05:04,212 SPECTROMETRY HAS ONE. EVEN 117 00:05:04,212 --> 00:05:08,249 AGAINST CRISPR. NOT A JOKE. IT 118 00:05:08,249 --> 00:05:12,253 IS A SERIOUS COMPETITION. 119 00:05:12,253 --> 00:05:12,787 IN MORE RECENT YEARS HE HAS 120 00:05:12,787 --> 00:05:13,621 UPPING HIS GAME WITH 121 00:05:13,621 --> 00:05:14,188 TRANSLATIONAL RESEARCH AND 122 00:05:14,188 --> 00:05:14,756 DEEPER CONNECTION TO HUMAN 123 00:05:14,756 --> 00:05:15,156 DISEASE. 124 00:05:15,156 --> 00:05:18,092 THE TITLE OF BEN'S TODAY IS 125 00:05:18,092 --> 00:05:18,659 QUANTITATIVE PROTEOMICS FOR 126 00:05:18,659 --> 00:05:19,193 UNDERSTANDING EPIGENETICS 127 00:05:19,193 --> 00:05:23,831 MECHANISMS. PLEASE HELP ME 128 00:05:23,831 --> 00:05:24,932 WELCOME DR. BENJAMIN GARCIA. 129 00:05:24,932 --> 00:05:35,109 >> [APPLAUSE] 130 00:05:36,210 --> 00:05:44,285 >> DR. GARCIA: THANK YOU 131 00:05:44,285 --> 00:05:44,786 ALEXANDRA, FOR A GREAT 132 00:05:44,786 --> 00:05:45,686 INTRODUCTION AND BEING A 133 00:05:45,686 --> 00:05:46,287 FANTASTIC HOST. IT HAS BEEN A 134 00:05:46,287 --> 00:05:50,091 WONDERFUL DAY, MEETING MANY 135 00:05:50,091 --> 00:05:50,758 INCREDIBLE SCIENTISTS AND 136 00:05:50,758 --> 00:05:52,627 TRAINEES HERE. I HAVE REALLY 137 00:05:52,627 --> 00:05:55,696 ENJOYED IT SO FAR. 138 00:05:55,696 --> 00:05:56,264 AND SO WHAT I AM HOPING TO DO 139 00:05:56,264 --> 00:05:58,499 TODAY IS GIVE EVERYONE A LITTLE 140 00:05:58,499 --> 00:06:02,103 BIT OF KIND OF A TASTE OF SOME 141 00:06:02,103 --> 00:06:02,770 OF THE TECHNOLOGY, AND 142 00:06:02,770 --> 00:06:04,605 APPROACHES THAT WE HAVE BEEN 143 00:06:04,605 --> 00:06:05,206 DEVELOPING IN THE LAST COUPLE 144 00:06:05,206 --> 00:06:05,706 OF YEARS AND HOW WE ARE 145 00:06:05,706 --> 00:06:10,278 APPLYING THEM, ONE, TWO 146 00:06:10,278 --> 00:06:12,180 PROBLEMS IN CANCER EPIGENETICS 147 00:06:12,180 --> 00:06:12,814 AND THINK WE HAVE PUBLISHED AND 148 00:06:12,814 --> 00:06:13,714 A NEWER STORY THAT IS EMERGING 149 00:06:13,714 --> 00:06:16,751 AS WELL. 150 00:06:16,751 --> 00:06:19,287 I GUESS I WILL PROBABLY USE ... 151 00:06:19,287 --> 00:06:21,489 LET ME SEE. 152 00:06:21,489 --> 00:06:23,624 IF I USE THIS, WILL PEOPLE 153 00:06:23,624 --> 00:06:25,359 ONLINE BE ABLE TO SEE THAT? OR 154 00:06:25,359 --> 00:06:26,694 NO. 155 00:06:26,694 --> 00:06:29,464 >> (OFF MIC) 156 00:06:29,464 --> 00:06:33,734 >> OKAY GREAT. YEAH. RIGHT 157 00:06:33,734 --> 00:06:35,102 HERE? YOU CAN SEE IT? 158 00:06:35,102 --> 00:06:37,638 >> (OFF MIC) 159 00:06:37,638 --> 00:06:46,347 >> DR. GARCIA: AND SO YEAH, 160 00:06:46,347 --> 00:06:46,948 MAYBE FIRST BEFORE I GET INTO 161 00:06:46,948 --> 00:06:49,484 THE TECHNOLOGY AND APPLICATIONS 162 00:06:49,484 --> 00:06:50,051 OF WANT TO GIVE A COUPLE OF 163 00:06:50,051 --> 00:06:54,755 SLIDES ON THE BACKGROUND. AND 164 00:06:54,755 --> 00:06:56,424 I KNOW NOT EVERYONE WORKS ON 165 00:06:56,424 --> 00:06:57,358 CHROMATIN AND EPIGENETIC 166 00:06:57,358 --> 00:06:59,093 MECHANISMS. THAT IS OKAY. 167 00:06:59,093 --> 00:07:00,962 NOBODY IS PERFECT. 168 00:07:00,962 --> 00:07:01,496 BUT TO GIVE A LITTLE BIT OF 169 00:07:01,496 --> 00:07:05,399 BACKGROUND WE HAVE METER 170 00:07:05,399 --> 00:07:06,767 MOUNTED DNA THAT HAVE TO BE 171 00:07:06,767 --> 00:07:07,368 COMPACTED INTO SMALL AREA IN 172 00:07:07,368 --> 00:07:09,904 THE NUCLEUS, AND THE WAY THAT 173 00:07:09,904 --> 00:07:10,505 NATURE DOES THIS IS BY TAKING 174 00:07:10,505 --> 00:07:11,072 THE DNA AND WINDING IT ON A 175 00:07:11,072 --> 00:07:21,516 BALL IS CALLED HISTONES, 176 00:07:22,483 --> 00:07:23,050 SIMILAR TO WANTING A STRING OF 177 00:07:23,050 --> 00:07:23,684 SPOOL, SO YOU HAVE TO COPIES OF 178 00:07:23,684 --> 00:07:25,453 EACH OF THE CORE HISTONES AND 179 00:07:25,453 --> 00:07:34,395 ABOUT 146 PAIRS MAKE UP THE 180 00:07:34,395 --> 00:07:34,962 NUCLEUS. ESSENTIALLY FOLDED 181 00:07:34,962 --> 00:07:35,530 INTO CHROMATIN FIBER IN OUR 182 00:07:35,530 --> 00:07:37,265 CHROMOSOMES. 183 00:07:37,265 --> 00:07:38,199 IT WAS THOUGHT THAT THESE 184 00:07:38,199 --> 00:07:39,800 HISTONE PROTEINS WERE VERY 185 00:07:39,800 --> 00:07:40,334 STRUCTURAL COMPONENTS OF 186 00:07:40,334 --> 00:07:42,770 CHROMATIN. BUT OVER THE LAST 187 00:07:42,770 --> 00:07:45,640 2+ DECADES IT HAS BEEN FOUND 188 00:07:45,640 --> 00:07:48,776 THAT THESE HISTONES HAVE A HUGE 189 00:07:48,776 --> 00:07:49,343 ARRAY OF POST-TRANSLATIONAL 190 00:07:49,343 --> 00:07:52,346 MODIFICATIONS. OUR DYNAMIC 191 00:07:52,346 --> 00:07:52,914 PROTEINS THAT HELP REGULATE 192 00:07:52,914 --> 00:07:54,749 GENE EXPRESSION PATTERNS. 193 00:07:54,749 --> 00:07:59,020 AND SO, KIND OF FAST FORWARD 194 00:07:59,020 --> 00:07:59,620 NOW BY MANY METHODS INCLUDING 195 00:07:59,620 --> 00:08:04,592 MASS SPECTROMETRY. SEVERAL 196 00:08:04,592 --> 00:08:05,026 POST-TRANSLATIONAL 197 00:08:05,026 --> 00:08:06,761 MODIFICATIONS OF THESE SYSTEMS 198 00:08:06,761 --> 00:08:07,628 HAVE BEEN DESCRIBED. YOU CAN 199 00:08:07,628 --> 00:08:08,162 SEE A WHOLE HOST OF MANY 200 00:08:08,162 --> 00:08:08,596 DIFFERENT CHEMICAL 201 00:08:08,596 --> 00:08:14,669 MODIFICATIONS ACROSS MANY 202 00:08:14,669 --> 00:08:15,269 CITES. ESSENTIALLY THE ENTIRE 203 00:08:15,269 --> 00:08:16,637 PROTEIN OF THESE HISTONES. 204 00:08:16,637 --> 00:08:19,707 HOWEVER, DEFINITELY I WOULD SAY 205 00:08:19,707 --> 00:08:20,308 THE MAJORITY OF MODIFICATIONS 206 00:08:20,308 --> 00:08:22,877 ARE FOUND ON THE INTERNAL 207 00:08:22,877 --> 00:08:26,480 REGIONS OF THE PROTEINS. AND 208 00:08:26,480 --> 00:08:27,315 MORE HI STOKE GEOMETRIC 209 00:08:27,315 --> 00:08:35,656 MODIFICATIONS. 210 00:08:35,656 --> 00:08:36,190 YOU CAN SEE HERE THERE SOME 211 00:08:36,190 --> 00:08:36,724 SITES THAT HAVE MULTIPLE 212 00:08:36,724 --> 00:08:38,192 MODIFICATIONS 10 OR 11 213 00:08:38,192 --> 00:08:38,626 DIFFERENT TYPES OF 214 00:08:38,626 --> 00:08:49,070 MODIFICATIONS ON THE SAME 215 00:08:56,978 --> 00:09:05,286 RESIDUE. 216 00:09:05,286 --> 00:09:05,753 AND ONE THAT KEEPS AN 217 00:09:05,753 --> 00:09:06,654 ANALYTICAL CHEMIST LIKE MYSELF 218 00:09:06,654 --> 00:09:13,494 VERY BUSY. 219 00:09:13,494 --> 00:09:14,061 AND THESE GENE REGULATE GENE 220 00:09:14,061 --> 00:09:15,963 EXPRESSION IN THESE 221 00:09:15,963 --> 00:09:16,497 MODIFICATIONS, SHOWING IN 222 00:09:16,497 --> 00:09:18,599 CARTOON FORM SOME OF THE MORE 223 00:09:18,599 --> 00:09:19,200 ABUNDANT MODIFICATIONS LIKE 224 00:09:19,200 --> 00:09:21,168 ACETYLATION. AND THESE 225 00:09:21,168 --> 00:09:26,574 MODIFICATIONS HAVE BY 226 00:09:26,574 --> 00:09:27,174 BIOLOGISTS ONE AT A TIME HAVE 227 00:09:27,174 --> 00:09:27,775 BEEN LINKED TO VERY IMPORTANT 228 00:09:27,775 --> 00:09:28,376 PROCESSES ESPECIALLY TURNING 229 00:09:28,376 --> 00:09:32,747 GENES ON OR OFF. AND ALSO IN 230 00:09:32,747 --> 00:09:35,650 THE DNA DAMAGE EVENTS AND 231 00:09:35,650 --> 00:09:36,183 THINGS LIKE THAT AND THE 232 00:09:36,183 --> 00:09:36,717 HISTORY ON ACETYLATION IN 233 00:09:36,717 --> 00:09:37,918 GENERAL IS LINKED TO GENE 234 00:09:37,918 --> 00:09:40,888 ACTIVATION. HISTONE ACETYLATION 235 00:09:40,888 --> 00:09:41,489 IS A LITTLE BIT MORE COMPLEX. 236 00:09:41,489 --> 00:09:43,257 THERE'S DIFFERENT DEGREES OF 237 00:09:43,257 --> 00:09:44,191 METHYLATION. IT CAN ALSO BE 238 00:09:44,191 --> 00:09:51,065 LINKED TO BOTH GENE ACTIVATION 239 00:09:51,065 --> 00:09:51,699 OR GENE SILENCING DEPENDING ON 240 00:09:51,699 --> 00:09:52,333 WHETHER MODIFICATIONS FOUND. IN 241 00:09:52,333 --> 00:09:54,435 SO FOR EXAMPLE THIS WILL HERE 242 00:09:54,435 --> 00:09:55,036 IS FOUND ON ACTIVE GENES AND 243 00:09:55,036 --> 00:09:57,004 THE GENOME, PROMOTERS OF ACTIVE 244 00:09:57,004 --> 00:10:00,941 GENE AND THIS ON THE SAME 245 00:10:00,941 --> 00:10:01,509 HISTONE PROPERTY JUST A FEW 246 00:10:01,509 --> 00:10:02,143 AMINO ACID DOWN FROM THIS OTHER 247 00:10:02,143 --> 00:10:05,513 MODIFICATION ... LOST THE 248 00:10:05,513 --> 00:10:15,690 POINTER. NOT SURE IF I HAVE 249 00:10:15,690 --> 00:10:18,225 THAT POINTER ANYMORE. THERE IT 250 00:10:18,225 --> 00:10:19,860 IS. 251 00:10:19,860 --> 00:10:27,134 AND SO, THIS MODIFICATION, SAME 252 00:10:27,134 --> 00:10:27,635 METHYLATION ON THE SAME 253 00:10:27,635 --> 00:10:28,202 PROTEIN, A FEW AMINO ACIDS 254 00:10:28,202 --> 00:10:32,707 DOWN, THIS IS ACTUALLY BEEN 255 00:10:32,707 --> 00:10:33,307 FOUND IN THE HEADER CHROMATIC 256 00:10:33,307 --> 00:10:34,809 REGIONS OF THE GENOME. AND 257 00:10:34,809 --> 00:10:36,343 BECAUSE OF THIS RELATIONSHIP 258 00:10:36,343 --> 00:10:36,911 THAT A LOT OF THEORIES AND 259 00:10:36,911 --> 00:10:38,312 HYPOTHESES HAVE BEEN PUT FORTH 260 00:10:38,312 --> 00:10:38,879 TO TRY TO EXPEND HOW THESE 261 00:10:38,879 --> 00:10:41,082 MODIFICATIONS MAY REGULATE GENE 262 00:10:41,082 --> 00:10:41,716 EXPRESSION. ONE OF THIS IS THE 263 00:10:41,716 --> 00:10:45,686 HISTONE CODE OR PTM CODING 264 00:10:45,686 --> 00:10:46,287 HYPOTHESIS THAT STATES WHICH 265 00:10:46,287 --> 00:10:51,058 THESE MODIFICATIONS KIND OF-- 266 00:10:51,058 --> 00:10:51,659 WELL IF YOU THINK ABOUT IT IF 267 00:10:51,659 --> 00:10:52,293 THERE IS A CODE THAT HAS TO BE 268 00:10:52,293 --> 00:10:53,894 A WAY TO INTERPRET THIS CODE. 269 00:10:53,894 --> 00:10:54,395 IT IS THOUGHT THAT THESE 270 00:10:54,395 --> 00:10:56,130 MODIFICATIONS ACT AS A CODE BY 271 00:10:56,130 --> 00:10:59,800 BRINGING PROTEINS THAT THEN 272 00:10:59,800 --> 00:11:00,401 READ OR INTERPRET THE CODE TO 273 00:11:00,401 --> 00:11:03,471 SPECIFIC PARTS OF THE GENOME. 274 00:11:03,471 --> 00:11:04,038 AND SO IT IS NOT THAT THESE 275 00:11:04,038 --> 00:11:04,872 MODIFICATIONS ACT AS A PLATFORM 276 00:11:04,872 --> 00:11:07,775 FOR RECRUITING THESE PROTEINS 277 00:11:07,775 --> 00:11:09,510 TO ACTIVATE GENES; AND IN 278 00:11:09,510 --> 00:11:10,111 SUPPORT SEVERAL PROTEINS WITH 279 00:11:10,111 --> 00:11:11,178 SPECIALIZED DOMAINS HAVE BEEN 280 00:11:11,178 --> 00:11:12,379 FOUND TO BIND SPECIFIC SPOTS ON 281 00:11:12,379 --> 00:11:16,917 THESE HISTONES AND THE GENOME. 282 00:11:16,917 --> 00:11:19,186 AND THERE'S A LOT OF INTEREST 283 00:11:19,186 --> 00:11:21,188 RIGHT NOW IN KIND OF 284 00:11:21,188 --> 00:11:21,789 UNDERSTANDING-- ESPECIALLY IN 285 00:11:21,789 --> 00:11:23,023 HUMAN DISEASES-- HOW THIS 286 00:11:23,023 --> 00:11:24,325 EPIGENETIC MECHANISMS CAN GO 287 00:11:24,325 --> 00:11:32,366 AWRY. NOW WITH A DECADES' 288 00:11:32,366 --> 00:11:34,969 WORTH OR MORE OF LARGE-SCALE 289 00:11:34,969 --> 00:11:37,304 GENOMIC EFFECTS, SEVERAL 290 00:11:37,304 --> 00:11:37,938 EFFECTS THAT MODIFY HISTONES OR 291 00:11:37,938 --> 00:11:42,743 PROTEINS THAT INTERACT WITH 292 00:11:42,743 --> 00:11:45,045 HISTONES OR EVEN THE HISTONES 293 00:11:45,045 --> 00:11:45,646 THEMSELVES BEEN FOUND MUTATED 294 00:11:45,646 --> 00:11:48,149 IN VARIOUS CANCERS. SEVERAL 295 00:11:48,149 --> 00:11:48,783 COMPANIES INCLUDING VERY LARGE 296 00:11:48,783 --> 00:11:49,316 COMPANIES HAVE EPIGENETIC 297 00:11:49,316 --> 00:11:51,385 ONCOLOGY DIVISIONS. AND THERE 298 00:11:51,385 --> 00:11:52,753 ARE SMALLER COMPANIES THAT ONLY 299 00:11:52,753 --> 00:11:54,421 FOCUS ON THIS WITH EPIGENETIC 300 00:11:54,421 --> 00:11:55,523 INHIBITORS MAKING THEIR WAY 301 00:11:55,523 --> 00:11:57,324 THROUGH CLINICAL TRIALS AND FDA 302 00:11:57,324 --> 00:11:59,927 APPROVALS. 303 00:11:59,927 --> 00:12:00,394 AND SO THERE'S A LOT OF 304 00:12:00,394 --> 00:12:01,462 INTEREST IN UNDERSTANDING IF 305 00:12:01,462 --> 00:12:06,100 YOU KNOW, WHAT YOU ARE SEEING 306 00:12:06,100 --> 00:12:06,667 IN TERMS OF TRANSCRIPTIONAL 307 00:12:06,667 --> 00:12:07,234 CHANGES INVOLVED EPIGENETIC 308 00:12:07,234 --> 00:12:09,537 MECHANISMS. IN HUMAN DISEASE 309 00:12:09,537 --> 00:12:12,473 AND BIOLOGY. 310 00:12:12,473 --> 00:12:13,073 BUT I ALWAYS CANNOT TELL PEOPLE 311 00:12:13,073 --> 00:12:15,509 THAT YOU REALLY KNOW YOU ARE 312 00:12:15,509 --> 00:12:16,143 WORKING IN A GREY FIELD WHEN IT 313 00:12:16,143 --> 00:12:16,677 MAKES IT INTO BLOCKBUSTER 314 00:12:16,677 --> 00:12:23,617 MOVIES. AND SO HERE'S A FILM 315 00:12:23,617 --> 00:12:24,251 STILL OF ONE OF THE SPIDER FILM 316 00:12:24,251 --> 00:12:28,455 MOVIES FROM A LONG TIME AGO. SO 317 00:12:28,455 --> 00:12:29,056 YOU SEE ALL THE SIGNS ON THE 318 00:12:29,056 --> 00:12:29,657 BOARD EVERYTHING YOU NEED TO 319 00:12:29,657 --> 00:12:31,392 KNOW TO GET SUPERHUMAN POWER 320 00:12:31,392 --> 00:12:34,628 YOU MAY RECOGNIZE PHRASES OR 321 00:12:34,628 --> 00:12:35,262 SCHEMATICS AND THINGS LIKE THAT 322 00:12:35,262 --> 00:12:35,863 AND IF YOU LOOK RIGHT AT THE 323 00:12:35,863 --> 00:12:36,497 BOTTOM HERE THERE IS SOMETHING 324 00:12:36,497 --> 00:12:36,964 REALLY FAMILIAR. AND 325 00:12:36,964 --> 00:12:42,369 NUCLEOSOME. WITH HISTONE HS3 K 326 00:12:42,369 --> 00:12:46,407 27 METHYLATION ON IT SO WHOEVER 327 00:12:46,407 --> 00:12:47,007 KNEW THEY HAD TO PUT THE MOST 328 00:12:47,007 --> 00:12:49,476 IMPORTANT SIGNS ON THE BOARD IN 329 00:12:49,476 --> 00:12:50,144 THIS MOVIE KNEW THEY HAD TO 330 00:12:50,144 --> 00:12:53,881 INCLUDE EPIGENETIC MECHANISMS. 331 00:12:53,881 --> 00:12:54,348 AND SO THERE'S A LOT OF 332 00:12:54,348 --> 00:12:54,882 DIFFERENT WAYS TO LOOK AT 333 00:12:54,882 --> 00:12:59,987 HISTONE MODIFICATIONS. THE 334 00:12:59,987 --> 00:13:00,588 MOST COMMON APPROACHES USING 335 00:13:00,588 --> 00:13:01,222 SITE-SPECIFIC ANTIBODIES WHICH 336 00:13:01,222 --> 00:13:01,956 IF YOU USE ANTIBODIES CAN BE 337 00:13:01,956 --> 00:13:05,659 BOTH A BLESSING AND A CURSE. IF 338 00:13:05,659 --> 00:13:06,260 YOU HAVE A GOOD ANTIBODY YOU 339 00:13:06,260 --> 00:13:06,827 GET ONE SINGLE MODIFICATION 340 00:13:06,827 --> 00:13:11,632 DOES NOT EXIST IN YEAST, AND 341 00:13:11,632 --> 00:13:14,134 THIS IS WHAT YOU WILL SEE. YOU 342 00:13:14,134 --> 00:13:16,470 CAN SEE THE CROSS-REACTIVITY 343 00:13:16,470 --> 00:13:18,038 WITH ANOTHER PROTEIN THAT HAS A 344 00:13:18,038 --> 00:13:18,639 SIMILAR EPITOPE. AND YOU WILL 345 00:13:18,639 --> 00:13:21,642 SEE A COUPLE OF OTHER BANDS 346 00:13:21,642 --> 00:13:24,044 HERE. AND THIS RECOGNIZES THE 347 00:13:24,044 --> 00:13:27,548 COMMON -- NOT RECOGNIZING THE 348 00:13:27,548 --> 00:13:28,282 MODIFICATION AND SO THIS IS WHY 349 00:13:28,282 --> 00:13:31,752 BIOLOGISTS ONLY SHOW THE LITTLE 350 00:13:31,752 --> 00:13:32,353 SLIVER OF THE WESTERN BLOCK, 351 00:13:32,353 --> 00:13:32,920 NOT THE WHOLE THING BECAUSE 352 00:13:32,920 --> 00:13:34,922 THEN YOU WILL SEE ALL THE OTHER 353 00:13:34,922 --> 00:13:36,624 PROTEINS THAT IT IS NOT 354 00:13:36,624 --> 00:13:37,224 REACTING WITH YOU HAVE TO BE 355 00:13:37,224 --> 00:13:45,199 CAREFUL. 356 00:13:45,199 --> 00:13:45,633 ESPECIALLY WITH SUCH 357 00:13:45,633 --> 00:13:46,200 MODIFICATION-DENSE PROTEINS 358 00:13:46,200 --> 00:13:46,767 SUCH AS SYSTEMS TO REALLY 359 00:13:46,767 --> 00:13:47,868 UNDERSTAND WHAT EPISODE -- AND 360 00:13:47,868 --> 00:13:49,470 NOT GET WILL THAT IT IS ANOTHER 361 00:13:49,470 --> 00:13:53,274 HISTONE OR SOMETHING ELSE. 362 00:13:53,274 --> 00:13:53,874 THE OTHER ISSUE WITH ANTIBODIES 363 00:13:53,874 --> 00:13:56,143 AND HISTONE MODIFICATIONS IS I 364 00:13:56,143 --> 00:13:56,744 SHOWED YOU HOW MANY DIFFERENT 365 00:13:56,744 --> 00:13:57,378 MODIFICATIONS THERE ARE ON THIS 366 00:13:57,378 --> 00:14:00,547 HISTONES. AND SO YOU CAN 367 00:14:00,547 --> 00:14:01,982 IMAGINE THAT SOMEBODY'S 368 00:14:01,982 --> 00:14:02,583 MODIFICATIONS MIGHT ACTUALLY 369 00:14:02,583 --> 00:14:03,083 BLOCK THE ANTIBODY FROM 370 00:14:03,083 --> 00:14:03,751 RECOGNIZING THE INTENDED SITE. 371 00:14:03,751 --> 00:14:05,119 AND HERE IS AN EXAMPLE OF THIS. 372 00:14:05,119 --> 00:14:07,955 JUST THE PEPTIDE-- ANTIBODY 373 00:14:07,955 --> 00:14:13,928 AGAINST SERIES 10 374 00:14:13,928 --> 00:14:17,831 PHOSPHORYLATION, THESE ARE -- 375 00:14:17,831 --> 00:14:18,299 VERY GOOD RECOGNIZING 376 00:14:18,299 --> 00:14:18,933 PHOSPHORYLATION BY ITSELF BUT 377 00:14:18,933 --> 00:14:23,570 STRUGGLES ACTUALLY MODIFIED 378 00:14:23,570 --> 00:14:29,743 PEPTIDE WITH EITHER THE METHIL 379 00:14:29,743 --> 00:14:30,377 OR SUBTLE GROUP AND DOES EXIST 380 00:14:30,377 --> 00:14:32,379 IN VIVO BUT ABUNDANTLY. MUCH 381 00:14:32,379 --> 00:14:42,923 MORE EASIER DETECTED WITH MASS 382 00:14:47,194 --> 00:14:47,661 SPECTROMETRY THAT YOU CAN 383 00:14:47,661 --> 00:14:48,295 ANTIBODIES. AND YOU CAN DO THIS 384 00:14:48,295 --> 00:14:50,364 WITH --. 385 00:14:50,364 --> 00:14:50,898 AND TO UNDERSTAND DIFFERENT 386 00:14:50,898 --> 00:14:58,572 ASPECTS OF PROTEIN PTN 387 00:14:58,572 --> 00:14:59,173 MODIFICATIONS AS WELL AND SO 388 00:14:59,173 --> 00:14:59,640 TRYING TO CREATE VERY 389 00:14:59,640 --> 00:15:00,174 QUANTITATIVE HIGH THROUGH 390 00:15:00,174 --> 00:15:03,744 METHODS TO ANALYZE ADMINISTER 391 00:15:03,744 --> 00:15:08,015 MODIFICATIONS AT A TIME. 392 00:15:08,015 --> 00:15:08,582 TRYING TO UNDERSTAND WHICH 393 00:15:08,582 --> 00:15:09,183 COMBINATIONS OR MODIFICATION 394 00:15:09,183 --> 00:15:10,884 OCCUR TOGETHER BECAUSE OUR DATA 395 00:15:10,884 --> 00:15:11,518 SHOWS THAT ONE MODIFICATION ON 396 00:15:11,518 --> 00:15:12,119 THE HISTONE AT THE TIME JUST 397 00:15:12,119 --> 00:15:13,520 DOES NOT EXIST. IS ALWAYS IN 398 00:15:13,520 --> 00:15:14,088 COMBINATION WITH SOMETHING 399 00:15:14,088 --> 00:15:18,392 ELSE. BUT IF YOU'RE ONLY USING 400 00:15:18,392 --> 00:15:18,926 ANTIBODIES YOU ARE ONLY 401 00:15:18,926 --> 00:15:19,393 RECOGNIZING THAT ONE 402 00:15:19,393 --> 00:15:20,561 MODIFICATION. 403 00:15:20,561 --> 00:15:21,095 WE HAVE DEVELOPED A LOT OF 404 00:15:21,095 --> 00:15:22,596 METABOLIC LABELING APPROACHES, 405 00:15:22,596 --> 00:15:24,765 COMBINING THAT WITH PROTEOMICS 406 00:15:24,765 --> 00:15:25,366 TO BE ABLE TO UNDERSTAND THE 407 00:15:25,366 --> 00:15:33,774 HISTONE DYNAMICS AND GET UP THE 408 00:15:33,774 --> 00:15:34,375 KINETICS OF THESE MEDICATION 409 00:15:34,375 --> 00:15:35,009 BECAUSE THEY'RE NOT STATIC THEY 410 00:15:35,009 --> 00:15:35,642 ARE CONSTANTLY BEING PUT ON AND 411 00:15:35,642 --> 00:15:37,544 OFTEN COMBINING GENOMICS WITH 412 00:15:37,544 --> 00:15:38,078 THE DOWNSTREAM PROTEOMICS 413 00:15:38,078 --> 00:15:41,582 READOUT SO HOPEFULLY ONE DAY WE 414 00:15:41,582 --> 00:15:42,182 CAN GET TO THE POINT WHERE WE 415 00:15:42,182 --> 00:15:44,318 ARE PULLING OUT OUR FAVORITE 416 00:15:44,318 --> 00:15:47,121 GENE, AND IDENTIFYING ALL THE 417 00:15:47,121 --> 00:15:48,055 PROTEINS AND HISTONE 418 00:15:48,055 --> 00:15:50,524 MODIFICATIONS THAT ARE ALL THAT 419 00:15:50,524 --> 00:15:51,158 GENE, YOU KNOW IN BOTH KIND OF 420 00:15:51,158 --> 00:15:51,759 THE PHYSIOLOGICAL AND DISEASE 421 00:15:51,759 --> 00:15:54,161 STATE. 422 00:15:54,161 --> 00:15:56,463 SO, FOR THIS FIRST PART OF THE 423 00:15:56,463 --> 00:15:57,064 TALK I WAS JUST GOING TO GIVE 424 00:15:57,064 --> 00:15:59,533 AN UPDATE ON SOME NEWER 425 00:15:59,533 --> 00:16:00,134 APPROACHES AND NEW TECHNOLOGY 426 00:16:00,134 --> 00:16:01,869 THAT THE LAB HAS BEEN KIND OF 427 00:16:01,869 --> 00:16:02,503 EVALUATING OVER THE LAST YEAR 428 00:16:02,503 --> 00:16:06,373 +. WE ARE ALWAYS KIND OF ON 429 00:16:06,373 --> 00:16:08,742 THE LOOKOUT FOR NEW TECHNOLOGY 430 00:16:08,742 --> 00:16:12,780 THAT GIVES US SOME ADVANTAGES, 431 00:16:12,780 --> 00:16:13,414 AND ALLOWS US TO DO THINGS THAT 432 00:16:13,414 --> 00:16:14,882 WE WERE NOT ABLE TO DO BEFORE. 433 00:16:14,882 --> 00:16:19,720 AND ONE OF THE CRUTCHES OF 434 00:16:19,720 --> 00:16:22,289 QUANTITATIVE PROTEOMICS, YOU 435 00:16:22,289 --> 00:16:22,890 KNOW, EXCEPT FOR IN THE LAST 436 00:16:22,890 --> 00:16:26,427 YEAR OR SO, WAS THAT IT IS NOT 437 00:16:26,427 --> 00:16:27,061 VERY HIGH THROUGHPUT. IT TAKES 438 00:16:27,061 --> 00:16:32,499 A LONG TIME RUNNING AN HOUR OR 439 00:16:32,499 --> 00:16:33,067 TWO HOUR GRADIENTS ON THESE 440 00:16:33,067 --> 00:16:33,600 INSTRUMENTS. AND OUR GOLD 441 00:16:33,600 --> 00:16:36,870 STANDARD IS RUN ON AN 442 00:16:36,870 --> 00:16:39,706 INSTRUMENT TO DETECT HISTONE 443 00:16:39,706 --> 00:16:40,340 MODIFICATIONS. IT WOULD TAKE 60 444 00:16:40,340 --> 00:16:41,442 TO 90 MINUTES SO NOT A HIGH 445 00:16:41,442 --> 00:16:42,910 THROUGHPUT APPROACH. 446 00:16:42,910 --> 00:16:43,410 BUT NOW YOU HAVE A LOT OF 447 00:16:43,410 --> 00:16:44,711 INSTRUMENTS THAT ARE COMING 448 00:16:44,711 --> 00:16:47,881 OUT, BASED ON DIFFERENT MASS 449 00:16:47,881 --> 00:16:50,184 ANALYZERS, AND ORBIT TRAPS 450 00:16:50,184 --> 00:16:51,151 WHICH ARE HIGH-RESOLUTION 451 00:16:51,151 --> 00:16:51,685 INSTRUMENTS THAT ARE MUCH 452 00:16:51,685 --> 00:16:52,719 FASTER SCANNING INSTRUMENT SO 453 00:16:52,719 --> 00:16:53,353 WE WERE INTERESTED TO EVALUATE 454 00:16:53,353 --> 00:16:56,190 A FEW OF THESE. ONE WE ENDED UP 455 00:16:56,190 --> 00:16:58,826 REALLY LIKING, NOT JUST FOR THE 456 00:16:58,826 --> 00:17:01,662 HIGH THROUGHPUT ABILITIES WHICH 457 00:17:01,662 --> 00:17:02,296 I WILL SHOW YOU IN A SECOND BUT 458 00:17:02,296 --> 00:17:03,964 ALSO BECAUSE THE SYSTEM IS 459 00:17:03,964 --> 00:17:07,067 EQUIPPED WITH A DIFFERENT 460 00:17:07,067 --> 00:17:11,805 FRAGMENTATION MODE. ELECTRON 461 00:17:11,805 --> 00:17:12,372 INACTIVATED DISASSOCIATION 462 00:17:12,372 --> 00:17:13,006 WHICH ALLOWS US TO DO SOMETHING 463 00:17:13,006 --> 00:17:14,608 THAT WE CANNOT DO IN TERMS OF 464 00:17:14,608 --> 00:17:15,242 FRAGMENTATION SHOW SHOW YOU HOW 465 00:17:15,242 --> 00:17:15,876 WE ARE DOING THAT IN DIFFERENT 466 00:17:15,876 --> 00:17:18,278 WAYS AS WELL. 467 00:17:18,278 --> 00:17:18,846 TO READ ABOUT THE TIME WE GOT 468 00:17:18,846 --> 00:17:21,849 THIS INSTRUMENT, YOU KNOW, OUR 469 00:17:21,849 --> 00:17:26,753 FRIENDS AT SIAX (SOUNDS LIKE)-- 470 00:17:26,753 --> 00:17:27,387 I WANT TO DISCLOSE I DON'T HAVE 471 00:17:27,387 --> 00:17:27,888 A RESEARCH AGREEMENT OR 472 00:17:27,888 --> 00:17:29,590 ANYTHING LIKE THAT IN PLACE. WE 473 00:17:29,590 --> 00:17:30,190 ARE HAPPY CUSTOMERS. BUT OUR 474 00:17:30,190 --> 00:17:35,596 FRIENDS AT SIAX PUBLISHES 475 00:17:35,596 --> 00:17:36,230 PAPERS SAYING THAT ESSENTIALLY 476 00:17:36,230 --> 00:17:36,830 IT COULD QUANTIFY ABOUT 1000 477 00:17:36,830 --> 00:17:37,431 PROTEINS PER MINUTE ON THIS 478 00:17:37,431 --> 00:17:39,633 INSTRUMENT. 479 00:17:39,633 --> 00:17:40,200 KIND OF HARD TO READ THIS BUT 480 00:17:40,200 --> 00:17:43,003 THIS IS A FIVE-MINUTE GRADIENT. 481 00:17:43,003 --> 00:17:43,604 YOU CAN KIND OF SEE THAT SO 482 00:17:43,604 --> 00:17:44,671 YOU ARE TALKING REALLY FAST 483 00:17:44,671 --> 00:17:47,741 QUANTITATIVE PROTEOMICS. ALMOST 484 00:17:47,741 --> 00:17:49,209 TOO GOOD TO BE TRUE. AND 485 00:17:49,209 --> 00:17:50,444 HONESTLY, WHEN STUFF LIKE THIS 486 00:17:50,444 --> 00:17:53,747 COMES OUT, ARE USUALLY KIND OF 487 00:17:53,747 --> 00:17:54,381 SAY, I DON'T KNOW IF I WANT TO 488 00:17:54,381 --> 00:17:55,616 BELIEVE THIS PROPAGANDA. I NEED 489 00:17:55,616 --> 00:17:57,951 TO DO IT KIND OF FOR MYSELF. SO 490 00:17:57,951 --> 00:17:58,986 I GOT ALL OF THE PARAMETERS 491 00:17:58,986 --> 00:18:04,258 FROM PEOPLE AT SIAX, AND WAS 492 00:18:04,258 --> 00:18:06,960 ABLE TO REPRODUCE. I HAD THIS 493 00:18:06,960 --> 00:18:07,561 POSTDOC IN MY GOOGLE WAS ABLE 494 00:18:07,561 --> 00:18:09,963 TO REPRODUCE THIS. 495 00:18:09,963 --> 00:18:10,497 THIS IS OUR DATA HERE AGAIN 496 00:18:10,497 --> 00:18:14,067 FIVE-MINUTE GRADIENTS. AND 497 00:18:14,067 --> 00:18:14,635 ESSENTIALLY WE GET ANYWHERE 498 00:18:14,635 --> 00:18:15,269 FROM 4500 TO 5000 PROTEINS IN 499 00:18:15,269 --> 00:18:17,804 FIVE MINUTES DETECTED WITH 3 TO 500 00:18:17,804 --> 00:18:18,438 4 DIFFERENT POINTS ACROSS THE 501 00:18:18,438 --> 00:18:23,377 PEAK. AND SO THIS IS ACTUALLY 502 00:18:23,377 --> 00:18:23,844 A REALLY, REALLY FAST 503 00:18:23,844 --> 00:18:25,479 INSTRUMENT. 504 00:18:25,479 --> 00:18:28,882 AND NOW THIS IS KIND OF OUR QC. 505 00:18:28,882 --> 00:18:29,483 BEFORE WE WOULD RUN EITHER A 506 00:18:29,483 --> 00:18:31,919 WHOLE PROTEOMIC AND KIND OF 507 00:18:31,919 --> 00:18:32,519 RUNNER TO OUR GREATER MAYBE A 508 00:18:32,519 --> 00:18:34,721 FEW SYNTHETIC STANDARDS AND RUN 509 00:18:34,721 --> 00:18:36,790 LIKE HALF HOUR, AND NOW WE RUN 510 00:18:36,790 --> 00:18:38,358 FIVE MINUTES QC BEFORE EVERY 511 00:18:38,358 --> 00:18:42,930 BATCH OF SAMPLES. WE DO NOT GET 512 00:18:42,930 --> 00:18:44,932 4500 TO 5000 PROTEINS IN FIVE 513 00:18:44,932 --> 00:18:45,566 MINUTES, THEN THE INSTRUMENT IS 514 00:18:45,566 --> 00:18:46,166 TO BE CLEANER SOMETHING NEEDS 515 00:18:46,166 --> 00:18:47,100 TO BE ADJUSTED. SO WE WERE 516 00:18:47,100 --> 00:18:48,802 QUITE HAPPY WITH THAT. 517 00:18:48,802 --> 00:18:50,270 AND AS I MENTIONED, OUR 518 00:18:50,270 --> 00:19:00,480 GRADIENT FOR NORMAL NANO LC MS 519 00:19:00,480 --> 00:19:00,981 ANALYSIS HISTONE SYSTEM 520 00:19:00,981 --> 00:19:01,515 MODIFICATIONS IS ABOUT 55 521 00:19:01,515 --> 00:19:04,384 MINUTE GRADIENT. WE CREATED 522 00:19:04,384 --> 00:19:06,153 SOME REALLY QUICK RATINGS TO 523 00:19:06,153 --> 00:19:06,920 SEE IF WE COULD GET VERY 524 00:19:06,920 --> 00:19:08,188 SIMILAR DATA FROM JUST THESE 525 00:19:08,188 --> 00:19:08,989 MUCH FASTER HIGH THROUGHPUT 526 00:19:08,989 --> 00:19:11,258 RUNS. 527 00:19:11,258 --> 00:19:11,858 THE BIG DIFFERENCE HERE IS THAT 528 00:19:11,858 --> 00:19:17,564 WE ARE USING MICRO FLOW LCS, 529 00:19:17,564 --> 00:19:18,131 FOR THOSE IN THE PROTEOMIC 530 00:19:18,131 --> 00:19:23,837 FIELD 5 TO 10 5L PER MINUTE, 531 00:19:23,837 --> 00:19:25,706 FLOW RATE INSTEAD OF 200 NM 532 00:19:25,706 --> 00:19:26,206 FLOWRATES, WE WANTED TO 533 00:19:26,206 --> 00:19:29,676 EVALUATE THESE SO WE SET UP A 534 00:19:29,676 --> 00:19:35,782 WHOLE DATA INDEPENDENT 535 00:19:35,782 --> 00:19:36,350 ACQUISITION, TO GET THROUGH 536 00:19:36,350 --> 00:19:40,787 SOME OF THESE TRANSITION MODES 537 00:19:40,787 --> 00:19:41,388 TO OPTIMIZE THE WINDS WE WERE 538 00:19:41,388 --> 00:19:41,855 ASSESSING IN THE MASS 539 00:19:41,855 --> 00:19:42,756 SPECTROMETRY A LITTLE BIT MORE 540 00:19:42,756 --> 00:19:45,192 DYNAMICALLY. 541 00:19:45,192 --> 00:19:45,792 SOME AREAS WE HAD MORE MODIFIED 542 00:19:45,792 --> 00:19:46,360 SYSTEMS AND BIGGER WINDOWS 543 00:19:46,360 --> 00:19:46,994 INSTEAD OF GOING THROUGH WITH 544 00:19:46,994 --> 00:19:52,032 EQUAL WINDOWS LIKE WE DID IN 545 00:19:52,032 --> 00:19:53,800 SYSTEMS WE COULD TAKE THE TIME 546 00:19:53,800 --> 00:19:54,368 TO TAILOR AND OPTIMIZE THE 547 00:19:54,368 --> 00:19:58,772 WINDOWS AND WE KNOW THE FOR THE 548 00:19:58,772 --> 00:20:02,776 (CORRECTION) BINS WERE THE MOST 549 00:20:02,776 --> 00:20:03,377 MODIFIED SYSTEMS COME OUT AND 550 00:20:03,377 --> 00:20:05,178 WE LOOKED AT THE QUANTITATIVE 551 00:20:05,178 --> 00:20:05,812 DATA WE WERE QUITE HAPPY WITH 552 00:20:05,812 --> 00:20:06,380 IT SO YOU HAVE THE 55 AT A 553 00:20:06,380 --> 00:20:10,584 NORMAL NANO C RUN AND THESE 554 00:20:10,584 --> 00:20:11,518 CRAZY FAST FIVE MINUTE 555 00:20:11,518 --> 00:20:12,119 GRADIENTS OR RUNS AND YOU CAN 556 00:20:12,119 --> 00:20:14,855 SEE HERE PEAK SHAPE LOOKS 557 00:20:14,855 --> 00:20:25,232 REALLY, REALLY STRONG. 558 00:20:31,238 --> 00:20:31,805 WHEN YOU HAVE A PEPTIDE THAT 559 00:20:31,805 --> 00:20:32,439 HAS MULTIPLE LYZENES AND HAVE A 560 00:20:32,439 --> 00:20:35,709 MODIFICATION AS ISOLATION, YOU 561 00:20:35,709 --> 00:20:36,343 CAN LOOK AT THE TRANSITION AND 562 00:20:36,343 --> 00:20:36,810 YOU CAN TELL WHETHER 563 00:20:36,810 --> 00:20:37,644 MODIFICATIONS ARE FOUND, UNTIL 564 00:20:37,644 --> 00:20:39,413 WE GET ENOUGH POINTS ACROSS THE 565 00:20:39,413 --> 00:20:39,846 PEAK TO BE ABLE TO 566 00:20:39,846 --> 00:20:40,947 DIFFERENTIATE THESE ISOMERIC 567 00:20:40,947 --> 00:20:41,515 UPSIDES. AND WE CAN DO THIS 568 00:20:41,515 --> 00:20:44,818 REALLY WELL. 569 00:20:44,818 --> 00:20:45,285 WE HAVE DEVELOPED A FEW 570 00:20:45,285 --> 00:20:46,920 SOFTWARE TO BE ABLE TO HANDLE 571 00:20:46,920 --> 00:20:47,888 THIS DATA AS WELL. IN TERMS OF 572 00:20:47,888 --> 00:20:48,422 TOTAL NUMBER OF PEPTIDES 573 00:20:48,422 --> 00:20:51,958 IDENTIFIED, OR THE CS PEPTIDES 574 00:20:51,958 --> 00:20:53,627 THEY ARE VERY CONSISTENT ACROSS 575 00:20:53,627 --> 00:20:55,629 ALL OF THESE DIFFERENT TYPES OF 576 00:20:55,629 --> 00:21:02,903 RUNS, OUR GOLD STANDARD, NANO C 577 00:21:02,903 --> 00:21:03,537 RUN, HOUR PLUS, AND THE TENANT 578 00:21:03,537 --> 00:21:07,040 FIVE MINUTES. FAMILIES MIGHT BE 579 00:21:07,040 --> 00:21:07,641 A LITTLE BIT QUICK SO WE LIKE 580 00:21:07,641 --> 00:21:10,277 THE 10 MINUTE GRADIENT. BUT YOU 581 00:21:10,277 --> 00:21:10,877 CAN SEE THE VERY SOLID ACROSS 582 00:21:10,877 --> 00:21:11,445 ALL THE WAY IN TERMS OF THE 583 00:21:11,445 --> 00:21:13,580 NUMBERS. 584 00:21:13,580 --> 00:21:16,049 INCREDIBLY ROBUST INSTRUMENT AS 585 00:21:16,049 --> 00:21:16,683 WELL. HERE YOU HAVE 100 REPEAT 586 00:21:16,683 --> 00:21:17,617 INJECTIONS OF THE SAME SAMPLE 587 00:21:17,617 --> 00:21:20,387 RIGHT ON TOP OF EACH OTHER. 588 00:21:20,387 --> 00:21:22,122 AND YOU CAN SEE HERE RETENTION 589 00:21:22,122 --> 00:21:24,691 TIME, THE NUMBER OF PEPTIDES IS 590 00:21:24,691 --> 00:21:26,593 PRETTY CONSISTENT ACROSS LARGE 591 00:21:26,593 --> 00:21:28,261 MAJORITY OF THESE RUNS. 592 00:21:28,261 --> 00:21:32,199 AND SO NOW, WE CAN GET TO THE 593 00:21:32,199 --> 00:21:32,833 POINT WHERE WE ARE ABLE TO WORK 594 00:21:32,833 --> 00:21:33,567 WITH OUR CLINICAL COLLEAGUES. 595 00:21:33,567 --> 00:21:34,034 WE HAVE MANY CLINICAL 596 00:21:34,034 --> 00:21:39,473 COLLEAGUES AT PENN AND AT WASH 597 00:21:39,473 --> 00:21:42,509 U, THAT CAME UP TO ME AND SAID 598 00:21:42,509 --> 00:21:43,110 WE HAVE BEEN FOLLOWING THESE 599 00:21:43,110 --> 00:21:43,677 PATIENTS FOR THE LAST FIVE 600 00:21:43,677 --> 00:21:44,277 YEARS TAKING SAMPLES, BLOOD 601 00:21:44,277 --> 00:21:47,013 SAMPLES EVERY MONTH. AND WE 602 00:21:47,013 --> 00:21:47,647 HAVE THOUSANDS AND THOUSANDS OF 603 00:21:47,647 --> 00:21:49,116 SAMPLES. WE WOULD LOVE TO 604 00:21:49,116 --> 00:21:51,251 RUFFLE FOR HISTONE 605 00:21:51,251 --> 00:21:52,686 MODIFICATIONS. I CAN'T RUN THAT 606 00:21:52,686 --> 00:21:53,320 MANY SAMPLES. IT WOULD TAKE TOO 607 00:21:53,320 --> 00:21:53,954 MUCH INSTRUMENT TIME. 608 00:21:53,954 --> 00:21:57,524 BUT NOW WITH THIS 10 MINUTE 609 00:21:57,524 --> 00:21:58,759 GRADIENT MAY BE ABOUT 17 610 00:21:58,759 --> 00:22:00,427 MINUTES TOTAL RUN, WE CAN GET 611 00:22:00,427 --> 00:22:02,329 TO THE POINT WHERE WE ARE 612 00:22:02,329 --> 00:22:02,929 RUNNING HUNDREDS OR THOUSANDS 613 00:22:02,929 --> 00:22:07,067 OF SAMPLES IN A WEEK. AND WE 614 00:22:07,067 --> 00:22:08,402 CAN TAKE ON SOME OF THESE VERY 615 00:22:08,402 --> 00:22:09,035 LARGE-SCALE CLINICAL PROJECTS. 616 00:22:09,035 --> 00:22:10,871 OR THESE POPULATION TYPE OF 617 00:22:10,871 --> 00:22:15,475 STUDIES AS WELL. 618 00:22:15,475 --> 00:22:16,810 AND MENTIONED THIS ELECTRON 619 00:22:16,810 --> 00:22:17,444 ACTIVATED DISASSOCIATION. THIS 620 00:22:17,444 --> 00:22:20,614 IS VERY DIFFERENT. SOMETHING 621 00:22:20,614 --> 00:22:21,214 THAT NOT ALL INSTRUMENTS HAVE 622 00:22:21,214 --> 00:22:22,783 THE ABILITY TO USE ELECTRONS TO 623 00:22:22,783 --> 00:22:24,885 FRAGMENT PEPTIDES. MOST 624 00:22:24,885 --> 00:22:26,286 INSTRUMENTS USE COLLISIONS 625 00:22:26,286 --> 00:22:28,422 WITHIN THE INNER GAS. 626 00:22:28,422 --> 00:22:29,022 AND SO HERE YOU CAN SEE IF YOU 627 00:22:29,022 --> 00:22:32,292 INCREASE THE ENERGY, YOU GET 628 00:22:32,292 --> 00:22:35,495 THE FRAGMENTATION. IT GIVES YOU 629 00:22:35,495 --> 00:22:36,029 VERY COMPLETE COVERAGE IN 630 00:22:36,029 --> 00:22:36,596 BETWEEN ALMOST EVERY AMINO 631 00:22:36,596 --> 00:22:38,064 ACID. YOU CAN TELL EXACTLY 632 00:22:38,064 --> 00:22:40,834 WHERE THE MODIFICATION IS. 633 00:22:40,834 --> 00:22:41,468 WHAT WE FOUND THAT WE ACTUALLY 634 00:22:41,468 --> 00:22:43,270 WERE ABLE TO USE THIS IN A 635 00:22:43,270 --> 00:22:43,904 DIFFERENT ROUTE TO HELP SOLVE A 636 00:22:43,904 --> 00:22:45,772 REALLY DIFFICULT PROBLEM. 637 00:22:45,772 --> 00:22:47,741 AND SO I HAD A COLLABORATOR AT 638 00:22:47,741 --> 00:22:50,710 YALE, MATT SIMON, WHO SAID 639 00:22:50,710 --> 00:22:51,278 ABOUT OTHER METHODS HE HAD 640 00:22:51,278 --> 00:23:00,320 EVIDENCE THAT A LYSINE RESIDUE 641 00:23:00,320 --> 00:23:00,921 WOULD HAVE AN ACETO GROUP AND 642 00:23:00,921 --> 00:23:05,225 MONO GROUP TOGETHER. I NEVER 643 00:23:05,225 --> 00:23:06,760 HEARD OF TWO MEDICATIONS ON THE 644 00:23:06,760 --> 00:23:12,299 SAME LYSINE AT THE SAME TIME SO 645 00:23:12,299 --> 00:23:12,933 HE WANTED TO KNOW WE COULD GET 646 00:23:12,933 --> 00:23:14,301 MORE DIRECT EVIDENCE. AND THIS 647 00:23:14,301 --> 00:23:18,939 IS THE SAME MASS AS ANOTHER 648 00:23:18,939 --> 00:23:21,308 MODIFICATION CALL PROPIANO. WE 649 00:23:21,308 --> 00:23:21,908 CANNOT TELL THEM APART IS BY 650 00:23:21,908 --> 00:23:25,345 MASS WHEN WE FRAGMENT THESE 651 00:23:25,345 --> 00:23:25,879 PEPTIDE TOGETHER FROM THE 652 00:23:25,879 --> 00:23:26,513 SAMPLES WE SAW THERE WERE TWO 653 00:23:26,513 --> 00:23:28,281 SLIGHTLY OVERLAPPING PEAKS. WE 654 00:23:28,281 --> 00:23:28,882 FRAGMENTING THEM TOGETHER IT 655 00:23:28,882 --> 00:23:29,449 BASICALLY GAVE US COMPLETE 656 00:23:29,449 --> 00:23:30,217 COVERAGE. AND SO SINCE THESE 657 00:23:30,217 --> 00:23:36,089 ARE THE EXACT SAME OSIBERIC 658 00:23:36,089 --> 00:23:36,690 (SOUNDS LIKE) MATH WE CANNOT 659 00:23:36,690 --> 00:23:37,691 ALL DIFFERENT BUT THE NICE 660 00:23:37,691 --> 00:23:39,059 THING ABOUT THIS ASSOCIATION IS 661 00:23:39,059 --> 00:23:41,461 THAT IT IS VERY TUNABLE. YOU 662 00:23:41,461 --> 00:23:42,963 CAN CRANK UP THE ENERGY AND DO 663 00:23:42,963 --> 00:23:47,167 THINGS THAT YOU CANNOT DO 664 00:23:47,167 --> 00:23:47,767 ANOTHER INSTRUMENTS THAT HAVE 665 00:23:47,767 --> 00:23:48,401 FRAGMENTATION LOADS WE CRANKED 666 00:23:48,401 --> 00:23:49,035 UP THE ENERGY TO THE POINT WE 667 00:23:49,035 --> 00:23:49,503 WERE BREAKING OFF THE 668 00:23:49,503 --> 00:23:52,372 SIDECHAINS OF THE AMINO ACIDS. 669 00:23:52,372 --> 00:23:53,006 AND WE BROKE OFF THE SIDECHAINS 670 00:23:53,006 --> 00:24:01,548 OF THIS LYSINE THAT HAD THE 671 00:24:01,548 --> 00:24:02,182 ACETYLMETHOL, WE SAW THAT THIS 672 00:24:02,182 --> 00:24:10,357 WAS AN ACETYLMETHOL, AND THIS 673 00:24:10,357 --> 00:24:14,327 WAS THE PROPIANIC GROUP AND WE 674 00:24:14,327 --> 00:24:15,996 SAW THIS MEDICATION TOGETHER AT 675 00:24:15,996 --> 00:24:16,630 ANY PROTEIN THAT IS INTERESTED 676 00:24:16,630 --> 00:24:18,098 YOU AT THE SAME TIME. 677 00:24:18,098 --> 00:24:21,001 AND SO NOW I WANT TO GET INTO 678 00:24:21,001 --> 00:24:21,635 TWO PROJECT TO SHOW YOU HOW WE 679 00:24:21,635 --> 00:24:25,138 ARE USING THE TECHNOLOGY. THIS 680 00:24:25,138 --> 00:24:25,739 ONE WE HAVE GONE A LITTLE BIT 681 00:24:25,739 --> 00:24:28,174 FARTHER AND PUBLISHED. WE HAVE 682 00:24:28,174 --> 00:24:28,808 BEEN INTERESTED IN SOFT TISSUE 683 00:24:28,808 --> 00:24:31,411 SARCOMA FOR A LONG TIME. AND 684 00:24:31,411 --> 00:24:36,249 THERE'S A WHOLE HOST OF 685 00:24:36,249 --> 00:24:36,816 MUTATIONS TO MANY DIFFERENT 686 00:24:36,816 --> 00:24:37,450 TYPES OF SOFT TISSUE SARCOMAS, 687 00:24:37,450 --> 00:24:38,218 THAT EITHER AFFECT CHROMATIN 688 00:24:38,218 --> 00:24:47,961 REMODELING PROTEINS, WHICH IS 689 00:24:47,961 --> 00:24:48,528 THE POLICONE OR HAVE THESE 690 00:24:48,528 --> 00:24:53,099 INTERESTING FUSION PROTEINS, A 691 00:24:53,099 --> 00:24:53,600 MODIFIED ENZYME USED IN 692 00:24:53,600 --> 00:24:54,801 TRANSCRIPTION FACTORS OR 693 00:24:54,801 --> 00:24:58,338 SOMETHING LIKE THIS. NOT REALLY 694 00:24:58,338 --> 00:24:58,872 CLEAR WITH THE MOLECULAR 695 00:24:58,872 --> 00:24:59,472 MECHANISMS FOR HOW THIS LEADS 696 00:24:59,472 --> 00:25:00,006 TO THE PROGRESSION OF THE 697 00:25:00,006 --> 00:25:00,407 CANCER. 698 00:25:00,407 --> 00:25:01,708 AND THERE IS EVEN HISTONES GET 699 00:25:01,708 --> 00:25:12,218 MUTATED THEMSELVES. YOU CAN 700 00:25:13,153 --> 00:25:13,620 IMAGINE IF YOU HAD LYSINE 701 00:25:13,620 --> 00:25:14,187 RESIDUE AND NOW YOU HAVE A 702 00:25:14,187 --> 00:25:14,788 ADDITION TO SOMETHING OF YOU 703 00:25:14,788 --> 00:25:17,090 WIPEOUT MUTILATION. AND SO 704 00:25:17,090 --> 00:25:18,858 THERE OF INTERVIEWS THAT OCCUR 705 00:25:18,858 --> 00:25:19,859 IN ONE OF THE THING WE ARE 706 00:25:19,859 --> 00:25:20,393 INTO THAT IS A MALIGNANT 707 00:25:20,393 --> 00:25:28,802 PERIPHERAL NERVE SHEATH TUMOR 708 00:25:28,802 --> 00:25:36,810 MPNST, MOST PATIENTS HAVE AN 709 00:25:36,810 --> 00:25:38,478 UNDERLYING MUTATION TO THE 710 00:25:38,478 --> 00:25:42,515 FIBROMA GENE AND THEN WE GET 711 00:25:42,515 --> 00:25:43,149 INVITATION TO CANCER AND OLDER 712 00:25:43,149 --> 00:25:43,750 PATIENTS CAN SPORADICALLY GET 713 00:25:43,750 --> 00:25:51,257 THOSE SECOND MUTATIONS AND 714 00:25:51,257 --> 00:25:51,858 PROGRESSES TO CANCER. IT IS A 715 00:25:51,858 --> 00:25:52,425 RARE TUMOR, MAY BE SEVERAL 716 00:25:52,425 --> 00:25:53,393 HUNDRED A YEAR THAT ARE 717 00:25:53,393 --> 00:25:53,927 DESCRIBED. BUT IT IS VERY 718 00:25:53,927 --> 00:25:54,461 AGGRESSIVE. LESS THAN 50% 719 00:25:54,461 --> 00:25:55,729 FIVE-YEAR SURVIVAL RATE. 720 00:25:55,729 --> 00:25:57,931 THERE'S NOT A LOT OF OPTIONS TO 721 00:25:57,931 --> 00:25:58,531 TREAT. BASICALLY JUST SURGERY 722 00:25:58,531 --> 00:26:00,667 AND RADIATION. AND ABOUT HALF 723 00:26:00,667 --> 00:26:03,837 OF THE PATIENTS HAVE A RELAPSE. 724 00:26:03,837 --> 00:26:04,904 THERE'S A LOT KNOWN ABOUT THE 725 00:26:04,904 --> 00:26:11,811 MUTATIONS THAT LEAD TO MPNST. 726 00:26:11,811 --> 00:26:12,445 BUT ABOUT 80% OF PATIENTS HAVE 727 00:26:12,445 --> 00:26:15,815 A MUTATION TO ONE OR TWO 728 00:26:15,815 --> 00:26:17,283 COMPONENTS OF THE POLYCHROME 729 00:26:17,283 --> 00:26:18,752 REPRESSIVE COMPLEX, AND THIS IS 730 00:26:18,752 --> 00:26:19,452 ACCOMPLISHED IN CREATE 731 00:26:19,452 --> 00:26:24,557 TRI-METHYLATION AT HISTONE H3. 732 00:26:24,557 --> 00:26:28,662 AND THIS IS A PATHOLOGIST 733 00:26:28,662 --> 00:26:29,295 DIFFERENTIATE DIFFERENT MPNSTS 734 00:26:29,295 --> 00:26:33,233 BUT STAINING. AND SO IT HAS A 735 00:26:33,233 --> 00:26:33,867 DIFFERENT MUTATION AND YOU CAN 736 00:26:33,867 --> 00:26:36,236 SEE THE BROWN STANDING OF THE 737 00:26:36,236 --> 00:26:39,139 METHYLATION. AND YOU CAN SEE 738 00:26:39,139 --> 00:26:45,779 THE LOSS OF THAT METHYLATION. 739 00:26:45,779 --> 00:26:46,379 AND, THE LOSS IS PROBABLY ALSO 740 00:26:46,379 --> 00:26:49,149 THE ONE THAT YOU KNOW LEADS TO 741 00:26:49,149 --> 00:26:51,818 POOR SURVIVAL. AND SO YOU CAN 742 00:26:51,818 --> 00:26:55,221 SEE BECKY FROM PATIENTS WITH 743 00:26:55,221 --> 00:26:55,789 THE LOSS MUTATION VERSUS A 744 00:26:55,789 --> 00:27:00,393 DIFFERENT MUTATION. IT RETAINS 745 00:27:00,393 --> 00:27:02,529 P53 OR SOME OTHER TYPE OF 746 00:27:02,529 --> 00:27:03,563 MUTATION. 747 00:27:03,563 --> 00:27:08,168 HOWEVER, LOOKING AT HISTONE 748 00:27:08,168 --> 00:27:10,804 H3K27 ME3 IS PROBABLY THE 749 00:27:10,804 --> 00:27:11,404 MARKET FOR THIS PROBABLY NOT 750 00:27:11,404 --> 00:27:13,907 THE BEST THING TO DO BECAUSE 751 00:27:13,907 --> 00:27:17,110 K27 ME3 IS PROBABLY 752 00:27:17,110 --> 00:27:17,610 OVEREXPRESSED A LOT OF 753 00:27:17,610 --> 00:27:19,379 DIFFERENT CANCERS, ESPECIALLY 754 00:27:19,379 --> 00:27:20,013 CANCERS THAT ARE VERY DIFFICULT 755 00:27:20,013 --> 00:27:24,084 TO DISTINGUISH VERSUS MPNST, 756 00:27:24,084 --> 00:27:24,651 RELEASE FROM THE PATHOLOGY 757 00:27:24,651 --> 00:27:27,420 STANDPOINT. 758 00:27:27,420 --> 00:27:28,021 AND SO STARTING THIS PROJECT WE 759 00:27:28,021 --> 00:27:30,957 HAD A COUPLE OF QUESTIONS. ONE, 760 00:27:30,957 --> 00:27:32,292 WE CAN UNDERSTAND THE FIRST 761 00:27:32,292 --> 00:27:33,927 INITIAL LOSS. WE HAVE MUTATION 762 00:27:33,927 --> 00:27:40,166 AND THE POLYCOM LOSING K27 ME3, 763 00:27:40,166 --> 00:27:42,736 HOW DOES THIS FURTHER 764 00:27:42,736 --> 00:27:43,403 REPROGRAMMED THE EPIGENOME? 765 00:27:43,403 --> 00:27:44,037 WANTED TO ANSWER THIS WITH THE 766 00:27:44,037 --> 00:27:52,545 PROFILING. AND WE ARE 767 00:27:52,545 --> 00:27:53,146 EXPECTING BIG CHANGES HERE IN 768 00:27:53,146 --> 00:27:53,780 TERMS OF THE EIPGENOMES. AND SO 769 00:27:53,780 --> 00:27:57,050 HOW DOES THIS AFFECT 770 00:27:57,050 --> 00:27:57,584 TRANSCRIPTION? ESPECIALLY 771 00:27:57,584 --> 00:27:59,853 PROTEIN TRANSCRIPTION? AND SO 772 00:27:59,853 --> 00:28:05,625 WE HAVE TWO GREAT POSTDOCS. 773 00:28:05,625 --> 00:28:06,226 JOHN STARTED THE PROJECT AND 774 00:28:06,226 --> 00:28:06,860 JOANNA IS CURRENTLY WORKING THE 775 00:28:06,860 --> 00:28:10,096 PROJECT. 776 00:28:10,096 --> 00:28:10,697 THE FIRST THING WE HAD TO DO IS 777 00:28:10,697 --> 00:28:11,297 CONVINCE HERSELF TO GET HIGH 778 00:28:11,297 --> 00:28:15,869 QUALITY PROTEOMIC DATA FROM 779 00:28:15,869 --> 00:28:22,142 PARAFFIN- EMBEDDED SAMPLES, AND 780 00:28:22,142 --> 00:28:22,742 A LOT OF TIMES MORE THE CELLS 781 00:28:22,742 --> 00:28:26,813 ARE FROZEN. BUT PARAFFIN 782 00:28:26,813 --> 00:28:27,480 EMBEDDED IN THESE WAX CHUNKS SO 783 00:28:27,480 --> 00:28:29,282 WE HAD TO WORK OUT A LOT OF 784 00:28:29,282 --> 00:28:34,487 METHODOLOGY TO GET HIGH QUALITY 785 00:28:34,487 --> 00:28:35,088 HISTONES AND PROTEOMES OUT OF 786 00:28:35,088 --> 00:28:37,557 THE SAMPLES. BUT IT DID TAKE A 787 00:28:37,557 --> 00:28:39,425 FAIR AMOUNT OF OPTIMIZATION. 788 00:28:39,425 --> 00:28:40,593 THIS IS GONNA TRIVIAL TO REALLY 789 00:28:40,593 --> 00:28:42,095 RETRIEVING YOU KNOW PROTEIN 790 00:28:42,095 --> 00:28:48,701 SAMPLES FROM THESE FFP BLOCK TO 791 00:28:48,701 --> 00:28:49,636 THE BEEN SITTING HERE FOR 10 792 00:28:49,636 --> 00:28:50,069 PLUS YEARS. 793 00:28:50,069 --> 00:28:52,005 AND SO JUMPING INTO THE DATA, 794 00:28:52,005 --> 00:28:57,277 IF WE TOOK FFP SAMPLES FROM 795 00:28:57,277 --> 00:28:57,844 PATIENTS JUST OF THE NEURAL 796 00:28:57,844 --> 00:29:01,481 FIBROMA MUTATION, ONES THAT HAD 797 00:29:01,481 --> 00:29:05,752 POLICONE LOSS OR RETAIN YOU SEE 798 00:29:05,752 --> 00:29:06,386 SOMETHING VERY DIFFERENT ABOUT 799 00:29:06,386 --> 00:29:09,189 WANT TO HAVE THE POLY CONE 800 00:29:09,189 --> 00:29:11,524 LOSS, LOOKING AT ABOUT 150 801 00:29:11,524 --> 00:29:14,160 MODIFICATIONS, CLUSTER TOGETHER 802 00:29:14,160 --> 00:29:14,727 IN THIS PRINCIPAL COMPONENT 803 00:29:14,727 --> 00:29:15,295 ANALYSIS AND YOU CAN SEE A 804 00:29:15,295 --> 00:29:16,896 LITTLE BIT BETTER IN THE 805 00:29:16,896 --> 00:29:19,265 VOLCANO PLOT. SO THESE ARE ALL 806 00:29:19,265 --> 00:29:28,107 THE HISTONES MARKS UPREGULATED. 807 00:29:28,107 --> 00:29:28,741 IN THE PATIENTS WITH POLYCLONAL 808 00:29:28,741 --> 00:29:30,476 LOSS ARE DOWN RELATED, 809 00:29:30,476 --> 00:29:33,313 ESPECIALLY FROM THE 810 00:29:33,313 --> 00:29:34,514 NEUROFIBROMATOSIS POLY CONE 811 00:29:34,514 --> 00:29:35,114 RETAINED, AND THE SAMPLES AND 812 00:29:35,114 --> 00:29:40,119 USING SEE THE LOSS OF THE K27 813 00:29:40,119 --> 00:29:40,720 DIE. AND OTHER THINGS ARE UP 814 00:29:40,720 --> 00:29:41,754 AND DOWN REGULATED. AND WE HAVE 815 00:29:41,754 --> 00:29:42,288 SEEN THAT IN THEIR OTHER 816 00:29:42,288 --> 00:29:49,462 HISTONE MARKS SUCH AS THE 817 00:29:49,462 --> 00:29:56,269 K4H420 METHYLATION THAT IS DOWN 818 00:29:56,269 --> 00:29:56,870 REGULATED. AND THEN YOU HAVE 819 00:29:56,870 --> 00:29:57,470 ALL OF THESE METHYLATION AND 820 00:29:57,470 --> 00:29:57,937 ACETYLATION THAT ARE 821 00:29:57,937 --> 00:29:59,906 UPREGULATED AS WELL. 822 00:29:59,906 --> 00:30:01,941 IN BLUE IS THE POLYCHROME 823 00:30:01,941 --> 00:30:04,377 RETAIN. IN RED IS POLYCHROME 824 00:30:04,377 --> 00:30:05,478 LAWSON WHEN YOU LOSE YOU HAVE 825 00:30:05,478 --> 00:30:16,022 HIGHER LEVELS OF H4 ACETILATION 826 00:30:19,025 --> 00:30:20,260 ISOFORMS. 827 00:30:20,260 --> 00:30:22,896 IT IS ONLY PARTIALLY TRUE. WHAT 828 00:30:22,896 --> 00:30:23,496 WE ENDED UP FINDING FROM THE 829 00:30:23,496 --> 00:30:24,130 DATA WAS THAT THERE'S PEPTIDES 830 00:30:24,130 --> 00:30:32,405 THAT HAVE K27 MONO DIE AND TRI 831 00:30:32,405 --> 00:30:33,039 DIE. IT WILL LOOK AT THE TOTAL 832 00:30:33,039 --> 00:30:38,144 LEVELS OF K36 METHYL, THEY ARE 833 00:30:38,144 --> 00:30:44,284 FOUND OFTEN WITH K27 MONODINE 834 00:30:44,284 --> 00:30:46,085 TRY, K27. IN THE DISEASE STATE 835 00:30:46,085 --> 00:30:49,622 YOU LOSE THE K27 METHYLATION. 836 00:30:49,622 --> 00:30:51,691 AND SO BASICALLY YOU JUST HAVE 837 00:30:51,691 --> 00:30:53,126 K36 ALONE WITH THE UNMODIFIED 838 00:30:53,126 --> 00:31:00,867 K27. 839 00:31:00,867 --> 00:31:06,973 >> IT IS LOSING THE K 27 WE 840 00:31:06,973 --> 00:31:12,245 SEEK A 36 INCREASING. 841 00:31:12,245 --> 00:31:12,812 WHAT THE DATA IS TELLING US 842 00:31:12,812 --> 00:31:14,447 WERE GOING FROM A SILENCING 843 00:31:14,447 --> 00:31:17,684 MARK OF K 27 BOTH BY ITSELF IT 844 00:31:17,684 --> 00:31:18,184 MAY BE WITH SOME ACTIVE 845 00:31:18,184 --> 00:31:24,390 HISTONE MARKS THAT THERE IT 846 00:31:24,390 --> 00:31:24,958 ITSELF IS INTERESTING BUT IN 847 00:31:24,958 --> 00:31:25,525 THE DISEASE STATE YOU LOSE A 848 00:31:25,525 --> 00:31:31,531 LOT OF KEY 27 THERE IS GLOBAL 849 00:31:31,531 --> 00:31:34,467 DOMINANT WITH HISTONE MARKS 850 00:31:34,467 --> 00:31:40,340 FOUND WE DID SEE THAT K 27 851 00:31:40,340 --> 00:31:40,873 DIMETHYL AND TRIMETHYL ARE 852 00:31:40,873 --> 00:31:48,581 BOTH DOWN REGULATED. 853 00:31:48,581 --> 00:31:49,182 THAT IS INTERESTING ITSELF AS 854 00:31:49,182 --> 00:31:49,782 WELL BECAUSE AND OTHER CANCERS 855 00:31:49,782 --> 00:31:51,184 I TOLD YOU K 27 AND TRIMETHYL 856 00:31:51,184 --> 00:31:54,887 BUT AND OTHERS DIMETHYL IS NOT. 857 00:31:54,887 --> 00:31:57,857 THESE TWO ARE VERY SPECIFIC 858 00:31:57,857 --> 00:32:05,531 MARKERS. 859 00:32:05,531 --> 00:32:05,999 AND JOHN IS A RESIDENT 860 00:32:05,999 --> 00:32:06,566 PATHOLOGIST HAS GONE BACK TO 861 00:32:06,566 --> 00:32:07,133 THE CLINICS AND USING THESE 862 00:32:07,133 --> 00:32:12,171 TWO MARKS TO DIFFERENTIATE. 863 00:32:12,171 --> 00:32:13,272 SO WE KNOW WHAT HAPPENS TO THE 864 00:32:13,272 --> 00:32:17,944 APPLE GENOME IT GOES FOR CLOSE 865 00:32:17,944 --> 00:32:18,511 DATE TO OPEN ACTIVE BASED ON 866 00:32:18,511 --> 00:32:21,447 THE HISTONE MARKS. 867 00:32:21,447 --> 00:32:23,049 WHAT ABOUT THE PROTEINS OR 868 00:32:23,049 --> 00:32:25,952 GENE EXPRESSION? 869 00:32:25,952 --> 00:32:30,923 ANOTHER VAULT A NO PLOT AND 870 00:32:30,923 --> 00:32:32,258 YOU CAN SEE A LOT OF THINGS 871 00:32:32,258 --> 00:32:35,461 ARE UPREGULATED AND WHEN WE DO 872 00:32:35,461 --> 00:32:45,972 THE ANALYSIS ALL PROTEINS ARE 873 00:32:51,811 --> 00:32:52,245 INVOLVED TO KEEP AN OPEN 874 00:32:52,245 --> 00:32:52,712 ACTIVE CHROMATIN WITH 875 00:32:52,712 --> 00:32:53,312 REMODELERS AS WELL AS PROTEINS 876 00:32:53,312 --> 00:32:53,846 INVOLVED IS TRANSCRIPTION. 877 00:32:53,846 --> 00:32:54,447 THIS MATCHES REALLY WELL WITH 878 00:32:54,447 --> 00:32:54,981 HISTONE DATA GOING FROM A 879 00:32:54,981 --> 00:32:55,581 CLOSED CHROMATIN TO MORE OPEN 880 00:32:55,581 --> 00:32:55,882 AND ACTIVE. 881 00:32:55,882 --> 00:32:56,482 AND IT SUPPORTS THAT WE HAVE 882 00:32:56,482 --> 00:32:57,917 PROTEINS THAT MAINTAIN 883 00:32:57,917 --> 00:33:00,520 TRANSCRIPTION. 884 00:33:00,520 --> 00:33:01,087 BUT AS YOU CAN SEE A LOT OF 885 00:33:01,087 --> 00:33:05,625 PROTEINS ARE DOWN REGULATED. 886 00:33:05,625 --> 00:33:08,661 THEY ARE INVOLVED IN IMMUNE 887 00:33:08,661 --> 00:33:10,997 RESPONSE SO THAT COMPLEX 888 00:33:10,997 --> 00:33:15,935 PROCESSING INTERFERON 889 00:33:15,935 --> 00:33:17,003 SIGNALING THAT POTENTIALLY 890 00:33:17,003 --> 00:33:18,771 COULD LEAD TO A PATIENT BEING 891 00:33:18,771 --> 00:33:23,409 MORE IMMUNOCOMPROMISED. 892 00:33:23,409 --> 00:33:24,811 JUST LOOK AT CLASS ONE AND 893 00:33:24,811 --> 00:33:30,750 CLASS TWO PROTEINS YOU CAN SEE 894 00:33:30,750 --> 00:33:33,820 A HIGHER LEVEL OF THE PROTEIN 895 00:33:33,820 --> 00:33:36,823 GROUPS. 896 00:33:36,823 --> 00:33:38,524 YOU CAN SEE THIS JUST BY BIRDS 897 00:33:38,524 --> 00:33:44,464 EYE STAINING FOR BOTH OF THE 898 00:33:44,464 --> 00:33:44,797 HLA MOLECULES. 899 00:33:44,797 --> 00:33:54,373 HIGHER STATING GOES TO LOWER, 900 00:33:54,373 --> 00:33:54,874 SO THE DATA TELLS US THE 901 00:33:54,874 --> 00:33:55,441 HISTONE MARKS THERE IS MORE 902 00:33:55,441 --> 00:33:55,975 ACTIVE OPEN HISTONE MARKS. 903 00:33:55,975 --> 00:34:01,280 AND A SUBSET ALSO THAT IS 904 00:34:01,280 --> 00:34:01,814 UPREGULATED AS WELL MOSTLY 905 00:34:01,814 --> 00:34:02,482 INVOLVED WITH KEEPING THE OPEN 906 00:34:02,482 --> 00:34:05,318 ACTIVE CHROMATIN THAN WITH THE 907 00:34:05,318 --> 00:34:08,187 ANTIGEN PATHWAY IS DOWN 908 00:34:08,187 --> 00:34:10,756 REGULATED. 909 00:34:10,756 --> 00:34:11,891 'S AND NOW THE QUESTION ARISES 910 00:34:11,891 --> 00:34:14,060 YOU LOSE A REPRESSOR WITH A 911 00:34:14,060 --> 00:34:14,460 SILENCING PROTEIN. 912 00:34:14,460 --> 00:34:21,167 I UNDERSTAND HOW YOU CAN LEAD 913 00:34:21,167 --> 00:34:23,669 TO ACTIVATION AND TARGETS, BUT 914 00:34:23,669 --> 00:34:27,406 HOW DO YOU GET MORE 915 00:34:27,406 --> 00:34:27,707 OPPRESSION? 916 00:34:27,707 --> 00:34:28,641 SO JOHN WENT BACK TO THE DATA 917 00:34:28,641 --> 00:34:34,780 AND FOUND THE PROTEIN WAS ALSO 918 00:34:34,780 --> 00:34:41,220 UPREGULATED. 919 00:34:41,220 --> 00:34:41,754 AND IN FACT THESE PATIENTS 920 00:34:41,754 --> 00:34:46,659 HAVE HYPER METHYLATION AND 921 00:34:46,659 --> 00:34:48,661 HIGHER LEVELS OF DNA 922 00:34:48,661 --> 00:34:50,863 METHYLATION SO WE HAVE 923 00:34:50,863 --> 00:34:57,236 DEVELOPED METHODS TO ANNALEE 924 00:34:57,236 --> 00:34:57,770 ANALYZED A CLAY CASTLE AND 925 00:34:57,770 --> 00:35:00,206 MODIFICATIONS WE SEE 926 00:35:00,206 --> 00:35:03,075 METHYLATION'S EVEN THEN DNA 927 00:35:03,075 --> 00:35:03,643 DAMAGE MARKERS THAT ARE ALL 928 00:35:03,643 --> 00:35:04,243 UPREGULATED IN THESE PATIENTS 929 00:35:04,243 --> 00:35:09,916 AS WELL. 930 00:35:09,916 --> 00:35:10,483 IF YOU LOOK AT WHERE IN THE 931 00:35:10,483 --> 00:35:13,052 GENOME HYPER METHYLATION IS 932 00:35:13,052 --> 00:35:19,425 FOUND RED IS LOW AND BLUE IS 933 00:35:19,425 --> 00:35:19,625 HIGH. 934 00:35:19,625 --> 00:35:20,159 LOW METHYLATION TOO HIGH. 935 00:35:20,159 --> 00:35:20,760 YOU CAN SEE HIGHER METHYLATION 936 00:35:20,760 --> 00:35:27,967 IN THE PATIENT'S AND ALL WITH 937 00:35:27,967 --> 00:35:28,868 THE PROMOTERS OF THINGS WE SAW 938 00:35:28,868 --> 00:35:32,905 DOWN REGULATED AND THEN 939 00:35:32,905 --> 00:35:33,472 INTERFERE ON THE PATHWAY AND 940 00:35:33,472 --> 00:35:36,309 ALL THE IMMUNE RESPONSE 941 00:35:36,309 --> 00:35:36,576 PATHWAYS. 942 00:35:36,576 --> 00:35:44,617 SO THE PICTURE IS NOW NOW WITH 943 00:35:44,617 --> 00:35:45,184 LOSSES OPEN ACTIVE CHROMATIN 944 00:35:45,184 --> 00:35:45,718 IT IS EXPECTED BUT IMMUNE 945 00:35:45,718 --> 00:35:56,229 RESPONSES DOWN REGULATED THEN 946 00:35:59,031 --> 00:35:59,465 YOU HAVE DNA METHYLATION 947 00:35:59,465 --> 00:35:59,999 ANOTHER SILENCING MARK AND 948 00:35:59,999 --> 00:36:00,499 PROMOTERS OF THE GENES. 949 00:36:00,499 --> 00:36:00,967 SO WHAT HAPPENS IF WE 950 00:36:00,967 --> 00:36:01,534 MANIPULATE THE PATHWAYS AND 951 00:36:01,534 --> 00:36:09,742 RESTORE ACTIVITY? 952 00:36:09,742 --> 00:36:10,309 OR IF WE KNOCKED DOWN OTHER 953 00:36:10,309 --> 00:36:10,876 ENZYMES OF THE HISTONE MARKS 954 00:36:10,876 --> 00:36:13,813 THAT ARE REGULATED OR DNA 955 00:36:13,813 --> 00:36:19,452 METHYLATION? 956 00:36:19,452 --> 00:36:20,052 SO I TOLD YOU BEFORE MAYBE IN 957 00:36:20,052 --> 00:36:20,686 THE NORMAL STATE YOU HAVE K 27 958 00:36:20,686 --> 00:36:21,854 A SILENCING MARK AND IN THE 959 00:36:21,854 --> 00:36:27,426 DISEASE STATE YOU LOSE THE K 960 00:36:27,426 --> 00:36:27,994 27 METHYLATION SO YOU HAVE K 961 00:36:27,994 --> 00:36:32,765 36 OR OTHER MARKS FOUND ALONE. 962 00:36:32,765 --> 00:36:33,766 WHAT HAPPENS IF WE RESTORE THE 963 00:36:33,766 --> 00:36:42,508 MUTATED SUBUNITS AND TRY TO IF 964 00:36:42,508 --> 00:36:48,714 WE REMOVE THE K 36 AND IF WE 965 00:36:48,714 --> 00:36:59,091 CAN GET TO THE STATE. 966 00:37:01,394 --> 00:37:02,561 AND THEN WE CAN HAVE THE 967 00:37:02,561 --> 00:37:04,330 ENTIRE COMPLEX TOGETHER THAT 968 00:37:04,330 --> 00:37:06,766 IS WHAT WE COULDN'T DO WITH 969 00:37:06,766 --> 00:37:10,536 THE MUTATION WE CAN'T PUT OUT 970 00:37:10,536 --> 00:37:21,013 THE ENTIRE COMPLEX AND THEN 971 00:37:21,914 --> 00:37:22,948 THE METHYLATION LEVELS COME 972 00:37:22,948 --> 00:37:25,351 BACK. 973 00:37:25,351 --> 00:37:35,861 WE CAN KNOCKED DOWN AND ASK T. 974 00:37:36,262 --> 00:37:43,369 WE KNOCKED DOWN THAT NSD WE 975 00:37:43,369 --> 00:37:46,339 HAVE CREATED THAT'S LOOK AT 976 00:37:46,339 --> 00:37:51,310 THE PROFILES OR THE NSD 977 00:37:51,310 --> 00:37:51,844 KNOCKDOWN IS BASICALLY THE 978 00:37:51,844 --> 00:37:52,611 SAME GENES WHAT WE SAW BEFORE 979 00:37:52,611 --> 00:37:54,847 BEING AFFECTED SUCH AS THE 980 00:37:54,847 --> 00:37:56,682 INTERFERON GENES AND OTHER 981 00:37:56,682 --> 00:38:01,487 TRANSCRIPTIONAL BUT BASICALLY 982 00:38:01,487 --> 00:38:02,021 THIS SAYS THESE DIFFERENT 983 00:38:02,021 --> 00:38:02,621 HISTONE MARKS ARE FOUND ON THE 984 00:38:02,621 --> 00:38:08,060 SAME GENOME. 985 00:38:08,060 --> 00:38:08,627 HOW SUSCEPTIBLE ARE THESE 986 00:38:08,627 --> 00:38:12,164 CELLS TO THE KNOCKDOWN OR 987 00:38:12,164 --> 00:38:14,667 REMOVAL OF K 36 METHYLATION? 988 00:38:14,667 --> 00:38:17,837 THOSE THAT HAVE A DIFFERENT 989 00:38:17,837 --> 00:38:18,971 MUTATION THE CELLS ARE NOT 990 00:38:18,971 --> 00:38:21,440 SENSITIVE. 991 00:38:21,440 --> 00:38:31,250 BUT IN THOSE THAT LOSE YOU CAN 992 00:38:31,250 --> 00:38:31,851 SEE HOW SENSITIVE THEY ARE TO 993 00:38:31,851 --> 00:38:32,451 LOSE NSD WE CAN DO A TRICK OF 994 00:38:32,451 --> 00:38:33,052 REMOVING ACROSS THE GENOME IT 995 00:38:33,052 --> 00:38:36,455 IS THE SAME STORY. 996 00:38:36,455 --> 00:38:39,425 THE CELLS HAVE A DIFFERENT 997 00:38:39,425 --> 00:38:43,629 MUTATION THOSE THAT HAVE THE 998 00:38:43,629 --> 00:38:44,163 LOST MUTATION DO CARE THEY 999 00:38:44,163 --> 00:38:44,430 LOSE K 36. 1000 00:38:44,430 --> 00:38:54,807 IT'S A DIFFERENT WAY. 1001 00:38:58,511 --> 00:38:59,011 THIS COULD BE A THERAPEUTIC 1002 00:38:59,011 --> 00:38:59,578 VULNERABILITY IN THE FUTURE. 1003 00:38:59,578 --> 00:39:00,079 WITH THE DNA METHYLATION 1004 00:39:00,079 --> 00:39:00,679 INHIBITOR YOU WILL INCREASE OR 1005 00:39:00,679 --> 00:39:01,247 KILL ANY CELLS THAT HAVE DNA 1006 00:39:01,247 --> 00:39:01,847 METHYLATION AND INHIBITION IF 1007 00:39:01,847 --> 00:39:02,448 YOU INCREASE THE CONCENTRATION 1008 00:39:02,448 --> 00:39:03,582 YOU CAN SEE HOW MUCH MORE 1009 00:39:03,582 --> 00:39:10,689 SENSITIVE EVEN THE CELLS THAT 1010 00:39:10,689 --> 00:39:11,190 CAN KILL THEM BUT I TELL 1011 00:39:11,190 --> 00:39:14,193 SENSITIVE THEY ARE TO THE 1012 00:39:14,193 --> 00:39:19,565 METHYLATION THEN YOU HAVE 1013 00:39:19,565 --> 00:39:20,099 EXPRESSION OF THOSE GENES 1014 00:39:20,099 --> 00:39:20,599 WHICH CONFIRMS WHICH WE 1015 00:39:20,599 --> 00:39:24,737 THOUGHT WAS THE MECHANISM WHEN 1016 00:39:24,737 --> 00:39:26,305 IT COMES INTO SILENCE THE 1017 00:39:26,305 --> 00:39:33,312 IMMUNE RESPONSE AND THEN YOU 1018 00:39:33,312 --> 00:39:33,846 GET UP REGULATION OF THOSE 1019 00:39:33,846 --> 00:39:34,346 THAT WERE DOWN REGULATED 1020 00:39:34,346 --> 00:39:34,580 BEFORE. 1021 00:39:34,580 --> 00:39:41,187 BUT THE PROBLEM IS TO RESTORE 1022 00:39:41,187 --> 00:39:41,687 PROTEIN ACTIVITY WITH A 1023 00:39:41,687 --> 00:39:42,288 PATIENT IS DIFFICULT AND THERE 1024 00:39:42,288 --> 00:39:45,257 IS NO NSD INHIBITOR THERE ARE 1025 00:39:45,257 --> 00:39:53,933 NOT VIABLE OPTIONS. 1026 00:39:53,933 --> 00:39:54,500 BUT THERE IS A LOT OF OTHER 1027 00:39:54,500 --> 00:39:54,967 HISTONE MARKS THAT ARE 1028 00:39:54,967 --> 00:39:55,501 EXPRESSED ESPECIALLY WITH 1029 00:39:55,501 --> 00:39:56,535 ACETYLATION. 1030 00:39:56,535 --> 00:40:02,608 AND WITH HISTONE THEY SHARE A 1031 00:40:02,608 --> 00:40:08,147 MECHANISM OF ACTION. 1032 00:40:08,147 --> 00:40:08,714 WE THOUGHT WE COULD PUSH THE 1033 00:40:08,714 --> 00:40:13,219 CELLS TO A HYPER ACETYLATED 1034 00:40:13,219 --> 00:40:13,752 STATE TO REDUCE TOXICITY. 1035 00:40:13,752 --> 00:40:17,189 WE CAN DO THIS SO WE CAN DO 1036 00:40:17,189 --> 00:40:20,659 THIS WITH THE AGE THREE AND 1037 00:40:20,659 --> 00:40:24,296 EACH FOR ACETYLATION THOSE 1038 00:40:24,296 --> 00:40:27,299 LEVELS ARE READY HIRE AT A 1039 00:40:27,299 --> 00:40:30,736 BASELINE LEVEL AND WHEN WE ADD 1040 00:40:30,736 --> 00:40:31,337 THE INHIBITOR THE CELLS REALLY 1041 00:40:31,337 --> 00:40:32,538 GO HIGH. 1042 00:40:32,538 --> 00:40:33,739 SO WE DO FEEL WE ARE GETTING 1043 00:40:33,739 --> 00:40:37,643 TO THIS POINT. 1044 00:40:37,643 --> 00:40:48,153 GETTING TO THE KILL CURVES IN 1045 00:40:48,454 --> 00:40:52,224 TERMS OF THE INCREASING 1046 00:40:52,224 --> 00:40:55,461 CONCENTRATION THESE THREE IN 1047 00:40:55,461 --> 00:40:56,228 RED HAVE THE MUTATION AND ARE 1048 00:40:56,228 --> 00:41:02,334 MUCH MORE SENSITIVE THAN THEN 1049 00:41:02,334 --> 00:41:02,935 WITH THE INHIBITOR WE CAN HAVE 1050 00:41:02,935 --> 00:41:09,208 A EXPRESSION OF ALL OF THE HLA 1051 00:41:09,208 --> 00:41:10,776 MOLECULES THEY HAD REPRESSED 1052 00:41:10,776 --> 00:41:18,250 IN THE DISEASE STATE. 1053 00:41:18,250 --> 00:41:18,951 AND YOU CAN SEE THAT SHIFTS 1054 00:41:18,951 --> 00:41:23,522 THE ABILITY. 1055 00:41:23,522 --> 00:41:23,989 MORE WITH THE A STACK 1056 00:41:23,989 --> 00:41:25,558 INHIBITOR BUT USING A 1057 00:41:25,558 --> 00:41:32,097 COMBINATION AND A SINGLE DOSE 1058 00:41:32,097 --> 00:41:32,698 OF DNA METHYLATION AND YOU CAN 1059 00:41:32,698 --> 00:41:35,935 SEE THE TWO TOGETHER ARE MORE 1060 00:41:35,935 --> 00:41:36,502 IMPORTANT THING AND KILLING 1061 00:41:36,502 --> 00:41:39,371 THE CELLS THAN JUST THE AGE 1062 00:41:39,371 --> 00:41:42,308 DECK INHIBITOR ALONE. 1063 00:41:42,308 --> 00:41:45,311 WITH THE QUANTITATIVE POINT OF 1064 00:41:45,311 --> 00:41:55,754 VIEW WE LEARNED A LOT ABOUT 1065 00:41:55,754 --> 00:41:56,288 THE SYSTEM WITH EPIGENETIC 1066 00:41:56,288 --> 00:41:56,822 REPROGRAMMING AND HOW THIS 1067 00:41:56,822 --> 00:41:57,389 COULD LEAD TO PROGRESSION OF 1068 00:41:57,389 --> 00:41:57,623 CANCER. 1069 00:41:57,623 --> 00:41:58,190 WE PUBLISH THE INITIAL PAPER 1070 00:41:58,190 --> 00:42:02,094 YEARS AGO. 1071 00:42:02,094 --> 00:42:12,571 QUICKLY AND WANT TO SHARE A 1072 00:42:16,809 --> 00:42:17,343 NEW PROJECT IT IS IN PROCESS. 1073 00:42:17,343 --> 00:42:17,910 NOT A COMPLETE STORY BUT WE 1074 00:42:17,910 --> 00:42:18,477 ALWAYS HAD A QUESTION IF YOU 1075 00:42:18,477 --> 00:42:19,078 HAD A HIGHLY MODIFIED HISTONE 1076 00:42:19,078 --> 00:42:19,612 AND YOU WANT TO REMOVE THE 1077 00:42:19,612 --> 00:42:20,179 MODIFICATIONS HOW DO YOU DO 1078 00:42:20,179 --> 00:42:23,649 THIS EFFICIENTLY? 1079 00:42:23,649 --> 00:42:24,116 AND THEN TO REMOVE THE 1080 00:42:24,116 --> 00:42:24,783 MODIFICATION THAT SEEMS LIKE A 1081 00:42:24,783 --> 00:42:29,421 LOT OF WORK. 1082 00:42:29,421 --> 00:42:29,989 BUT WE ALWAYS THOUGHT IF YOU 1083 00:42:29,989 --> 00:42:30,422 WANT TO REMOVE THOSE 1084 00:42:30,422 --> 00:42:32,324 MODIFICATIONS THEN CLICK THIS 1085 00:42:32,324 --> 00:42:35,327 AND THEN THE HISTONE WOULD BE 1086 00:42:35,327 --> 00:42:37,129 EXCHANGED. 1087 00:42:37,129 --> 00:42:37,630 THERE COULD BE A LOT OF 1088 00:42:37,630 --> 00:42:39,064 CONSEQUENCES HERE WITH 1089 00:42:39,064 --> 00:42:40,633 MODIFICATIONS AND OTHER 1090 00:42:40,633 --> 00:42:47,006 PROTEINS THAT ARE ATTRACTED 1091 00:42:47,006 --> 00:42:47,606 BUT THEN YOU CAN REMOVE THESE 1092 00:42:47,606 --> 00:42:47,940 INTERACTIONS. 1093 00:42:47,940 --> 00:42:58,283 AND IN FACT HISTONE 1094 00:43:01,787 --> 00:43:02,287 CARDIOLOGISTS HAS BEEN SHOWN 1095 00:43:02,287 --> 00:43:02,888 IN MANY CONTEXT TO BE INVOLVED 1096 00:43:02,888 --> 00:43:03,489 IN A FEW DIFFERENT PROSTHESES 1097 00:43:03,489 --> 00:43:03,889 ESPECIALLY CANCER 1098 00:43:03,889 --> 00:43:04,189 PROGRESSION. 1099 00:43:04,189 --> 00:43:04,723 PROTEASE IS THAT HAS BEEN 1100 00:43:04,723 --> 00:43:10,729 IDENTIFIED AS A HISTONE AND 1101 00:43:10,729 --> 00:43:17,469 THEN WITH THOSE SPECIFIC SITES 1102 00:43:17,469 --> 00:43:18,070 ON THE PROTEIN AND ALSO THERE 1103 00:43:18,070 --> 00:43:28,480 HAS BEEN AND AS A WAY TO 1104 00:43:30,282 --> 00:43:30,783 REMOVE TALES AS METHYLATION. 1105 00:43:30,783 --> 00:43:31,350 THERE IS A A FEW PAPERS HERE 1106 00:43:31,350 --> 00:43:37,056 AND THERE IS INTERESTING WE 1107 00:43:37,056 --> 00:43:37,656 SHOULD SPENT A LOT OF TIME ON 1108 00:43:37,656 --> 00:43:38,257 NUCLEUS BUT THERE ARE PAPERS 1109 00:43:38,257 --> 00:43:42,728 THAT SHOW TRANSCRIPTION 1110 00:43:42,728 --> 00:43:43,228 FACTORS AND THIS AFFECTS 1111 00:43:43,228 --> 00:43:45,698 DIFFERENTIATION AS WELL. 1112 00:43:45,698 --> 00:43:55,574 SEE YOU CAN SEE HERE IT 1113 00:43:55,574 --> 00:43:56,141 AFFECTS THE MOUSE COLOR AND 1114 00:43:56,141 --> 00:44:04,783 HAIR SO ARE THERE ANY OTHER 1115 00:44:04,783 --> 00:44:05,317 SUBSTRATES IN THE NUCLEUS? 1116 00:44:05,317 --> 00:44:05,884 FOCUSING ON HISTONES BUT WE 1117 00:44:05,884 --> 00:44:07,920 HAVE DATA ON OTHERS AS WELL. 1118 00:44:07,920 --> 00:44:10,322 WHAT WE ACTUALLY FOUND LOOKING 1119 00:44:10,322 --> 00:44:16,762 BACK THROUGH SOME DATA WE 1120 00:44:16,762 --> 00:44:17,362 DIDN'T CHOP UP WITH A PROTEASE 1121 00:44:17,362 --> 00:44:18,263 BUT BACK DIRECTLY WE FOUND A 1122 00:44:18,263 --> 00:44:24,937 LOT OF SITES THAT THIS IS 1123 00:44:24,937 --> 00:44:32,111 HISTONE H2 A AND THEN WE 1124 00:44:32,111 --> 00:44:32,678 INCUBATED THAT AND SAW THERE 1125 00:44:32,678 --> 00:44:40,252 WERE SOME SITES THAT WERE 1126 00:44:40,252 --> 00:44:40,853 HIGHLY PREFERRED WHICH MATCHED 1127 00:44:40,853 --> 00:44:50,929 THE IN VIVO DATA. 1128 00:44:50,929 --> 00:44:51,530 SO LOOKING AT ANY PROCESS THAT 1129 00:44:51,530 --> 00:44:54,800 HAD A DIFFERENTIATION OF THE 1130 00:44:54,800 --> 00:44:58,570 HISTONE FRAGMENT EVENTS AND 1131 00:44:58,570 --> 00:45:01,373 GOING TO MOUSE EMBRYONIC STEM 1132 00:45:01,373 --> 00:45:02,441 CELLS THEY COULD BE MANY 1133 00:45:02,441 --> 00:45:10,249 DIFFERENT LINEAGES AND THE 1134 00:45:10,249 --> 00:45:10,816 EMBRYONIC DIFFERENTIATION SO 1135 00:45:10,816 --> 00:45:11,416 WE WENT TO VISIT THIS WITH THE 1136 00:45:11,416 --> 00:45:13,485 QUANTITATIVE POINT OF VIEW TO 1137 00:45:13,485 --> 00:45:17,489 MAKE EMBRYONIC BODIES AND THE 1138 00:45:17,489 --> 00:45:19,792 NEURON CELLS AND WHAT WE FOUND 1139 00:45:19,792 --> 00:45:23,662 IS IT IS HARD TO SEE THAT 1140 00:45:23,662 --> 00:45:31,036 THERE IS A LIGHTER FRAGMENT 1141 00:45:31,036 --> 00:45:31,637 THAT IS LIGHTING UP HERE AND 1142 00:45:31,637 --> 00:45:32,204 TO LOOK AT THE FRAGMENTS WE 1143 00:45:32,204 --> 00:45:35,107 CAN SEE THAT DURING A FOUR-DAY 1144 00:45:35,107 --> 00:45:40,979 DIFFERENTIATION WE HAVE 1145 00:45:40,979 --> 00:45:42,514 CLEAVAGE SITES ESPECIALLY ON 1146 00:45:42,514 --> 00:45:44,750 DAY FOUR. 1147 00:45:44,750 --> 00:45:49,755 THERE IS CLEAVAGE AND 1148 00:45:49,755 --> 00:45:55,727 FRAGMENTS WE THOUGHT SO 1149 00:45:55,727 --> 00:45:56,328 BECAUSE THERE IS SIMILARITY IN 1150 00:45:56,328 --> 00:46:02,701 THE MAJOR SITES WITH H2 A BUT 1151 00:46:02,701 --> 00:46:04,636 INCREASING DIFFERENTIATION 1152 00:46:04,636 --> 00:46:09,107 EXPRESSION IS A MATURE FORM 1153 00:46:09,107 --> 00:46:09,675 YOU CAN SEE ON DAY FOR IT IS 1154 00:46:09,675 --> 00:46:14,646 PRETTY HIGH. 1155 00:46:14,646 --> 00:46:15,214 SO WE KNOCK IT DOWN WITH THE 1156 00:46:15,214 --> 00:46:15,781 EMBRYONIC STEM CELLS AND WE 1157 00:46:15,781 --> 00:46:18,050 CAN SEE SEVERAL SITES THAT GO 1158 00:46:18,050 --> 00:46:20,686 DOWN ESPECIALLY ALL 23 IT 1159 00:46:20,686 --> 00:46:24,022 DECREASES IN ABUNDANCE SOME 1160 00:46:24,022 --> 00:46:29,127 DON'T CHANGE BUT OTHER 1161 00:46:29,127 --> 00:46:31,930 PROTEASE COULD AFFECT THAT MAY 1162 00:46:31,930 --> 00:46:32,464 BE PROCESSING ARTIFACTS OF 1163 00:46:32,464 --> 00:46:33,966 WHAT WE ARE DOING? 1164 00:46:33,966 --> 00:46:36,668 SPENT SOME SHOW 1165 00:46:36,668 --> 00:46:40,205 DIFFERENTIATION ON OF 1166 00:46:40,205 --> 00:46:43,242 CLEAVAGE. 1167 00:46:43,242 --> 00:46:47,012 SO WHY WOULD YOU NEED THE 1168 00:46:47,012 --> 00:46:47,646 PROTEASE CRACKS IF YOU HAVE 1169 00:46:47,646 --> 00:46:51,917 MODIFICATIONS IT'S A GREAT WAY 1170 00:46:51,917 --> 00:46:52,284 TO REMOVE THOSE 1171 00:46:52,284 --> 00:46:52,618 MODIFICATIONS. 1172 00:46:52,618 --> 00:46:56,054 LOOK AT H2 A IT HAS THOSE THAT 1173 00:46:56,054 --> 00:46:58,357 CAN BE ACETYLATED AT A LOWER 1174 00:46:58,357 --> 00:47:02,094 LEVEL. 1175 00:47:02,094 --> 00:47:02,661 AND OVER DIFFERENTIATION YOU 1176 00:47:02,661 --> 00:47:07,099 CAN SEE THE ABUNDANCE GOES UP 1177 00:47:07,099 --> 00:47:07,666 FOR ALL OF THESE AND THEN IT 1178 00:47:07,666 --> 00:47:10,836 DIPS A DAY FOR SO WHAT HAPPENS 1179 00:47:10,836 --> 00:47:18,277 WHEN WE KNOCK IT DOWN HERE 1180 00:47:18,277 --> 00:47:18,844 DURING THE DIFFERENTIATION? 1181 00:47:18,844 --> 00:47:19,378 AND THE CONTROL DAY FOR IT 1182 00:47:19,378 --> 00:47:23,048 GOES DOWN BUT THEN THE LEVELS 1183 00:47:23,048 --> 00:47:25,083 DON'T GO DOWN ANYMORE AND THE 1184 00:47:25,083 --> 00:47:30,489 NUMBER FOUR YOU CAN SEE THE 1185 00:47:30,489 --> 00:47:31,056 LEVELS DROP AND THEN WHEN WE 1186 00:47:31,056 --> 00:47:31,657 KNOCK IT DOWN THE LEVELS DON'T 1187 00:47:31,657 --> 00:47:34,226 DROP ANYMORE. 1188 00:47:34,226 --> 00:47:37,195 THIS TOLD US THAT CAPS ON IS A 1189 00:47:37,195 --> 00:47:46,204 PROTEASE. 1190 00:47:46,204 --> 00:47:47,205 WE WANT TO SEE WHERE CANINE 1191 00:47:47,205 --> 00:47:49,841 ACETYLATION WAS BOTH IN THE 1192 00:47:49,841 --> 00:47:56,315 CONTROL AND DOWN SO HERE WHAT 1193 00:47:56,315 --> 00:47:56,882 YOU CAN SEE BETWEEN DAY TWO 1194 00:47:56,882 --> 00:47:58,483 AND DAY FOR IN THE CONTROL 1195 00:47:58,483 --> 00:48:02,354 THEY HAVE WHAT DECREASES A 1196 00:48:02,354 --> 00:48:06,992 LITTLE BIT YOU CAN SEE THIS 1197 00:48:06,992 --> 00:48:12,331 BUT WHEN YOU KNOCK DOWN IN 1198 00:48:12,331 --> 00:48:12,931 SOME CASES IT GOES UP IS WHAT 1199 00:48:12,931 --> 00:48:15,968 YOU WOULD EXPECT SO 1200 00:48:15,968 --> 00:48:19,137 ESSENTIALLY WHEN YOU COMPARE 1201 00:48:19,137 --> 00:48:23,709 DAY TO THROUGH DAY FOR THOSE 1202 00:48:23,709 --> 00:48:26,712 THAT GO DOWN DO NOT GO DOWN 1203 00:48:26,712 --> 00:48:30,315 ANYMORE AND THE EXPRESSION 1204 00:48:30,315 --> 00:48:35,721 LEVELS THE NUMBER OF PEAKS DAY 1205 00:48:35,721 --> 00:48:36,321 TO THROUGH DAY FOR YOU CAN SEE 1206 00:48:36,321 --> 00:48:38,757 THEM GOING DOWN BUT INCREASE 1207 00:48:38,757 --> 00:48:43,161 HERE THE ANALYSIS SHOWS ALL 1208 00:48:43,161 --> 00:48:43,729 THINGS INVOLVED IN CELLULAR 1209 00:48:43,729 --> 00:48:44,329 DIFFERENTIATION ESPECIALLY TO 1210 00:48:44,329 --> 00:48:53,071 THE NERVOUS SYSTEM NOT A LOT 1211 00:48:53,071 --> 00:48:54,172 IS KNOWN ABOUT ACETYLATED H2 1212 00:48:54,172 --> 00:49:04,649 WAY BUT THE WHOLE IDEA IT IS 1213 00:49:07,019 --> 00:49:07,519 PROBABLY THERE IS SOMETHING 1214 00:49:07,519 --> 00:49:08,086 RECOGNIZE THE MODIFICATION. 1215 00:49:08,086 --> 00:49:08,687 WE THOUGHT TO DO AN EXPERIMENT 1216 00:49:08,687 --> 00:49:09,287 WHAT COULD BE RECOGNIZE SO WE 1217 00:49:09,287 --> 00:49:11,656 SAW SYNTHETIC PEPTIDES EITHER 1218 00:49:11,656 --> 00:49:12,090 WITH OR WITHOUT THE 1219 00:49:12,090 --> 00:49:14,659 ACETYLATION AND THE PULL DOWNS 1220 00:49:14,659 --> 00:49:16,361 FROM EXTRACTS TO THE PRETTY 1221 00:49:16,361 --> 00:49:20,999 ONYX WHERE WE COMPARED H2 A 1222 00:49:20,999 --> 00:49:21,600 ALLOSTATIC, CANINE OR THE 1223 00:49:21,600 --> 00:49:31,977 COMBINATION TOGETHER. 1224 00:49:34,913 --> 00:49:35,981 AND FOR THOSE THAT MIGHT 1225 00:49:35,981 --> 00:49:40,619 RECOGNIZE THESE IT IS THE 1226 00:49:40,619 --> 00:49:49,027 COMPLEX SEVERAL DOMAINS AND 1227 00:49:49,027 --> 00:49:50,162 THEN THEY COULD BIND THE 1228 00:49:50,162 --> 00:49:56,735 ACETYLATED SITES. 1229 00:49:56,735 --> 00:49:57,335 IT DOESN'T PULL THEM AS STRONG 1230 00:49:57,335 --> 00:50:07,646 AS HE ACETYLATED. 1231 00:50:11,616 --> 00:50:12,150 BUT THIS IS ALL UNIT SPECIFIC 1232 00:50:12,150 --> 00:50:12,517 TO THE COMPLEX. 1233 00:50:12,517 --> 00:50:14,719 AND P BETH IS KNOWN TO PLAY A 1234 00:50:14,719 --> 00:50:22,094 ROLE AND THEN TO SEE 1235 00:50:22,094 --> 00:50:22,594 POTENTIALLY WITH H2A AND 1236 00:50:22,594 --> 00:50:26,832 EMBRYONIC STEM CELLS. 1237 00:50:26,832 --> 00:50:30,502 AND THEN TO BE TURNED ON A 1238 00:50:30,502 --> 00:50:34,873 STAPLE WANT TO DIFFERENTIATE 1239 00:50:34,873 --> 00:50:35,474 AND THEN IT COMES UP WITH THE 1240 00:50:35,474 --> 00:50:42,114 GENE REGULATION AND ALLOWS THE 1241 00:50:42,114 --> 00:50:42,747 CELLS TO DIFFERENTIATE INTO 1242 00:50:42,747 --> 00:50:52,390 DIFFERENT LINEAGES. 1243 00:50:52,390 --> 00:50:55,093 SO AS YOU CAN SEE HERE THESE 1244 00:50:55,093 --> 00:51:04,002 ARE THE PEAKS PR BM ONE WITH 1245 00:51:04,002 --> 00:51:12,644 H2 A CANINE. 1246 00:51:12,644 --> 00:51:15,714 SO YOU HAVE A LARGER NUMBER OF 1247 00:51:15,714 --> 00:51:18,817 PEAKS WITH A CANINE AS THE 1248 00:51:18,817 --> 00:51:22,621 PROMOTER. 1249 00:51:22,621 --> 00:51:33,131 IN TERMS OF THE EXPRESSION AND 1250 00:51:36,968 --> 00:51:37,469 THEN WITH THE SETTLE IN THE 1251 00:51:37,469 --> 00:51:38,069 SAME WITH THE PR BM EXPRESSION 1252 00:51:38,069 --> 00:51:38,670 IS NOT AS STRONG AS THOSE THAT 1253 00:51:38,670 --> 00:51:43,642 HAVE IT WITH THE H2A. 1254 00:51:43,642 --> 00:51:46,611 HE WENT TO DO EXPERIMENTS TO 1255 00:51:46,611 --> 00:51:50,482 SEE IF IT WOULD MANIPULATE TO 1256 00:51:50,482 --> 00:51:55,554 CLIP H2A. 1257 00:51:55,554 --> 00:52:02,594 WE EXPRESS FULLY INTO CELLS 1258 00:52:02,594 --> 00:52:07,032 AND THAT SHOWS IT IS MISSING A 1259 00:52:07,032 --> 00:52:10,902 LOT OF THE PROTEINS AND THERE 1260 00:52:10,902 --> 00:52:14,372 IS ACTUALLY PROTEINS THAT ARE 1261 00:52:14,372 --> 00:52:22,914 UPREGULATED SO THESE ARE 1262 00:52:22,914 --> 00:52:27,152 EXCHANGE FACTORS OR NUCLEOSOME 1263 00:52:27,152 --> 00:52:28,186 ASSEMBLY FACTORS OF THESE 1264 00:52:28,186 --> 00:52:29,921 COULD BE THE PROTEINS THAT 1265 00:52:29,921 --> 00:52:35,093 RECOGNIZE REMOVING THEM. 1266 00:52:35,093 --> 00:52:36,127 AND WITH THOSE NUCLEOSOME'S 1267 00:52:36,127 --> 00:52:37,963 WHEN YOU LOOK AT THE FULL ONE 1268 00:52:37,963 --> 00:52:46,438 WITH THE CLUB. 1269 00:52:46,438 --> 00:52:47,038 AND WHEN YOU HAVE EXPERIMENTS 1270 00:52:47,038 --> 00:52:54,412 YOU CAN SEE HERE THAT THE 1271 00:52:54,412 --> 00:52:54,946 CONTROL KNOCKDOWN ARE THE 1272 00:52:54,946 --> 00:52:56,548 LEVELS OF THE PR BM AND THEN 1273 00:52:56,548 --> 00:53:03,455 YOU CAN SEE THESE ARE LEVELS 1274 00:53:03,455 --> 00:53:08,827 WITH THE HISTONE MARK AND IF 1275 00:53:08,827 --> 00:53:09,861 YOU JUST PULL OUT A TRACK YOU 1276 00:53:09,861 --> 00:53:20,305 CAN SEE HERE WHEN WE KNOCK 1277 00:53:21,539 --> 00:53:21,973 THIS OUT THE PEAK GETS A 1278 00:53:21,973 --> 00:53:22,540 LITTLE STRONGER FROM H2 WAY. 1279 00:53:22,540 --> 00:53:22,941 AND HERE AS WELL. 1280 00:53:22,941 --> 00:53:23,875 YOU CAN SEE PR BM. 1281 00:53:23,875 --> 00:53:26,678 SO IN THE KNOCKDOWN WE GET 1282 00:53:26,678 --> 00:53:36,955 STRONGER H2 WAY. 1283 00:53:41,226 --> 00:53:48,667 AND CLIPPING TO THE ABILITY TO 1284 00:53:48,667 --> 00:53:49,267 BE FOUND AT SPECIFIC GENES AND 1285 00:53:49,267 --> 00:53:51,670 THE GENOME THAT HAVE H2A. 1286 00:53:51,670 --> 00:53:58,743 LASTLY WITH THE FULL LINKAGE 1287 00:53:58,743 --> 00:53:59,311 TO A WE CAN SEE THE HISTONE 1288 00:53:59,311 --> 00:54:03,181 MARKS ARE QUITE DIFFERENT AND 1289 00:54:03,181 --> 00:54:03,782 THE CLIPPED H2 A IS FOUND WITH 1290 00:54:03,782 --> 00:54:07,585 MORE ACTIVE GENES. 1291 00:54:07,585 --> 00:54:12,390 WE KNOW IT'S GETTING CLIPPED. 1292 00:54:12,390 --> 00:54:21,232 WITH MORE ACETYLATED H2A. 1293 00:54:21,232 --> 00:54:22,400 AND THOSE THAT ARE MORE ACTIVE 1294 00:54:22,400 --> 00:54:26,905 MARKS. 1295 00:54:26,905 --> 00:54:28,073 AND THOSE THAT ARE MORE LINKED 1296 00:54:28,073 --> 00:54:36,815 TO GENE SILENCING. 1297 00:54:36,815 --> 00:54:37,349 THE CLIPPED H2 WAY YOU CAN 1298 00:54:37,349 --> 00:54:37,982 IMAGINE IT CANNOT SAY IN THE 1299 00:54:37,982 --> 00:54:45,623 NUCLEOSOME FOREVER SO IF YOU 1300 00:54:45,623 --> 00:54:46,124 DO A POST CHASE TYPE OF 1301 00:54:46,124 --> 00:54:47,559 EXPERIMENT AND LOOK AT THE 1302 00:54:47,559 --> 00:54:50,862 INCORPORATION OR THE CLIPPED 1303 00:54:50,862 --> 00:55:01,373 H2 WAY WE CAN SEE ONCE WE ADD 1304 00:55:04,209 --> 00:55:04,676 USING SOME PRO RODEO PSALM 1305 00:55:04,676 --> 00:55:05,243 INHIBITORS THESE DO NOT HAVE 1306 00:55:05,243 --> 00:55:05,810 IT BUT THEY ARE INHIBITING A 1307 00:55:05,810 --> 00:55:08,880 PROTEIN SYNTHESIS YOU CAN SEE 1308 00:55:08,880 --> 00:55:09,881 THE LEVELS STAY FOR SEVERAL 1309 00:55:09,881 --> 00:55:19,991 HOURS. 1310 00:55:23,128 --> 00:55:23,528 IS NOT AS STABLE FOR A 1311 00:55:23,528 --> 00:55:24,129 FULL-LENGTH THAT IS A WORK IN 1312 00:55:24,129 --> 00:55:24,662 PROGRESS WITH A DIFFERENT 1313 00:55:24,662 --> 00:55:26,231 MECHANISM THAT COULD BE 1314 00:55:26,231 --> 00:55:30,668 INVOLVED AND TO RECOGNIZE LOW 1315 00:55:30,668 --> 00:55:39,444 LEVEL ACETYLATION. 1316 00:55:39,444 --> 00:55:40,712 SHOWING THE TECHNOLOGY 1317 00:55:40,712 --> 00:55:49,053 ADVANCES. 1318 00:55:49,053 --> 00:55:49,554 TO FOLLOW THE EPIGENETIC 1319 00:55:49,554 --> 00:55:50,155 CHANGES THAT OCCUR BECAUSE OF 1320 00:55:50,155 --> 00:55:50,722 THESE MUTATIONS AND THE SOFT 1321 00:55:50,722 --> 00:55:57,395 TISSUES. 1322 00:55:57,395 --> 00:55:57,962 THAT GIVES A LOT OF TARGETS 1323 00:55:57,962 --> 00:55:58,530 THAT TRY TO GET DATA IN THE 1324 00:55:58,530 --> 00:56:00,498 MOUSE MODELS AND KEEP MARCHING 1325 00:56:00,498 --> 00:56:04,335 TOWARDS WITH THE THERAPEUTIC 1326 00:56:04,335 --> 00:56:09,707 INTERVENTION. 1327 00:56:09,707 --> 00:56:11,075 IT IS ON THE BASIC SCIENCE 1328 00:56:11,075 --> 00:56:21,119 SIDE BUT AND TO REGULATE 1329 00:56:21,119 --> 00:56:21,719 HISTONE MODIFICATIONS DUE TO A 1330 00:56:21,719 --> 00:56:22,754 MUCH MORE PERMANENT TYPE OF 1331 00:56:22,754 --> 00:56:24,255 WAY. 1332 00:56:24,255 --> 00:56:25,557 AND WAS STEM CELL 1333 00:56:25,557 --> 00:56:29,160 DIFFERENTIATION. 1334 00:56:29,160 --> 00:56:35,800 THANK YOU TO THE GROUP. 1335 00:56:35,800 --> 00:56:36,935 AND CONTRIBUTED TO THE 1336 00:56:36,935 --> 00:56:42,907 DIFFERENT PROJECTS AND ALSO 1337 00:56:42,907 --> 00:56:47,745 ONE OF THE PEOPLE WORKING ON 1338 00:56:47,745 --> 00:56:51,616 PROJECT AND THOSE WORKING ON 1339 00:56:51,616 --> 00:56:56,354 THE TECHNOLOGY ADVANCES AS 1340 00:56:56,354 --> 00:56:59,357 WELL AND I'LL BE HAPPY TO 1341 00:56:59,357 --> 00:57:00,425 ANSWER ANY QUESTIONS. 1342 00:57:00,425 --> 00:57:07,298 THANK YOU. 1343 00:57:07,298 --> 00:57:07,565 [APPLAUSE] 1344 00:57:07,565 --> 00:57:08,466 >> IT LOOKS LIKE WE HAVE SOME 1345 00:57:08,466 --> 00:57:16,541 QUESTIONS. 1346 00:57:16,541 --> 00:57:17,876 >> I HAVE A COUPLE OF 1347 00:57:17,876 --> 00:57:21,846 TECHNICAL QUESTIONS AND THEN 1348 00:57:21,846 --> 00:57:32,357 TO DESCRIBE THE QUANTIFICATION 1349 00:57:34,792 --> 00:57:35,260 OF THE POST- TRANSLATIONAL 1350 00:57:35,260 --> 00:57:35,827 MODIFICATION OF HISTONES IS 1351 00:57:35,827 --> 00:57:36,294 EXTREMELY INTERESTING. 1352 00:57:36,294 --> 00:57:36,828 AS YOU POINTED OUT IS ALSO 1353 00:57:36,828 --> 00:57:37,228 VERY CHALLENGING. 1354 00:57:37,228 --> 00:57:37,795 THE NUMBER OF MODIFICATIONS 1355 00:57:37,795 --> 00:57:38,496 AND DENSITY AND THE NUMBER OF 1356 00:57:38,496 --> 00:57:45,203 POSSIBLE COMBINATIONS. 1357 00:57:45,203 --> 00:57:45,770 I WONDER WHAT YOUR THOUGHTS 1358 00:57:45,770 --> 00:57:46,404 ARE ON THE RELATIVE MERITS OF 1359 00:57:46,404 --> 00:57:47,772 TOP-DOWN ANALYSIS VERSUS 1360 00:57:47,772 --> 00:57:49,774 BOTTOM-UP? 1361 00:57:49,774 --> 00:57:52,310 AND WHAT IS THE THINKING WITH 1362 00:57:52,310 --> 00:57:54,145 REGARD TO BOTTOM-UP ANALYSIS 1363 00:57:54,145 --> 00:58:00,285 AS TO WHAT PROTEASE TO USE? 1364 00:58:00,285 --> 00:58:00,818 THAT COULD BE PROBLEMATIC. 1365 00:58:00,818 --> 00:58:05,223 >> GOOD QUESTION. 1366 00:58:05,223 --> 00:58:05,690 IT REALLY GETS BACK TO 1367 00:58:05,690 --> 00:58:08,293 EVERYTHING I SHOWED YOU WAS 1368 00:58:08,293 --> 00:58:09,761 BOTTOM-UP. 1369 00:58:09,761 --> 00:58:10,762 SMALLER PEPTIDES. 1370 00:58:10,762 --> 00:58:20,038 BUT IT REALLY DEPENDS ON THE 1371 00:58:20,038 --> 00:58:20,572 QUESTIONS YOU ARE ASKING. 1372 00:58:20,572 --> 00:58:21,172 IF YOU ASK ABOUT COMBINATIONS 1373 00:58:21,172 --> 00:58:21,739 OF MODIFICATIONS AND WANT TO 1374 00:58:21,739 --> 00:58:22,307 LOOK AT A BIGGER STRETCH YOU 1375 00:58:22,307 --> 00:58:29,347 HAVE TO DO MIDDLE OR TOP DOWN. 1376 00:58:29,347 --> 00:58:29,881 THERE ARE CHALLENGES THERE 1377 00:58:29,881 --> 00:58:30,481 BOTTOM-UP IS MUCH MORE FASTER 1378 00:58:30,481 --> 00:58:31,082 AND QUANTITATIVE AND EASIER TO 1379 00:58:31,082 --> 00:58:31,616 DO TOP-DOWN BASICALLY THE 1380 00:58:31,616 --> 00:58:39,290 OPPOSITE. 1381 00:58:39,290 --> 00:58:39,857 WE HAVE DONE A LOT AND BOTH 1382 00:58:39,857 --> 00:58:40,091 WORLDS. 1383 00:58:40,091 --> 00:58:40,592 BUT TO ANSWER BIOLOGICAL 1384 00:58:40,592 --> 00:58:41,092 QUESTIONS RIGHT NOW MOST 1385 00:58:41,092 --> 00:58:45,763 PEOPLE STILL PREFER BOTTOM-UP. 1386 00:58:45,763 --> 00:58:46,264 YOU CAN GET OUT WHAT IS 1387 00:58:46,264 --> 00:58:46,764 HAPPENING IF YOU DO SEE 1388 00:58:46,764 --> 00:58:53,504 SOMETHING HINTING AT A 1389 00:58:53,504 --> 00:58:54,105 COMBINATION WE SAW A PEPTIDE K 1390 00:58:54,105 --> 00:59:01,179 27 K 36 METHYLATION CHANGING. 1391 00:59:01,179 --> 00:59:01,779 I DIDN'T SHOW YOU THE DATA BUT 1392 00:59:01,779 --> 00:59:02,380 WE WANT TO EXTEND THAT TO SEE 1393 00:59:02,380 --> 00:59:02,981 WHAT OTHER METHYLATION WE HAVE 1394 00:59:02,981 --> 00:59:04,916 DONE OTHER TOP-DOWN AND EXTEND 1395 00:59:04,916 --> 00:59:12,557 TO OTHER MODIFICATIONS SO IT 1396 00:59:12,557 --> 00:59:13,157 IS A LOCAL BINARY COMBINATIONS 1397 00:59:13,157 --> 00:59:15,493 WHICH IS DEPENDS ON THE 1398 00:59:15,493 --> 00:59:16,961 QUESTION. 1399 00:59:16,961 --> 00:59:17,595 BUT DEFINITELY GOING TO MIDDLE 1400 00:59:17,595 --> 00:59:18,963 AND TOP-DOWN YOU BETTER BE 1401 00:59:18,963 --> 00:59:20,031 PREPARED TO SPEND MORE EFFORT 1402 00:59:20,031 --> 00:59:30,174 ON THAT. 1403 00:59:30,675 --> 00:59:31,009 AND THE PROTEASE. 1404 00:59:31,009 --> 00:59:31,609 I DIDN'T GO OVER THE COMPLETE 1405 00:59:31,609 --> 00:59:32,110 PLATFORM OF WHAT WE DO. 1406 00:59:32,110 --> 00:59:32,543 BUT FOR BOTTOM-UP WE 1407 00:59:32,543 --> 00:59:33,277 CHEMICALLY DERIVATIZED THE 1408 00:59:33,277 --> 00:59:36,547 HISTONE TO BLOCK THE LYSINE. 1409 00:59:36,547 --> 00:59:40,918 IT WILL BE NATURALLY BLOCKED 1410 00:59:40,918 --> 00:59:43,788 SO WE CHEMICALLY MODIFY TO 1411 00:59:43,788 --> 00:59:54,298 BLOCK THE UNMODIFIED AND THEN 1412 00:59:55,566 --> 00:59:56,100 THEY ARE NICELY SPREAD OUT SO 1413 00:59:56,100 --> 00:59:56,701 WITH THIS APPROACH YOU CAN GET 1414 00:59:56,701 --> 00:59:58,636 A DEAR 90 PERCENT SEQUENCE 1415 00:59:58,636 --> 01:00:02,674 COVERAGE. 1416 01:00:02,674 --> 01:00:05,443 >> I MAY HAVE MISSED IT BUT IF 1417 01:00:05,443 --> 01:00:10,148 IT IS TARGETED TO ALL HISTONES 1418 01:00:10,148 --> 01:00:12,884 OR WHAT IS TARGETING IT? 1419 01:00:12,884 --> 01:00:19,057 >> A PREVIOUS PAPER THAT I 1420 01:00:19,057 --> 01:00:19,657 MENTIONED DOES SHOW IT CAN HIT 1421 01:00:19,657 --> 01:00:24,495 HISTONE AGE THREE BUT FROM OUR 1422 01:00:24,495 --> 01:00:25,063 OWN DATA IT SEEMS H2 WAY WE 1423 01:00:25,063 --> 01:00:31,402 SEE MORE FUNDED FRAGMENTS. 1424 01:00:31,402 --> 01:00:36,174 BUT WE DO SEE A LITTLE BIT 1425 01:00:36,174 --> 01:00:37,375 EVEN IN OUR SAMPLES AND LITTLE 1426 01:00:37,375 --> 01:00:43,448 BIT OF EACH TO BE BUT IT IS 1427 01:00:43,448 --> 01:00:44,449 PROMISCUOUS AND HITTING A 1428 01:00:44,449 --> 01:00:47,685 LITTLE BIT OF EVERYTHING. 1429 01:00:47,685 --> 01:00:48,219 >> IS TARGETED TO CERTAIN 1430 01:00:48,219 --> 01:00:49,887 PARTS OF THE GENOME? 1431 01:00:49,887 --> 01:00:53,591 >> I THINK SO. 1432 01:00:53,591 --> 01:00:59,864 IT LOOKS LIKE IT IS MOSTLY 1433 01:00:59,864 --> 01:01:03,634 AFFECTING PATTERNS THAT HAVE 1434 01:01:03,634 --> 01:01:04,168 THE ACETYLATED H2 WAY AND 1435 01:01:04,168 --> 01:01:06,437 THAT'S NOT EVERYWHERE IN THE 1436 01:01:06,437 --> 01:01:10,308 GENOME. 1437 01:01:10,308 --> 01:01:10,908 AND HOW IT IS TARGETED TO THAT 1438 01:01:10,908 --> 01:01:11,476 WE HAVE NO IDEA BUT IT SEEMS 1439 01:01:11,476 --> 01:01:19,317 TO BE TARGETED TO THE 1440 01:01:19,317 --> 01:01:19,884 ACETYLATED FORM THAT IS LESS 1441 01:01:19,884 --> 01:01:21,285 THAN HALF A PERCENT OF THE 1442 01:01:21,285 --> 01:01:24,355 ENTIRE H2 WAY MARK SO THERE IS 1443 01:01:24,355 --> 01:01:24,989 SOMETHING VERY SPECIFIC ABOUT 1444 01:01:24,989 --> 01:01:27,358 IT BEING TARGETED BUT WE HAVE 1445 01:01:27,358 --> 01:01:32,096 NO CLUE WHAT THAT IS WE'VE 1446 01:01:32,096 --> 01:01:32,630 TRIED TO DO SOME IP TO SEE 1447 01:01:32,630 --> 01:01:33,197 WHAT IS BINDING BUT NOTHING 1448 01:01:33,197 --> 01:01:43,641 THAT MAKES A LOT OF SENSE. 1449 01:01:45,209 --> 01:01:45,610 MAYBE WE NEED TO USE A 1450 01:01:45,610 --> 01:01:46,177 CROSS-LINK SO WE DON'T LOSE 1451 01:01:46,177 --> 01:01:46,711 WHATEVER IT IS INTERACTING 1452 01:01:46,711 --> 01:01:46,911 WITH. 1453 01:01:46,911 --> 01:01:47,411 THAT IS A GOOD QUESTION. 1454 01:01:47,411 --> 01:01:47,979 I DON'T KNOW HOW IT IS BEING 1455 01:01:47,979 --> 01:01:48,246 TARGETED. 1456 01:01:48,246 --> 01:01:51,549 >> I HAVE A QUESTION BUT IT'S 1457 01:01:51,549 --> 01:02:01,793 MORE GENERAL. 1458 01:02:43,067 --> 01:02:43,501 WE SEE A LOT FRAGMENTS. 1459 01:02:43,501 --> 01:02:44,001 THERE COULD BE A LOT OF 1460 01:02:44,001 --> 01:02:44,535 PROTEASE GETTING INTO THE 1461 01:02:44,535 --> 01:02:47,238 NUCLEUS AND SPECIFICALLY -- -- 1462 01:02:47,238 --> 01:02:52,076 SPECIFICALLY CHROMATIN. 1463 01:02:52,076 --> 01:02:54,178 >> I THINK WE WILL CONCLUDE. 1464 01:02:54,178 --> 01:02:54,612 THANK YOU VERY MUCH. 1465 01:02:54,612 --> 01:03:04,789 [APPLAUSE]