Decoding the Non-coding Genome in Blood Development and Disorders

Transcript

• 00:00Alright everyone, good afternoon,
• 00:02let's go ahead and get started so
• 00:06everyone can get on with their day.
• 00:09It is our great pleasure today to welcome
• 00:11Doctor Jian Xu as our distinguished speaker
• 00:14of Hematology for that Yale Cooperative
• 00:16Center of Excellence in Hematology.
• 00:19We are in enjoyed very glad to
• 00:21welcome him not just on behalf of
• 00:24the Yellow Cooperative Center of
• 00:26Excellence of Hematology,
• 00:27but also for all of the Centers of excellence
• 00:31in hematology across the United States.
• 00:33As you can see, the title of his talk
• 00:36is decoding the noncoding genome in
• 00:39blood development and disorders.
• 00:40He was trained at UCLA.
• 00:42He did a postdoc at Boston Children's,
• 00:45and he's now an associate
• 00:47professor at you T Southwestern.
• 00:49His lab studies the molecular
• 00:50mechanisms that regulate gene
• 00:52expression and hematopoiesis and cancer,
• 00:54especially transcriptional enhancers
• 00:55and epigenetic regulatory regions.
• 00:56So we're just very excited to have him work.
• 01:00We're grateful he's allowed
• 01:01us to record his talk today.
• 01:03I'm at the end of his talk.
• 01:05We ask that if you have questions,
• 01:07you put your questions in the
• 01:09Q&A or in the chat.
• 01:10I think I will have difficulty unmuting you,
• 01:12so if you type your question I will
• 01:14read it and then he can answer it.
• 01:16So again,
• 01:17thank you so much and we were quite
• 01:19excited about your talk today.
• 01:21Thank you so much Jeannie for that
• 01:25really nice introduction and I want
• 01:27to sort of start up before sinking
• 01:29Pat Gallagher Anadan across for the
• 01:32opportunity and a kind invitation to a
• 01:35virtually visitor cooperative center
• 01:37of excellence of hematology at year and,
• 01:39and I'm excited to share some more
• 01:42recent work related to the study of
• 01:45noncoding genome in the context of
• 01:47blood development and disorders.
• 01:49So just a brief introduction of my lab,
• 01:53so we started the mechanism that
• 01:56regulate gene expression during
• 01:58blood cell differentiation,
• 01:59and how deregulation these process on the
• 02:03lines of development of blood disorders,
• 02:06and more specifically we aim to understand
• 02:09how we need specific transcription factors.
• 02:12Epigenetic regulators cooperate with
• 02:14environmental signals to control
• 02:17cell identity by acting on a set
• 02:19of non coding regulatory elements
• 02:21such as transcriptional enhancers,
• 02:23which can be the central focus
• 02:26of my talk today.
• 02:29So as we all know that a noncoding
• 02:32genome occupies nearly 99% of the
• 02:35human genomics space and consistent
• 02:38various regulatory elements as well
• 02:40as many of the pulley characters.
• 02:43Repetitive element an other genomic DNA is,
• 02:46on the other hand,
• 02:47from genetic studies we know vast
• 02:50majority of disease associated genetic
• 02:52variants are located in a noncoding genome,
• 02:56but identify the causal mechanism.
• 02:59Has remained a significant
• 03:00challenge for the field.
• 03:02An an.
• 03:03In select cases we know our careful
• 03:06dissection of the underlying pathways
• 03:08can often lead to new insights in Human
• 03:12Genetics and an even therapeutics,
• 03:15for example,
• 03:16by associating common genetic
• 03:18variations with certain block
• 03:20trades such as feeding him a globin
• 03:23expression levels a gene called BCL
• 03:2611 A was identified more than 10
• 03:28years ago. OK, I did as opposed to
• 03:31Federal in Boston Children's with walking
• 03:34and together with Vijay Sankar Anadem.
• 03:37Bawa have shown that this genetic
• 03:40variants that were identified forms
• 03:43you are status actually do not affect
• 03:45the coding sequences of BCL 11 A.
• 03:48But instead that a tissue specific
• 03:51enhancer for this transcription factor,
• 03:53so normally as nicely illustrated
• 03:55by Los Harrison Global Bill,
• 03:57that PC anime is activated in.
• 04:00Definitive hematopoietic cells
• 04:01by this tissue,
• 04:03especially enhancer to request fitting
• 04:06hemoglobin expression and impatient when
• 04:08the adult beta globin gene is mutated,
• 04:11such as in the context of sickle cell anemia,
• 04:15and this is permissive for
• 04:18developping as sickle cell disease.
• 04:20The GEOHASH variant basically
• 04:23functions to attenuate enhance
• 04:25activity leads to less of a PCR.
• 04:28My expression an and reactivation
• 04:30of feeling hemoglobin.
• 04:32Question 2A milli.
• 04:33Ameliorated disease symptoms.
• 04:35So this is how the genetics works,
• 04:38but this fine is not only
• 04:41established the underlying genetic
• 04:43basis of hemoglobin switching,
• 04:45but also raise the possibility that
• 04:48teach you specific enhancer elements.
• 04:51Maybe potential therapeutic targets
• 04:53and this idea actually has led
• 04:56to an ongoing clinical trials by
• 04:59Christmas Therapeutics in partnership.
• 05:01Vertex Pharmaceuticals.
• 05:02To target PCR 11 Enhancer in sickle
• 05:05cell disease and beta thalassemia
• 05:08patients using CAS 9 genome editing.
• 05:11So when you walk since first
• 05:14to mobilize our city.
• 05:16Sorry for positive hematopoietic
• 05:18stem progenitor cells from affecting
• 05:20individuals and transducer cells with
• 05:23rubber nuclear proteins containing
• 05:25cost 9 and a single godani that
• 05:27target the PCL away enhancer,
• 05:29which leads to disruption of
• 05:31the enhancer function,
• 05:33and then these edited cells.
• 05:35We are well reinfused back to the same
• 05:39patient and observe for disease phenotypes.
• 05:43As you may know,
• 05:44the result of the first 2 patient one
• 05:47sickle cell patient in one Peter Self well,
• 05:51recently reported in a paper at
• 05:53publishing Union General Medicine
• 05:55with more cases recently reported in
• 05:57the Ash American Society Hematology
• 05:59Annual Meeting just a couple weeks ago.
• 06:02So as one example showing here that
• 06:05here's the result for the first bit of
• 06:08cell patients that are treated by CAS 9,
• 06:11editing of the PCL 11 Enhancer.
• 06:14As you can see on the left graph
• 06:18before infusion of the edited cells,
• 06:21the patient had a hemoglobin level
• 06:23of nine .0 gram per deciliter with
• 06:27feeding him global level only .3 grams
• 06:30per deciliter but just a couple months
• 06:34after infusion of the edited cells,
• 06:36the feeling hemoglobin level
• 06:38increased to 6.5 gram per deciliter
• 06:41an letter 1113.1 gram per deciliter.
• 06:44With total hemoglobin increased to
• 06:4714.1 and this is accompanied by nearly
• 06:51100% F cells or these feeding him
• 06:54globin positive cells and up to 18
• 06:57months of follow up so this patient
• 07:00is clinically cured technically,
• 07:03therefore this proof of principle
• 07:06studies are very encouraging not only
• 07:09to show that therapeutic targeting
• 07:12disease associated enhancer elements can.
• 07:14Likely provide a cure for the most
• 07:17common monogenic disease that was first
• 07:20described more than a century ago,
• 07:23but also open up new ideas
• 07:25and opportunities for people
• 07:27to understand biology and more important,
• 07:30it develop approaches to target this disease.
• 07:33Associated noncoding regulatory elements.
• 07:36So. First question we ask when
• 07:39when I started my lap at you T
• 07:43Southwestern is what are enhancers?
• 07:45How enhancer was initially discovered?
• 07:47So Enhancer was first
• 07:48discovered nearly 40 years ago,
• 07:50an actually from a viral genome as a shot.
• 07:54Deer sequences in the SV 40 Gino
• 07:56that can enhance the expression of
• 07:59a rabbit bitter globin gene in the
• 08:01orientation independent manner.
• 08:03In a transient transfection
• 08:05assays soon after the 1st century
• 08:07enhancer was discovered.
• 08:09From the mouth immuno globin gene.
• 08:12In a tissue specific manner so therefore
• 08:15even from the very early days enhancer
• 08:18at defined as SESAC ingenious sequences
• 08:22that function at often at a distance to
• 08:25activate gene transcription in orientation
• 08:28independent and tissue specific manner.
• 08:30As such, it has been very
• 08:33difficult to studying answers,
• 08:34and an still remains very tickled.
• 08:37To identify enhancer target
• 08:38genes and their in vivo function,
• 08:41largely due to the lack
• 08:43of experimental tools.
• 08:44So therefore there are several
• 08:47important questions enhancer biology
• 08:48that remain to be addressed Anan.
• 08:51This is some of the questions that we are
• 08:53very particularly interesting excited about,
• 08:56and I would like to share some recent
• 08:59work that we have done trying to address
• 09:03some of these important questions.
• 09:05He has a biology and particularly
• 09:08how to identify enhances and housing,
• 09:10has a regular their target gene
• 09:13expression and how it has it themselves.
• 09:16Regulated and finally,
• 09:17how do he has alterations contribute
• 09:20to diseases,
• 09:21typically in the context of
• 09:25hematopoietic malignancy?
• 09:26So the first question is how
• 09:28to identify Hazard.
• 09:30This has been challenging,
• 09:31but with the advances in next Gen
• 09:34sequencing technologies coupled with an
• 09:36analysis of variety of chromatin features,
• 09:39we can now easily annotate these
• 09:42noncoding genome using a number of methods
• 09:45such as chip sequencing as you know,
• 09:48to examine protein,
• 09:49DNA interactions,
• 09:50air taxi or dinner sequencing to
• 09:52examine chromatin Accessibility as
• 09:54a surrogate for open property, or.
• 09:57Active transcription and more recently
• 09:59chromatin confirmation capture
• 10:01or three CBEST methods to examine
• 10:04high order chromatin structures.
• 10:06So now we can.
• 10:08Fairly easily to identify putative
• 10:10enhances other regulatory elements in
• 10:12a systematic manner and using these
• 10:15tools are we can use a simple combination.
• 10:19We enhance associated histone Marks
• 10:21and chromatin Accessibility to identify
• 10:23putative active enhancers or other
• 10:25regulatory elements across the Gina.
• 10:27So using our favorite local that human
• 10:30beta globin gene cluster as example.
• 10:33As you can see from this genome browser,
• 10:36tracks that we can easily identify
• 10:39the upstream enhancer.
• 10:40Pass this on Locus control region
• 10:42or else are by the presence of
• 10:45dinners will have sensitivity.
• 10:48He can 27 simulation and
• 10:50HCC for modern isolation.
• 10:51On the other side you can also
• 10:54identify active promoters by
• 10:56the presence of translation.
• 10:58At 3K four there's all done in primary
• 11:01humour as well progenitor cells.
• 11:04So using this approach,
• 11:06we and others have previously compared
• 11:08enhancer landscapes between human primary,
• 11:11hematopoietic stem progenitor cells
• 11:13and differentiated industry sales,
• 11:15and we notice that initially that
• 11:18enhances undergoes a pretty progressive
• 11:20turnover during limits differentiation,
• 11:23such that about 2/3 of the enhancer
• 11:26that were found in undifferentiated
• 11:28City City for positive cell or
• 11:31loss and replaced by a similar
• 11:34number of Linux specific enhancers.
• 11:37In just a few cell divisions,
• 11:40we then identified transcription
• 11:42factors and their combinations that
• 11:44are required for this image and a
• 11:47developmental stage specific enhance
• 11:49activities and I should say that similar
• 11:51results obtained by elegant studies
• 11:53for pack animals group here at the Yell
• 11:57and Ross Hardison, and many others,
• 12:00in different model systems.
• 12:01And this really has provided a
• 12:04useful resource for the community
• 12:06to understand the regulatory.
• 12:08Basketball hematopoietic
• 12:09cell differentiation.
• 12:10So with this increasing availability
• 12:13of this large genomic data set,
• 12:15so we wonder whether we could use
• 12:18this information to understand
• 12:19how it has a regular,
• 12:21their target genes blushing.
• 12:23Anne,
• 12:24this let us to focus a set of these
• 12:27linear specific enhancer clusters or
• 12:29super enhancers as some people call it,
• 12:32including one showing here that's located
• 12:35upstream of the gene call SLC 25 S 37,
• 12:38which is a includes iron transporter
• 12:41critical for hematopoietic cells.
• 12:43So as you can see,
• 12:45these tissue specific enhancer that
• 12:47are conserved between mouse and human
• 12:49are contains 3 individual enhancers
• 12:51that are virtually indistinguishable
• 12:53in terms of histone Marks and
• 12:55transcription factor binding.
• 12:56In this case got one.
• 12:58Inhuman amounts are extra cells so
• 13:01therefore the critical question
• 13:03we had initially was how do these
• 13:06individual constitute enhances
• 13:07contribute to the function of
• 13:09this super enhancer as a whole.
• 13:11So we decided to use CRISPR
• 13:13knockout to knockout each individual
• 13:15enhances and their combinations.
• 13:17We found that quite surprisingly that
• 13:20this thing has a seems to be organized
• 13:24as a functional hierarchy such that
• 13:27not card enhances 3 E 3 or the most.
• 13:29This thing has us completely
• 13:31abolished in has activity,
• 13:33whereas knockout the other two
• 13:35neighboring hazards had little effect.
• 13:37And this was also seen by others in
• 13:40different hazard clusters and cell types.
• 13:42So therefore the key question is what
• 13:45is unique about this enhanced E3?
• 13:47In fact they are hard to distinguish
• 13:49based on the chromatin features
• 13:51and how to identify.
• 13:53This seems to be functionally more
• 13:55important or or predominant enhancers in
• 13:57super enhancer function and more importantly.
• 14:00This list another important question
• 14:02that we've been studying is how
• 14:04enhancer themselves are regulated
• 14:06or organized in native quality.
• 14:08In, in, in, in prime itself.
• 14:11So you attempt to address this question.
• 14:14We thought that we will ideally be able
• 14:18to isolating hazard from this native
• 14:21quality environment and bisector.
• 14:23Regularly composed compositions.
• 14:24So we ended up engineering are
• 14:27inside your capture assay by
• 14:30leveraging CRISPR CAS 9 technology.
• 14:32So briefly many of new by combining a
• 14:36gene specific garden sequence with a
• 14:39nucleus in activity cost 9 or D CAS 9.
• 14:43The crisper cast 9 Rd CAS 9 can
• 14:45be targeted
• 14:46to the proximity of of any answer
• 14:49that you might be interested
• 14:51or other regulatory elements.
• 14:54We then met further
• 14:55modifications of the system,
• 14:57including adding an epitope tag
• 14:59that can be enabled by terminated,
• 15:02then using high affinity stood
• 15:04averaging biotin based application.
• 15:06We can isolate the enhancer complex and
• 15:09all associated molecular interactions
• 15:10and determine has associated proteins.
• 15:13Amaze and DNA complexes by proteomics
• 15:17and next generation sequences.
• 15:20So as a proof of principle this approach
• 15:23we started with human telomeres,
• 15:25which contains many copies of repetitive's
• 15:29at telemetric repeat sequence that
• 15:31can be targeted by a single God on it.
• 15:34Upon inside your capture,
• 15:36we found out that email can be
• 15:39labeled by image Ng if you fuse,
• 15:41because now with the GOP molecule
• 15:44for example also by Q PCR,
• 15:46we identified chilling your
• 15:48DNA are highly enriched,
• 15:49ANAN more important by Western blot and
• 15:52subsequent in mass spec based pre omics.
• 15:55We can identify many of the noise,
• 15:57artillery,
• 15:58associated proteins and many of the
• 16:00unknown factors that she annualizing
• 16:02to how telomere may be regulated.
• 16:04This is a highly repetitive sequences.
• 16:07What about a single copy?
• 16:09Locals in the human genome?
• 16:11So we choose to focus on our favorite.
• 16:14Locals are human beta globin gene
• 16:16cluster as you know that these
• 16:18cluster contains a set of five beta
• 16:20like globin genes that undergoes
• 16:23developmental switching doing.
• 16:25Insurance cell differentiation and all
• 16:27these things are controlled by a shield.
• 16:29Locals control raging or super
• 16:31enhancer if you wish and look at
• 16:34it upstream of this gene clusters.
• 16:37So using a single godani that
• 16:39are designed to be specific for
• 16:42the three Prime HS,
• 16:43one insulator element we found by
• 16:46chip sequencing of the DCAS 9 capture
• 16:49DNA that only three primary chest one
• 16:52and no other regions and across loci,
• 16:55is highly enriched.
• 16:56Similarly,
• 16:56if you use a gardener targeting HPV,
• 16:59you will see a single peak antagony
• 17:02HPP promoter regions we know in
• 17:05the human genome the gamma gene is.
• 17:08Duplicate it at pgy and beaches.
• 17:10SVG two, so therefore a single God,
• 17:13an actual capture both,
• 17:14and that's exactly what we're seeing,
• 17:17and then we can simply by design
• 17:19different God.
• 17:20Honest, having different regulatory elements,
• 17:22enhancers for example,
• 17:23and we can capture each individual enhances.
• 17:26Using this card on is,
• 17:28and more importantly,
• 17:29if we pull all these God honors
• 17:31together an echo expressing the same
• 17:33cell that we can capture all five
• 17:35enhancers suggestion that this system
• 17:37can be multiplexed to capture many
• 17:40enhances at the same time in the same cell.
• 17:44So based on these findings,
• 17:46but we went ahead,
• 17:47developed a capture proteomics
• 17:48to determine decathlon capture,
• 17:50look locals are specific podium and
• 17:52identify in this case again using
• 17:55beta globin gene cluster as example
• 17:57that we identify manufacturers
• 17:58that are social with,
• 18:00for example HS2 enhance at the
• 18:03LCR region including some familiar
• 18:05faces like God.
• 18:06I want a long beyond the four
• 18:08etc and but also some other new
• 18:11factors that we decided to follow
• 18:14up on. Such as a nuclear pore proteins
• 18:16and you can see some of these factors
• 18:19also present at the gamma globin gene
• 18:22promoters and other factors are present in.
• 18:25Beta globin gene clusters.
• 18:28So this analysis provides the initial
• 18:31evidence for the composition based
• 18:34organization of the beta globin
• 18:36gene enhancers and promoters.
• 18:39We all know that the human genome is
• 18:42organized into a multilayer structure.
• 18:44Units are organized by Long
• 18:46range dinner interactions,
• 18:47or chromatin looping,
• 18:48so we never saw that.
• 18:51Can we combine this DCAS 9 capture
• 18:53with three C analysis to identify local
• 18:56specific long range thing interactions?
• 18:58So the way it works is we first using
• 19:01in vivo bite Internet DCAS 9 to
• 19:04isolate enhances that you might be
• 19:07interested and Cromartie is crosslinked.
• 19:09Followed by enzyme digestion.
• 19:11Usually we use frequent Cadillac
• 19:14DPM two and this is followed
• 19:17by a proximity ligation of the.
• 19:19Adjusted genomic DNA Anan followed
• 19:22by fragmentation and then the
• 19:24enhancer can be directly isolated
• 19:26using strip having best purification,
• 19:29ANAN together with all the DF
• 19:32fragment that will really get it,
• 19:34and using pale and sequencing we
• 19:37can identify all the long range
• 19:40interactions that are social with
• 19:42this target enhances and more
• 19:45importantly by combining capture
• 19:46as we see analysis with capture.
• 19:49Baryonyx we hope to be able to
• 19:51identify causality.
• 19:52What are the factors that might
• 19:55be responsible for this Sweetie
• 19:57Genome organization?
• 19:58This is just showing one of the
• 20:00example of the data how this works,
• 20:03so here we're showing capture 3C
• 20:05analysis for the active HP Gigi.
• 20:07This was done in Cape RC2 sales.
• 20:10Now you will see that is active
• 20:12Edge BGM contains many long range
• 20:14interactions with other six element
• 20:16across the low side but not the
• 20:19nearby repressed genes.
• 20:20But first I if you capture the
• 20:22repressions HPB you will see the
• 20:24HPV forms of predominant shorter
• 20:26range interactions and downstream
• 20:28sweeper matches one insulator.
• 20:30There's no interaction between active
• 20:32and repressed genes on when we capture this.
• 20:35We next capture each individual
• 20:37in hazard within,
• 20:39else are an interesting and we
• 20:41found that H S3 has a contains
• 20:43many more long range interaction
• 20:45then other nearby enhancers,
• 20:47including access to and.
• 20:49We further validated this using
• 20:51independent God honest and we
• 20:53notice that H3 has a consistently
• 20:55contained many more interactions
• 20:57than the nearby actions to enhance,
• 21:00and this was interesting to us because
• 21:02it's just too has been sought to be
• 21:06the strongest enhancer within LCR.
• 21:08By transgenic enhancer Reporter,
• 21:10access YH Three was also shown
• 21:12to be important,
• 21:13but only in the context of native quality.
• 21:16So therefore our findings may help
• 21:19to explain these findings and
• 21:21support a model that the hierarchy
• 21:23organization with a beta globin LCR
• 21:25in which that itches to my function
• 21:28to recruit a trans acting factors and
• 21:30function more like a conventional enhancer,
• 21:33yhs 3 might might be more important,
• 21:36immediate and long ranged in actions of.
• 21:39Formative,
• 21:39loopy,
• 21:39and really,
• 21:40it's a combination posing hazard.
• 21:42Am I dictating what the locals control?
• 21:45Regional Super Enhancer will
• 21:47function in its native quality.
• 21:50So as a brief summary,
• 21:52so we think that this decathlon
• 21:54capture my provider are useful
• 21:56tool for multi omic dissection
• 21:59of this regulatory elements from
• 22:01their endogenous loci and this
• 22:03biotin scribbling based affinity
• 22:05capture provides high specificity
• 22:07and sensitivity and this SGI based
• 22:10targeting allows for Multiplex
• 22:12analysis which are going to show
• 22:14in a minute and more importantly
• 22:17the simultaneous analysis of local
• 22:19specific proteome and 3D interactions.
• 22:21Help to establish causality and
• 22:24the system should be brought it
• 22:26up application applicable to other
• 22:29genomic locals or model systems.
• 22:31However,
• 22:32one of the major limitation of
• 22:34the original capture Mesa was that
• 22:36it requires large number of cells
• 22:39and typically 10s of sometime
• 22:41hundreds of millions of sales for
• 22:43local specific proteomic studies,
• 22:45and thus was not applicable to primary
• 22:48cell types or male cell populations.
• 22:50So we wonder whether we could improve
• 22:53the design to increase capture
• 22:55efficiency so you know original design.
• 22:57We fuse the biotin tag to the
• 23:00N terminus of the kastein.
• 23:02We just close to the nucleotide
• 23:05protein interacting pocket that
• 23:06might cause arepa tomaski,
• 23:08whereas the C terminus of CAS 9
• 23:11proteins largely exposed an unstructured.
• 23:13So we have tried tagging the seat
• 23:16owners instead for by connection.
• 23:18Moreover, in our original design,
• 23:20we use the conventional every tag
• 23:23together with the bacteria puje biotin
• 23:25ligase for in vivo bike nation.
• 23:27So this will make a three vector system.
• 23:31However there are other.
• 23:32Peptide sequence that can be bought
• 23:35in an area using endogenous biting
• 23:38like is that expressing my man in
• 23:40sales so we have been testing some
• 23:42of those sequences and more recently
• 23:45approximately biting ligands such as APEX.
• 23:47Two was shown to be able to buy
• 23:49terminate nearby proteins on comity
• 23:51which may increase the signal to noise
• 23:54ratio for identification by proteomics.
• 23:56So we have been trying and
• 23:59adapting those systems for capture.
• 24:01So with this improved capture system
• 24:04with first attempted Multiplex capture,
• 24:06many answers in a single experiment,
• 24:08so we started with the LCI enhancer
• 24:11with 10 individual gardeners to
• 24:13capture all 5H S enhances and observe
• 24:16interactions social with all enhancers
• 24:18in a single experiment and more
• 24:20importantly you actually can resolve
• 24:22this data into a single enhancer resolution.
• 24:25Look at what other enhancer mediated
• 24:27by each individual enhances,
• 24:29as you may recall,
• 24:31the pattern looks almost the same.
• 24:33As we did previously for the
• 24:36individual capture,
• 24:36you negating that multiplicative
• 24:38multipliers capture.
• 24:39We tend the native permitting architecture.
• 24:42So based on this,
• 24:44we next performed much less capture
• 24:47of actually super enhancers.
• 24:49Using this super enhancer calling algorithm,
• 24:52and in this experiment we use nearly
• 24:542000 God on his targeting more
• 24:57than 150 super enhances containing
• 25:00more than 800 individual enhances.
• 25:03A very large panel.
• 25:04And that data shows that we can
• 25:07capture the vast majority of that
• 25:10enhances in a single experiment.
• 25:13So with these high resolution
• 25:16multiplus capture,
• 25:16we notice that some interesting features
• 25:19about this special configuration of
• 25:22these super enhancers, specifically,
• 25:23we can often identify one or few
• 25:26enhances within a super enhancer
• 25:29that have unusually higher frequency
• 25:31of interactions compared to other
• 25:34nearby enhances we call this.
• 25:36He has a happy hanses.
• 25:38We further developed a computational
• 25:40workflow and a fund at about
• 25:421/4 of the Super enhancers are
• 25:44organized as a hierarchy structure
• 25:46containing hopping answers,
• 25:48and here just showing a snapshot of
• 25:511 example of a hierarchy of super
• 25:53enhancer that contains a hopping
• 25:55answer that you can see that has
• 25:58more frequent interactions with
• 26:00other enhancers and promoters
• 26:02within the same genomic region,
• 26:04and what I won't show you the
• 26:06data we actually went ahead using
• 26:08crisper cast 9 to knockout.
• 26:11These hopping hazard.
• 26:13Versus the nearby non hop enhances and
• 26:17almost all the time that we can see,
• 26:21knockout hopping has at least a more
• 26:24profound effect and target gene
• 26:26expression comparing to knockout
• 26:29an unhappy hazards suggestion that
• 26:31this enhancer might be functionally
• 26:34more potent enhances that.
• 26:36Functioning within a super has a
• 26:39gene cluster. So as a brief summary.
• 26:42So here we showcase our several
• 26:44potential implications for the D
• 26:46cast net capture system for high
• 26:48resolution Multiplex analysis and
• 26:50local specific quality interactions.
• 26:52We hope that the capture system can
• 26:55enable us to determine a special
• 26:57configuration enhances and their
• 26:59target genes,
• 27:00as well as a temporal regulation
• 27:03during development.
• 27:04So with this,
• 27:05we started to one of the next important
• 27:08question that that would be interested in is.
• 27:11How do we have to actually control tagger?
• 27:15Jinx,
• 27:16brushing?
• 27:16Inside to it,
• 27:17particularly doing in people development.
• 27:20And this has not been trivial.
• 27:22Trivial as you can imagine,
• 27:24you can knockout this individual enhancers,
• 27:26but offering you're not getting answer.
• 27:29Look at in a cell grows best answer,
• 27:32you don't really see a difference
• 27:34is so we thought that to address
• 27:36this we will need a tool that
• 27:38allow us to systematically bisector
• 27:41functional requirement of this
• 27:43enhances in their native comity.
• 27:45Ideally doing in people development
• 27:47and such as human quality limits,
• 27:49differentiation so.
• 27:50We recently redesigned the Cast 9
• 27:53best so called epigenetic editing
• 27:56system that can efficiently perturb
• 27:59enhancer activities by modulating
• 28:01enhance associated chromatin features.
• 28:04Particularly careful and K 27 simulation
• 28:07specifically for enhanced activation,
• 28:09refused ecaster with P300,
• 28:11which catalyze H3K27
• 28:13assimilation within adapted,
• 28:15helping actimel Ms 2 on the
• 28:18SG and sequence to recruit.
• 28:21Another activated VP 64 similarly for
• 28:24the repression that refused the cast.
• 28:26Now with LC-1 which catalyze
• 28:28the removal of H3K,
• 28:30four born monogamous flashing
• 28:32and then as geometry,
• 28:34could another repressor correct,
• 28:36so therefore the main advantage of
• 28:39this system which we call increased
• 28:41by or increase for a or enhancer
• 28:44targeting increased bio quiz for a is
• 28:47that this system combines effectors
• 28:49with distinct mechanism for modulating.
• 28:51Enhancer associated genetic marks.
• 28:53So we have used this system in
• 28:56a variety of cell models and
• 28:59showing that the
• 29:00new system is superior than the
• 29:02original crisper I whisper in
• 29:05system for enhancing perturbations.
• 29:06However, the main challenges analysis of
• 29:08enhancer function doing illegal development.
• 29:11To address this, we are when I
• 29:13generate a knock in mouse model.
• 29:1610 D CAS. Nine care app converging
• 29:18under the docs inducible promoter
• 29:20in College in one in one low side.
• 29:24So we then developed by in Vivo Enhancer
• 29:27perturbation asset you then there's no
• 29:29key mouse to determine hazard function.
• 29:32Doing him at opposes so briefly the
• 29:34way it works is that we reasoned
• 29:37that by ABBA genetic modulation
• 29:38of Venus physic enhancers in HCS
• 29:42followed by pulmonary transplantation.
• 29:44We could access the HSE deriving
• 29:46mature cell images.
• 29:47As a read out for the functional
• 29:49impact of he has a population
• 29:52doing HSC Danish differentiation.
• 29:54So that way you would do it.
• 29:57You will isolate him out of politics.
• 29:59Tampa general sales from this
• 30:01knocking Mouse and then transduced
• 30:03with God only library with Garnet
• 30:05design against each individual
• 30:07enhancer that you might be interested
• 30:09followed by bone marrow transplant.
• 30:11Then D Kastner Kara will be induced
• 30:14by docs admin administration in
• 30:16the period of 12 to 16 weeks.
• 30:19Then you want to isolate a
• 30:22mature cells cells and perform
• 30:25amplicon sequencing to determine.
• 30:28The abundance of SG and a in a starting
• 30:32population before transplant an the
• 30:34resulting population after transplantation.
• 30:37So as a proof of principle this we
• 30:39focus on several key hematopoietic
• 30:41transcription factors and their
• 30:43annotating enhances an and.
• 30:45Design multiplies got an A plus for
• 30:47in vivo enhancer population squeeze,
• 30:49so I would like to show you the CBF
• 30:52and locals as a first example which
• 30:55contains out for annotating hazards,
• 30:57look at it in a different upstream
• 31:00or downstream regions to the
• 31:01transcription start site by enhance
• 31:03in billing has a population.
• 31:05We found that SGML for CDL 4 plus.
• 31:08So disable.
• 31:10Enhancer all year for Enhancer in this.
• 31:17A significantly depleted in mylow
• 31:19cells but not in B&T cells.
• 31:21This is interesting,
• 31:23as previously has been shown that
• 31:25CBF on gene knockout or the plus 37
• 31:29Hanson AKA annoying to be required
• 31:31by more minor cell differentiation
• 31:34but not for lymphopoiesis,
• 31:35so our result not only validate
• 31:38these findings,
• 31:39but also show that the plus a
• 31:42keeping hazard is E2 enhancer
• 31:44showing over here are also important.
• 31:47For modeling differentiation,
• 31:48but the other two enhancer seems
• 31:50to be indispensable,
• 31:51and none of these enhancer seems to be
• 31:55important for B&T cell development.
• 31:57The second example is a SPI one
• 32:00locus or PU .1 G,
• 32:02and we observed this gene has a single
• 32:05enhancer located at 14 KP option.
• 32:07With this gene we notice that the
• 32:10three independent guard on it
• 32:12against its enhancer significantly
• 32:13depleted in my loiselle and B cells,
• 32:16but not in T cells, and this result,
• 32:19again are consistent with the role of SPI.
• 32:22One Pu .1 for normal Milo and
• 32:25busier development,
• 32:25but not for teacher development, so again.
• 32:28I did that this initial genetic studies.
• 32:32Using mouse models.
• 32:34And next time I want to show is that
• 32:37the wrong Swan Locust so runs well?
• 32:40Actually have two annotated
• 32:41transcription start site with three
• 32:43enhancers at different genomic regions.
• 32:45Again using this perturbation we find that
• 32:47none of the enhancer seems to be important.
• 32:50Actually, in myeloid B&T cells,
• 32:51but the promoter garden is
• 32:53somewhat you reached in my low
• 32:55self but depleted and B&T cells,
• 32:57so this is one of the first example.
• 33:00We actually see the opposite
• 33:01phenotype in different cell images
• 33:03and this is interesting because
• 33:05ranks will knock out your mouth.
• 33:07Has shown to to develop Mylar
• 33:10preffective phenotype characterized
• 33:12by myeloid enhanced my lawyer.
• 33:15Refreshing but defective B&T cell
• 33:18maturation so therefore our results
• 33:21also consistent with this and
• 33:24recapture the phenotype of ranks.
• 33:26One deficiency showcasing that this
• 33:29crisp Ohio increased by best epigenetic
• 33:33editing and can be convenient assay too.
• 33:37Isaca regulatory elements that
• 33:39are required for this linear
• 33:41specific transcription factors.
• 33:43And finally,
• 33:44we perform a Multiplex perturbation
• 33:46by pulling all the God are
• 33:49made in a single experiment.
• 33:51Anan by this analysis.
• 33:52Now you can have a ranking order of
• 33:55different enhancers and promoters that
• 33:58are specifically enriched or depleted.
• 34:00In my lawyer B&T cells,
• 34:02for example with Dan Ified that
• 34:04CB back on cartoon has a required
• 34:07for myeloid differentiation,
• 34:09while ranks one enhancer promoters
• 34:11are required for PMT sales.
• 34:13Therefore, this enhanced CRISPR editing.
• 34:15I could provide a useful tool for
• 34:18functional interrogation of SIS
• 34:20element doing illegal development
• 34:22and more importantly by combining
• 34:24these knocking mouse model with
• 34:26other disease models we might be
• 34:29able to study enhancer function
• 34:31doing different biological process
• 34:33of pathological process and this is
• 34:36something that we're very excited about
• 34:38and certainly looking forward to any.
• 34:41Collaborations whom I simply
• 34:43store might be useful for their
• 34:45respective disease models.
• 34:47So as a brief summary for for this part,
• 34:51so we've shown that enhance the
• 34:54control limits, differentiation,
• 34:56disease,
• 34:56phenotype and undergoes profound
• 34:58turnover during development.
• 34:59We've developed the Constine best capture
• 35:02tools for multimeric analysis of local,
• 35:05specific quality interactions.
• 35:06We also redesign enhancing target,
• 35:08increase pain response system that
• 35:10will enable in vivo functional
• 35:12interrogation of enhancer.
• 35:14As we have other success element doing in.
• 35:18We want development and we're
• 35:19happy to share the tools or the
• 35:22construct has been deposited action.
• 35:23And if you are any of your
• 35:26colleagues are interested,
• 35:27feel free to reach out.
• 35:30So in the last few minutes I want
• 35:32to switch gears a little bit and I
• 35:35want to discuss some of the recent
• 35:38effort we trying to address the last
• 35:41question that is how to pathological
• 35:43enhance alterations contribute to diseases,
• 35:45particularly the development block answers.
• 35:48As we know,
• 35:49much of our knowledge on cancer driver
• 35:52mutations is based on alterations
• 35:54of protein coding sequences and
• 35:57little is knowing whether man,
• 35:59how noncoding alterations may contribute
• 36:02to disease on passive biology,
• 36:04especially in the developer
• 36:06hematopoietic malignancies.
• 36:07So we started this rather
• 36:09ambitious project several years ago
• 36:12with the goal to identify leukemia,
• 36:15associating handsome mutations
• 36:16by targeted sequencing.
• 36:18So the way it works is that we first
• 36:21annotated plot Vinny specific enhancers
• 36:24based on chip sequencing and a taxi,
• 36:27just like everybody else is doing that,
• 36:30I introduced earlier and then we can gather.
• 36:34This game is almost twenty 2000s
• 36:36of enhancers that are present
• 36:39in variety of different normal.
• 36:42Gmail cell lines that we have gathered
• 36:44and then we can design target sequencing
• 36:48panel to specifically resequence
• 36:50the enhancer sequences in a panel
• 36:53of human hematopoietic malignancy
• 36:55is a particularly in an email.
• 36:58An NDS conditions.
• 37:00We also included some informal samples
• 37:03an acute lymphoblastic leukemia and,
• 37:05importantly, thirty. One of these samples.
• 37:08We actually have tumor normal
• 37:10pairs that we can.
• 37:12Identify somatic mutations in
• 37:14the in the non coding Gina.
• 37:17Pen and if you do this and like other
• 37:20people doing a protein coding sequence
• 37:22as well you you can easily identify
• 37:26thousands of recurrently mutated mutations.
• 37:29In this case we identify almost
• 37:31slightly over a 4000 frequently
• 37:34mutated noncoding elements.
• 37:36We call them mutational Hotspot Ann and
• 37:39these overlays about 1800 enhances that
• 37:41we have identified from the initial steps.
• 37:45So these are the enhancement
• 37:47carries somatic mutations.
• 37:48That are frequently mutated in
• 37:50human hematopoietic malignancy.
• 37:52The key question is and the key challenges.
• 37:55How do you know whether they are
• 37:57functional an what is how to access their
• 38:01functional roles in cancer pathogenesis?
• 38:03So Fortunately with the crisper a quiz
• 38:05by system that we have engineered that
• 38:08especially for enhances so we could
• 38:10perform a functional interrogation of
• 38:13functional perturbation screens using God.
• 38:15RNAs that are designed to
• 38:17target this enhances.
• 38:19Then we perform the screening and
• 38:21multiple leukemia cell lines.
• 38:23And by this we can identify hundreds of
• 38:26enhances that seems to be a putative tumor.
• 38:29Suppressive or uncle genic.
• 38:31In other ways,
• 38:32we use cell growth as a reader so that
• 38:35that perturbation of this enhances often
• 38:38can lead to an enhanced or inhibited
• 38:41cell growth phenotype and this really
• 38:44have provided a number of candidate
• 38:47enhances an associated genetic loci.
• 38:49Subsequence studies.
• 38:49So I would like to focus on one
• 38:53of the enhancer that we follow up
• 38:56with more detailed analysis and
• 38:58this enhances located about 150
• 39:00KB upstream of the gene called
• 39:03carrots that contains several non
• 39:05coding variants in AML samples,
• 39:06and by chromatin profiling and 3D
• 39:09chromatin confirmation capture.
• 39:10We found this thing has it seems to
• 39:13be physically interact with Akira's
• 39:15promoter regions which is located about
• 39:17150 KB downstream of this enhancer.
• 39:20And more importantly,
• 39:21when we use CRISPR CAS 9 to knockout this,
• 39:25he has a in a leukemia cell line
• 39:27and we found that Carros expression
• 39:30was significantly down regulated.
• 39:32But none of these other genes within
• 39:34the same neighborhood affected
• 39:36suggestion at this.
• 39:37Enhancer is selectively required
• 39:40for the expression of casting.
• 39:42So is this was interesting because
• 39:45unlike any grass care US,
• 39:48protein coding mutations are rarely
• 39:50found in human animal patients.
• 39:53However, high care,
• 39:54high level of care as expression in
• 39:57AML is associated with poor survival
• 40:00using data from the TCG cohorts.
• 40:03So we thought that Miss May identify
• 40:07potential functional enhancer that
• 40:09plays a role in a male biology. So too.
• 40:13Establish the functionality of whether
• 40:15not is kerosene has is important for AML.
• 40:18We generate Caroline Hanson
• 40:20AKA AML cell line.
• 40:22This is Daniels is selling called Mkpo
• 40:25one and observe the significant less
• 40:28tumor burden in bone marrow cells and
• 40:31blood of the Xeno graft recipient.
• 40:33Using two independent enhancer
• 40:35single Cell developed in Hazen
• 40:38Okok looms as a control,
• 40:40we also knockout the carrots
• 40:42protein coding gene.
• 40:43And we see that he has a knockout
• 40:47almost recalculate the care as GM
• 40:49lockout in this access and just
• 40:52showing more data showing that this
• 40:55is reflected by Les Plus constant
• 40:57prefer block as well as less.
• 41:00And number of frequency of the
• 41:03Premier sales in xenograft animals
• 41:05in the bone marrow and blood.
• 41:08So this result demonstrator care us
• 41:10and this semantic enhance associated
• 41:12enhancer are required for AML cell
• 41:15grows in virtual and an illegal.
• 41:17So then we went ahead.
• 41:19Get the motif analysis and found
• 41:22this recurrent carers enhancer that
• 41:24we found the AML patient highly
• 41:26colocalized with binding site
• 41:28of interest in nuclear hormone
• 41:30receptors in particularly par,
• 41:32gamma and ice are showing over here.
• 41:35I'm so interested in the cameras
• 41:37in has are also found in has
• 41:40mutations are also found in other
• 41:43cancer types based on TCG and pain.
• 41:46Cancer pan cancer data set here showing
• 41:48this is a leukemia reputation that we found,
• 41:52but hole in has actually carries
• 41:54many recovery mutations.
• 41:55Many of these mutations are also
• 41:58overlap with predicted nuclear
• 41:59hormone binding sites,
• 42:01and this was interesting as nuclear
• 42:03hormones are family of lichen
• 42:05regulated transcription.
• 42:06Factors that are activated by hormones,
• 42:09ligands or growth factors and
• 42:12usually in the absence of ligands,
• 42:15PPL comma AXA dimer will recruit
• 42:18Corey Presser complex immediate
• 42:20derepression appan liggen binding.
• 42:22This time are actually cutco
• 42:25activist coactivators through
• 42:27activating transcription.
• 42:28So we wonder whether cameras enhance
• 42:31it might actually be regulated
• 42:33by nuclear hormone signaling,
• 42:35so to validate days we perform chip
• 42:39sequencing analysis in multiple AML
• 42:41cell line as well as non email.
• 42:44Tumor cell lines.
• 42:45We found a strong PPR gamma and I
• 42:49saw binding at the very associated
• 42:52enhancer element and if you zoom in
• 42:54you can see some of the speaker really.
• 42:57Directly overlapping the variant that
• 43:00that was found in the AML samples.
• 43:04To directly test whether this email
• 43:06associated non coding variants
• 43:08indeed modulate nuclear hormone
• 43:10binding and enhancer function,
• 43:12we went ahead,
• 43:13generate a knocking audio and this
• 43:16was not trivial.
• 43:18That efficiency is still not very high,
• 43:21but anyway we achieved by knocking
• 43:24by Christmas targeting of the either
• 43:26the white type or the mutant value in
• 43:29two K562 leukemia cells within screen.
• 43:32Single cell derived knocking
• 43:34clones and measure nuclear Homer
• 43:37by name by chip experiment.
• 43:39So the data showing on the top right
• 43:42corner so you will see in the input
• 43:45dinner you will see this expecting
• 43:47one to one ratio of the whiter
• 43:50versus knocking earlier because we
• 43:52generate the heroes actors knocking
• 43:54values in in this cell lines.
• 43:56However, if you look at abundance
• 43:58of this of the ratio of the
• 44:01knocking mutually versus Vytorin,
• 44:02the chipped in and you see the knocking
• 44:05mutant value are significantly enriched,
• 44:07and this suggests that the mutant value.
• 44:10Actually have stronger Association
• 44:12with the nuclear hormone receptors
• 44:14by chip experiment and consistent
• 44:16with this and nuclear agonist,
• 44:18I can list more enhanced CARROS expression
• 44:21in a knock in sales compared to the
• 44:24wild type cells against suggestion that
• 44:27these mutant assume associated cameras
• 44:30enhancer are regulated by nuclear
• 44:32hormone receptors and this recurrent
• 44:34mutations my function to enhance
• 44:36nuclear hormone receptor binding.
• 44:38To trans activate Cara sticks.
• 44:41Watching you email sales.
• 44:43So we also valid that is finding other
• 44:46enhances that I won't have time to show
• 44:49an including a interesting Lee has a
• 44:52controlling our security engine code,
• 44:53PO2 this is all publisher
• 44:55if you're interested.
• 44:56You're more than happy to
• 44:58read about the details so.
• 45:00Moreover, we are in a global analysis.
• 45:03We found his nuclear hormone,
• 45:05our finest artist seems to be frequent
• 45:08targets of noncoding mutations,
• 45:10in particular email,
• 45:11but also other hematological malignancy,
• 45:13suggestion,
• 45:13perhaps a more generalizable mechanisms.
• 45:16So therefore we seek our findings
• 45:18support model that is pathogenic and non
• 45:21coding variants are noncoding mutations,
• 45:23might cooperate with signal
• 45:25independent transcriptional machinery.
• 45:26In this particular case nuclear
• 45:28receptors to rewire signal dependent
• 45:30gene expression programs.
• 45:31Then my potentially promote are
• 45:34functionally that contribute to the
• 45:37development of hematopoetic malignancies.
• 45:40So as a final summary.
• 45:43Is that an explosion of genomic
• 45:46and epigenomic information?
• 45:48In recent years we have learned a
• 45:50great deal of how gene regulation
• 45:53controls normal development and
• 45:55how dysregulation of this process
• 45:58contribute to human diseases.
• 46:00How what.
• 46:01We currently know only represents
• 46:03a very small portion of the
• 46:06complicated complex.
• 46:07The human genome,
• 46:09and in retrospect and maybe
• 46:11more relevant to our studies.
• 46:13As we know,
• 46:15the first documented cases of
• 46:17sickle cell disease was described
• 46:19by an James Herrick and in 90.
• 46:22Early 90 app.
• 46:24Centuries and 9010 specifically,
• 46:27which was later dropped as a first
• 46:30molecular disease by a Linus Pauling.
• 46:33In 1947 an enhancer was not
• 46:35discovered until early 1980s
• 46:37and followed by the completion.
• 46:39The first draft of human
• 46:41genome in early 2000s.
• 46:43Now more than a century after
• 46:46discovering sickle cell disease and
• 46:4840 years of discovery of enhances,
• 46:51we might have a first enhancer targeting
• 46:54therapist for this molecular disease.
• 46:56Very soon,
• 46:57and we certainly hope that by
• 46:59focusing on your hands as another
• 47:02non coding regulatory at genomic
• 47:05elements that we might be able to
• 47:08identify new mechanism and genetic
• 47:10pathways that contribute to a normal
• 47:13blood cell development an animal.
• 47:15Long term that we might be
• 47:17able to develop our
• 47:18enhanced targeting therapeutics
• 47:20for blood disorders.
• 47:22So with that most important,
• 47:24I want to acknowledge all
• 47:26the people who met his work,
• 47:28possible the initial study PCL
• 47:30PCL Evan Hansen was done in
• 47:33Stockings Lab in collaboration
• 47:34with Stambaugh and Vicious Ankara,
• 47:37and profiling work done in primary
• 47:39hematopoietic cells was done in
• 47:41collaboration with the formal post or
• 47:43fellow John Huang from Stalking Slab.
• 47:46Now he has his own lab in Sherman University.
• 47:50The development of the cast net capture
• 47:53was spearheaded by a former poster in
• 47:56my lap as she knew now has his own
• 47:59app together with another poster fellow.
• 48:02Again using the developer of
• 48:04Enhancer targeting CRISPR,
• 48:05Aquifer was spearheaded by another
• 48:07poster fellow Kyle only think is
• 48:10one of the fellows over here.
• 48:12If I can move my brows anyway
• 48:14together with another Postal federal,
• 48:17most of them are transitioned
• 48:19to their independent positions.
• 48:20Together with other collaborators
• 48:21and we couldn't do this with a
• 48:24wonderful collaboration from Boston
• 48:25Children's and our local collaborators
• 48:27at you T Southwestern UT Dallas.
• 48:28I will stop here.
• 48:29I'm happy to take any questions
• 48:31that you might have.
• 48:33Thank you very much for your time.