Assembling the Genome: Hierarchical ... -...
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1.)Considerthefundamentalbuildingblocksforgenomearchitecture
2.)Problemsinassemblyandhow(wethink)someoftheseareovercome
3.) How(wethink)theseareorganizedtoproduceafunctioningtranscriptionalfactory
Group-basedactivity
Writeagrant
Plansforthismorning
CentralLibraryforInformation– theBlueprintsforCarbon-Copyreplicate– theProgramthatconvertsCell-TypeandFate
InsidetheNucleus
Largestorganelleinthecellat~6µm--focusofattentionformostgeneticists
GeneticistViewoftheCell
TwoLevelsofInformationStorage
1. CodingInformation(1.5%ofthehumangenome)
2. RegulatoryInformation(estimatesof8%- 40%)
Humangenome
Fundamentalcomponents
DNAProtein
Histones-Majorcomponents
Non-Histones-minorcomponentswhichincludetranscriptionfactorsandotherstructuralcomponents
Togetherthesecomposethechromatin(functional)componentofthegenome
EarlyEMworkformedinitialviewofchromatin
Millerspreads(1969)
Beadsonastringconfiguration‘Nu bodies’
OlinsandOlins(1973)
HistonestructureTetramerFormationNucleosomeProductions
N&C-terminaltails
Self-Assembly
Roles—CompactingDNACompartmentalizingDNA(Histonecode)
HowmuchDNAinacell?
InHumanFemale6.06x 109 bpMale5.96x109
Ecoli4.6x106
Yeast12x106Scerevisiae(1st eucaryotic genomesequenced)
Lungfish130x109(largestvert.genome)
Amoeboid670x109(Largestknowngenome)
~2metresofDNA/cellInitialsequencecost<$3billion
CompactionofTwoMetersofDNA(Human)
Packingratio5-10X
PackingratioApprox 50X
Proposedhierarchicalstructureofchromatin
Finch,J.T.&Klug,A.Solenoidalmodelforsuperstructureinchromatin.Proc.NatlAcad.Sci.USA73,1897–-1901(1976).
AbitoftheHistory
SchalchT,Duda S,SargentDF,RichmondTJ(2005).X-raystructureofatetranucleosome anditsimplicationsforthechromatinfibre.Nature.436 (7047):138–41
IsolatedratliverchromatinDialysedagainst0.5mMMgCl2.
Determinedbyreconstitutionofnucleosome(tetranucleosome)
Two-starthelix
Fig. 1 A fluorescent DNA-binding dye that catalyzes local DAB polymerization on chromatin in the nucleus.
Horng D. Ou et al. Science 2017;357:eaag0025
Traditionally,chromatinstainedwithelectrondense,heavymetalstainssuchasOsO4 whichdonotstainDNAselectively;preferRNA,lipidsandproteins
MembranepermeabledyethatselectivelybindsdsDNAandisusedforlivecellimaging
Twoinnovations- StainingtechniqueandEMTomography
Disordered 5- to 24-nm-diameter chromatin chains are flexible and can be packed together at different concentration densities in interphase nuclei and mitotic chromosomes.
Inbothmitoticandinterphase-Avediameter~14nmChromatinisindisorderedprimarypolymerchains
rangesfrom5-24nm
Interphase—packedathigherdensitiesSuggestthatscaffoldingfactorsconstrainarchitecture
BasicstructureDisordered 5- to 24-nm-diameter chromatin chains are Advantage—
Flexible and can be packed together at different concentration densities in interphase nuclei and mitotic chromosome
May explain speed at which chromosome condenseQuestions-
Heterochromatin assoc with high CVCIs [3D chromatin] a universal principle that determines functional activity & accessibility of genomic DNA
Packed in short interspersedintervals bent back on themselves
Extended curvilinear chains with infrequent contacts
Whyisthisimportant?
Chromatinisthefunctionalcomponentofthegenome
Chromatinstructureisimportantforgeneactivity
Thisnotionofchromatinstructureprovidesdifferentproblemstoconfront
Whyregulategenes?
Unicellularorganisms- Regulatorypromoters
Putsthegenomeintouchwithitsenvironment
Agreatevolutionaryinventionsthatenabledmulticellularity?
Distalcis-regulators
GeneralRoleofCis-regulators
1.Linksthegenometotheworldoutsidethecell.
2.Enablesthegenetorespondtomultiplecomplexsignals
Propertiesofanenhancer
Cis-actingelementthatincreases(thelikelihood)oftranscriptionofagene.ItstargetsaregenepromotersOperatesfromadistanceArefoundupstream,downstreamorinsideagene
Hongzhu Qu, Xiangdong Fang (2013) A Brief Review on the Human Encyclopedia of DNA Elements (ENCODE) Project
Genomics, Proteomics & Bioinformatics, Volume 11, Issue 3, 2013, 135–141
Multi-dimensionalregulationofgeneexpression
Exploitingpropertiesofcis-regulatoryelementsENCODE- Methodologiesusedtostudyenhancers
1.)clusteringofTFsites--200TFsusedbyChIP-Seq
2.)DNAse hypersensitivity– over150celllinesandtissues
3.)Histonemodifications– H3K4me1,H3K4me3,H3K27ac;inadditionp300.
>400,000cis-regulatoryelements
Calo E,Wysocka J.(2013)ModificationofEnhancerChromatin:What,How,andWhy?MolecularCell49,825.
Next-generationssequencingor
High-throughputsequencing
Illumina(Solexa)sequencingRoche454sequencingIontorrent:Proton/PGMsequencingSOLiD sequencing
Capacity—Wholegenomesequencing(30Xcoverage)overnight
WholegenomeanalysisdependentonNG-sequencing
(MinION sequencer– Handheld)
HiSeqX system(£700genome)COMINGsoon – NovaSeq 5000&6000(£100genome)
Manytechniquesexaminingchromatininteractionsusecrosslinkingwithformaldehyde
Hoffmanetal.2015FormaldehydeCrosslinking:AToolfortheStudyofChromatinComplexes THEJOURNALOFBIOLOGICALCHEMISTRY290,26404–26411,
Thesmallsizeofformaldehydedictatesitslinkageofgroupsthatareapproximately2Åapart,makingitwellsuitedforcaptureofInteractionsbetweenmacromoleculesthatareincloseproximity
70Å
25Åwidth
HypersensitivesitesareshortregionsofchromatinwhicharearedetectedbytheirsensitivitytoDnaseI orothernucleases
Targetislesscompactednucleosomal structure
DNAseI SensitivityisanAssayforDistalRegulatorsNucleasesorATACtechniques
DNAseI profiles
GregoryD.Bowman*†andMichaelG.Poirier.(2015)Post-TranslationalModificationsofHistonesThatInfluenceNucleosomeDynamicsChem Rev.Mar25;115(6):2274–2295.
EnhancerRNAs
PriortopromoteractivationEnhancersrecruitRNAPolII andTFstoformPICTranscribes1Dor2D- producesshortunstabletranscripts
NotallenhancershavebeenassociatedwitheRNAs (only25%of12,000neuronalenhancers)
Propertiesofanenhancer
DoesChromatinOrganisationPlayaRoleinGeneRegulation?
HowcanweassayHierarchicalStructureacrossthegenome?
E10.5
Insig1>En2>RBM33> Rnf32>Nom1>Ube3c>
<Cnpy1<Shh <Lmbr1Mnx1>
Long-RangeLocusCompositionDesign-A-Locus
1.6Mb
0.95Mb
RBM33> Rnf32>Nom1>
<Shh <Lmbr1SBE3SBE4 SBE2SLGESFPE1
SBE1
MACS1MRCS1
ZRSMFCS4SFPE2
ZPA
(Shh)
Limbasymmetry
?E17.5
Different3CApproaches
Specificinteraction
Allinteractionsfromoneviewpoint
Allinteractionoveralimiteddomain
Allinteractions
Figure 3. Organization of Chromatin into Topologically Associated Domains(A) Hi-C or 5C heatmaps visualize three-dimensional interactions or compartmentalization of chromosomes into TADs, visible as triangular blocks of increased interaction frequencies.(B) TA...
Hannah K. Long, Sara L. Prescott, Joanna Wysocka (2016) Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution Cell 167; 1170-1187.
OrganizationofChromatinintoTopologicallyAssociatedDomainsTADs
Loop-extrusionmodel
StabilizedbyCTCFandcohesin rings
OutliningtheBasicsforaResearchGrant
TheFrancisCrickFoundationhasannouncedanewinitiativetofundtheverybestofouryoungscientist.
Initiativeentitled“HealthylivingthroughGenetics”.Funding5yearprograms(worthupto£5million)toinvestigatebasicproblemsinunderstandinghowourgenomeswork
E10.5
Insig1>En2>RBM33> Rnf32>Nom1>Ube3c>
<Cnpy1<Shh <Lmbr1Mnx1>
1.6Mb
0.95Mb
RBM33> Rnf32>Nom1>
<Shh <Lmbr1SBE3SBE4 SBE2SLGESFPE1
SBE1
MACS1MRCS1
ZRSMFCS4SFPE2
ZPA
(Shh)
Limbasymmetry
ModelSystem
ShhanditsRegulatoryDomain
CTCFsites
Overarching(General)ScientificQuestion-
Hypotheses
Goals(SpecificAims)
ExperimentalProcedures
OutlineofaGrantProposal
Overarching(General)ScientificQuestion-1.)HowspecificaretheenhancersforShh promoter?2.)Howdotheenhancersactivatethepromoter?3.)Howaretheenhancersactivated4.)Howdoyourrestrainregulatoryactivitywithinthelocus5.)Whattranscriptionfactorsareworkingatoneoralloftheenhancers
Cantheseleadtoidentificationofthekeysignalingpathways6.)Whataretheboundaryelementsforthislocus
andareCTCFsitestheinsulatorsforthelocus
Hypotheses
Goals(SpecificAims)
ExperimentalProcedures