AssemblingtheGenome:HierarchicalStructurefromtheBasicBuildingBlocks

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)

OrganizationofcomponentsintoBuildingBlocks

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

Typesofsolenoidalstructure

Science (2017) 357, eaag0025

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

ChromatinImmunoprecipitation

eLife2017;6:e22631

HypersensitivesitesareshortregionsofchromatinwhicharearedetectedbytheirsensitivitytoDnaseI orothernucleases

Targetislesscompactednucleosomal structure

DNAseI SensitivityisanAssayforDistalRegulatorsNucleasesorATACtechniques

DNAseI profiles

Histonemodifications

H3K4me1Activeandpoisedenhancers

H3K27acActiveenhancers

H3K27me3Inactivegenes

GregoryD.Bowman*†andMichaelG.Poirier.(2015)Post-TranslationalModificationsofHistonesThatInfluenceNucleosomeDynamicsChem Rev.Mar25;115(6):2274–2295.

EnhancerRNAs

PriortopromoteractivationEnhancersrecruitRNAPolII andTFstoformPICTranscribes1Dor2D- producesshortunstabletranscripts

NotallenhancershavebeenassociatedwitheRNAs (only25%of12,000neuronalenhancers)

Propertiesofanenhancer

Modifiedfrom- NatureGenetics 32,555–556(2002)

HowDistalRegulatoryElementsActivateTargetGenes

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

ChromatinConformationCapture(3C)

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

Roleofnucleolus