How DNA Is Packed In The Cell: Chromosomes, Genes, Nucleosomes
Summer Course in BiophysicsJune 5, 2014How DNA Is Packed In The Cell:Chromosomes, Genes, NucleosomesBrian D. StrahlDepartment of Biochemistry & BiophysicsUNC-School of Medicine
OutlineI.Chromatin organization The DNA packaging problem Histones and nucleosome core particle Chromatin folding and nuclear organization Euchromatin vs HeterochromatinII. Factors that influence chromatin organization and gene function Histone post-translational modifications (PTMs) and the ‘histone code’ Histone variants DNA methylationIII. Tools and technologies leading the charge in chromatin research Modification-specific antibodies and chromatin immunoprecipitation High-throughput microarray/DNA sequencing technologies Proteomics and mass spectrometric analyses
The DNA packaging problem-E. Coli:1X1 million base pairs-Yeast genome:12X12 million base pairs-Fruit fly genome:122X122 million base pairs-Human genome:3400X3.4 billion base pairs(Chlamydia trachomatis)If our strands of DNA were stretched out in a line, the 46 chromosomes makingup the human genome would extend more than six feet ( 2 meters)
A Matter of Fitting In!0.0043meters(0.17 inches)8850 meters ( 5.5 miles)Mount Everest t/thehimalaya.htmpipet tip image: Biologix Research(slide provided by Raymond Reeves)
How is DNA packagingachieved?
Organization of eukaryotic chromatinDNA double n loop:Chromatin 100,000 bp DNA
First order of DNAcompaction
Nucleosomes are the buildingblocks of chromatin
Histone structure 2/3 of chromatin mass is protein 95% of chromatin protein are histonesH1NC“Tail” domain“Globular” domain Regulatory domain Involved in higher-order packing Histone-histone interactions DNA wrapping
Nucleosome organizationH3-H4 tetramersbuild a “wall” that is“capped” by H2AH2B dimers10
Blue H2A/H2BWhite H3/H411
H3H4H2AH3 ‘tail’H2BLuger et al, Nature 1997
H3H4H2AH2BLuger et al, Nature 1997
Second order of DNAcompaction
Secondary Structure H1 : essential for the solenoid structure
Third order of DNAcompaction
Histone-depleted metaphasechromosomeLoops of DNAProtein scaffold
Histone-depleted metaphasechromosomeScaffold/Matrix attachmentregions
A condensed metaphase human chromosome
Genome architecture: chromatin domains
Heterochromatin vs. Euchromatin Highly condensedRepetitive sequencesReplicates later in the cell cycleTranscriptionally OFF DecondensedSingle copy sequences (genes)Replicates early in the cell cycleTranscriptionally ON
OutlineI.Chromatin organization The DNA packaging problem Histones and nucleosome core particle Chromatin folding and nuclear organization Euchromatin vs HeterochromatinII. Factors that influence chromatin organization and gene function Histone post-translational modifications (PTMs) and the ‘histone code’ Histone variants DNA methylationIII. Tools and technologies leading the charge in chromatin research Modification-specific antibodies and chromatin immunoprecipitation High-throughput microarray/DNA sequencing technologies Proteomics and mass spectrometric analyses
Molecular mechanisms that influencechromatin structure and function1. Chromatin remodeling complexes (e.g. Swi/Snf)2. Histone modifications3. Histone variants (e.g. H2A.Z, CENP-A, etc.)4. DNA methylation
Molecular mechanisms that influencechromatin structure and function1. Chromatin remodeling complexes (e.g. Swi/Snf)2. Histone modifications3. Histone variants (e.g. H2A.Z, CENP-A, etc.)4. DNA methylation
Histone deChromatinregulatorTailGlobular
Histone deChromatinregulatorTailGlobular
Histone acetylation and chromatin structure“Off”“On”(Adapted from Wade & Wolffe - Current Biology, 1997)
Bromodomain-containing proteins can bind to acetylated histones(TBP)TATAA(Taken from E. Pennisi - Science, 2000)
Epigenetic ‘Toolkit’Gardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful languagewhere context matters. J. Mol. Biol. 409:36-46.
Histone Code romo bromoPHDbromo bromo bromo bromoPHD PHD(Figure from Abcam)PHDchromo chromotudortudorBAH BAH HMGDEXD HELICJMJD2A(demethylase)BAF180(SWI/SNF)CHD4(NuRD)
Histone deChromatinregulatorTailGlobular
Histone ADP-ribosylationUbiquitinationSumoylationHistone CodeHP1K9H3TailGlobular
Histone H3 methylationSUV39Set1 ARKSAPSTGGVK4Geneactivation9Gene repressionHeterochromatinX-inactivation27Gene repressionX-inactivation36Dot1K79Globular domainGeneactivation
Staining of female metaphase chromosomeswith site-specific methyl H3 antibodiesmethyl (Lys 9) H3(Taken from Boggs BA et al. - Nat Genet., 2002)methyl (Lys 4) H3
Roles of H3 lysines 4 and 9 methylationK9 MeK4 MeOnOff(Taken from Bannister et al. - Nature, 2001)
Post-translational modifications decorate histones
Molecular mechanisms that influencechromatin structure and function1. Chromatin remodeling complexes (e.g. Swi/Snf)2. Histone modifications3. Histone variants (e.g. H2A.Z, CENP-A, etc.)4. DNA methylation
HTZ1 (H2A.Z)Figure from Millipore/Upstate
Histone Variants(HTZ1)(Table from Henikoff and Ahmad, Annu. Rev. Cell Dev. Biol, 2005)
Molecular mechanisms that influencechromatin structure and function1. Chromatin remodeling complexes (e.g. Swi/Snf)2. Histone modifications3. Histone variants (e.g. H2A.Z, CENP-A, etc.)4. DNA methylation
DNA methylationOccurs in:(1) select organisms and (2) usually at CpG dinucleotideresidues1. Organisms found in:HumansMiceFrogsFlies*(low levels and CpT)2. Occurs on Cytosine:
How DNA methylation regulates gene repression?HDACsA)B) & C)D)By sterically blocking the binding of transcription factors (e.g. E2F, NF-kB, CTCFBy recruiting chromatin modifying activitiesBy affecting RNA Polymerase II transcription(figure from Klose & Bird, Trends Biochem Sci., 2006)
OutlineI.Chromatin organization The DNA packaging problem Histones and nucleosome core particle Chromatin folding and nuclear organization Euchromatin vs HeterochromatinII. Factors that influence chromatin organization and gene function Histone post-translational modifications (PTMs) and the ‘histone code’ Histone variants DNA methylationIII. Tools and technologies leading the charge in chromatin research Modification-specific antibodies and chromatin immunoprecipitation High-throughput microarray/DNA sequencing technologies Proteomics and mass spectrometric analyses
Histone modification-specific antibodies haveenabled the study of chromatin!methyl (Lys 9) H3(Taken from Boggs BA et al. - Nat Genet., 2002)methyl (Lys 4) H3
The ChIP-chip procedureCrosslink Chromatin with FormaldehydeShear Chromatin by SonicationIncubate with AntibodyReverse CrosslinksRecover IP DNAReverse CrosslinksRecover Input DNAAmplify, Label RedAmplify, Label GreenHybridize To Microarray(Provided by Jason Lieb, UNC)
Solexa Sequencing (Illumina)DNA(0.1-1.0 ug)3’ 23456789T G C T A C G A T Image acquisitionBase calling
ChIP-Seq Follow standard ChIP procedure Identify uniquely aligned sequences in human genomeEnriched by ChIP
Mass spectrometry is a vital tool in combinatorial PTM discoveryA. Bottom-up KDIQLARRIRGERA-134B. Top-down MSRP-HPLCH3H3HILIC(hydrophilic interactionliquid RA-134
Mass Spectrometry technologies have revealed novel histone‘marks’ and specific histone codes
Mass spectrometry is a vital tool in combinatorial PTM discovery
SILAC-based approaches are unlocking identification of novel(Stable isotope labeling byeffector proteinsamino acids in cell beledextractsUnlabeledextractspeptideBeadSDS/PAGE andtryptic digestionm/z
Bromodomain-containing proteins can bind to acetylated histonesPHD(TBP)K4TATAA(Taken from E. Pennisi - Science, 2000)
Semi-synthetic modified nucleosomes explore multivalentengagements in chromatinthioesterNT peptideScysteineHNH2NOROHSpeptide ligationHNNT peptideHNHNOOHNOOSHSHmethyl aminoethylhalideNative chemical ligation (NCL) andExpressed protein ligation(EPL)(Kent/Cole/Muir labs) NBrbaseHNHNOOSMethyl-lysine analogue (MLA)(Shokat et al.) N
Thank you!
The DNA packaging problem-E. Coli: 1X 1 million base pairs (Chlamydia trachomatis)-Yeast genome: 12X 12 million base pairs-Fruit fly genome: 122X 122 million base pairs-Human genome: 3400X 3.4 billion base pairs. If our strands of DNA were stretched out in a line, the 46 chromosomes making
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