How DNA Is Packed In The Cell: Chromosomes, Genes, …€¦ · 05/06/2014 · Chromosomes, Genes,...
Transcript of How DNA Is Packed In The Cell: Chromosomes, Genes, …€¦ · 05/06/2014 · Chromosomes, Genes,...
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How DNA Is Packed In The Cell: Chromosomes, Genes, Nucleosomes
Brian D. StrahlDepartment of Biochemistry & Biophysics
UNC-School of Medicine
Summer Course in Biophysics
June 5, 2014
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OutlineI. Chromatin organization
• The DNA packaging problem• Histones and nucleosome core particle• Chromatin folding and nuclear organization• Euchromatin vs Heterochromatin
II. Factors that influence chromatin organization and gene function• Histone post-translational modifications (PTMs) and the ‘histone code’• Histone variants• DNA methylation
III. 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
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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 up the human genome would extend more than six feet (~ 2 meters)
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Mount Everest image: http://www.unu.edu/mountains2002/photoexhibit/thehimalaya.htm
pipet tip image: Biologix Research
8850 meters (~5.5 miles)
0.0043 meters
(0.17 inches)
A Matter of Fitting In!
(slide provided by Raymond Reeves)
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How is DNA packaging achieved?
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Organization of eukaryotic chromatin
DNA double helix
NucleosomesHistones
Solenoid
Chromatin loop:~100,000 bp DNAChromatin
H3H2BH2AH4
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First order of DNA compaction
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Nucleosomes are the building blocks of chromatin
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Histone structure
• 2/3 of chromatin mass is protein• 95% of chromatin protein are histones
N CH1
“Tail” domain•Regulatory domain•Involved in higher-order packing
“Globular” domain•Histone-histone interactions•DNA wrapping
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Nucleosome organization
H3-H4 tetramers build a “wall” that is “capped” by H2A-H2B dimers
10
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Blue= H2A/H2BWhite= H3/H4
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Luger et al, Nature 1997
H3
H4
H2A
H2B H3 ‘tail’
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Luger et al, Nature 1997
H3
H4
H2A
H2B
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Second order of DNA compaction
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Secondary Structure
• H1 : essential for the solenoid structure
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Third order of DNA compaction
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Histone-depleted metaphase chromosome
Protein scaffold
Loops of DNA
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Histone-depleted metaphase chromosome
Scaffold/Matrix attachment regions
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A condensed metaphase human chromosome
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Genome architecture: chromatin domains
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Heterochromatin vs. Euchromatin
• Highly condensed• Repetitive sequences• Replicates later in the cell cycle• Transcriptionally OFF
• Decondensed• Single copy sequences (genes)• Replicates early in the cell cycle• Transcriptionally ON
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OutlineI. Chromatin organization
• The DNA packaging problem• Histones and nucleosome core particle• Chromatin folding and nuclear organization• Euchromatin vs Heterochromatin
II. Factors that influence chromatin organization and gene function• Histone post-translational modifications (PTMs) and the ‘histone code’• Histone variants• DNA methylation
III. 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
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1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Molecular mechanisms that influence chromatin structure and function
3. Histone variants (e.g. H2A.Z, CENP-A, etc.)
4. DNA methylation
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1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Molecular mechanisms that influence chromatin structure and function
3. Histone variants (e.g. H2A.Z, CENP-A, etc.)
4. DNA methylation
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Histone CodeChromatinregulator
Tail Globular
Histone Modifications
AcetylationPhosphorylationMethylationADP-ribosylationUbiquitinationSumoylation
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Histone CodeChromatinregulator
Tail Globular
Histone Modifications
AcetylationPhosphorylationMethylationADP-ribosylationUbiquitinationSumoylation
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Histone acetylation and chromatin structure
(Adapted from Wade & Wolffe - Current Biology, 1997)
“Off” “On”
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Bromodomain-containing proteins can bind to acetylated histones
(Taken from E. Pennisi - Science, 2000)
(TBP)
TATAA
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Gardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful language where context matters. J. Mol. Biol. 409:36-46.
Epigenetic ‘Toolkit’
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PHD bromoPHDDDT
PHDPHD tudor tudorJmjcJmjnJMJD2A
(demethylase)
BPTF(NURF)
Histone Code ‘readers’
bromo HMGBAHbromo bromo bromo bromo bromo BAH BAF180(SWI/SNF)
PHD chromo chromoPHD CHD4(NuRD)
DEXD HELIC
(Figure from Abcam)
PWWP
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Histone CodeChromatinregulator
Tail Globular
Histone Modifications
AcetylationPhosphorylationMethylationADP-ribosylationUbiquitinationSumoylation
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Histone CodeHP1
Tail Globular
Histone Modifications
K9
H3
AcetylationPhosphorylationMethylationADP-ribosylationUbiquitinationSumoylation
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Histone H3 methylation
Set1MLL
H34 9 27 36
N-ARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVK- Globular domain
Gene repressionHeterochromatin
X-inactivation
Dot1
K79
SUV39ESETG9a EZH2
Set2NSD1
Geneactivation
Gene repressionX-inactivation
Geneactivation
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Staining of female metaphase chromosomes with site-specific methyl H3 antibodies
methyl (Lys 9) H3 methyl (Lys 4) H3
(Taken from Boggs BA et al. - Nat Genet., 2002)
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(Taken from Bannister et al. - Nature, 2001)
On Off
K4 MeK9 Me
Roles of H3 lysines 4 and 9 methylation
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Post-translational modifications decorate histones
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1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Molecular mechanisms that influence chromatin structure and function
3. Histone variants (e.g. H2A.Z, CENP-A, etc.) 4. DNA methylation
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HTZ1 (H2A.Z)
Figure from Millipore/Upstate
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Histone Variants
(HTZ1)
(Table from Henikoff and Ahmad, Annu. Rev. Cell Dev. Biol, 2005)
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1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Molecular mechanisms that influence chromatin structure and function
3. Histone variants (e.g. H2A.Z, CENP-A, etc.)
4. DNA methylation
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DNA methylation
Occurs in:(1) select organisms and (2) usually at CpG dinucleotide
residues
1. Organisms found in:
HumansMiceFrogsFlies*(low levels and CpT)
2. Occurs on Cytosine:
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How DNA methylation regulates gene repression?
A) By sterically blocking the binding of transcription factors (e.g. E2F, NF-kB, CTCFB) & C) By recruiting chromatin modifying activitiesD) By affecting RNA Polymerase II transcription
HDACs
(figure from Klose & Bird, Trends Biochem Sci., 2006)
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OutlineI. Chromatin organization
• The DNA packaging problem• Histones and nucleosome core particle• Chromatin folding and nuclear organization• Euchromatin vs Heterochromatin
II. Factors that influence chromatin organization and gene function• Histone post-translational modifications (PTMs) and the ‘histone code’• Histone variants• DNA methylation
III. 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
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Histone modification-specific antibodies have enabled the study of chromatin!
methyl (Lys 9) H3 methyl (Lys 4) H3
(Taken from Boggs BA et al. - Nat Genet., 2002)
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The ChIP-chip procedureCrosslink Chromatin with Formaldehyde
Shear Chromatin by Sonication
Hybridize To Microarray
Reverse CrosslinksRecover Input DNA
Amplify, Label Green
Incubate with Antibody
Reverse Crosslinks Recover IP DNA
Amplify, Label Red
(Provided by Jason Lieb, UNC)
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DNA(0.1-1.0 ug)
Single molecule arraySample
preparation Cluster growth5’
5’3’
G
T
C
A
G
T
C
A
G
T
C
A
C
A
G
TC
A
T
C
A
C
C
TAG
CG
TA
GT
1 2 3 7 8 94 5 6
Image acquisition Base calling
T G C T A C G A T …
Sequencing
Solexa Sequencing (Illumina)
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Enriched by ChIP
ChIP-Seq• Follow standard ChIP procedure
• Identify uniquely aligned sequences in human genome
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Mass spectrometry is a vital tool in combinatorial PTM discovery
A. Bottom-up MS
H3H3
RP-HPLC Trypsin RP-HPLCMS
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA-134
B. Top-down MS
H3H3RP-HPLC HILIC
(hydrophilic interaction liquid chromatography)
MS
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA-134
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Mass Spectrometry technologies have revealed novel histone ‘marks’ and specific histone codes
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Mass spectrometry is a vital tool in combinatorial PTM discovery
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SILAC-based approaches are unlocking identification of novel effector proteins
Beadpeptide Beadpeptide
Unlabeled extracts
Isotope-labeled extracts
Beadpeptide Beadpeptide
SDS/PAGE and tryptic digestion
m / z
(Stable isotope labeling by amino acids in cell culture)
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Bromodomain-containing proteins can bind to acetylated histones
(Taken from E. Pennisi - Science, 2000)
(TBP)
TATAAK4
PHD
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Semi-synthetic modified nucleosomes explore multivalent engagements in chromatin
HN
O
SH
HN
O
N+
HN
O
S
HN
O
N+
Br
base
Methyl-lysine analogue (MLA)(Shokat et al.)
methyl aminoethylhalide
H2NO
HS
HN
S
O
R
HN
O
SH
HN
O
NT peptide
NT peptide
thioester cysteine
peptide ligation
Native chemical ligation (NCL) and Expressed protein ligation(EPL)
(Kent/Cole/Muir labs)
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Thank you!