How DNA Is Packed In The Cell:
Chromosomes, Genes, Nucleosomes
Brian D. Strahl
Department of Biochemistry & Biophysics
UNC...
Outline
I. Chromatin organization
• The DNA packaging problem
• Histones and nucleosome core particle
• Chromatin folding ...
The DNA packaging problem
-E. Coli: 1X 1 million base pairs
(Chlamydia trachomatis)
-Yeast genome: 12X 12 million base pai...
Mount Everest image:
http://www.unu.edu/mountains2002/photoexhibit/thehimalaya.htm
pipet tip image: Biologix Research
8850...
How is DNA packaging
achieved?
Organization of eukaryotic chromatin
DNA double helix
NucleosomesHistones
Solenoid
Chromatin loop:
~100,000 bp DNA
Chromat...
First order of DNA
compaction
Nucleosomes are the building
blocks of chromatin
Histone structure
• 2/3 of chromatin mass is protein
• 95% of chromatin protein are histones
N CH1
“Tail” domain
•Regulato...
10
Nucleosome organization
H3-H4 tetramers
build a “wall” that is
“capped” by H2A-
H2B dimers
11
Blue= H2A/H2B
White= H3/H4
Luger et al, Nature 1997
H3
H4
H2A
H2B H3 ‘tail’
Luger et al, Nature 1997
H3
H4
H2A
H2B
Second order of DNA
compaction
Secondary Structure
• H1 : essential for the solenoid structure
Third order of DNA
compaction
Histone-depleted metaphase
chromosome
Protein scaffold
Loops of DNA
Histone-depleted metaphase
chromosome
Scaffold/Matrix attachment
regions
A condensed metaphase human chromosome
Genome architecture: chromatin domains
Heterochromatin vs. Euchromatin
• Highly condensed
• Repetitive sequences
• Replicates later in the cell cycle
• Transcrip...
Outline
I. Chromatin organization
• The DNA packaging problem
• Histones and nucleosome core particle
• Chromatin folding ...
1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Molecular mechanisms that influence
chromatin st...
1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Molecular mechanisms that influence
chromatin st...
Histone CodeChromatin
regulator
Tail Globular
Histone Modifications
Acetylation
Phosphorylation
Methylation
ADP-ribosylati...
Histone CodeChromatin
regulator
Tail Globular
Histone Modifications
Acetylation
Phosphorylation
Methylation
ADP-ribosylati...
Histone acetylation and chromatin structure
(Adapted from Wade & Wolffe - Current Biology, 1997)
“Off” “On”
Bromodomain-containing proteins can bind to acetylated histones
(Taken from E. Pennisi - Science, 2000)
(TBP)
TATAA
Gardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful language
where context matters. J. Mol. Biol. 409:36-46....
PHD bromoPHDDDT
PHDPHD tudor tudorJmjcJmjn
JMJD2A
(demethylase)
BPTF
(NURF)
Histone Code ‘readers’
bromo HMGBAHbromo bromo...
Histone CodeChromatin
regulator
Tail Globular
Histone Modifications
Acetylation
Phosphorylation
Methylation
ADP-ribosylati...
Histone Code
HP1
Tail Globular
Histone Modifications
K9
H3
Acetylation
Phosphorylation
Methylation
ADP-ribosylation
Ubiqui...
Histone H3 methylation
Set1
MLL
H3 4 9 27 36
N-ARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVK- Globular domain
Gene repression
Heter...
Staining of female metaphase chromosomes
with site-specific methyl H3 antibodies
methyl (Lys 9) H3 methyl (Lys 4) H3
(Take...
(Taken from Bannister et al. - Nature, 2001)
On Off
K4 Me
K9 Me
Roles of H3 lysines 4 and 9 methylation
Post-translational modifications decorate histonesPost-translational modifications decorate histones
1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Molecular mechanisms that influence
chromatin st...
HTZ1 (H2A.Z)
Figure from Millipore/Upstate
Histone Variants
(HTZ1)
(Table from Henikoff and Ahmad, Annu. Rev. Cell Dev. Biol, 2005)
1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Molecular mechanisms that influence
chromatin st...
DNA methylation
Occurs in:
(1) select organisms and (2) usually at CpG dinucleotide
residues
1. Organisms found in:
Humans...
How DNA methylation regulates gene repression?
A) By sterically blocking the binding of transcription factors (e.g. E2F, N...
Outline
I. Chromatin organization
• The DNA packaging problem
• Histones and nucleosome core particle
• Chromatin folding ...
Histone modification-specific antibodies have
enabled the study of chromatin!
methyl (Lys 9) H3 methyl (Lys 4) H3
(Taken f...
The ChIP-chip procedure
Crosslink Chromatin with Formaldehyde
Shear Chromatin by Sonication
IP SAMPLE REFERENCE
Hybridize ...
DNA
(0.1-1.0 ug)
Single molecule array
Sample
preparation Cluster growth
5’
5’3’
G
T
C
A
G
T
C
A
G
T
C
A
C
A
G
T
C
A
T
C
A...
Enriched by ChIP
ChIP-Seq
• Follow standard ChIP procedure
• Identify uniquely aligned sequences in human genome
Mass spectrometry is a vital tool in combinatorial PTM discovery
A. Bottom-up MS
H3
H3
RP-HPLC Trypsin RP-HPLC
MS
ARTKQTAR...
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
effector proteins
Beadpeptide
Beadpeptide
Unlabeled
extracts
...
Bromodomain-containing proteins can bind to acetylated histones
(Taken from E. Pennisi - Science, 2000)
(TBP)
TATAA
K4
PHD
Semi-synthetic modified nucleosomes explore multivalent
engagements in chromatin
Methyl-lysine analogue (MLA)
(Shokat et a...
Thank you!
Brian strahl's 2013 Lecture in the 2013 Summer Course in Biophysics: Case Studies in the Physics of Life
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Brian strahl's 2013 Lecture in the 2013 Summer Course in Biophysics: Case Studies in the Physics of Life

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The Biophysical Society
2013 Summer Course in Biophysics: Case Studies in the Physics of Life
UNC Campus & Summer Course
by Brian Strahl

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  • Intro: The human genome contains 3 billion base pairs of DNA. This needs to fit into the size of 10 microns. This is the equivalent of taking a string that is as long as Mt. Everest is tall (~29,000 feet) and fitting it into a compartment the size of a pipett tip. Transition: Cells are able to package to fit the DNA into the relatively small space of the nucleus by wrapping the DNA around specialized proteins.
  • Histones: A group of positively charged small proteins; high content of lysine and arginine Generally small polypeptides; 10,000 to < 25,000 daltons Facilitates interactions with negatively charged phosphate backbone of DNA Linker DNA may vary anywhere from 8-114bp
  • Histones: A group of positively charged small proteins; high content of lysine and arginine Generally small polypeptides; 10,000 to < 25,000 daltons Facilitates interactions with negatively charged phosphate backbone of DNA Most abundant chromatin protein Linker DNA may vary anywhere from 8-114bp
  • Transcript of "Brian strahl's 2013 Lecture in the 2013 Summer Course in Biophysics: Case Studies in the Physics of Life "

    1. 1. How DNA Is Packed In The Cell: Chromosomes, Genes, Nucleosomes Brian D. Strahl Department of Biochemistry & Biophysics UNC-School of Medicine Summer Course in Biophysics June 6, 2013
    2. 2. Outline I. Chromatin organization • The DNA packaging problem • Histones and nucleosome core particle • Chromatin folding and nuclear organization • Euchromatin vs Heterochromatin I. Factors that influence chromatin organization and gene function • Histone post-translational modifications (PTMs) and the ‘histone code’ • Histone variants • DNA methylation I. 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
    3. 3. 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)
    4. 4. 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)
    5. 5. How is DNA packaging achieved?
    6. 6. Organization of eukaryotic chromatin DNA double helix NucleosomesHistones Solenoid Chromatin loop: ~100,000 bp DNA Chromatin H3 H2B H2A H4
    7. 7. First order of DNA compaction
    8. 8. Nucleosomes are the building blocks of chromatin
    9. 9. 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
    10. 10. 10 Nucleosome organization H3-H4 tetramers build a “wall” that is “capped” by H2A- H2B dimers
    11. 11. 11 Blue= H2A/H2B White= H3/H4
    12. 12. Luger et al, Nature 1997 H3 H4 H2A H2B H3 ‘tail’
    13. 13. Luger et al, Nature 1997 H3 H4 H2A H2B
    14. 14. Second order of DNA compaction
    15. 15. Secondary Structure • H1 : essential for the solenoid structure
    16. 16. Third order of DNA compaction
    17. 17. Histone-depleted metaphase chromosome Protein scaffold Loops of DNA
    18. 18. Histone-depleted metaphase chromosome Scaffold/Matrix attachment regions
    19. 19. A condensed metaphase human chromosome
    20. 20. Genome architecture: chromatin domains
    21. 21. 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
    22. 22. Outline I. Chromatin organization • The DNA packaging problem • Histones and nucleosome core particle • Chromatin folding and nuclear organization • Euchromatin vs Heterochromatin I. Factors that influence chromatin organization and gene function • Histone post-translational modifications (PTMs) and the ‘histone code’ • Histone variants • DNA methylation I. 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
    23. 23. 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
    24. 24. 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
    25. 25. Histone CodeChromatin regulator Tail Globular Histone Modifications Acetylation Phosphorylation Methylation ADP-ribosylation Ubiquitination Sumoylation
    26. 26. Histone CodeChromatin regulator Tail Globular Histone Modifications Acetylation Phosphorylation Methylation ADP-ribosylation Ubiquitination Sumoylation
    27. 27. Histone acetylation and chromatin structure (Adapted from Wade & Wolffe - Current Biology, 1997) “Off” “On”
    28. 28. Bromodomain-containing proteins can bind to acetylated histones (Taken from E. Pennisi - Science, 2000) (TBP) TATAA
    29. 29. Gardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful language where context matters. J. Mol. Biol. 409:36-46. Epigenetic ‘Toolkit’
    30. 30. PHD bromoPHDDDT PHDPHD tudor tudorJmjcJmjn JMJD2A (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
    31. 31. Histone CodeChromatin regulator Tail Globular Histone Modifications Acetylation Phosphorylation Methylation ADP-ribosylation Ubiquitination Sumoylation
    32. 32. Histone Code HP1 Tail Globular Histone Modifications K9 H3 Acetylation Phosphorylation Methylation ADP-ribosylation Ubiquitination Sumoylation
    33. 33. Histone H3 methylation Set1 MLL H3 4 9 27 36 N-ARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVK- Globular domain Gene repression Heterochromatin X-inactivation Dot1 K79 SUV39 ESET G9a EZH2 Set2 NSD1 Gene activation Gene repression X-inactivation Gene activation
    34. 34. 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)
    35. 35. (Taken from Bannister et al. - Nature, 2001) On Off K4 Me K9 Me Roles of H3 lysines 4 and 9 methylation
    36. 36. Post-translational modifications decorate histonesPost-translational modifications decorate histones
    37. 37. 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
    38. 38. HTZ1 (H2A.Z) Figure from Millipore/Upstate
    39. 39. Histone Variants (HTZ1) (Table from Henikoff and Ahmad, Annu. Rev. Cell Dev. Biol, 2005)
    40. 40. 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
    41. 41. DNA methylation Occurs in: (1) select organisms and (2) usually at CpG dinucleotide residues 1. Organisms found in: Humans Mice Frogs Flies*(low levels and CpT) 2. Occurs on Cytosine:
    42. 42. How DNA methylation regulates gene repression? A) By sterically blocking the binding of transcription factors (e.g. E2F, NF-kB, CTCF B) & C) By recruiting chromatin modifying activities D) By affecting RNA Polymerase II transcription HDACs (figure from Klose & Bird, Trends Biochem Sci., 2006)
    43. 43. Outline I. Chromatin organization • The DNA packaging problem • Histones and nucleosome core particle • Chromatin folding and nuclear organization • Euchromatin vs Heterochromatin I. Factors that influence chromatin organization and gene function • Histone post-translational modifications (PTMs) and the ‘histone code’ • Histone variants • DNA methylation I. 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
    44. 44. 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)
    45. 45. The ChIP-chip procedure Crosslink Chromatin with Formaldehyde Shear Chromatin by Sonication IP SAMPLE REFERENCE Hybridize To Microarray Reverse Crosslinks Recover Input DNA Amplify, Label Green Incubate with Antibody Reverse Crosslinks Recover IP DNA Amplify, Label Red (Provided by Jason Lieb, UNC)
    46. 46. DNA (0.1-1.0 ug) Single molecule array Sample preparation Cluster growth 5’ 5’3’ G T C A G T C A G T C A C A G T C A T C A C C T A G C G T A G T 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)
    47. 47. Enriched by ChIP ChIP-Seq • Follow standard ChIP procedure • Identify uniquely aligned sequences in human genome
    48. 48. Mass spectrometry is a vital tool in combinatorial PTM discovery A. Bottom-up MS H3 H3 RP-HPLC Trypsin RP-HPLC MS ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREI AQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA-134 B. Top-down MS H3 H3 RP-HPLC HILIC (hydrophilic interaction liquid chromatography) MS ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREI AQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA-134
    49. 49. Mass Spectrometry technologies have revealed novel histone ‘marks’ and specific histone codes
    50. 50. Mass spectrometry is a vital tool in combinatorial PTM discovery
    51. 51. 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)
    52. 52. Bromodomain-containing proteins can bind to acetylated histones (Taken from E. Pennisi - Science, 2000) (TBP) TATAA K4 PHD
    53. 53. Semi-synthetic modified nucleosomes explore multivalent engagements in chromatin Methyl-lysine analogue (MLA) (Shokat et al.) methyl aminoethylhalide thioester cysteine peptide ligation Native chemical ligation (NCL) and Expressed protein ligation(EPL) (Kent/Cole/Muir labs)
    54. 54. Thank you!

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