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

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. 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. 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. 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. How is DNA packaging
achieved?

6. Organization of eukaryotic chromatin
DNA double helix
NucleosomesHistones
Solenoid
Chromatin loop:
~100,000 bp DNA
Chromatin
H3
H2B
H2A
H4

7. First order of DNA
compaction

8. Nucleosomes are the building
blocks of chromatin

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
Nucleosome organization
H3-H4 tetramers
build a “wall” that is
“capped” by H2A-
H2B dimers

11. 11
Blue= H2A/H2B
White= H3/H4

12. Luger et al, Nature 1997
H3
H4
H2A
H2B H3 ‘tail’

13. Luger et al, Nature 1997
H3
H4
H2A
H2B

15. Second order of DNA
compaction

16. Secondary Structure
• H1 : essential for the solenoid structure

17. Third order of DNA
compaction

18. Histone-depleted metaphase
chromosome
Protein scaffold
Loops of DNA

19. Histone-depleted metaphase
chromosome
Scaffold/Matrix attachment
regions

20. A condensed metaphase human chromosome

21. Genome architecture: chromatin domains

22. 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

23. 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

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. 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

26. Histone CodeChromatin
regulator
Tail Globular
Histone Modifications
Acetylation
Phosphorylation
Methylation
ADP-ribosylation
Ubiquitination
Sumoylation

27. Histone CodeChromatin
regulator
Tail Globular
Histone Modifications
Acetylation
Phosphorylation
Methylation
ADP-ribosylation
Ubiquitination
Sumoylation

28. Histone acetylation and chromatin structure
(Adapted from Wade & Wolffe - Current Biology, 1997)
“Off” “On”

29. Bromodomain-containing proteins can bind to acetylated histones
(Taken from E. Pennisi - Science, 2000)
(TBP)
TATAA

30. Gardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful language
where context matters. J. Mol. Biol. 409:36-46.
Epigenetic ‘Toolkit’

31. 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

32. Histone CodeChromatin
regulator
Tail Globular
Histone Modifications
Acetylation
Phosphorylation
Methylation
ADP-ribosylation
Ubiquitination
Sumoylation

33. Histone Code
HP1
Tail Globular
Histone Modifications
K9
H3
Acetylation
Phosphorylation
Methylation
ADP-ribosylation
Ubiquitination
Sumoylation

34. 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

35. 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)

36. (Taken from Bannister et al. - Nature, 2001)
On Off
K4 Me
K9 Me
Roles of H3 lysines 4 and 9 methylation

37. Post-translational modifications decorate histonesPost-translational modifications decorate histones

38. 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

39. HTZ1 (H2A.Z)
Figure from Millipore/Upstate

40. Histone Variants
(HTZ1)
(Table from Henikoff and Ahmad, Annu. Rev. Cell Dev. Biol, 2005)

41. 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

42. 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:

43. 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)

44. 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

45. 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)

46. 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)

47. 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)

48. Enriched by ChIP
ChIP-Seq
• Follow standard ChIP procedure
• Identify uniquely aligned sequences in human genome

49. 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

50. Mass Spectrometry technologies have revealed novel histone
‘marks’ and specific histone codes

51. Mass spectrometry is a vital tool in combinatorial PTM discovery

52. 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)

53. Bromodomain-containing proteins can bind to acetylated histones
(Taken from E. Pennisi - Science, 2000)
(TBP)
TATAA
K4
PHD

54. 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)

55. Thank you!