How DNA Is Packed In The Cell: Chromosomes, Genes, Nucleosomes

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1 Summer Course in Biophysics June 5, 2014 How DNA Is Packed In The Cell: Chromosomes, Genes, Nucleosomes Brian D. Strahl Department of Biochemistry & Biophysics UNC-School of Medicine

2 Outline I. 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

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 A Matter of Fitting In! meters (0.17 inches) 8850 meters (~5.5 miles) Mount Everest image: pipet tip image: Biologix Research (slide provided by Raymond Reeves)

5 How is DNA packaging achieved?

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

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 H1 N C Tail domain Regulatory domain Involved in higher-order packing Globular domain Histone-histone interactions DNA wrapping

10 Nucleosome organization H3-H4 tetramers build a wall that is capped by H2A- H2B dimers 10

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

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

13 H3 H4 H2A H2B Luger et al, Nature 1997

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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 Loops of DNA Protein scaffold

19 Histone-depleted metaphase chromosome Scaffold/Matrix attachment regions

20 A condensed metaphase human chromosome

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22 Genome architecture: chromatin domains

23 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

24 Outline I. 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

25 Molecular mechanisms that influence chromatin structure and function 1. Chromatin remodeling complexes (e.g. Swi/Snf) 2. Histone modifications 3. Histone variants (e.g. H2A.Z, CENP-A, etc.) 4. DNA methylation

26 Molecular mechanisms that influence chromatin structure and function 1. Chromatin remodeling complexes (e.g. Swi/Snf) 2. Histone modifications 3. Histone variants (e.g. H2A.Z, CENP-A, etc.) 4. DNA methylation

27 Histone Modifications Acetylation Phosphorylation Methylation ADP-ribosylation Ubiquitination Sumoylation Histone Code Chromatin regulator Tail Globular

28 Histone Modifications Acetylation Phosphorylation Methylation ADP-ribosylation Ubiquitination Sumoylation Histone Code Chromatin regulator Tail Globular

29 Histone acetylation and chromatin structure Off On (Adapted from Wade & Wolffe - Current Biology, 1997)

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

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

32 Histone Code readers DDT PHD PHD bromo BPTF (NURF) PWWP Jmjn Jmjc PHD PHD tudor tudor JMJD2A (demethylase) bromo bromo bromo bromo bromo bromo BAH BAH HMG BAF180 (SWI/SNF) PHD PHD chromo chromo DEXD HELIC CHD4 (NuRD) (Figure from Abcam)

33 Histone Modifications Acetylation Phosphorylation Methylation ADP-ribosylation Ubiquitination Sumoylation Histone Code Chromatin regulator Tail Globular

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

35 Histone H3 methylation Set1 MLL SUV39 ESET G9a EZH2 Set2 NSD1 Dot1 H3 K79 N-ARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVK- Globular domain Gene activation Gene repression Heterochromatin X-inactivation Gene repression X-inactivation Gene activation

36 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)

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

38 Post-translational modifications decorate histones

39 Molecular mechanisms that influence chromatin structure and function 1. Chromatin remodeling complexes (e.g. Swi/Snf) 2. Histone modifications 3. Histone variants (e.g. H2A.Z, CENP-A, etc.) 4. DNA methylation

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

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

42 Molecular mechanisms that influence chromatin structure and function 1. Chromatin remodeling complexes (e.g. Swi/Snf) 2. Histone modifications 3. Histone variants (e.g. H2A.Z, CENP-A, etc.) 4. DNA methylation

43 DNA methylation Occurs in: (1) select organisms and (2) usually at CpG dinucleotide residues 1. Organisms found in: 2. Occurs on Cytosine: Humans Mice Frogs Flies* (low levels and CpT)

44 How DNA methylation regulates gene repression? HDACs 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 (figure from Klose & Bird, Trends Biochem Sci., 2006)

45 Outline I. 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

46 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)

47 (Provided by Jason Lieb, UNC) The ChIP-chip procedure Crosslink Chromatin with Formaldehyde Incubate with Antibody Shear Chromatin by Sonication Reverse Crosslinks Recover IP DNA Reverse Crosslinks Recover Input DNA Amplify, Label Red Amplify, Label Green Hybridize To Microarray

48 Solexa Sequencing (Illumina) 3 5 DNA ( ug) Sample preparation Single Cluster molecule growtharray A C T C T G C T G A A G 5 T G C T A C G A T A C C C G A T C G A T Sequencing T G C T A C G A T Image acquisition Base calling

49 ChIP-Seq Follow standard ChIP procedure Identify uniquely aligned sequences in human genome Enriched by ChIP

50 Mass spectrometry is a vital tool in combinatorial PTM discovery A. Bottom-up MS H3 RP-HPLC Trypsin RP-HPLC H3 MS ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIA QDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA-134 B. Top-down MS H3 RP-HPLC H3 HILIC (hydrophilic interaction liquid chromatography) MS ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIA QDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA-134

51 Mass Spectrometry technologies have revealed novel histone marks and specific histone codes

52 Mass spectrometry is a vital tool in combinatorial PTM discovery

53 SILAC-based approaches are unlocking identification of novel effector proteins (Stable isotope labeling by amino acids in cell culture) peptide Bead peptide Bead Unlabeled extracts Isotopelabeled extracts peptide Bead peptide Bead SDS/PAGE and tryptic digestion m / z

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

55 Semi-synthetic modified nucleosomes explore multivalent engagements in chromatin NT peptide thioester S R cysteine HN H 2 N O O HS peptide ligation NT peptide H N HN O O H N HN O O SH SH methyl aminoethylhalide Native chemical ligation (NCL) and Expressed protein ligation(epl) (Kent/Cole/Muir labs) Br base N + H N HN O O S Methyl-lysine analogue (MLA) (Shokat et al.) N +

56 Thank you!