Chapt 13: Chromatin Structure and Its Effects on Transcription 13-1 Chromatin in developing human spermatid Student learning outcomes : Explain relationship among activators, chromatin structure and gene activity Describe basic structure of the nucleosome Explain how histones interact with DNA and other proteins to control transcription

Describe how position of nucleosomes can result in repression, and how remodeling permits activation. Explain how modification of histones can affect gene expression Diagram two new techniques: DNase hypersensitivity assay Chromatin immunoprecipitation (ChIP) Describe how heterochromatin is condensed, genetically inactive form Important Figures: 1, 2, 3, 7, 9, 12, 13*, 16*, 21, 22, 24, 26*, 27, 29*, 32*, 34*, 35*, 36*; Table 1 Review problems: 1, 7, 8, 9, 12, 13, 14, 16, 18; Anal Q 2,

Histones in Eukaryotic cells –H121.5 kD –H2A14.0 –H2B13.8 –H315.4 –H411.3 Abundant proteins: mass in nuclei nearly equals that of DNA Pronounced positive charge at neutral pH: 20% lys and arg Each type not homogenous –Gene reiteration –Posttranslational modifications (Ac, Me, PO 4 -) Fig. 1 Histones from calf thymus on SDS-PAGE

Not single copy genes: repeated many times Some copies are identical; Others are different H4 has only had 2 variants ever reported ** Originally viewed as scaffolds for DNA: regulatory role for gene expression is more recent

Nucleosomes Each chromosome is 1 long, thin DNA molecule Will tangle if not carefully folded 1 st order of folding: nucleosome: beads on string –X-ray diffraction shows repeats of structure at 100Å intervals –Approximates nucleosomes spaced at 110Å intervals

13-6 Histones in the Nucleosome Chemical cross-linking in solution: –H3 to H4; H2A to H2B H3 and H4 form tetramer (H3-H4) 2 Chromatin: roughly equal masses of DNA, histones: 1 histone octamer per 200 bp of DNA Octamer composed of: 2 each H2A, H2B, H3, H4 DNA wrapped on outside [1 each of H1 binding to linker region between core nucleosomes -> ‘beads on string’ ] Fig. 3

Fig Fig. 4 Nucleosome core structure; DNA on outside; unstructured histone N-terminal tails; (H3-H4) 2 tetramer

13-8 Nucleosome Structure Central (H3-H4) 2 core attached to H2A-H2B dimers Grooves on surface define a left-hand helical ramp – a path for DNA winding –DNA winds almost twice around histone core, condensing DNA length 6- to 7-fold –Core histones contain a histone fold: 3  -helices linked by 2 loops Extended tail ~ 28% of core histone mass Tails are unstructured

13-9 H1 and Chromatin Trypsin or high salt buffer removes histone H1 Leaves chromatin looking like “beads-on-a-string” Beads are nucleosomes –Core histones form ball with DNA around outside –DNA on outside minimizes bending –H1 also lies on outside of nucleosome Fig. 6 H1 and chromatin

13-10 The 30-nm Fiber 2 nd order of chromatin folding produces fiber 30 nm in diameter –String of nucleosomes condenses to form 30-nm fiber in solution of increasing ionic strength –Condensation results in another 6- to 7-fold condensation of nucleosome itself 4 nucleosomes condense into 30-nm fiber, form zig-zag structure Fig. 7 tetranucleosome

13-11 Formation of 30-nm Fiber Two stacks of nucleosomes form left-handed helix –Two helices of polynucleosomes –Zig-zags of linker DNA Role of histone H1? –30-nm fiber can’t form without H1 –H1 crosslinks to other H1 more often than to core histones Fig. 8 model of 30-nm fiber

13-12 Model: Higher Order Chromatin Folding 30-nm fibers are most of chromatin in typical interphase nucleus Further folding needed in mitotic chromosomes Model for higher order folding is radial loops Can be supercoiling in loops Fig. 9 higher order folding Source: Adapted from Marsden, M.P.F. and U.K. Laemmli, Metaphase chromosome structure: Evidence of a radial loop model. Cell 17:856, 1979.

Chromatin Structure and Gene Activity Histones, especially H1, repressive effect on gene activity in vitro Two families of 5S rRNA genes studied in Xenopus laevis (pol III type 1) –Oocyte genes expressed only in oocytes About 20,000 gene copies –Somatic genes expressed both in oocytes and somatic cells About 400 copies –Somatic genes form more stable complexes with transcription factors, prevent nucleosomes forming complex with the internal control region

13-14 Transcription Factors and Histones Control 5S rRNA expression Genes are active when TFIIIs prevent formation of nucleosome stable complexes with internal control region Stable complexes require histone H1 and exclude TFIIIs once formed, so genes are repressed Fig. 13Fig

13-15 Effects of Histones on Transcription of Class II Genes (pol II) Core histones assemble nucleosome cores on naked DNA Transcription of reconstituted chromatin (average of 1 nucleosome / 200 bp DNA): Exhibits 75% repression relative to naked DNA Remaining 25% activity is due to promoter sites not covered by nucleosome cores

13-16 Histone H1 and Transcription Histone H1 causes further repression of template activity, in addition to that of core histones H1 repression counteracted by transcription factors Sp1 and GAL4 act as both: –Antirepressors prevent histone repression –Transcription activators GAGA factor: –Binds to GA-rich sequences in Krüppel promoter –An antirepressor – prevents repression by histones

13-17 Model of Transcriptional Activation: position of nucleosome is critical Fig. 16 Source: Adapted from Laybourn, P.J. and J. T. Kadonaga, Role of nucleosomal cores and histone H1 in regulation of transcription by polymerase II. Science 254:243, Fig. 16; Yellow is H1

13-18 Nucleosome Positioning Model of activation and antirepression: Transcription factors can cause antirepression by: –Removing nucleosomes that obscure promoter –Preventing initial nucleosome binding to promoter Both actions are forms of nucleosome positioning: activators force nucleosomes to positions around, not within, promoters Detect nucleosome-free zones by: –Electron microsopy after restriction digest –DNase hypersensitivity assay

13-19 Detecting nucleosome-free zones Nucleosome positioning should give nucleosome-free zones in control regions of active genes: SV40 virus model system Assessment in circular chromosome difficult without some type of marker – use restriction enzymes Figs. 17,18, 19 a-c, BamHI, d-f, BglI

13-20 Detecting DNase-Hypersensitive Regions Active genes tend to have DNase- hypersensitive control regions Part of hypersensitivity is absence of nucleosomes Detect as cleavage products on gels with probe Fig. 22 shows analysis of globin gene expression

13-21 Acetylation of Histone tails activates gene expression Histone acetyltransferase (HAT) adds acetyl group Nuclear acetylation of core histone N-terminal tails: –Catalyzed by HAT A on specific lysines (HAT B cytoplasm) H3 (K9, 13, 18); H4 (K5, 8, 12, 16) –Correlates with transcription activation (ex. TR/RXR) –Coactivators of HAT A may loosen association between nucleosomes and gene’s control region –Attracts proteins like TAF , essential for transcription Some coactivators have HAT A activity : GCN5, CBP/p300; TAF

13-22 Histone Deacetylation represses genes Fig. 24 Transcription repressors bind to DNA sites, interact with corepressors, which bind histone deacetylases Deacetylation of histones: basic histone tails bind strongly to DNA Repressors include: unliganded nuclear receptors Mad-Max Corepressors include : NCoR/SMRT SIN3 Histone deacetylases : HDAC 1, 2

13-23 Chromatin, Activation and Repression Deacetylation of core histones removes binding sites for HAT A coactivator proteins that are essential for transcription activation **Fig. 26

13-24 Chromatin Remodeling Activation of many eukaryotic genes requires chromatin remodeling (loosening, repositioning) Several protein complexes do remodeling –All have ATPase activity: use energy from ATP hydrolysis to remodel nucleosomes –Alter structure of nucleosome core to make more accessible to activators, nucleases Ex. SWI/SNF from yeast (mating type switch) also in mammals

13-25 Model SWI/SNF Chromatin Remodeling SWI/SNF : In mammals, protein BRG1 is ATPase 9-12 BRG1-associated factors (BAFs); a highly conserved BAF is BAF 155 or 170 its SANT domain binds histones - helps SWI/SNF bind nucleosomes Fig. 27

13-26 Mechanism of Chromatin Remodeling –Mobilization of nucleosomes from starting position –Loosen association between DNA, core histones –Open up promoters to transcription factors Formation of distinct conformations of nucleosomal DNA/core histones - contrast with: –Uncatalyzed DNA exposure in nucleosomes –Simple nucleosome sliding along a DNA stretch

Fig Testing model of nucleosome remodeling Movement of nucleosomes opens up different sequences to restriction enzyme digestion - Time after addition of ATP and SWI/SNF

13-28 Chromatin Immunoprecipitation technique Fig. 29 Identifies specific sequence bound by a specific protein Steps include: Crosslink cell proteins to DNA with formaldehyde in vivo Isolate chromatin; Carefully shear DNA Precipitate desired protein with antibody and beads Reverse crosslink; remove proteins Use PCR with specific primers to see if particular region was bound

13-29 Ex. Remodeling in Yeast HO Gene Activation Chromatin immunoprecipitation (ChIP) reveals order of factors binding to specific gene during activation Yeast have 2 mating types: a and  ; can switch As HO gene is activated (mating type switch): –First factor to bind is Swi5 –Followed by SWI/SNF and SAGA (containing HAT GCN5) –Next general transcription factors and other proteins Chromatin remodeling is among first steps in activation of gene Order could be different in other genes

Timing of histone acetyloation after activation of Human IFN-  Gene ChIP analysis: Infect with virus to activate IFN Antibodies to precipitate specific proteins Analyze mRNA, TBP B. Deplete HAT activity -> no acetylation of H Fig. 30a

13-31 Remodeling Human IFN-  Gene: ex. Histone Code The Histone Code: –Combination of modifications on nucleosome near gene’s control region affects efficiency of transcription –Code is epigenetic, not affect sequence of DNA 1) Activators in IFN-  enhanceosome recruit GCN5 HAT –HAT acetylates some Lys on H3 and H4 in nucleosome at promoter –Protein kinase phosphorylates Ser on H3 –Permits acetylation of another Lys on H3 –Ac-Lys recruits SWI/SNF, remodels nucleosome

13-32 Remodeling Human IFN-  Gene: 2) TF Binding Remodeled nucleosome allows TFIID to bind 2 Ac-Lys via bromodomain in TAF II 250 (domain that binds Ac- lys) TFIID binding: Bends DNA (TBP), Moves remodeled nucleosome aside, paves way for transcription to begin Fig. 32

13-33 Heterochromatin Euchromatin: relatively extended and open potentially active Heterochromatin: very condensed, DNA inaccessible –Repressive character can silence genes 3 kb away – centromeres – telomeres

13-34 Heterochromatin at tips of yeast chromosomes (telomeres) silences nearby genes: telomere position effect (TPE) Requires binding of specific proteins –RAP1 to telomeric DNA –Recruitment of proteins in order: SIR3 SIR4 SIR2 Heterochromatin and Silencing at telomeres Fig. 34

13-35 SIR Proteins Heterochromatin at other locations in chromosome also depends on SIR proteins (silencing information regulator) SIR3 and SIR4 interact directly with histones H3 and H4 in nucleosomes –Acetylation of Lys 16 on H4 in nucleosomes prevents interaction with SIR3 –Blocks heterochromatin formation Histone acetylation (HATs) which acetylate histones promote gene activity

13-36 Histone Methylation Methylation of Lys 9 in tail of H3 attracts HP1 Recruits a histone methyltransferase (HMTase) –Methylates Lys 9 on neighboring nucleosome –Propagates repressed, heterochromatic state Methylation of Lys and Arg side chains in core histones can have repressive or activating effects Fig. 35

13-37 Modification Combinations Methylations occur in a given nucleosome in combination with other histone modifications: –Acetylations –Phosphorylations –Ubiquitylations Each particular combination can send a different message to the cell about activation or repression of transcription One histone modification can also influence other, nearby modifications

Fig Histone tail modifications can be repressive or activating; permit fine level of control on gene expression

Modifications interactions Fig Ubiquitination of H2B K123 by the rad6 protein is required for methylation of H3 K79 or K4 Antibodies specific for different modifications Western blot after separation of proteins by SDS- PAGE

13-40 Modification Interactions Modifications shown above histone tail activate –Ser phosphorylation –Lys acetylation Modification below tail (Lys methylation) represses Fig. 38

13-41 Nucleosomes and Transcription Elongation An important transcription elongation facilitator is FACT (facilitates chromatin transcription) –2 subunits: Spt16 binds to H2A-H2B dimers –acid-rich C-terminus is essential for these nucleosome remodeling activities SSRP1 binds to H3-H4 tetramers –Facilitates transcription through nucleosome by promoting loss of at least one H2A-H2B dimer Acts as histone chaperone promoting re-addition of H2A-H2B dimer to nucleosome that has lost dimer

Review questions 1. Diagram nucleosome, showing rough positions of histones; on another drawing, show position of the DNA 9. Present two models for antirepression by transcription activators, one in which the gene’s control region is not blocked by a nucleosome, the other in which it is. 12. Diagram technique for detecting DNase hypersensitive region on DNA 18. Describe how you could use Chromatin immunoprecipitation to detect proteins associated with a particular gene at various points in cell cycle of yeast