The Necessity For DNA Condensation The human genome (haploid) is 3  10 9 base pairs. The helical rise of dsDNA per base pair is 0.3  10 –9 meters. If all of the DNA from a single human cell were laid end-to- end, it would stretch ~ 2 m.

Chromatin Structure and Properties 1. Structure of the histone, the nucleosome, and higher-order chromatin structure 2. Roles of nucleosomes in repressing transcription 3. Local and global methods to identify nucleosome positions 4. Transcription regulation by histone acetylation

SDS-PAGE of Histone Proteins Small and highly basic Highly abundant Highly conserved All except H1 in core nucleosome 20 kDa 15 kDa 10 kDa

Electron Micrograph of Nucleosomes (1975) Pierre Chambon and co-workers Removed histone H1 with trypsin or high salt concentration Named the bead-like structures nucleosomes 500 nm

Structure of the Nucleosome (1991) x-ray crystallography provided moderate resolution structure of the histone core (3.1 Å) suggested a possible path for DNA H2A/H2B H3/H4 Evangelos Moudrianakis and co-workers

Structure of the Nucleosome (1997) First nucleosome structure with DNA 147 base pairs visible along with histone octamer (H1 was not present) Timothy Richmond and co-workers

Fig EM: 30 nm Fiber of Chromatin Panels A-C: Low ionic strength Beads on a string Panel D: Moderate ionic strength Panels E-G: Higher ionic strength Formation of 30 nm fiber (100 mM NaCl) 100 nM

Fig Structure of a Tetranucleosome (2005) Again, Richmond and co-workers Structure is only 9 Å resolution, but model constructed from higher resolution structures of single nucleosomes

Structural Model of the 30 nm Fiber Idealized structure from arranging tetranucleosomes so that angles between units are constant and steric overlap is avoided Structure leaves unclear the role of histone H1

Model For Higher-order Chromatin Structure Nuclease studies suggested ‘circular’ DNA, even for eukaryotes Estimates for loop size range from 35 kb to 83 kb

Inactive Chromatin Lacks H1 (1984) A. Histones in accidentally de-repressed chromatin B. Histones in purposely de- repressed chromatin, treated to remove histone H1 Certain 5S rRNA genes are transcriptionally active in Xenopus oocytes but inactive in somatic cells (Transcription is by RNA polymerase III) 0.6 M KCl

Competition Between Histones and Transcription Factors Both complexes are apparently long-lived, so whichever complex forms first ‘wins’ for controlling transcription

High Concentrations of Core Histones Can Also Repress Transcription (1991) Here, RNA polymerase II transcription followed, of a Drosophila gene Physiological core histone level gave 4-fold repression (lane 5) James Kadonaga and co-workers

Competing Effects of Activators and Histones Addition of H1 in addition to core histones further decreased transcription (odd numbered lanes) Activator GAL4-VP16 had larger effect in the presence of H1, effectively reversing H1 effect

A Model of Transcriptional Activation Core histones Histone H1

Mapping Nucleosome-free Zones

Genomic Methods of Nucleosome Mapping CHA1 promoter region Oliver Rando and co-workers, 2005 Microarrays used to determine positions of nucleosome throughout yeast genome Linker Positioned nucleosome Delocalized nucleosome Most nucleosomes in yeast are well-positioned

Promoter Regions Are Nucleosome-depleted Depleted regions

Nucleosome Position Preferences Are Present In vitro Yeast genomic DNA isolated and reassembled with purified chicken histones Positions of histones largely match those determined in vivo using same method Suggests that nucleosome positions are globally determined by DNA sequence Eran Segal, Jonathan Widom and co- workers, 2008

An Additional Layer of Complexity: Histones Are Modified by Acetylation Histone acetylation activates transcription, reducing the ability of nucleosomes to repress Nuclear acetylation of core histone N-terminal tails (histone acetyltransferase, HAT) –Catalyzed by HAT A (isolated in 1996) –Acetylate lysines within tails of core histones –Some HAT As are also transcriptional activators, suggesting that they bind near transcription start site and acetylate nearby histones H H +

An Additional Layer of Complexity: Histones Are Modified by Acetylation Nuclear acetylation of core histone N-terminal tails (histone acetyltransferase, HAT) –Catalyzed by HAT A (isolated in 1996) –Acetylate lysines within tails of core histones –Some HAT As are also transcriptional activators, suggesting that they bind near transcription start site and acetylate nearby histones H3CH3C Histone acetylation activates transcription, reducing the ability of nucleosomes to repress

An Additional Layer of Complexity: Histones Are Modified by Acetylation Cytoplasmic acetylation carried out by HAT B –Prepares histones for incorporation into nucleosomes –Acetyl groups later removed in nucleus Nuclear acetylation of core histone N-terminal tails (histone acetyltransferase, HAT) –Catalyzed by HAT A (isolated in 1996) –Acetylate lysines within tails of core histones –Some HAT As are also transcriptional activators, suggesting that they bind near transcription start site and acetylate nearby histones Histone acetylation activates transcription, reducing the ability of nucleosomes to repress

Model For Repression By Histone Deacetylase Retinoic acid receptor heterodimer

Histone Acetylation How does acetylation increase transcription? Reduces positive charge on lysine-rich regions, presumably reducing affinity of nucleosome for DNA May weaken or disrupt interactions between nucleosomes Can recruit transcription factors (bromodomain)

Key Points 1. Cellular DNA is packaged with histone proteins into a higher order structure called chromatin. The first level of chromatin organization is the nucleosome, and a higher order structure is the 30 nm fiber. 2. The packaging, or condensation, of DNA into chromatin represses transcription because the transcription machinery cannot efficiently access the promoter. 4. Modification of histones by acetylation activates transcription. 3. Nucleosomes are globally depleted near transcription start sites, and their positions appear to be greatly influenced by DNA sequences.