1. The role of histone H1 1 mM NaCl 5 mM NaCl H1 binds to the nucleosome where the DNA enters and exits the core. - H1 + H1 H1 is needed to form the zig-zag structure.

2. Role of H1 (cont.) • Evidence indicates that H1 also functions in forming the 30 nm fiber, and that it interacts with other H1 molecules H1-H1 interaction proven by cross-linking experiments.

3. What is the effect of histones on transcription in vitro? • Assemble core histones on a plasmid (1/200 bp), nucleosomes inhibit transcription by blocking promoter binding sites. • Addition of H1 further represses transcription (by binding to the linker DNA), but this can be overcome by activators such as Sp1. • There are regulatory proteins, such as the glucocorticoid-receptor complex, that can remove histones from certain promoters.

4. In Vivo Studies • Promoters of active genes are often deficient in nucleosomes SV40 virus minichromosomes with a nucleosome-free zone at its twin promoters. Can also be shown for cellular genes by DNase I digestion of chromatin – promoter regions are hypersensitive to DNase I. From Fig. 13.17

5. 3 ways to a clear (or nucleosome-deficient) promoter • Transcription factor(s) (for the respective RNA polymerase) bind the promoter before histones – there is competition to bind newly synthesized DNA 2. If H1, which binds linker DNA, is blocking the promoter, it can be displaced by some transcription factors (e.g., SP1). 3. If a nucleosome does form on the key part of the promoter, it has to be removed by special complexes to get transcriptional activation - a.k.a. “chromatin remodeling” .

6. 2 Models for Transcriptional Activation Similar to Fig. 13.16 Nucleosome covers promoter, still repressed after H1 removed. Remove nucleosome with special “remodeling” factors. H1 (yellow) covers promoter, remove it and bind activators (factors).

7. What about elongation by RNA polymerase? • How does RNA polymerase transcribe through regions with nucleosomes? • 2 possibilities: 1. It could partially open nucleosomes and slide around the DNA, which is on the outside. Or 2. It could completely displace nucleosomes.

8. Experimental Strategy • Construct DNA with only 1 nucleosome on it, downstream of a promoter. • Transcribe DNA in vitro. • Determine if the nucleosome moves to a new position*. • * DNA specifically bound in a nucleosome can be recovered after Micrococcal Nuclease digestion – which degrades all DNA not protected by the nucleosome.

9. Fig. 13.44 DNA protected by the nucleosome core is recovered after micrococcal nuclease digestion, radiolabeled, and then hybridized to restriction enzyme digests of pB18. Conclusion: some of the nucleosome (blue oval) is repositioned on pB18 after transcription in vitro with the viral RNA polymerase. (a) Restriction map of pB18, and (b) expected DNA fragments with different enzyme combinations. Fig. 13.43

10. Eukaryotic chromosomal DNA also has supercoiled regions. Fig. 13.10

11. 3’ Right handed Writhing structures Writhing – double helical strand passing over itself

12. Writhing structures

13. Equation for Supercoiling Lk = Tw + Wr Lk = Linking number (# times that strands cross) Tw = Twist (# of times that strands cross, excluding writhe, or # of helical turns) Wr = Writhe (# of times that double helix crosses itself)

14. Topoisomerase I relaxes DNA one link at a time Reduces Lk one unit at a time. + Topo I

15. Type I Topoisomerases change Ln by 1, breaks 1 strand.

16. Type II Topoisomerases – change L by 2, cuts both strands