Biol/Chem 473

1. Biol/Chem 473 Schulze lecture 6: Introduction to chromatin structure

2. Control regions upstream of pair-rule genes are complex General principle: regulatory regions of eukaryotic genes are complex; the promoter of any given gene has to integrate a large amount of combinatorial inputs that will define its activity depending on the context of the cell in which the gene resides. (Are bicoid levels high or low? What about Kruppel? Etc. )

3. Eukaryotic gene expression through the occluding effects of chromatin

4. What is chromatin? • It is a solution to a eukaryote’s packaging problem….

5. Eukaryotes have a BIG packaging problem • How do you fit approximately 2 meters (human diploid nucleus) into a space that averages maybe 5 millionths of a meter wide? • How do you replicate, repair and transcribe tightly packaged DNA?

6. Solution: Chromatin! • Chromatin is DNA packaged with specialised proteins (and even some RNA!) that serve to control the degree to which DNA sequences are accessible for synthesis and transcription • These proteins include specialized structural proteins and enzymes

7. Chromatin packaging heirarchy Level 1: nucleosome formation Level 2: 30 nm fiber Level 3: Nuclear scaffolding Level 4: Mitotic (metaphase) chromosome

8. Level One: Building blocks of chromatin: nucleosomes • “string on a bead” for obvious reasons • Linker can vary (8-114bp or more) • Compaction ratio is approx 7 fold

9. N C H1 Nucleosomes are composed of histones • 2/3 of chromatin mass is protein • 95% of chromatin protein are histones • H1, H2A, H2B, H3, H4

10. Nucleosome structure Chromatosome: octamer of histones plus ~146 bp DNA AND linker histone H1 (this term rarely used now) Nucleosome core particle: octamer of histones plus ~146 bp DNA

11. Nucleosome core particle

12. Level Two: the 30nm fiber • Requires Histone H1 • Compaction ratio approx 100 fold Lehninger

13. Level three: nuclear scaffolding • Not well understood • Organization is not random; involved sequence elements (red dots), more non-histone chromatin proteins and tethering to the nuclear envelope and matrix

14. Genome contortions during the cell cycle Time for replication, transcription Time for cell division: no gene expression

15. Metaphase chromatin: level 4 packaging: fully condensed

16. Interphase chromatin: levels 1-3 relatively decondensed chromosomes • Heterochromatin: dark-staining, condensed (mostly simple-sequence DNA) • Euchromatin: light-staining, less condensed (complex sequence DNA: e.g. genes)

17. Summary: chromatin • DNA plus protein • Enables extraordinary condensation and packaging of eukaryotic genomes • Fundamental unit is the nucleosome • Nucleosome consists of an octamer of histone proteins: 2XH2A, 2X H2B, 2XH3 and 2XH4 • Between nucleosomes, a fifth histone, H1, acts as a linker (among other mysterious things) • Gene expression in eukaryotes takes place in the context of highly packaged chromatin • Regulation of gene expression by chromatin structure is epigenetic regulation

18. Heterochromatin • WHY?? WHY?? WHY?? • It is a pain to work with (lots of repetitive DNA) • It KILLS gene expression (transcriptionally repressive) • It’s boring.

19. Turning genes OFF may be more important that turning genes ON • Inverse dose response • Delete a chunk of chromosome and background gene expression tends to go UP (suggesting most of the deleted genes are repressors) • Differential gene expression in development • Coming attraction! • Genome surveillance • Keep parasitic (middle repetitive) DNA from wreaking havoc in the genome

20. Gene silencing X Genes

21. Genome architecture: chromatin domains

22. Stains darkly (highly condensed) Repetitive sequences Replicates later in the cell cycle Little or no recombination Transcriptionally repressive: silences gene expression Stains lightly (decondensed) Single copy sequences (genes) Replicates early in the cell cycle Recombines Transcriptionally active: permissive for gene expression Heterochromatin vs Euchromatin

23. How do eukaryotes replicate their linear DNA? Primase makes primers for okazaki fragments as well as first primer DNA synthesis is continuous on the leading strand, but discontinuous on the lagging strand

24. Solution: telomerase!

25. Solution: telomerase!

26. Solution: telomerase!

27. Heterochromatin summary • Localized to telomeres and regions flanking centromeres. • Consists of repetitive sequences (mostly). • Transcriptionally repressive. • Study of heterochromatin revealed how chromatin affects gene expression. • Many of the silencing mechanisms operating constitutively in heterochromatin are used by euchromatin as well to locally regulate gene expression.

28. Why is there heterochromatin? • Structural role? • Homolog pairing at meiosis? • “graveyard” for potentially parasitic elements? • No reason – just too much trouble to get rid of?

29. It all started with flies….

30. w+ w Drosophila gene nomenclature • Early days of Drosophila research: a gene was named after the phenotype that resulted when that gene was mutant. (Example: the white gene results in loss of red pigmentation in the eye, so the eye is white) • Now it a lot more complicated (and less consistent). Genes tend to be named after the products they encode. (Example: DEAD box protein 80 = Dbp80)

31. Position Effect Variegation (PEV) in Drosophila • A euchromatic gene relocated next to or within heterochromatin will variegate (show variable silencing) • The sequence of the gene has not changed, only its position • Therefore this is an epigenetic (“beyond genetic”) effect

32. w+ w+ The wild type white gene variegates because of its position (PEV)

33. Fly with chromosomal rearrangement placing the wild type white gene next to het Fly with chromosomal rearrangement placing the wild type white gene next to het AND a mutation in a background suppressor Fly with chromosomal rearrangement placing the wild type white gene next to het AND a mutation in a background enhancer Genetic screen for modifiers of PEV Wallrath LL, Cur. Opin. Genet. Dev. 1998, 8:147

34. Chromatin associated proteins • Su(var) 2-5 encodes Heterochromatin Protein 1 • a chromatin structural protein which recognizes methylated histone H3 and can also recognize and bind to itself • Su(var) 3-9 encodes a histone methyl-transferase • an enzyme which transfers a methyl group onto a specific lysine residue on Histone H3 • Su(var) 326 encodes a histone deacetylase • an enzyme which removes acetyl groups from histones; deacetylated histones are correlated with repressed chromatin • these proteins (and others) are reading a HISTONE CODE

35. The histone code hypothesis …would it make a good movie? Is Tom Hanks available???

36. So what’s this histone code all about? • Histones are subjected to a variety of post translational modifications (most often on the N-terminal tails) • These modifications are generated by specific enzymes • These modifications are recognized by proteins that can influence gene expression and other chromatin functions

37. Histone modifications cont… From: Khorasanizadeh, 2004.

38. CHROMO Chromatin organization modifier CHROMO SHADOW DOMAIN SET Su(var)3-9-E(z)-TRX Su(var) 2-5 (HP1) Su(var) 3-9 Su(var) 326 (Rpd3) Protein domains in chromatin associated proteins SMART (simple modular architectural research tool) http://smart.embl-heidelberg.de/

39. There are “new” histone modifications being discovered…