Biol/Chem 473

1. Biol/Chem 473 Schulze lecture 7: Eukaryotic gene regulation: Epigenetic regulation of homeotic genes

2. 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

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

4. 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/

5. Histone acetyltransferases, Kinases Histone deacetylases Heterochromatin Protein 1 (Su(var)2-5) Histone methyltransferases

6. Heterochromatin vs. euchromatin adapted from Jenuwein & Allis (2001) Science 293, 1074

7. In prokaryotes, the ground state for gene regulation is non-restrictive. In eukaryotes, the ground state is restrictive, and the silenced state is even more restrictive.

8. Conservation of the histone code S. cerevisiaeS.pombehuman Deacetylation: Sir2 Clr3, Clr6 HDAC1, 5 histone binding: Sir4, 3 Swi6 HP1 (α,β,γ) H3K9 methylation: - Clr4 SUV39H1

9. Mechanism of heterochromatic silencing using the histone code in S. pombe “spreading” model of heterochromatin assembly

10. w+ Spreading of heterochromatin in flies (and humans!)

11. 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).

12. Summary: chromatin • Gene expression in eukaryotes takes place in the context of highly packaged chromatin. • Regulation of gene expression by chromatin structure is epigenetic regulation. • Epigenetic regulation is achieved by interpretation of a histone code consisting of covalent groups attached to the N-terminally extended tails of histone proteins. • The structural proteins and enzymes responsible for generating this code were discovered by genetic analysis of position effect variegation in flies. • These proteins are all highly conserved across taxa!

13. Chromatin structure and the regulation of homeotic genes • A case study for epigenetics: “cellular (or transcriptional) memory” • Paradigm of differential gene expression • Hox (homeotic) genes, development and evolution • Maintenance genes: Polycomb and trithorax group • Connections to the histone code and heterochromatic silencing

14. Differential gene expression ? …..not to scale…..

15. Differential gene expression ? …..not to scale…..

16. Differential gene expression • All the information to make a human (fly) resides in every cell • But not every cell can make a human (fly) • WHY???

17. Gene silencing X Genes

18. Early development: overlapping concentration gradients of transiently expressed transcriptional regulators

19. End of the line: segment identity is specified by homeotic genes • Homeotic genes are activated as segments are determined • Homeotic genes were first identified as dominant mutations that changed the identity of structures. • Homeosis: wholescale transformation of one body part into another • The homeotic genes encode transcriptional regulators that act as genetic switches, turning complex programs of cellular differentiation on or off within each segment Antennapedia-Complex Bithorax-Complex

20. Homeotic mutants are striking Wild type

21. Homeotic genes are conserved • Mutations in plant HOX genes cause elaborate flower structures (among other things)

22. Homeotic genes are conserved

23. Homeotic genes are famous

24. Homeotics have evolutionary importance

25. Transcriptional memory • Positional information in the early embryo is set up by maternally supplied or early zygotic transcriptional regulators • But these regulators are expressed only TRANSIENTLY • How do the cells remember which segment they belong to and what they are supposed to become? • The homeotic genes themselves are regulated epigenetically by the products of genes belonging to two groups: the Polycomb and trithorax group of genes

26. How does a cell “remember” which genes are supposed to be on or off through the course of cell division?

27. Mutations in homeotic genes change segment identities

28. Example of “ectopic expression”: gene (or genetic program) is expressing in the wrong time and place Mutations in Polycomb group genes change many segment identities

29. Many of these proteins interact to form complexes that also interact with chromatin in the vicinity of homeotic genes POLYCOMB GROUP GENES • additional sex combs • chameau • cramped • enhancer of zeste • Enhancer of Polycomb • extra sexcombs • pipsqueak • pleiohomeotic • Polycomb • Polycomblike • polyhomeotic distal • polyhomeotic proximal • Posterior sexcombs • Sex combs extra • Sex combs on midleg • Suppressor of zeste 2 • Su(z)2(D) - (See Posterior sexcombs)

30. REPRESSORS Mutations in trithorax group genes also change many segment identities

31. Many of these proteins interact to form complexes that also interact with chromatin in the vicinity of homeotic genes TRITHORAX GROUP GENES • absent, small, or homeotic discs 1 • absent, small, or homeotic discs 2 • brahma • eyelid (also known as osa) • ISWI • kismet • lola like • modifier of mdg4 • moira • Snf5-related 1 • trithorax • Trithorax like • zeste

32. Polycomb and trithorax group genes have antagonistic functions • Mutations in PcG genes mimic OVEREXPRESSION of homeotic genes, so PcGs (when wild type) are therefore repressors of homeotic loci • Mutations in the trxG genes mimic LOSS OF FUNCTION of homeotics, so trxGs (when wild type) are therefore activators of homeotic loci • PcG and trxG proteins associate in large multi-subunit protein complexes that modify chromatin structure.

33. How do PcG and trxG proteins function? • Both Polycomb and trithorax group proteins act to alter the accessibility of DNA to factors required for gene transcription • Polycomb group genes are involved in chromatin based gene silencing • Trithorax group genes counteract the silencing effects of chromatin to maintain gene activity – we will hear more about them when discussing chromatin remodeling • Together, these gene products maintain the expression patterns set up early in development that specify segment identity • This maintenance is stable through mitosis: CELLULAR or TRANSCRIPTIONAL MEMORY

34. Polycomb group protein domains Polycomb Sex combs extra (AKA dRING) Extra sex combs Enhancer of zeste

35. Spreading of heterochromatin in flies (and humans!) K9 of H3

36. Spreading of silenced chromatin on a homeotic gene E(z) (SET) K27 of H3 Polycomb Polycomb Polycomb Polycomb-complexed chromatin

37. Three big questions… • How are the PcG proteins targeted to their cognate sequences? • What is the maintenance “mark” that functions to keep specific genes active/inactive? • How is the maintenance mark maintained through cell division?

38. Deposition of histones during replication During replication, parental histones are distributed randomly onto the replicated DNA strands, and newly synthesized histones fill the gaps. http://www.umassmed.edu/faculty/show.cfm?start=0&faculty=912

39. Repressive effect of chromatin: HP1 and Polycomb GENOME ARCHITECTURE Heterochromatin & Position Effect Variegation (PEV) DIFFERENTIAL GENE EXPRESSION Polycomb, trithorax & “transcriptional memory” Regulation of specific developmental programs Regulation of chromatin domains Similarities? Heterochromatin protein 1 Polycomb