1. Analysis of cis action of PRE/TRE in the regulation of cellular memory An example of a functional molecular study Data from: Dejardin, J., and Cavalli, G. (2004). Chromatin inheritance upon Zeste-mediated Brahma recruitment at a minimal cellular memory module. Embo J 23, 857-868.

2. Fab-7UAS-lacZ white Hsp 70 GAL4 Driver construct Reporter construct The GAL4 system for the study of PRE/TRE function: mimicking the developmental pathway leading to maintenance of homeotic gene expression Check Eye color Light Eye color white repressed Embryo Larva Pupa 1st2nd3rd instar HsGAL4 pulse during early development Experimental approach ? Maintenance of active states GAL4

3. Transient transactivator If there is maintenance of cellular memory: sustained gene expression after decay of the primary transactivator Expression levels embryogenesis L1L2L3 pupa adulthood If no memory: loss of transient activation is expected Chromosomal elements maintaining memory of active and repressed chromatin states are defined as “Cellular Memory Modules” larva

4. Fab-7: a cellular memory module (CMM) that maintains active as well as silenced chromatin throughout development +HS -HS lacZwhite Fab-7 G 3,6 kb Gal4 hsp70 GAL4 HS UAS Beta-gal stains to study lacZ expression

5. lacZwhite UAS Ab-Fab (217 bp) The 219 bp Ab-Fab fragment is a minimal CMM Embryonic pulse of GAL4

6. Activated state correlates with Zeste recruitment DAPIFISHZesteMergeMerge split Ab-Fab No GAL4 Ab-Fab Low GAL4 Ab-Fab GAL4 pulse in embryos

7. Ab-Fab minimal CMM Still a CMM ? Still a PRE ?  Z-Fab gg zzzppp z Molecular swicth for activation/repression: the role of Zeste protein PREs/TREs contain binding sites for the Zeste protein. Zeste was suggested to be a trxG member on the basis of its ability to recruit the Brm chromatin remodeling complex in vitro and of genetic evidence on the effect of z mutations on PREs in transgenes … but Zeste was also shown to be a component of PcG complexes in vitro Is Zeste involved in silencing or in activation at PREs/TREs in vivo?

8. PH Mutation of Zeste binding sites does not impair PcG recruitment

9. Ab∆Z-Fab lines Gaf Pho Embryonic Pulse of GAL4 Zeste binding site mutation abolishes maintenance of active chromatin states

10. no HS HSE Zeste stabilizes PH recruitment at Fab-7 PH HSE PH Wild type Deletion of Zeste sites

11. zeste gene behaves like a trxG gene if Zeste sites are present, The action of brm parallels the effect of zeste  Z-Fab WTtrx E2 brm 2 Ab-Fab zaza

12. Activated state correlates with BRM recruitment + brm 2 + WT - Embryonic activation  Z-Fab;hsGAL4 31A 31F1 32A 33A 30A DAPIFISHBRMMERGESplit merge Ab14 Ab-Fab;hsGAL4 43A 44A 42A 42F1 43A 42F1 44A 42A 43A 42F1 42A 44A + + -

13. GAF Summary mechanism of CMM action an equilibrium between silencing and activation dependent on early developmental cues CBP CMM dRing PH PC Pho PRC1/other PcG related complexes PSC Z Z Z Z PRE TRE BAP111 osa mor BRM-C Brm ? trx TAC-1 ? Z

14. When PRE is favored during early development… dRing PH PC Z PSC dRing PH PC Z PSC CMM dRing PH PC PSC PRE CBP Z Z Z Z Z BAP111 osa mor Brm trx TRE Pho

15. … and when TRE function is activated CBP trx CMM dRing PH PC Z PSC Z Z Z Z Z Z Z PRE TRE BAP111 osa mor Brm CBP trx BAP111 osa mor Brm BAP111 osa mor Brm Pho