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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.

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  1. Analysis of cis action of PRE/TRE in the regulation of cellular memoryAn 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. Déjardin, J., Rappailles, A., Cuvier, O., Grimaud, C., Decoville, M., Locker, D., and Cavalli, G. (2005). Recruitment of Drosophila Polycomb Group proteins to chromatin by DSP1. Nature, (2005) 434, 533-538

  2. Maintenance of active states The GAL4 system for the study of PRE/TRE function: mimicking the developmental pathway leading to maintenance of homeotic gene expression GAL4 Hsp 70 GAL4 Driver construct white Fab-7 UAS-lacZ Reporter construct Experimental approach HsGAL4 pulse during early development ? Embryo 1st 2nd 3rd instar Pupa Larva Light Eye color white repressed Check Eye color

  3. Chromosomal elements maintaining memory of active and repressed chromatin states are defined as “Cellular Memory Modules” Transient transactivator If there is maintenance of cellular memory: sustained gene expression after decay of the primary transactivator Expression levels If no memory: loss of transient activation is expected adulthood L1 L2 L3 pupa larva embryogenesis

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

  5. UAS lacZ white 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 DAPI FISH Zeste Merge Merge split Ab-Fab No GAL4 Ab-Fab Low GAL4 Ab-Fab GAL4 pulse in embryos

  7. Still a PRE ? 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? z z p z g g p p z minimal CMM Ab-Fab DZ-Fab Still a CMM ?

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

  9. Zeste binding site mutation abolishes maintenance of active chromatin states Ab∆Z-Fab lines Pho Gaf Embryonic Pulse ofGAL4

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

  11. zeste gene behaves like a trxG gene if Zeste sites are present, The action of brm parallels the effect of zeste WT trxE2 brm2 za Ab-Fab DZ-Fab

  12. WT brm2 Activated state correlates with BRM recruitment DAPI FISH BRM MERGE Split merge 44A Ab14 43A 42F1 42A Embryonic activation 44A - - + + 43A 42F1 WT Ab-Fab;hsGAL4 42A 44A + 43A 42F1 42A 30A DZ-Fab;hsGAL4 + 31A 31F1 32A 33A

  13. Analysis of recruitment of PcG proteins at PREs Jérome Déjardin Collaborator: Daniel Locker University of Orléans France

  14. Fab7 3,6 kb Boundary PRE abd-A Abd-B p d Minimal DNA sequence requirements for inheritance of silencing In Fab-7 there are 3 binding sites for Pho, 3 sites for zeste and two sites for GAF within a small region of about 200 nucleotides: is this sufficient to convey memory of repressed chromatin states?

  15. A minimal Fab-7 silencer Fab-7 3.6 Kb CONSTRUCT SILENCING PH BINDING YES YES Midi-Fab YES YES Ab-Fab NO NO Ab- DPho NO NO Ab- DGaga Mini-Fab NO NO NO NO Syn-Fab merge Eye color FISH PH Ab-Fab line

  16. mcp core PRE(1) C A A T G T C G G A A A A A G A G G C C A T G T Pho site at 8 bp mcp core PRE(2) C T G C C T C T G A A A A C A T G G C C T C T T Pho site at 34 bp bxd (1) A C C G A A A A G A A A A A A C A G C A G A G G bxd (2) A A A C C G A A A A G A C A A A A C A T T G C T Pho site at 12 bp bxd (3) A A A G T G C C G A T A A C T C A A A A A G A G Pho site at 4 bp iab7core PRE G C C G A G C T G A A A A T G A A G A A G A A G En core PRE T A G G C C A G G A A A A T G G C T G C C T T G Ph-p PRE418 (1) G A T G G C A C G A A A A A A G A A G A T G A A Pho site at 4 bp G C A A A A A A G A A A A G G A A A C G A G A C Ph-p PRE418 (2) A T G G C A A G A A G A A A A G C G C A T G C T C G Ph-p PRE418 (3) Pho site at 89 bp Antp-P1 PRE(1) G T G G A G C G G A A A A A G A A C A A C A A C C Antp-P1 PRE(2) A T T C C G C A T A G A A A A T T T A T G C G G C C The GA motif is present in all known PRE, often close to Pho sites

  17. Constructs ‘ PRE activity ’ yes Ab-Fab Mini-Fab no Synthetic-Fab no SynGA-Fab ? Testing the GA motif by adding it to the synthetic sequence GA motif (15 nucleotides added)

  18. The GA motif restores patterned variegation Constructs PRE activity yes Ab-Fab no Synthetic-Fab yes (weak) SynGA-Fab

  19. DAPI PH 89B5 w1118 PH+ FISH 89B5 SynGA23 72B Syn26 The GA motif recruits PH to SynGA-Fab in vivo SynGA-Fab Syn-Fab Signal weaker than the one observed for Ab-Fab Other unknown determinants in Ab-Fab may be important for PRE strength No Pairing Sensitive Silencing

  20. Mutation of the GA motif disrupts PcG silencing and recruitment AbGAmut Ab-Fab AbGAmut-Fab DAPI Polyhomeotic FISH Merge Ab-Fab AbGAmut

  21. DAPI DSP1 FISH Merge Ab-Fab AbGAm The DSP1 protein is recruited at Ab-Fab in a GA motif -dependent manner In conclusion, we have reconstituted a synthetic PRE and have defined minimal requirements of PcG proteins. However, part of the silencing function is still missing, and we still need to understand how are the trxG activators recruited…

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

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

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

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