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The Drosophila Trithorax protein is a coactivator required to prevent re-establishment of Polycomb silencing

Sylvain Poux, Béatrice Horard*, Christian J. A. Sigrist{dagger} and Vincenzo Pirrotta{ddagger}

Department of Zoology, University of Geneva, 30 quai Ernest Ansermet, CH-1211 Geneva, Switzerland
* Present address: LBMC-ENS Lyon, 46, allée d’ Italie, F-69364 Lyon Cedex 07, France
{dagger} Present address: Swiss Institute of Bioinformatics (SIB-ISB), 1, rue Michel Servet, CH-1211 Geneva, Switzerland



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  		Fig. 1. Transposon constructs. (A) The YGfPfMG construct is shown schematically with the PRE, gypsy, miniwhite and the yellow gene as a marker. FRT sites flanking the PRE allow its excision in the presence of the FLP enzyme, to give YGfMG. (B) The LexA-PC and the GAL-TRX expression constructs: the expression is driven by the hsp70 promoter and the yellow gene serves as a marker. (C) BHL4G4 contains LexA and GAL4 binding sites. The expression of the Ubx-lacZ gene is directed by the embryonic BX enhancer and the 2212H1 imaginal disc enhancer of Ubx. The marker gene is miniwhite. In BHL4G4PRE, the bxd PRE is added. The arrows indicate the direction of transcription. The yellow, lacZ and trx genes are not shown to scale.

 


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  		Fig. 2. Eye pigmentation in presence or absence of the bxd PRE. (A) Eye pigmentation is repressed by the PRE in line 8 (left) and is increased by deleting the PRE (right). (B) PRE repression is decreased by a heterozygous Pc mutation (right) compared to the wild type (left). (C) In line 91, eye pigmentation is stronger in presence of the PRE (left) than in its absence (right). (D) The stimulating effect of the PRE is due to TRX. In flies heterozygous for a trx mutation, eye pigmentation decreases (left) to a level comparable to lines without the PRE (right). Eye pigmentation in absence of the PRE is much stronger in line 91 compared to line 8 (compare A, C, right).

 


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  		Fig. 3. Immunoprecipitation of PRE fragments. (A) Map of the bxd PRE with subfragments produced by HinfI (Hf), AvaII (Av), BglI (Bg), PstI (P) and StyI (St) and their sizes in bp. GAGA and PHO binding sites are indicated by G and P. Immunoprecipitation of a fragment with anti-PC or anti-TRX is indicated by +, no precipitation by –. (B) LexA binding assays. Anti-PC immunoprecipitates the LexA probe from nuclear extracts containing LexA-PC but anti-TRX does not, showing that LexA-PC does not recruit TRX. A control fragment present in the binding reaction is indicated by *. Lanes : –, no antibody; pc, anti-PC; trx, anti-TRX. The immunoprecipitated fragments were analysed on an acrylamide gel together with an aliquot of the input mixture (i). (C) The BP subfragment (left) is precipitated by anti-PC, anti-TRX and anti-PHO but TRX binding is lost in the presence of GAGAG oligonucleotide competitor (ga). Mutation of all GAGAG sequences in this fragment (BPmut) impairs the binding of both proteins (right) while PHO still binds the mutated fragment. A diagram of BP and BPmut is shown above each panel. (D) Co-precipitation of GAGA and TRX. Nuclear extracts were immunoprecipitated with no antibody (mock IP) or with anti-TRX ({alpha}trx IP). Western blots of the IPs were probed with anti-GAGA or anti-TRX. The arrowhead indicates the IgGs in the immunoprecipitate.

 


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  		Fig. 4. TRX prevents re-establishment of repression. (A) BHL4G4PRE expression is maintained anterior to PS6 throughout embryonic development. In situ hybridization in late embryos shows that BX-driven expression ends in the ectoderm and H1-driven expression is limited to dorsolateral spots (arrows). In larvae, expression is repressed in the wing disc (w) and limited to the posterior haltere (h) and third leg (l) discs. (B) In homozygous trx embryos, the level of expression is reduced. In heterozygous trx larvae, expression in the posterior haltere is variegated and almost absent in the third leg disc. The arrows show the repressed domains in the haltere disc and the weak residual expression in the leg disc.

 


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  		Fig. 5. Effect of targeted TRX. (A) The pattern of expression of embryos carrying both BHL4 and hs-LexA-PC heat shocked at 2-3 hours is the same in a wild-type and in a homozygous trx background (trx). (B) BHL4G4 expression in early embryos (left) and after germ band retraction (right) with no induction of GAL-TRX (no hs). When GAL-TRX is induced at 2-3 hours, expression is enhanced only in regions where the gene was active (parasegments 6, 8, 10 and 12). In older embryos, after ectopic expression appears in the thorax, expression is stimulated everywhere. (C) The effect of GAL-TRX is transient and its induction at blastoderm does not stimulate expression in larvae (hs at 2-3 hours). A series of heat shocks 2 days before staining strongly enhances expression in leg discs (series hs). D) When both GAL-TRX and LexA-PC proteins are induced, LexA-PC recruits a repressive complex in the thorax (left), while GAL-TRX stimulates expression in the abdomen. Embryos heat shocked as in (B).

 


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  		Fig. 6. PRE silencing is destabilized by TRX or GAL4. GAL-TRX (A-D) and GAL4 (E-F) counteract silencing when targeted to the BHL4G4PRE reporter gene. When GAL-TRX is induced at the blastoderm stage, it interferes with the formation of the repressive complex in old embryos as well as in larvae (A). When GAL-TRX is induced after 5-6 hours (B) or during larval development (C), repression is not affected. When the TRX level is reduced, targeting GAL-TRX (D) at blastoderm to the BHL4G4PRE leads to a partial derepression in embryos and larvae. However, the residual repression is well maintained throughout development. Targeting GAL4 to the BHL4G4PRE gave similar results (E), with a partial derepression in a trx mutant background (F).

 


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  		Fig. 7. GAL-GCN5 at blastoderm causes loss of silencing memory in larvae. (A) BHL4G4 expression in early embryos (left) and after germ band retraction (right) with no induction of the GAL-GCN5 protein (no hs). GAL-GCN5 induction at 2-3 hours does not affect the expression of BHL4G4. (B) When both GAL-GCN5 and hs-LexA-PC (hs-LexA-PC) proteins are produced at 2-3 hours, repression induced by hs-LexA-PC is prevented by GAL-GCN5, resulting in a strong ectopic expression in late embryos. In contrast, when {alpha}1-tubulin-LexA-PC ({alpha}1T-LexA-PC) is provided maternally, GAL-GCN5 does not interfere with PcG silencing. (C) When targeted to BHL4G4PRE in the 2-3 hours embryo, GAL-GCN5 does not interfere with repression in late embryos but results in loss of repression in larval imaginal discs. When the TRX level is reduced (trx), targeting GAL-GCN5 to BHL4G4PRE leads only to a very weak and partial derepression in larvae.

 

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© The Company of Biologists Ltd 2002