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First published online 1 September 2005
doi: 10.1242/dev.02014

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Toutatis, a TIP5-related protein, positively regulates Pannier function during Drosophila neural development

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Department of Developmental Biology, CNRS/INSERM/ULP, Boite Postale 10142, 67404 Illkirch Cedex, France

View larger version (27K): [in a new window]   Fig. 1. Tou displays homology with members of the WAL family of chromatin remodelling factors. (A) Physical map of the tou locus. Structure of the tou gene and locations of the transposon insertions in EP662, tou1 and EY08961 are shown. tou2 and touE44.1 were generated by imprecise excision of tou1 and EY08961, respectively. The transposon in EP622 contains UAS sequences that allow overexpression of tou with the UAS/Gal4 system. Noncoding and coding regions of the tou transcript are depicted as open and filled boxes, respectively. (B) Tou shares functional domains with proteins expressed in Drosophila (Dm Acf1) and in human (Hs TIP5; Hs WSTF) and involved in chromatin remodelling. Tou carries a MBD domain (methyl-CpG binding domain), a DDT domain (after DNA binding homeobox and different transcription factors), a WAKZ motif (WSTF/Acf1/KIAA0314/ZK783.4), two PHD fingers (plant homeodomain) and a C-terminal bromodomain (BROMO). TouA, isolated during the yeast two-hybrid screening, contains the MBD and DDT domains. Dm Acf1 and Hs WSTF lack the MBD but display a N-terminal WAC motif (WSTF; Acf1; cbp146).

View larger version (108K): [in a new window]   Fig. 2. tou mutants genetically interact with pnr and Chip and exhibit loss of DC bristles. (A) Wild-type flies have four DC bristles (1-4). (B) Loss of function tou alleles [tou1, touE44.1 (not shown), tou2] results in phenotypes similar to pnr mutants, which lack DC bristles (asterisk). (C,D) tou2 behave as dominant suppressor (tou2; pnrD1/+) of the excess of DC bristles (arrowheads) observed with pnrD1 encoding a constitutive activator of ac-sc (pnrD1/+). (E,F) tou2 behave as dominant enhancer (ChipE tou2) of the loss of DC bristles associated with ChipE. (G) In contrast, overexpressed tou in the domain of apterous expression (apGal4/EP622) leads to an excess of DC bristles (arrowheads) associated with extra DC precursors in the imaginal disc (J), as revealed by A101 staining, in comparison with staining of wild-type disc (I). (H) Overexpressed tou suppresses the loss of DC bristles associated with ChipE (apGal4ChipE/EP622ChipE) and produces extra sensory organs (arrowheads). (K) tou and osa are antagonistic during neural development. Lowering the dosage of Osa suppresses the loss of DC bristles characteristic of tou2 (tou2; osa616/+). (L) Reducing Iswi function (Iswi2/+; pnrD1/+) also suppresses excess DC bristles associated with pnrD1. (M) Overexpression of the dominant-negative Iswi (apGal4/IswiK159R) in the domain of ap expression leads to loss of multiple sensory organs, including the DC bristles (asterisks). (N,O) The loss of DC bristles (asterisks) resulting from overexpressed IswiK159R in the domain of pnr expression (pnrGal4/IswiK159R) is aggravated when tou function is simultaneously reduced (tou2; pnrGal4/IswiK159R), reinforcing the hypothesis that Tou and Iswi could be subunits of a multiprotein complex regulating neural development.

View larger version (31K): [in a new window]   Fig. 3. Tou physically interacts with Pnr. (A) Structural features of the Pnr and Tou proteins used in the present study. The DNA binding domain of Pnr (PnrDBD), containing the two zinc fingers (grey boxes) and the C-terminal domain (PnrCT) containing the two amphipathic -helices (black boxes). The mutations associated with pnrD1 and with pnrVX1 are localized within the N-terminal zinc finger and in the N terminus of the helices respectively. A schematic drawing of the Tou domains used throughout this study (TouA to TouK). The functional domains of Tou are schematized as in Fig. 1. (B) Tou physically interacts with Pnr in yeast, through both the DBD and the C-terminus of Pnr. Expression vectors for unfused LexADBD(-) or LexADBDPnrDBD, LexADBDPnrCT, LexADBDPnrWT, LexADBDPnrD1, LexADBDPnrVX1 were introduced into L40 cells together with the unfused VP16AD (not shown) or VP16ADTouA. Protein extracts made from cultured L40 transformants were assayed for ß-galactosidase activity (expressed in nanomoles of substrate/mn/mg of protein). Values (±10%) are the averages of three independent experiments. (C,D) Tou physically interacts with (C) full-length Pnr, (D) the DBD of Pnr and the C terminus (CT) of Pnr in transfected cells. In each case, an immunoblot of a representative set of transfected cell extracts is shown. The transfected expression vectors are shown at the top of the panels. The B10 monoclonal antibody used to immunoprecipitate the extracts is shown on the left of the panels. The antibodies used to reveal the blots are indicated at the bottom. Pnr is recognized by the 2B8 antibody, GSTPnrDBD and GSTPnrCT are detected by the D10 antibody and the B10-tagged TouA is recognized by the B10 antibody. The locations of the proteins including the immunoglobulin heavy chain [IgG(H)] are indicated at the sides. (E) Tou directly interacts both with the DBD and the CT of Pnr in vitro. (E) Autoradiographs of SDS-PAGE gels from representative affinity chromatography experiments performed with GST control beads (lane 2), GSTPnrDBD beads (lane 3) and GSTPnrCT beads (lane 4) and in vitro translated 35S proteins as indicated on the left. One-tenth of the 35S input is shown in lane 1. Luciferase is used as a negative input. Experiments were performed three times and, with all proteins except luciferase, 50-fold more protein bound to GSTPnrDBD and GSTPnrCT than to GST control. (F) The N terminus of the MBD domain of Tou mediates interaction with Pnr. Expression vectors for unfused LexADBD (not shown) or LexADBDPnrWT were introduced in L40 cells together with the unfused VP16AD (not shown) or VP16ADTouA, VP16ADTouC, VP16ADTouD, VP16ADTouJ or VP16ADTouK. Protein extracts made from cultured L40 transformants were assayed for ß-galactosidase activities (expressed in nanomoles of substrate/mn/mg of protein). Values (±10%) are the averages of three independent experiments.

View larger version (31K): [in a new window]   Fig. 4. Tou physically interacts with Chip. (A) Structural features of the Chip proteins used in this study: the N-terminal homodimerization domain (DDT) of Chip (NTChip; black box) and the C-terminal LIM-interacting domain (LID) of Chip (CTChip; grey box). (B) Tou interacts with Chip in yeast through the N-terminal homodimerization domain. Expression vectors encoding the unfused LexADBD (-) or the LexADBDChip, the LexADBDNTChip, the LexADBDCTChip were introduced in L40 cells together with the unfused VP16AD (not shown) or VP16ADTouA. Protein extracts made from L40 transformants, grown in liquid medium, were assayed for ß-galactosidase activities, which are expressed as in Fig. 3B. (C) Tou interacts with Chip in transfected cells. The layout is as in Fig. 3C. The flagged Chip is detected with the M2 antibody whereas the B10-tagged proteins (B10-Chip, B10-NTChip, B10-CTChip) are recognized by the B10 antibody. (D) Tou directly interacts with Chip in vitro. (D) Autoradiographs of SDS-PAGE gels from representative affinity chromatography experiments performed with GST control beads (lane 2) and GST Chip beads (lane 3) and in vitro translated 35S proteins as indicated on the left. One-tenth of the 35S input is shown in lane 1. Luciferase was used as a negative input. Experiments were performed three times and 50-fold more 35S labelled TouA bound to GST Chip than to GST control. (E) The DDT domain of Tou mediates interaction with Chip. Expression vectors encoding the unfused LexADBD (not shown) or the LexADBDChip were introduced into L40 cells together with the unfused VP16AD (not shown) or the VP16ADTouA, VP16ADTouC, VP16ADTouD, VP 16ADTouL, VP16ADTouM or VP16ADTouN. Protein extracts made from L40 transformants, grown in liquid medium, were assayed for ß-galactosidase activities (expressed as in B).

View larger version (21K): [in a new window]   Fig. 5. The trimer Pnr-Chip-Tou can exist in living cells. The ternary complex is revealed by double-immunoprecipitation of protein extracts made from transfected Cos cells. (A) Immunoblot of a representative set of protein extracts. The transfected expression vectors are shown at the top. The asterisk denotes an artifactual band. (B) Double-immunoprecipitation of the protein extracts described in panel A. The M2 (IP 1) and B10 (IP 2) mouse antibodies are used to immunoprecipitate the flagged TouA domain (F.TouA) and the tagged full-lenght Chip (B10.Chip), respectively. They are shown on the left of the panels. After immunoprecipitation with the M2 antibody, the selected proteins were recovered by elution with the Flag peptide (IP 1 mAB M2+Elution). The antibodies used to reveal the blots are indicated at the bottom of the panels. Pnr, the F-TouA domain and the B10-Chip are recognized by the 2B8, the M2 and the B10 antibody, respectively. The locations of the proteins, including the IgG(H) of the B10 antibody, are indicated by arrows.

View larger version (84K): [in a new window]   Fig. 6. Tou regulates proneural expression through the DC-specific enhancer of the ac-sc complex. Late third instar thoracic imaginal discs show lacZ expression at the DC site (Garcia-Garcia et al., 1999). In each case, the reaction was left for one hour at 22°C. (A) Wild-type pattern with lacZ expression in the DC area (arrow) (w1118/+; DC: ac-lacZ/+). (B,C) Decrease of tou function results in a reduction in lacZ activity at the DC site (B: tou2; DC: ac-lacZ/+; C: touE44.1; DC: ac-lacZ/+). (D,E) pnrD1 behave as constitutive activator of proneural expression (pnrD1/DC: ac-lacZ/+) and causes increased lacZ activity. Reducing tou function in pnrD1 suppresses excess lacZ activity at the DC site (tou2/tou2; pnrD1/DC: ac-lacZ/+), demonstrating that Pnr and Tou regulate ac-sc expression through the DC enhancer. (F) Reduced Iswi function leads to a severe decrease of lacZ activity at the DC site (Iswi1/Iswi2; DC: ac-lacZ/+). (G,H,I) Overexpressed Tou activates lacZ expression driven by the DC enhancer (apGal4/EP622; DC: ac-lacZ/+) whereas overexpression of the dominant-negative Iswi (IswiK159R) is associated with a decrease in lacZ activity (apGal4/IswiK159R; DC: aclacZ/+).

View larger version (27K): [in a new window]   Fig. 7. Iswi associates with Tou, Pnr and Chip and directly regulates ac-sc expression in vivo. (A) Iswi interacts with Tou in yeast. Expression vectors encoding the unfused LexADBD or the LexADBDIswi were introduced into L40 cells together with the VP16AD (data not shown) or the VP16ADTouA. Protein extracts were made from L40 transformants, grown in liquid medium, and assayed for ß-galactosidase activity (expressed as in Fig. 3B). (B) Iswi interacts with Tou in transfected cells. The transfected expression vectors are shown at the top of the panels. The layout is as in Fig. 3C. F-Iswi and B10-TouA are recognized by the M2 antibody and B10 antibody, respectively. (C) Iswi directly interacts with Pnr and Chip in vitro. (C) Autoradiographs of SDS-PAGE gels from representative affinity chromatography experiments performed with GST control beads (lane 2), GST DBD Pnr (lane 3), GST CT Pnr (lane 4), GST Chip beads (lane 5) and in vitro translated 35S proteins as indicated on the left. One-tenth of the 35S input is shown in lane 1. Luciferase is used as a negative input. (D) Model on how Tou, Iswi and the Brm complex regulate activity of the proneural complex during enhancer-promoter communication at the ac-sc locus. Tou and Iswi positively regulate proneural expression and may belong to a complex with antagonistic activity to that of the Brm complex (Heitzler et al., 2003). The complex containing Tou and Iswi regulates activity of Pnr and Chip during enhancer-promoter communication, possibly through chromatin remodelling.