Targets of Activity

The primary structure of Tou reveals that, among the members of the WAL family of chromatin remodelling factors, it is most closely related to TIP5. The fact that TIP5 associates in vivo with SNF2h, the mammalian homologue of Drosophila Iswi (Strohner, 2001) prompted an investigation of whether Iswi associates with Tou and plays a role during neural development (Vanolst, 2005).

Genetic interactions between Iswi and pnr were investigated. Use of Iswi1 and Iswi2, which are both characterized by a point mutation that introduces a premature stop codon and are consequently predicted to encode truncated Iswi. However, Iswi1 and Iswi2 behave as null alleles since truncated Iswi1 and Iswi2 are not detected in western analyses, suggesting that the C terminus is required to stabilize Iswi (Deuring, 2000). The Iswi2/+ flies are wild type (2.00 DC bristle/heminotum) whereas the pnrD1/+ flies have excess DC bristles (3.34±0.1 DC bristle/heminotum). However, this excess is lowered when Iswi function is simultaneously reduced [Iswi2/+; pnrD1/+ flies display 2.84±0.18 DC bristles/heminotum]. Conversely, the lack of DC bristles associated with pnrVX1 (1.86±0.03 DC bristles/heminotum) is aggravated when Iswi function is simultaneously reduced (1.61±0.07 DC bristles/heminotum). It was also observed that the loss of DC bristles associated with ChipE (1.6±0.07 DC bristles/heminotum) is accentuated when Iswi function is simultaneously reduced (1.29±0.07 DC bristles/heminotum). Thus, loss-of-function Iswi alleles behave like loss-of-function tou alleles, implying that Iswi is also required during neural development and suggesting that Iswi and Tou may act as subunits of a multiprotein complex (Vanolst, 2005).

It is not possible to generate flies lacking both maternal and zygotic Iswi function (Deuring, 2000). Hence, to assess the consequences of the loss of Iswi function on neural development overexpression experiments of a dominant negative Iswi were performed. The UAS-IswiK159R (Deuring, 2000) line was used that allows overexpression of a dominant negative Iswi through the UAS/Gal4 system. A conserved lysine in the ATP-binding site of the ATPase domain is replaced with an arginine. This K159R substitution eliminates ATPase and chromatin-remodelling activities of Iswi in vitro, but does not affect the stability of Iswi and its incorporation into high molecular mass complexes. Hence IswiK159R behaves as a strong dominant negative protein (Vanolst, 2005).

Overexpression of IswiK159R in the precursor cells using the scaGal4 driver provokes a loss of multiple sensory bristles (Deuring, 2000) and it has been proposed that Iswi has a late function during neural development, essential for either viability or division of the precursor cells. Widespread expression of IswiK159R was induced in the dorsal compartment of the imaginal wing disc using Gal4-apMD544. Overexpression of IswiK159R is therefore induced early during development and results in a lack of sensory organs, including a frequent loss of DC bristles (Vanolst, 2005).

Tou and Iswi promote DC development and could be subunits of a multiprotein complex. However, it can also be hypothesized that Tou can substitute for Iswi function and vice versa. To address this issue, IswiK159R was overexpressed in either a wild-type genetic background or in conditions of loss of tou function. Overexpressed IswiK159R in the domain of pnr expression reduces DC bristles (1.56±0.11 bristle/heminotum). The loss of DC bristles is aggravated when the tou function is simultaneously reduced. Indeed, tou2/+; pnrGal4/UASIswiK159R and tou2; pnrGal4/UASIswiK159R flies have 1.14±0.12 bristle/heminotum and 0.75±0.11 bristle/heminotum, respectively. This observation reinforces the hypothesis that Tou and Iswi could be subunits of a complex during neural development (Vanolst, 2005).

Whether overexpressed IswiK159R affects the activity of the DC enhancer was investigated. Overexpression of IswiK159R leads to reduced expression of the lacZ reporter driven by the ac minimal promoter fused to the DC enhancer (line DC: aclacZ). The consequences were examined of loss of Iswi function associated with the Iswi1/Iswi2 transheterozygous combination, which dies during late larval stages (Deuring, 2000). It was observed that loss of Iswi function leads to decreased lacZ expression. These observations indicate that Iswi is also necessary for activation of ac-sc expression at the DC site, although the possibility that this interaction is indirect cannot be ruled out (Vanolst, 2005).

Protein Interactions

Previous experiments in vertebrates have demonstrated that the function of GATA factors during development is regulated by dimerization of their DNA binding domain (DBD) with cofactors. Hence, the yeast two-hybrid system was used to identify Pnr-interacting proteins and a bait was used in which the DBD of Pnr was fused to the DBD of LexA. A screen of 106 primary transformants of a Drosophila embryonic cDNA expression library yielded 35 potential positive clones. Among the different clones, a domain of Tou, designated as TouA was identified (Vanolst, 2005).

TouA physically interacts with the DBD of Pnr in yeast. Since the C terminus of Pnr also mediates physical interactions with cofactors, whether TouA interacts with the C terminus of Pnr was investigated in yeast. Expression vectors encoding either the C terminus of Pnr fused to the LexA DBD (LexADBDPnrCT) or the unfused LexA DBD (LexADBD) were introduced into the L40 yeast strain together with the vector for the unfused VP16 activation domain (VP16AD) or for TouA fused to VP16AD (VP16ADTouA). L40 cells contain a lacZ reporter driven by eight LexA binding sites. Protein extracts were prepared from L40 transformants grown in liquid medium and were assayed for ß-galactosidase activity. No reporter activity was observed in extracts made from yeast expressing LexADBDPnrCT/VP16AD, LexADBD/VP16ADTouA or LexADBD/VP16AD. In contrast, a robust activation of the reporter was detected with yeast expressing LexADBDPnrCT/VP16ADTouA, indicating that TouA associates with the C terminus of Pnr. Then, both the DBD and the C terminus of Pnr physically interact with TouA. Accordingly, it was found that TouA interacts with wild-type Pnr (PnrWT) in which both the DBD and the C terminus of Pnr are present and with PnrVX1, a truncated Pnr lacking the C terminus. TouA also associates with PnrD1, a mutated Pnr in which the structure of the DBD is probably disrupted since a coordinating cysteine of the N terminal zinc finger has been replaced by a tyrosine (Vanolst, 2005).

Whether TouA interacts with Pnr was analyzed in a cultured cell line. Immunoprecipitations were performed of protein extracts made from Cos cells transfected with expression vectors for TouA and either Pnr or GST fusion proteins containing the DBD of Pnr (GSTPnrDBD) or the C terminus of Pnr (GSTPnrCT). Pnr, GSTPnrDBD and GSTPnrCT were found to coimmunoprecipitate with TouA. Finally, the abilities were tested of in vitro-translated 35S-labelled TouA to bind to GSTPnrDBD or GSTPnrCT attached to glutathione-bearing beads. TouA interacts both with the DBD of Pnr and the C terminus of Pnr (Vanolst, 2005).

To investigate the interactions between Tou and Pnr in more detail, several expression vectors were constructed encoding in yeast contiguous domains of Tou. The various segments of Tou were fused with VP16AD and introduced into yeast together with unfused LexADBD or LexADBDPnrWT. Activation of the reporter was observed only in yeast expressing LexADBDPnrWT/VP16ADTouC and in yeast expressing LexADBDPnrWT/VP16ADTouJ, showing that the amino terminus of the MBD domain of Tou mediates physical interactions with Pnr in yeast (Vanolst, 2005).

Chip is an essential cofactor of Pnr since it facilitates enhancer/promoter communication necessary for ac-sc expression during Pnr-driven neural development. Since TouA associates with Pnr in yeast, use of the yeast was made of the two-hybrid system to ask whether TouA also associates with Chip. Chip was fused to LexADBD and assayed for interaction with VP16ADTouA. A strong increase of ß-galactosidase activity was observed in extracts made from yeast expressing LexADBDChip/VP16ADTouA above the background level seen with extracts made from yeast expressing LexADBD/VP16ADTouA or LexADBDChip/VP16AD, indicating that TouA associates with Chip (Vanolst, 2005).

Sequence comparison between the Ldb proteins from various species reveals two conserved functional domains, involved in protein-protein interactions. Thus, Chip contains a N-terminal homodimerization domain, also involved in interactions with Pnr and Osa and a C-terminal LIM interacting domain (LID) mediating heterodimerization with LIM-homeodomain proteins (LIM-HD) and basic helix-loop-helix proteins (Vanolst, 2005).

The interaction between TouA and Chip was examined. Assays for ß-galactosidase activity revealed that the interaction between TouA and Chip is mediated by the N-terminal homodimerization domain. Whether TouA can interact with Chip in a cultured cell line was investigated by immunoprecipitating proteins extracts of Cos cells transfected with expression vectors encoding TouA and Chip, the N-terminal domain of Chip (NT Chip) or the C-terminal domain of Chip (CT Chip). TouA physically interacts with Chip and the interaction is mediated by the N-terminal domain of Chip. In vitro translated 35S-labelled TouA interacted with GST Chip attached to glutathione-bearing beads (Vanolst, 2005).

The interaction between Tou and Chip was investigated in more detail. The segments of Tou were fused with the VP16AD and introduced into yeast together with unfused LexADBD or LexADBDChip. Only TouD containing the DDT domain associates with Chip. Moreover, the physical interaction is mediated by the DDT domain itself (Vanolst, 2005).

Tou functionally cooperates with Pnr and Chip during neural development. Since Tou physically interacts with Pnr and Chip, it was then asked whether a ternary complex containing Tou, Pnr and Chip can exist in living cells. Double immunoprecipitations were performed of extracts made from Cos cells containing Pnr, Tou and Chip. The extract was first immunoprecipitated with the M2 antibody recognizing the flagged TouA and the precipitated proteins were recovered by elution with the Flag peptide. The eluate was then immunoprecipitated with the B10 antibody directed against the tagged Chip. Pnr was observed to coimmunoprecipitate with Chip and TouA, indicating that the trimer Chip-Tou-Pnr can be formed. Moreover, this suggests that Chip and Pnr act together to recruit Tou and to target its activity to the ac-sc promoter sequences. This observation is reminiscent to what has observed on the role of Osa during neural development. Osa belongs to the Brm complex and it was suggested that Pnr and Chip cooperate to recruit Osa and to target activity of the Brm complex to the ac-sc promoter sequences, leading to negative regulation of enhancer-promoter communication. However, Tou and Osa display antagonistic activities during regulation of ac-sc and may probably define distinct chromatin remodelling complexes since the loss of DC bristles associated with reduced tou function (tou2 flies have 1.4±0.05 DC bristles/heminotum) is suppressed by lowering the dosage of osa. Indeed, tou2; osa616/+ flies have 1.97±0.07 DC bristles/heminotum (Vanolst, 2005).

Since Tou, Pnr, Chip and Iswi are required to activate proneural expression at the DC site, whether Iswi can interact with Tou, Pnr and Chip was examined. It was found that Iswi can interact with Tou in both yeast and Cos cells, in agreement with previous reports showing that Iswi can interact with Tou-related proteins in insect (referring to Acf1; Fyodorov, 2002) and in mouse cells (Strohner, 2001). An expression vector encoding LexADBDIswi was introduced in yeast together with a vector for the unfused VP16AD or VP16AD fused to one of the various segments of Tou. Measurement of ß-galactosidase activity revealed that only protein extracts made from yeast expressing LexADBDIswi and either VP16ADTouA or VP16ADTouC display activity above background levels. Hence, TouA associates in yeast with Pnr, Chip and Iswi. Finally, immunoprecipitation of protein extracts revealed that TouA and Iswi also associate in transfected Cos cells (Vanolst, 2005).

Whether Iswi can interact with Chip and Pnr was examined by testing the abilities of in vitro translated 35S-labelled Iswi to bind to GST-Chip attached to glutathione-bearing beads. It was found that Iswi associates with full-length Chip. Similarly, it was observed that Iswi can also associate both with the DBD and the C terminus of Pnr. Since Pnr and Chip directly regulate ac-sc expression at the DC site, these findings suggest that Iswi and Tou may belong to a complex, which, in vivo, regulates the activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

toutatis: Biological Overview | Developmental Biology | Effects of Mutation | References

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