The GATA factor Pannier (Pnr) activates proneural expression through binding to a remote enhancer of the achaete-scute (ac-sc) complex. Chip associates both with Pnr and with the (Ac-Sc)-Daughterless heterodimer bound to the ac-sc promoters to give a proneural complex that facilitates enhancer-promoter communication during development. Using a yeast two-hybrid screening, Toutatis (Tou; see Teutates the supposed deified spirit of male tribal unity in ancient Celtic polytheism, best known under the name Toutatis, through the Gaulish catchphrase "By Toutatis!", invented for the Asterix comics by Goscinni and Uderzo), which physically interacts with both Pnr and Chip, was identified. Loss-of-function and gain-of-function experiments indicate that Tou cooperates with Pnr and Chip during neural development. Tou shares functional domains with chromatin remodelling proteins, including TIP5 (termination factor TTFI-interacting protein 5) of NoRC (nucleolar remodelling complex), which mediates repression of RNA polymerase 1 transcription. In contrast, Tou acts positively to activate proneural gene expression. Moreover, Iswi associates with Tou, Pnr and Chip, and is also required during Pnr-driven neural development. The results suggest that Tou and Iswi may belong to a complex that directly regulates the activity of Pnr and Chip during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

Transcriptional activation of many developmentally regulated genes is mediated by proteins binding to enhancers scattered over the genome, raising the question on how long-range activation is restricted to the relevant target promoter. Numerous studies have highlighted the essential role of boundaries, which maintain domains independent of their surrounding (Vanolst, 2005).

The patterning of the large sensory bristles (macrochaetae) on the thorax of Drosophila melanogaster is a powerful model to study how enhancers communicate with promoters during regulation of gene expression. Each macrochaeta derives from a precursor cell selected from a group of equivalent ac-sc-expressing cells, the proneural cluster. ac and sc encode basic helix-loop-helix proteins (bHLH) that heterodimerize with Daughterless (Da) to activate expression of downstream genes required for neural fate. Transcription of ac and sc in the different sites of the imaginal disc is initiated by enhancers of the ac-sc complex and the expression is maintained throughout development by autoregulation mediated by the (Ac-Sc)-Da heterodimers binding to E boxes within the ac-sc promoters. Each enhancer interacts with specific transcription factors that are expressed in broader domains than the proneural clusters and define the bristle prepattern. Thus, the GATA factor Pannier (Pnr) binds to the dorsocentral (DC) enhancer and activates proneural expression to promote development of DC sensory organs. The Drosophila LIM-domain-binding protein 1 (Ldb1), Chip physically interacts both with Pnr and the (Ac-Sc)-Da heterodimer to give a multiprotein proneural complex which facilitates the enhancer-promoter communication (Vanolst, 2005 and references therein).

Chromatin plays a crucial role in control of eukaryotic gene expression and is a highly dynamic structure at promoters. In Drosophila, the polycomb (Pc) group and the trithorax (Trx) group proteins are chromatin components that maintain stable states of gene expression and are involved in various complexes. The Pc group proteins are required to maintain repression of homeotic genes such as Ultrabithorax, presumably by inducing a repressive chromatin structure. Members of the Trx group were identified by their ability to suppress dominant Polycomb phenotypes. Evidence has been provided that enhancer-promoter communication during Pnr-driven proneural development is negatively regulated by the Brahma (Brm) chromatin remodelling complex (Heitzler, 2003; Treisman, 1997; Collins, 1999), homologous to the yeast SWI/SNF complex (Vanolst, 2005).

Evidence is presented that Toutatis (Tou), a protein that associates both with Pnr and Chip and that positively regulates activity of the proneural complex encompassing Pnr and Chip during enhancer-promoter communication. Tou has been previously identified in a genetic screen for dominant modifiers of the extra-sex-combs phenotype displayed by mutant of polyhomeotic (ph), a member of the Pc group in Drosophila (Fauvarque, 2001). Tou shares functional domains with Acf1, a subunit of both the human and Drosophila ACF (ATP-utilizing chromatin assembly and remodelling factor) and CHRAC (chromatin accessibility complex), and with TIP5 of NoRC (nucleolar remodelling complex; Strohner, 2001). Hence, Tou regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling. Moreover, Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is also necessary for activation of ac-sc and neural development. Since Iswi is shown to physically interact with Tou, Pnr and Chip, it is suggested that a complex encompassing Tou and Iswi directly regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

In Drosophila, Chip has been postulated to be a facilitator required both for activity of the DC enhancer of the ac-sc complex (Ramain, 2000). Enhancer-promoter communication at the ac-sc complex is negatively regulated by the Brm complex whose activity is targeted to the ac-sc promoter sequences through dimerization of the Osa subunit with both Pnr and Chip (Heitzler, 2003). The Brm complex is thought to remodel chromatin in a way that represses transcription (Vanolst, 2005).

Tou and Iswi appear to act together as subunits of a multiprotein complex to positively regulate activity of Pnr and Chip during enhancer-promoter communication. Tou and Iswi therefore display opposite activity to that of the Brm complex, raising questions about their molecular function during neural development. Tou shares essential functional domains with members of the WAL family of chromatin remodelling proteins, including Acf1 of ACF and CHRAC. Importantly, Acf1 and TIP5 associate in vivo with Iswi (Ito, 1999; Strohner, 2001), showing that Iswi can mediate both activation and repression of gene expression. Tou positively regulates Pnr/Chip function during the period of ac-sc expression in neural development, and it associates with Iswi. Since Iswi also positively regulates Pnr/Chip function, it is hypothesized that a complex encompassing Tou and Iswi acts during long-range activation of proneural expression, possibly through chromatin remodelling. Further studies will help to resolve this issue (Vanolst, 2005).

Interestingly, Chip and Pnr seem to play similar roles both during recruitment of the Brm complex and recruitment of Tou and Iswi, since they dimerize with Osa, Tou and Iswi. In addition, Pnr and Chip apparently cooperate to strengthen the physical association with Osa and Tou. However, Osa, on the one hand, and Tou and Iswi, on the other, display antagonistic activities during neural development. Since they are ubiquitously expressed, accurate regulation of ac-sc expression would require a strict control of the stoichiometry between Osa, Tou and Iswi. It remains to be investigated whether the functional antagonism between Osa and Tou/Iswi relies on a molecular competition for association with Pnr and Chip. Determination of this would require a complete molecular definition of the putative complex encompassing Tou and Iswi, together with a full understanding of how this complex and the Brm complex molecularly interact with the proneural complex to regulate enhancer-promoter communication during development (Vanolst, 2005).

Biochemical analysis of Iswi and Iswi-containing complexes, together with genetic studies of Iswi and associated proteins in flies and in budding yeast, has revealed roles for Iswi in a wide variety of nuclear processes, including transcriptional regulation, chromosome organization and DNA replication (Corona, 2004). Accordingly, Iswi was found to be a subunit of various complexes, including NURF (nucleosome remodelling factor) (Badenhorst, 2002), ACF and CHRAC. Iswi-containing complexes were primarily recognized as factors that facilitate in vitro transcription from chromatin templates. However, genetic analysis in Drosophila and in Saccharomyces cerevisiae have provided evidence that Iswi-containing complexes are involved in both transcriptional activation and repression in vivo. For example, immunostaining of Drosophila polytene chromosomes of salivary glands showed that Iswi is associated with hundreds of euchromatic sites in a pattern that is non-overlapping with RNA polymerase II. It suggests that Iswi may play a general role in transcriptional repression. In contrast, it was also demonstrated that expression of engrailed and Ultrabithorax are severely compromised in Iswi-mutant Drosophila larvae. Recent studies have also shown that a mouse Iswi-containing complex, NoRC, plays an essential role during repression of transcription of the rDNA locus by RNA polymerase I (Strohner, 2001; Santoro, 2002; Zhou, 2002). Tou, a protein that is structurally related to the TIP5 subunit of NoRC (Santoro, 2002). Tou positively regulates enhancer-promoter communication during Pnr-driven proneural development and its activity is targeted to the ac-sc promoter sequences through dimerization with Pnr and Chip. Evidence is provided that Iswi is required during neural development. Overexpression of Iswi^K159R in the precursor cells of the sensory organs using the sca^Gal4 driver (Deuring, 2000) leads to flies lacking multiple bristles, suggesting that Iswi functions late during neural development, essential for either cell viability or division of the precursor cell. Using the Iswi¹/Iswi² transheterozygous combination and individuals overexpressing Iswi^K159R in earlier stages of development and in less restricted patterns, it has been shown that Iswi also regulates ac-sc expression. Interestingly, the regulation is probably direct since Iswi associates with the transcription factors Pnr and Chip, known to promote ac-sc expression at the DC site (Garcia-Garcia, 1999; Ramain, 2000). Since Iswi interacts with Tou, it is proposed that Tou and Iswi may positively regulate activity of Pnr and Chip during enhancer-promoter communication, possibly as subunits of a multiprotein complex involved in chromatin remodelling (Vanolst, 2005).

GENE STRUCTURE

cDNA clone length - 10497 bp (isoform A)

Bases in 5' UTR - 309

Exons - 5

Bases in 3' UTR - 945

PROTEIN STRUCTURE

Amino Acids - 3080

Structural Domains

Tou is predicted to encode a 3080 amino acids sequence protein that contains a Methyl-CpG binding (MBD) domain, a DDT domain, a WAKZ motif, two PHD zinc fingers and a C-terminal bromodomain. Tou shares essential functional domains with members of the WAL family of chromatin remodelling proteins, including Acf1 of ACF (see Drosophila Acf1) and CHRAC (Ito, 1999) and also TIP5 (Strohner, 2001) of NoRC (nucleolar remodelling complex), involved in repression of the rDNA (Santoro, 2002) and human WSTF (Williams syndrome transcription factor; Lu, 1998) (Vanolst, 2005).

date revised: 5 March 2006

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