Effects of Mutation or Deletion

Members of the Polycomb group (Pc-G) and trithorax group (trx-G) of genes, as well as the enhancers of trx-G and Pc-G (ETP), function together to maintain segment identity during Drosophila development. In order to obtain new marked P mutations in these genes, a screen was performed for dominant modifiers of the extra-sex-combs phenotype displayed by males mutant for the polyhomeotic (ph) gene, a member of the Pc-G group. Five P(lacW) insertions in four different genes were found to stably suppress ph: two are allelic to trithorax, one is the first allele specific to the Minute(2)21C gene, and the remaining two define new trx-G genes, toutatis (tou) in 48A and taranis (tara) in 89B10-13. tou is predicted to encode a 3109 amino acid sequence protein (TOU), which contains a TAM DNA-binding domain, a WAKZ motif, two PHD zinc fingers and a C-terminal bromodomain, and as such is likely to be involved in regulation of chromatin structure as a subunit of a novel chromatin remodelling complex. In a previous study, insertion of a P(ph) transposable element containing ph regulatory sequences was found to creat a high frequency of mutations modifying ph homeotic phenotypes. One such insertion enhanced the ph phenotype and it was found to be a new allele of UbcD1/eff, a gene encoding a ubiquitin-conjugating enzyme that is involved in telomere association and potentially in chromatin remodelling (Fauvarque, 2001).

Pannier (Pnr) promotes development of DC sensory organs. Tou physically interacts with Pnr in yeast and tou mRNA was found to be ubiquitously expressed in the wing imaginal disc. tou mRNA is expressed in the wing pouch, in agreement with the wing defects associated with tou alleles (Fauvarque, 2001) and in the dorsal-most region of the thorax, covering the site of appearance of the dorsocentral (DC) bristles and the domain of pnr expression. Hence, it was asked whether Tou is also required during development of DC sensory organs (Vanolst, 2005).

tou1 corresponds to a PlacW transposon insertion within the large intron of tou at 836 bases pairs (bp) upstream of the second exon of tou that contains the ATG translational start (Fauvarque, 2001). tou2 was generated by imprecise excision of the tou1 insertion, in which 200 bp of the intron was replaced by a 7 kb sequence from the P-element. Oregon wild-type flies have 2.00 DC bristles per heminotum. Both tou1 and tou2 give rise to homozygous escapers lacking DC bristles. This suggests that tou is necessary for DC neural development. tou1 and tou2 are associated with molecular lesions affecting intronic sequences and display an abnormal bristle phenotype, suggesting that tou1 and tou2 disrupt functioning of an important regulatory element. Accordingly, the level of tou expression is strongly reduced in tou2 thoracic discs, suggesting that tou2 is likely to be a loss of function allele. This hypothesis is reinforced by the fact that the loss of DC bristles is more severe for tou1/Df(2R)en-SFX31 and tou2/Df(2R)en-SFX31 than for tou1 and tou2. Df(2R)en-SFX31 is a deficiency that spans tou and other genes between 48A1 and 48B5. Moreover, the requirement for tou to promote DC neural development is further demonstrated by analysis of touE44.1. touE44.1 was generated by imprecise excision of the transposon insertion in EY08961 and encodes a mutant protein lacking the C terminus, containing the PHD fingers and the bromodomain. Since the touE44.1 flies lack DC bristles and because PHD fingers and the bromodomain are believed to play important functions, it is concluded that touE44.1 is a loss-of-function allele and that Tou is required to promote neural development (Vanolst, 2005).

Genetic interactions between the pnr and tou alleles were analyzed. Use of tou1 and tou2, which are loss-of-function alleles and whether they interact with pnrD1 was addressed. pnrD1 encodes a mutant protein carrying a single point mutation in the DBD that disrupts interaction with the Ush antagonist. Hence, PnrD1 constitutively activates ac-sc at the DC site, leading to excess DC sensory organs. pnrD1/+ flies have 3.34±0.1 DC bristles per heminotum and this excess is suppressed when tou function is simultaneously reduced. For example, tou2; pnrD1/+ flies have 2.17±0.08 DC bristles per heminotum. Conversely, pnrVX1 encodes a truncated protein, lacking the domain of interaction with Chip, which is required for enhancer-promoter communication during ac-sc expression and development of DC sensory organs. Hence, pnrVX1 displays a loss of DC bristles (1.86±0.03 DC bristles/heminotum) and this loss is aggravated when tou function is simultaneously reduced. tou2/+; pnrVX1/+ flies display 1.57±0.05 DC bristles/heminotum. ChipE carries a single point mutation that disrupts the enhancer-promoter communication and is associated with reduced proneural expression at the DC site and loss of sensory organs (1.6±0.07 DC bristles/heminotum). The loss of DC bristles was accentuated when tou function was simultaneously reduced as in tou2ChipE flies, which display 1.11±0.05 DC organs/heminotum. These observations indicate that Tou probably cooperates with Pnr and its Chip cofactor during development of the DC bristles (Vanolst, 2005).

The effects of overexpressed tou on neural development were analyzed. Use of the UAS/Gal4 system and tou was overexpressed in the dorsal compartment of the disc Gal4-apMD544. Gal4-apMD544/EP622 flies have excess DC sensory organs (2.78±0.12 DC bristles/heminotum). Moreover, the loss of DC bristles associated with ChipE is suppressed when Tou is overexpressed. Indeed, ChipE apGal4/ChipE EP622 flies have 2.23±0.08 DC bristles/heminotum (Vanolst, 2005).

The development of extra DC sensory organs, revealed by phenotypic analysis, was compared with the positions of DC bristle precursors detected with a lacZ insert, A101, in the neuralized gene that exhibits staining in all sensory organs. In EP622/Gal4-apMD544, additional DC precursors are seen that lead to excess DC bristles. Hence, it is concluded that Tou cooperates with Pnr and Chip during neural development (Vanolst, 2005).

To investigate whether the interactions between Pnr, Chip and Tou function in vivo, effects of both loss-o-function and gain-of-function of tou on were examined expression of a lacZ reporter driven by a minimal ac promoter fused to the DC enhancer [transgenic line DC:ac-lacZ. Expression is strongly impaired in tou2 flies and in touE44.1 flies, in agreement with the lack of DC bristles. In contrast, overexpressed Tou (apGal4/EP622) leads to increased lacZ expression at the DC site, consistent with the excess of DC bristles. The excess of DC bristles in pnrD1 correlates with expanded ac-sc expression at the DC site. Since loss of tou function suppresses the excess DC bristles associated with pnrD1, the consequence of loss of tou function on expression of the lacZ reporter in pnrD1/+ flies was examined. Expanded lacZ expression associated with pnrD1 is decreased when tou function is simultaneously reduced. It is concluded conclude that Pnr and Tou cooperate during neural development and regulate ac-sc expression through the DC enhancer (Vanolst, 2005).


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

date revised: 5 March 2006

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