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. 2012 Jul;32(13):2407-15.
doi: 10.1128/MCB.06311-11. Epub 2012 Apr 30.

Functional association between eyegone and HP1a mediates wingless transcriptional repression during development

Lara Salvany  1 , David Requena, Natalia Azpiazu
Affiliations

Functional association between eyegone and HP1a mediates wingless transcriptional repression during development

Lara Salvany et al. Mol Cell Biol. 2012 Jul.

Abstract

The eyegone (eyg) gene encodes Eyg, a transcription factor of the Pax family with multiple roles during Drosophila development. Although Eyg has been shown to act as a repressor, nothing is known about the mechanism by which it represses its target genes. Here, we show that Eyg forms a protein complex with heterochromatin protein 1a (HP1a). Both proteins bind to the same chromatin regions on polytene chromosomes and act cooperatively to suppress variegation and mediate gene silencing. In addition, Eyg binds to a wingless (wg) enhancer region, recruiting HP1a to assemble a closed, heterochromatin-like conformation that represses transcription of the wg gene. We describe here the evidence that suggests that Eyg, encoded by eyegone (eyg), represses wingless (wg) during eye development by association with HP1a. We show that Eyg forms a protein complex with HP1a and both proteins colocalize on salivary gland polytene chromosomes. Using position effect variegation (PEV) experiments, we demonstrated that eyg has a dose-dependent effect on heterochromatin gene silencing and identified a genetic interaction with HP1a in this process. We further demonstrated that HP1a binds to the same wg enhancer element as Eyg. DNase I sensitivity assays indicated that this enhancer region has a closed heterochromatin-like conformation, which becomes open in eyg mutants. In these mutants, much less HP1a binds to the wg enhancer region, as shown by ChIP experiments. Furthermore, as previously described for Eyg, a reduction in the amount of HP1a in the eye imaginal disc derepresses wg. Together, our results suggest a model in which Eyg specifically binds to the wg enhancer region, recruiting HP1a to that site. The recruitment of HP1a prevents transcription by favoring a closed, heterochromatin-like structure. Thus, for the first time, we show that HP1a plays a direct role in the repression of a developmentally regulated gene, wg, during Drosophila eye development.

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Figures

Fig 1
Fig 1
Eyg is required for heterochromatic gene silencing. Effects of mutations in eyg and Su(var)205 on the white+ variegation induced by DX1 transgene repeats (a to e) or a control transgene (f to j), are shown as changes in red eye pigmentation. Altering the dosage of eyg (b) or HP1a (c) suppresses variegation in DX1 flies. Note the increased red eye pigmentation in DX1 flies (d) heterozygous for both eyg and Su(var)205 genes. (e and j) Changes in pigmentation were determined by measuring absorbance at a wavelength of 480 nm. The data are expressed as means ± standard errors of the mean (SEM) (*, P < 0.001 compared with +/+ control; Student's t test; n = 60 per group). control is a single P[white+] element inserted in a euchromatic region that does not induce heterochromatin formation. (k to o) Variegation of Sb in a T(2;3)Sb[V] rearranged chromosome (7) is suppressed by partial depletion of Eyg and/or HP1a. (o) Suppression of Sb variegation is shown as changes in the Sb-to-WT macrochete ratio. The data are expressed as means ± SEM (*, P < 0.005 compared with an Sb[V] control; Student's t test; n = 40 per group).
Fig 2
Fig 2
Eyg and HP1a colocalize to heterochromatic regions. (a to c) Eyg and HP1a colocalize to the heterochromatic regions of salivary gland polytene chromosomes (arrows in panels b and c). (a′ to c′) Enlargement of the chromocenter of the chromosome. (d to f) Eyg and HP1a also colocalize in some interband regions of salivary gland polytene chromosomes (arrows in panels e and f). (g to i) HP1a localizes to the wg genomic region, marked by a fluorescent DNA probe (asterisks in panels h and i). (j) Enrichment of constitutive heterochromatic regions 1360 and H23 when immunoprecipitated with anti-Eyg, expressed as the percentage of input (1:100 dilution) in ChIP experiments. HP1a binding to the same heterochromatic regions was also observed. Control refers to chromatin immunoprecipitated with guinea pig preimmune serum. An isotype control for mouse antibody gave similar results (not shown). The same immunoprecipitated DNA was amplified with primers for the labial genomic region, the wg promoter, and the act5c promoter region, which were used as negative controls. (k) Eyg and HP1a belong to the same protein complex. Western blot analyses detected HP1a protein in salivary gland extracts immunoprecipitated with anti-Eyg antibody and Eyg protein in salivary gland extracts immunoprecipitated with anti-HP1a antibody. In the control lanes, salivary gland extracts were immunoprecipitated with guinea pig preimmune serum or with a monoclonal anti-α-tubulin antibody (control). (l) An EygPSSLN mutant for the PSVLL motif is able to bind HP1a in S2 cells, as does wild-type Eyg (EygPSVLL). negative, no antibody. 1 and 2 refer to two independent coimmunoprecipitation experiments.
Fig 3
Fig 3
Eyg recruits HP1a and mediates the assembly of a heterochromatic-like structure in the wg enhancer region. (a) Schematic view of the wg genomic region. The wg enhancer region that drives Wg expression in the eye imaginal disc is located at the 3′ end of the genomic region (wg 2.10, black bar). (b) Formation of a DNase I-resistant structure in the wg enhancer region. The act5c and H23 loci were used as controls for open and closed genomic regions, respectively. The bars represent changes in CT values (ΔCT) in the wg enhancer region of wild-type, eyg20MD1/+, and HP1a mutant act>HP1aRNAi eye-antennal disc extracts after treatment with 50 U DNase I. In eyg20MD1/+ heterozygous discs, as well as in act>HP1aRNAi, the wg enhancer region became almost as sensitive to DNase I as the euchromatic actin locus. The H23 locus remained mostly closed for wild-type control and eyg20MD1/+ and open and DNase I sensitive for HP1 mutant extracts. (c) Analysis of HP1a and Eyg binding to the wg enhancer region using ChIP. A similar enrichment in binding to the wg enhancer region was found in wild-type control chromatin immunoprecipitated with an anti-Eyg or an anti-HP1a antibody. Binding of Eyg and, to some extent, HP1a was reduced in the chromatin of eyg20MD1/+ eye-antennal disc extracts.
Fig 4
Fig 4
HP1a controls Wg expression in the eye imaginal disc and in the hinge region of the wing disc. (a and b) wg expression (red) in third-instar eye imaginal disc clones of cells with reduced HP1a levels induced using the act>Gal4 line (green). The arrow points to ectopic wg activation. (c) Expression of wg in a wild-type third-instar eye disc. (d) Schematic depiction of a third-instar eye imaginal disc showing the disc domains in which ectopic activation of wg was more frequent after reducing HP1a levels. Of the UAS-HP1aRNAi clones that showed ectopic wg activation, 90% of ectopic activation sites lay in the middle portion of the disc. (e) Expression domain of the 248Gal4 line in the eye disc (green). (f) wglacZ activity in an eye disc of the genotype 248Gal4<UAS-HP1aRNAi. (g) Expression of wglacZ in a wild-type eye imaginal disc. (h) Wild-type eye. (i) Eye phenotype after driving UAS-HP1aRNAi;UAS-GFP under the control of the 248Gal4 line. (j) Adult eye of a fly in which both HP1a and Wg levels were reduced in the same domain. Note that the eye size is rescued to almost wild-type levels (compare with panel i). (k to m) Generation of HP1aRNAi clones (green) in the wing disc. (l) Reducing HP1a levels in the cells of the wing hinge activated Wg expression (red). However, the distribution of Wg protein was not altered in the notum. (m) Distribution of Wg protein in a wild-type wing disc. (n and o) No ectopic wg expression (red) was observed in clones of cells (green fluorescent protein [GFP]-positive cells) expressing a mutated form of dpp, UAS-dpp*RNAi (15), commonly used as an RNAi control line.
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References

    1. Aagaard L, et al. 1999. Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J. 18:1923–1938 - PMC - PubMed
    1. Ainsztein AM, Kandels-Lewis SE, Mackay AM, Earnshaw WC. 1998. INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1. J. Cell Biol. 143:1763–1774 - PMC - PubMed
    1. Aldaz S, Morata G, Azpiazu N. 2003. The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of Drosophila. Development 130:4473–4482 - PubMed
    1. Chi N, Epstein JA. 2002. Getting your Pax straight: Pax proteins in development and disease. Trends Genet. 18:41–47 - PubMed
    1. Comet I, et al. 2006. PRE-mediated bypass of two Su(Hw) insulators targets PcG proteins to a downstream promoter. Dev. Cell 11:117–124 - PubMed

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