A GAL4-HP1 fusion protein targeted near heterochromatin promotes gene silencing

Abstract

We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin. These rearrangements induce variegation of both white and lacZ. Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3-7 and Su(var)2-5. Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain. Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2-5, is shown to enhance variegation of Heidi and Tell. Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera. Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes.

Drosophila GAGA factor directs histone H3.3 replacement that prevents the heterochromatin spreading

“…However, conventional promoters do not have a barrier function against heterochromatin silencing. For example, the presence of GAL4 (or E2F) on a promoter carrying GAL4 (or E2F)-binding site did not modify PEV of the attached reporter gene (Seum et al 2000). Genome-wide profiling of H3.3 replacement in Drosophila has revealed the clear dip of H3.3-containing nucleosomes at immediately upstream of the transcription start sites of active genes (Mito et al 2005).…”

“…After raising at 18°C, the eye colors of male offsprings were observed within 6 h after eclosion. To examine the effect of d1 on w variegation, the flies in which d1 had been flipped out or not were crossed with GHP1-6 (Seum et al 2000), then the fusion protein GAL4-HP1 was expressed by heat shock at day 7 AEL for 30 min in a 37°C water bath, and the eye colors were observed after raising at 25°C. To measure eye pigmentation, 10 (w m4 lines in Fig.…”

Does heterochromatin protein 1 always follow code?

“…A Gal4-HP1 fusion protein tethered upstream of a reporter gene caused silencing at only one of six genomic locations tested (51,81). Interestingly, the site that supported silencing was flanked by middle repetitive DNA sequences, reminiscent of heterochromatin domains.…”

Histone chaperone ASF1 cooperates with the Brahma chromatin-remodelling machinery

“…This chromosomal rearrangement juxtaposes a P-element transposon containing the miniwhite reporter gene next to centromeric heterochromatin on the second chromosome (Seum et al 2000) and leads to the mosaic inactivation of the miniwhite expression in the eye (Fig. 1C).…”