Position-effect variegation in Drosophila: the modifier Su(var)3-7 is a modular DNA-binding protein

Abstract

An increase in the dose of the Su(var)3-7 locus of Drosophila augments heterochromatin-promoted variegated silencing. The deduced protein sequence of Su(var)3-7 reveals seven widely spaced zinc fingers. We found that Su(var)3-7 has affinity for DNA in vitro and that the minimal protein sequence requirement for DNA binding is any module containing two zinc fingers and the interval between them. As Su(var)3-7 is a heterochromatin-associated protein, we tested its affinity for various satellite DNA sequences in vitro. The AATAT and 353-bp elements have the highest affinity. If affinity for satellite DNAs contributes to the presence of Su(var)3-7 in heterochromatin, a general affinity for DNA, or sequences yet to be determined, suggests a function in the genomic silencing of position-effect variegation: expansion of heterochromatin, whether continuous by spreading or discontinuous by pairing with sequence elements scattered through euchromatin, could use the affinity of Su(var)3-7 for DNA.

Fig. 1. Binding of different DNA probes by GST–Su(var)3-7. (A) Each lane of a 2.5% agarose gel corresponds to a digest of a ³²P-labelled recombinant plasmid. The satellite DNA insert is marked by a star and identified above each lane. The arrows indicate the plasmid fragments used to estimate the relative affinity. (B) Fragments selected by the GST–Su(var)3-7 fusion protein in the absence of poly(dI–dC). Same reaction and probe as in (A). (C) Western blot of the GST–Su(var)3-7 constructs containing seven (lane 2) or six (lane 1) zinc fingers. Both lanes show a minor band of the correct size (110 and 100 kDa, respectively). The major products are truncated proteins, one of 80 kDa (corresponding to a protein with five zinc fingers) and one of 45 kDa (ending just after the second finger). (D) Competition for binding of labelled plasmid pBS(353 bp) by GST–Su(var)3-7. The binding reaction mixtures contain poly(dI–dC) at a final concentration of 0.1 µg/µl and ∼10 ng of labelled plasmid, and 0, 1.0 or 10.0 µg of the same, but unlabelled, digested plasmid was added. In this experiment, the pBS(353 bp) plasmid was cut with EcoRI, HindIII and DdeI, the latter splitting the insert into two fragments.

Fig. 2. Binding of the AATAT satellite by different GST–Su(var)3-7 derivatives. (A) The Su(var)3-7 protein is represented as a black bar within which the zinc fingers appear as grey boxes. (B) Western blot of the different recombinant proteins schematized in (A), bound to glutathione–Sepharose 4-B. Lanes 0–6 correspond to proteins with zero to six zinc fingers revealed with anti-GST antibody. (C) Autoradiogram of the agarose gel after electrophoresis of the 300-bp AATAT repeats fragment retained by the same proteins. The amounts of protein fragments in the binding assay were those deposited on the western blot in (B), and the experiment was performed in the presence of 0.1 µg/µl of the non-specific competitor poly(dI–dC).

Fig. 3. Binding of the AATAT and 353-bp satellites by different zinc finger pairs derived from Su(var)3-7. (A) Different parts of the fusion proteins are schematized as in Figure 2. (B) Western blot of the proteins corresponding to (A), after purification on glutathione–Sepharose 4-B, revealed with an anti-GST antibody. Lanes are named according to the pair of fingers present in the fusion proteins. (C) Autoradiography of the agarose gel after electrophoresis of the 300-bp AATAT repeats fragment and the 500-bp insert containing the 353-bp unit bound by the GST-tagged protein constructs. The amounts of protein fragments in the binding assay were those deposited on the western blot in (B), and the experiment was carried out in the presence of 0.1 µg/µl of the non-specific competitor poly(dI–dC).