Prod is a novel DNA-binding protein that binds to the 1.686 g/cm(3) 10 bp satellite repeat of Drosophila melanogaster

Abstract

The proliferation disrupter (prod) gene of Drosophila melanogaster encodes a novel protein associated with centromeric chromosomal regions that is required for chromatin condensation and cell viability. We have examined the binding of the Prod protein to DNA in vitro. Co-immunoprecipitation experiments demonstrate that Prod is a DNA-binding protein that specifically recognizes the 10 bp AGAATAACAT satellite repeat of D.melanogaster. Footprinting experiments show that the protein interacts with a 5-8 bp target sequence in each 10 bp repeat and suggest that it can mediate condensation of this satellite into a superhelix. Gel retardation experiments indicate that Prod does not have a well defined DNA-binding domain and it binds the satellite in a co-operative manner, probably forming Prod multimers. Since Prod localizes to both heterochromatin and euchromatin in vivo, we discuss the possibility that the ability of pre-existing euchromatic proteins to bind DNA in a co-operative manner, might be a prerequisite of satellite compaction and satellite amplification, thereby providing a basic factor in heterochromatin evolution.

Figure 1

(A) The different GST–Prod fusion protein constructs used in our studies. Open rectangles, GST coding region; solid bars, Prod coding region; thin lines, non-coding regions of the Prod cDNA. Overlapping sections of the constructs are aligned above each other. Small numbers above the lines show amino acid (aa) positions in the Prod protein, numbers under the lines represent base pair (bp) positions in the prod cDNA. The localization of predicted coiled-coil is depicted at the bottom. (B) Western blot of the different purified GST–Prod fusion proteins illustrated in (A), revealed with an anti-GST antibody.

Figure 2

Co-immunoprecipitation with the different GST fusion constructs. Autoradiogram of labeled random λ DNA fragments co-immunoprecipitated with the GST–Prod fusion proteins designated above the lanes. GST, glutathione S-transferase protein without Prod; lab, labeled DNA before immunoprecipitation, constructs 13, 15, 25 and P (Prod) are described in Figure 1.

Figure 3

Co-immunoprecipitation of different satellite DNA clones. (A) Autoradiogram of end-labeled second and third chromosome-specific satellite DNA clones separated by agarose gel electrophoresis. Numbered lanes contain the following sequence repeats (11): AAGAC (lane 1); AATAACATAG (lane 2); 359 bp (1.688 g/cm³) repeat (lane 3); AATAG (lane 4); AATAT (lane 5); AAGAGAG (lane 6); AAGAG (lane 7). (B) Autoradiogram of the same satellite clones after co-immunoprecipitation with the GST–Prod fusion protein under stringent binding conditions (170 mM NaCl, 100 µg competitor DNA).

Figure 4

DNase I footprint of the Prod protein. Lanes labeled C, G, T and A are sequencing reactions, the sequence is shown on the left of the gel. Lane K is a control where no protein was added to the DNase I reaction, lane P contained the GST–Prod fusion protein and lanes labeled 25 contained the GST–25 fusion protein (Fig. 1A). Superscripts denote the protein concentration (M). Open boxes, unprotected bases; dark boxes, well protected bases; gray boxes, bases where the protection is uncertain. The arrow points at the C residue that is poorly cleaved in the control lane but well cleaved in the presence of Prod.

Figure 5

Capability of the different Prod fusion constructs to bind DNA. Autoradiogram of a gel-shift made with the GST–Prod fusion constructs using the 4.5 Prodsat probe. The sample in lane C corresponds to the free DNA probe (control lane, no protein added). Samples in the other lanes contained the fusion protein designated above each lane (Fig. 1A). A wide variety of different binding conditions had been tested before for each construct (also see text) and this figure shows only the optimized binding conditions achieved. Binding reactions with GST, 28, 19, 16, 13 and 15 contained 1 µg carrier DNA while reactions with P and 25 contained 3 µg carrier DNA for optimal results. Lane P contains 0.5 µg protein and the relative amounts of proteins added to the other reactions were as follows: P:1, 25:2, 28:4, 19:100, 16:200, 13:200, 15:200 and GST:200.

Figure 6

Gel-shift titration of the GST–Prod fusion proteins. (A, B and C) Gel-shifts of the 4.5× Prodsat probe by the GST–P, GST–25 and GST–28 proteins, respectively. Protein concentrations (M) are indicated above each lane. Arrows, predominant and minor faster-migrating complexes formed at low protein concentrations. Note that the reactions shown for the P and 25 contained 3 µg carrier DNA, while all 28 reactions contained 1 µg carrier DNA only. (D) The titration curves extracted from Phosphorimager quantitations of the radioactivity present as free DNA in the gels of (A), (B) and (C) (18). The fractional saturation (the ratio of DNA molecules complexed with the protein) is plotted as a function of the logarithm of the protein concentration.