Identification of a new POP-1 target using a computational search for Helper site-HMG site clusters.

Abstract

<p>(A) Schematic depicting the <i>K08D12.3</i> locus (Gene ID: 176979) with black boxes representing exons and the gray box the flanking gene <i>pbs-1</i>. The start codon is marked by ‘M’. The white box indicates the genomic region used to construct the GFP transcriptional reporter (nucleotides −579 to +14; first nucleotide of TlSS represents +1), with the asterisk indicating where the <i>K08D12.3</i> start codon was mutated to allow GFP to be read in the correct frame. The location of the HMG and Helper sites are indicated in red and blue respectively. Fluorescence (B–D; B′–D′) and Brightfield (B″–D″) images of live late L4 larvae extrachromosomally expressing the <i>K08D12.3::VENUS</i> reporter. Strong expression was seen in the head muscles, pharyngeal muscles, posterior intestine and hindgut (arrowheads) and moderate expression in the midgut (arrows). (B) Wildtype, (C) HMG mutant and (D) Helper mutant worms were scored based on the VENUS expression in the head muscles, pharyngeal muscles and intestine. (E) Histogram showing the expression analysis of late L4 larvae from three independent lines carrying either the WT, HMG mutant or Helper mutant <i>K08D12.3::VENUS</i> reporters, grouped into strong, intermediate or weak expressers, represented by the images in panels B, C & D, respectively. (F) Competition analysis using EMSA with POP-1 protein with a 90 bp probe (sequence shown in panel A) containing the three functional Helper sites and the functional HMG site from the <i>K08D12.3</i> WRE. The POP-1 dependent shift (lane 2) is competed by an excess of unlabeled WT probe (lanes 3, 4), while unlabeled HMG mutant probe (lanes 5, 6) or the Helper3 mutant probe (lanes 7, 8) does not compete even at 200 fold excess competitor levels. Unlabeled Helper1 mutant (lanes 9, 10) and Helper2 mutant (lanes 11, 12) probes displayed a moderate level of competition. The black arrowhead represents the DNA-protein complex and the white arrowhead represents unbound probe. The data is representative of three independent experiments.</p