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First published online 16 January 2008
doi: 10.1242/dev.014779

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The role of Polycomb-group response elements in regulation of engrailed transcription in Drosophila

Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20982, USA.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Eight kilobases of upstream en regulatory DNA includes sequences with PRE activity. (A) Consensus binding sites for DNA-binding proteins implicated in PRE activity are shown (Brown et al., 2005) along with the location of PSE1 and PSE2. The line below shows the extent of deletion present in the en mutant, en530. (B) DNA constructs used in these experiments. Arrows indicate the start of transcription. Black boxes indicate P element ends. Green boxes indicate en DNA. Red boxes indicate mini-white DNA. Blue boxes indicate lacZ DNA. White boxes indicate DNA deleted from the construct. Sequences deleted from PSE2 extend from -395 to -576 bp upstream of the start of en transcription. Sequences deleted from both extend from -395 bp to -2407 bp. Misexpression (ME) is defined as detection of β-gal protein between the stripes. Number of lines with misexpression/total number of lines stained. Misexpression in P[en1] and P[PSE2] lines was much less extensive than that seen in both (see Fig. 2). (C) En (green) and β-gal protein (red) in a P[en1] line in a wild type and a ph-d401 ph-p602 double mutant embryo. Anterior is leftwards, dorsal is upwards. Ten hours AEL (after egg laying). DL, double label. En is present in a single PNS cell between the stripes, but β-gal is not.

View larger version (44K): [in this window] [in a new window]   Fig. 2. Both PSE1 and PSE2 contribute to PRE activity. (A) Construct P[en2] contains 8 kb of upstream en sequences with PSE1 and PSE2 flanked by LoxP (L) and FRT (F) sites, respectively. (B-I) Immunoperoxidase staining detecting β-gal protein in embryos from a P[en2] line and its derivatives obtained by treating with Flp and/or Cre recombinase. Embryos shown are 6 hours AEL (B-E) and 10 hours AEL (F-I); anterior is leftwards, dorsal is upwards. All are lateral views, except C, which is a ventrolateral view. Expression is initiated correctly in all lines, then β-gal is misexpressed in cells between the stripes in PSE2, PSE1 and both lines. Deletion of PSE2 causes relatively minor misexpression (G). Deletion of PSE1 is more extensive (H), and deletion of both causes the most misexpression between the stripes (I).

View larger version (40K): [in this window] [in a new window]   Fig. 3. PRE action over a distance. (A) Location of P[en2] in line P[en2]-E(Pc). Thick line indicates genomic DNA. Arrows below the line indicate the extent and direction of transcription of E(Pc), inv and en. Black box above the line indicates the location of the PSE1 and PSE2 DNA in the genomic en gene. Arrow above the line denotes the location of P[en2] in the E(Pc) gene. (B) Immunoperoxidase staining showing β-gal protein in the starting line P[en2]-E(Pc) and in P[en2]-E(Pc)both. β-Gal is expressed in only a few cells between the stripes in P[en2]-E(Pc)both, in contrast to the massive misexpression seen at other chromosomal locations (see Fig. 2I). Embryos are anterior leftwards, dorsal upwards, 10 hours AEL. (C) X-gal staining of wing discs from P[en2]-E(Pc) and its Flp and/or Cre recombinase-treated derivatives. β-Gal activity in the posterior compartment (where en is normally expressed) is largely dependent on the presence of PSE1 or PSE2, although a small amount of β-gal activity remains in the both derivative.

View larger version (44K): [in this window] [in a new window]   Fig. 4. P[en3] inserted in tou is expressed in an en-like manner. (A) The P[en3] construct contains en DNA from -2407 bp to +188 bp. PSE1 is flanked by LoxP sites (L), PSE2 is flanked by FRT sites (F). (B) Thick line indicates genomic DNA. Arrows below the line indicate the transcription units and the direction of transcription of en and tou. The start of tou transcription is 10 kb upstream of what is shown. Arrows above the line indicate the position of insertion of P[en3] in two independent lines, P[en3]-en and P[en3]-tou. (C,D) Immunoperoxidase staining to detect β-gal protein in embryos and X-gal staining to detect β-gal activity in wing discs of lines P[en3]-en (C) and P[en3]-tou (D). Embryos are oriented with anterior leftwards and dorsal upwards. Upper embryos are 6 hours AEL. Lower embryos are 10 hours AEL. β-Gal expression in stripes is not initiated until about 6 hours AEL in P[en3]-tou embryos, and staining is apparent within individual cells of stripes before the entire stripe is stained. Wing discs were stained for equivalent amounts of time.

View larger version (66K): [in this window] [in a new window]   Fig. 5. PSE1 and PSE2 can act as positive regulatory elements. Left column shows immunoperoxidase staining to detect β-gal protein in line P[en3]-tou and its derivatives and from P[en3]-enboth (embryos 10 hours AEL; anterior is leftwards, dorsal is upwards). Right column indicates wing discs stained for β-gal activity. Two things happen in embryos when the PSEs are removed from P[en3]-tou: (1) the intensity of the stripes decreases and (2) the β-gal background levels increase. In wing discs, removal of PSE2 causes some misexpression of β-gal in the anterior compartment in the wing pouch in P[en3]-touPSE2 wing discs. Similar results were seen in other discs. Deletion of PSE1 from P[en3] caused most of the β-gal activity in wing discs to be lost. Similar results were seen in other discs (data not shown). Deletion of PSE1 and PSE2 from P[en3]-en had no effect on the expression of β-gal at any stage examined.

View larger version (80K): [in this window] [in a new window]   Fig. 6. en PSEs can mediate both positive and negative signals from many enhancers and silencers. P[en3] is inserted into the capulet transcription unit. β-Gal activity in proventriculus (PV), brain and wing disc (WD) is shown. In the PV, loss of both PSE1 and PSE2 leads to ubiquitous expression of β-gal, whereas in the brain, loss of both PSE1 and PSE2 decreases the expression. PSE2, which is present in PSE1, seems to repress transcription in the proventriculus and wing disc, but activates it in the brain.

View larger version (127K): [in this window] [in a new window]   Fig. 7. Deletion of 530 bp including PSE2 in the endogenous en gene leads to abnormalities in the posterior compartment in wings. (A) Wild-type wing. (B) Wing from en530 homozygotes. Arrows indicate the intersection of the anterior crossvein with the IV longitudinal vein where a defect is seen in the en530 homozygotes. Arrowheads indicate the intersection of longitudinal vein with the wing margin. This vein splits before the intersection with the margin in en530 homozygotes.

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