Figure 1.
Corto and CycG interact in vitro.
A. Two-hybrid tests. Three Corto sub-fragments comprising amino-acids 1 to 324 (C1/324), 325 to 550 (C325/550), and 127 to 203 (C127/203) (chromodomain) were fused with LexA DNA binding domain. B42AD-CycG was isolated from an embryonic library using LexA-C1/324 as bait. Interaction was observed only between LexA-C1/324 and B42-CycG. Negative controls: LexA versus B42AD-CycG and LexA-C1/324 versus B42AD. Positive control: LexA53 (LexA/p53 fusion) versus B42AD-T (B42AD/large T-antigen fusion). B. GST pull-down assays. Top: Coomassie staining of fusion proteins (shown by arrows). Middle: Autoradiography. Input (CycG-35S) shows two isoforms (asterisks). Bottom: schematic representation of Corto full-length and truncated forms (black box: chromodomain). C. GST pull-down assays. Top: Coomassie staining of the fusion proteins (shown by arrows). Middle: Autoradiography. Input (Corto 35S). Bottom: schematic representation of CycG full-length and truncated forms (grey box: cyclin domain).
Figure 2.
Alignment of cyclin domains of Drosophila and mammalian CycG proteins.
A. Alignment of the cyclin domains of Drosophila CycG (NM 524609), human CycG1 (NP 954854), mouse CycG1 (NP 033961), rat CycG1 (NP 037055), human CycG2 (NM 004345), mouse CycG2 (NP 031661) and rat CycG2 (NP 446451) (dark grey: identical amino-acids, light grey: similar amino-acids). B. Developmental Northern blot. Poly(A+) RNA from w1118 embryos, third instar larvae (LIII) and adult flies were probed with 32P-labelled CycG cDNA (left). Arrows point to five mRNA species ranging from 2.0 to 3.5 kb in size that may correspond to CG11525 RA-RE transcripts predicted in Flybase. The same filter was hybridized with 32P-labelled 18S and 28S probes (right) for loading control. C. Total protein (left) and chromatin (right) extracts were prepared from w1118 0–14 h embryos and analysed using rabbit anti-CycG antibodies.
Figure 3.
CycG is an essential gene in Drosophila.
A. Validation of RNAi efficiency by in situ hybridization. Leg imaginal discs from ptc::Gal4>UAS::dsCycG3 third instar larvae were hybridized using CycG as a probe. Expression of the CycG mRNA was specifically reduced in the ptc expression domain along the antero-posterior border (arrow). B. Validation of RNAi efficiency by immuno-labelling. Leg imaginal discs from en::Gal4>UAS::dsCycG3 were immuno-labelled using rabbit anti-CycG antibodies. The expression of CycG was specifically reduced in the en expression domain i.e. in the posterior compartment (arrow), whereas it was ubiquitous in the control (UAS::dsCycG3).
Table 1.
Lethality of RNAi CycG flies.
Figure 4.
Corto and CycG interact in vivo.
A. CycG co-immunoprecipitates with Corto in embryonic extracts. Protein extracts from 0–14 h embryos were incubated with rabbit anti-Corto antibodies (IP) or rabbit preimmune serum (mock). Western blot analysis was performed using rat anti-Corto antibodies (left) or rat anti-CycG antibodies (right). Note specific precipitation of Corto (70 kDa) and the 68 kDa CycG species (arrows). The asterisks label unspecific IgG signals in all panels. B. Myc-CycG co-immunoprecipitates with Corto-Flag in S2 cell extracts. S2 cells were co-transfected with pAct::Corto-Flag and pAct::Myc-CycG. Proteins extracts were incubated with either mouse anti-Flag antibodies (IP) or mouse anti-HA antibodies (mock). Western blot analysis was performed using mouse anti-Flag antibodies (left) or mouse anti-Myc antibodies (right). Specific precipitation of Corto-Flag (left) or Myc-CycG (right) is indicated by arrows. C. Corto-Flag co-immunoprecipitates with Myc-CycG in S2 cell extracts. S2 cells were co-transfected with pAct::Corto-Flag and pAct::Myc-CycG. For immunoprecipitation we used either mouse anti-Myc antibodies (IP) or mouse anti-HA antibodies (mock), and for detection, mouse anti-Myc antibodies (left) or mouse anti-Flag antibodies (right). Specific precipitation of Myc-CycG (left) or Corto-Flag (right) is indicated by arrows. 4% of the starting material used in each IP (input) and 50% of the immunoprecipitated material were loaded onto the gel in all our assays.
Figure 5.
Corto, CycG and PH partially co-localize in vivo.
Simultaneous detection of Corto (red) and CycG (green) (A), PH (red) and CycG (green) (B), PH (red) and Corto (green) (C) on polytene chromosomes stained with DAPI (blue). Close-ups of chromosome 3 L ends showing 3 loci simultaneously bound by PH (Red) and CycG (green) (D), and the same 3 loci simultaneously bound by PH (red) and Corto (green) (E). Overlaps appear yellow.
Figure 6.
Expression of Hox genes in corto mutant embryos.
A. Anti-Ubx antibody staining of w1118 embryos, or embryos depleted of maternal and zygotic Corto. Upper row, lateral view and lower row, dorsal view. (B) Dorsal view. In wild-type embryos (left), Ubx is expressed from parasegment PS5 to PS12 with a higher expression in PS6. In corto embryos (right), the anterior boundary of Ubx is not modified but Ubx is strongly downregulated in PS11 and PS12 (arrowheads) . Parasegment numbers are indicated in white and the anterior border of Ubx expression by arrows. B. Anti-AbdB antibody staining of w1118 embryos, or embryos depleted of maternal and zygotic Corto. Upper row, close-ups of the ventral nerve chord. In wild-type embryos (left), Abd-B is expressed in a decreasing gradient from PS13 to PS10. In corto embryos (right), this gradient extends anteriorly to PS9 and PS8. Moreover, expression of Abd-B seems to be higher in PS13 to PS10. Lower row, dorsal view of whole mount embryos showing ectodermal Abd-B expression; note ectopic expression of Abd-B in anterior parasegments in corto mutant embryos (arrows).
Figure 7.
Corto and CycG bind the iab-7 PRE and the promoter of Abd-B.
Schematic representation of the Abd-B locus (numbers and transcript names refer to SEQ89/U31961) [44]. Corto or CycG XChIP was performed using total chromatin from 0–14 h embryos. Fragments around 500 bp from the iab-7 PRE (p9, p8) and overlapping the iab-7 PRE (p7) or the Abd-B B promoter (p10) were amplified with specific primers using the immuno-precipitated DNA [42], [45]. A fragment from the rp49 gene was used as a negative control. The 31st, 33rd and 35th PCR cycle samples are shown. Relative enrichment was estimated for the 33rd PCR cycle sample from the ratio between Corto or CycG immunoprecipitations and mock signals from three independent experiments. The input track shows amplification of DNA from total chromatin with the same primers (Mock: rabbit preimmune serum, Corto: rabbit anti-Corto, CycG-R: rabbit anti-CycG, CycG-GP: guinea-pig anti-CycG). The iab-6, iab-7 and iab-8 cis-regulatory domains are indicated.