Figure 4
The presence of dCas9-VPR in the bxI enhancer generates a typical loss of function phenotype of Ubx. (A) Genomic organization of the Ubx locus showing the position of the gRNAs designed inside the bxI enhancer localized inside the third intron of the gen; the open chromatin regions were mapped using previously published ATAC-seq data49. The genome coordinates of the gRNAs position are indicated. (B) Homeotic transformations generated by the occupancy of the dCas9-VPR in the bxI enhancer. The arrows indicate the location of the halters in the Act5C-GAL4 control fly (black) and the transformation of the metanotum (yellow) and the halteres to wings in the Act5C-GAL4;dCas9-VPR flies (blue). (C) Transformation of the halteres to wings comparing the size versus the control structures. (D) Immunostainings of Ubx in haltere discs when dCas9-VPR is sent to the bxI enhancer. The arrows mark cells that do not express Ubx (blue), express low levels of Ubx (green) and normal levels of Ubx (yellow). (E) RT-qPCR of the transcripts that surround the occupancy of the dCas9-VPR in the bxI enhancer and of the nascent Ubx transcript. At least three independent experiments were performed (see material and methods). Transcript levels of rp49 were used as a reference. The positions in the Ubx gene of the amplicons analyzed are indicated in the figure and its location is indicated in Sup. Table 2. *p < 0.05; **p < 0.01.
