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Fig. 6. Roe binds E(spl)-C regulatory DNA sequences both in vitro and in vivo. (A) Schematic presentation of the relative position of the 0.3 kb fragment used as a probe for EMSA or amplified in in vivo chromatin immunoprecipitation (ChIP) assays with respect to the E(spl)m gene. Coordinates are based upon `Release 5' of the Drosophila genome sequence (BDGP, http://www.fruitfly.org/). 60 bp fragments denoted as P1-P9 correspond to the probes used in EMSAs described in Fig. S2 in the supplementary material. (B) Representative EMSA experiment using the 0.3 kb fragment (A) as a probe. Lanes are as follows: (1) Free probe (FP) +2.5 µg Su(H), (2) FP + 5 µg Su(H), (3) FP+2.5 µg Su(H) + 2.5 µg Roe, (4) FP + 2.5 µg Su(H) + 2.5 µg Roe + excess cold probe (ECP) as competitor, (5) FP + 2.5 µg Roe, (6) FP + 5 µg Roe, and (7) FP + 2.5 µg Roe + ECP. Shifts caused by Su(H) and Roe (lanes 1,2 and 5,6, respectively) are unaffected when both proteins are simultaneously present (lane 3). (C) In vivo ChIP from a wild-type eye imaginal discs. A band corresponding to the 0.3 kb fragment is amplified when the sample is immunoprecipitated with anti-Roe antibody (Roe-ChIP lane, compare with INPUT lane) but not when preimmune NGS was used for IP (mock-ChIP lane). As a control for specificity, anti-Roe did not co-immunoprecipitate DNA from the AttacinA (AttA) promoter. (D) In vivo ChIP from homozygous roe null (rn¹⁶) eye discs. No specific ChIP band was amplified when the sample was immunoprecipitated either with anti-Roe antibody or preimmune NGS (mock-IP). Same results were obtained with DNA from the AttA promoter. In both C and D, weak bands can be observed occasionally, owing to nonspecific binding of DNA to the resin used during the IP process. (E) Model for Roe action on N targets. In the absence of N-signaling activity, N target genes are repressed by DNA-bound Su(H) together with transcriptional co-repressors. Upon N activation, N_intra translocates to the nucleus and, together with Mam and other transcriptional co-activators (not shown), binds to Su(H) to turn ON the expression of target genes. We propose a third scenario in which N self-modulates its transcriptional activity by upregulating Roe. Roe binds to regulatory sequences of N target genes independently of Su(H), leading to an attenuated transcriptional activity.

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