(A) There is an Atro peak that overlaps with Trl peak within the sbb locus (left peak, Atro and Trl Peak in B). There is an Atro peak that does not overlap with Trl about 20 kb upstream of the sbb locus (right peak, Atro Only Peak in B). The red rectangle marks the region used as negative control in B. (B) ChIP-re-ChIP qPCR results. ChIP-re-ChIP samples are labeled as the sequence of antibody used (e.g. IgG, Atro means rabbit IgG ChIP followed by Atro re-ChIP). Negative controls for the ChIP-re-ChIP are any ChIPs with IgG. Atro, Trl ChIP-re-ChIP enriches the Atro and Trl Peak (purple bar) but it does not enrich the Atro only peak (same enrichment as Atro, IgG). Mean Ct value was used to calculate percent input and standard deviation of the Ct values was carried over in calculations and used as error bars. (C) Trl and Atro ChIP peaks coincide at the same loci upstream of en. (D) TrlR85 imaginal disc clones have decreased En levels (arrow, wing disc clone shown here). (E) Trl and Atro ChIP peaks coincide at the tkv locus. (F) TrlR85 wing disc clones have decreased pMad levels (arrow), possibly due to a decrease of tkv expression. All clones are marked by the absence of GFP; all figures have posterior to the left, dorsal up. Scale bars are 50 μm. (G) Model of how Atro and Trl function together to regulate expression of target genes. Trl is required to activate the transcription of its target genes. In the absence of Atro (Left), the target gene will express at a higher level than normal. Atro binds to the same site as Trl either directly or via some unknown cofactors (X?, Right). Atro modulates the expression of its target gene by counteracting Trl; potentially Atro is doing so by recruiting Histone deacetylase 1 (HDAC1) and G9a, a histone methyl transferase. Thus, target genes are expressed at the correct levels.