(A) Low level Dam:Hb expression is non-toxic. Control 1, sca-gal4/sca-gal4; control 2, sca-gal4 UAS-HA::UPRT (Miller et al., 2009); Dam, sca-gal4 UAS-LT3-Dam; Dam:Hb, sca-gal4 UAS-LT3-Dam:Hb, Dam:Hb, Da-gal4 UAS-LT3-Dam:Hb (n = 300 for each genotype). (B) Dam:Hb retains Hb function and can induce ectopic Eve+ U neurons. Anterior up; midline, dashed line. Left hemisegment shows a single ectopic Eve+ neuron (yellow) to comprise six total U neurons, whereas the right hemisegment has the normal five U neurons. Below, quantification. Wild type (y w) represents 68 hemisegments from six embryos; Dam:Hb (da-Gal4 UAS-LT3-Dam:Hb, second ORF) represents 8 of 232 hemisegments from 15 embryos with an ectopic U neuron. ELs, Eve lateral neurons. (C) Dam:Hb binding is reproducible. Left, three biological replicates of genomic binding sites showing high Pearson correlation coefficients. Right, Dam:Hb binding over 1341 kb on chromosome IV is highly similar in all three biological replicates. Genotype da-Gal4 UAS-LT3-Dam:Hb in stage 17 embryos. Data range: −2.84–7.07. (D–G) Dam:Hb-bound loci correlate with Hb ChIP loci. (D) Alignment of Dam:Hb and Hb ChIP binding sites over 766 kb of genomic DNA near the Hb locus, where Hb is known to bind. Data range for Hb ChIP: −1.01–6.23; Data range for Dam:Hb: −2.63–5.3. (E) Alignment of Dam:Hb and Hb ChIP binding sites at the Krüppel (Kr) locus. Data range for Hb ChIP: −1.66–9.04; Data range for Dam:Hb: −0.63–5.68. (F) Dam:Hb peaks for three replicates (blue, cyan, yellow) are correlated with Hb ChIP signal. Plot shows the Hb ChIP signal ±10 kb of the center of all the peaks identified by Dam:Hb analysis in the three replicates. (G) Dam:Hb signal is enriched at sites of Hb ChIP binding (blue), but not that of Bcd (cyan) or Ftz (yellow). Plot shows the Dam:Hb signal ±5 kb of the center of all the peaks identified by ChIP-chip analysis.