Validation of Hoxa9 and Meis1 binding sites identified by ChIP-Seq. (A) For each binding site, enrichment profiles are shown for 2 replicates of Hoxa9 and Meis1 ChIP-Seq, with corresponding genomic annotation displayed as UCSC mm8 tracks at the Aff3, Flt3, and Lmo2 loci. A locus is deemed a high-confidence Hoxa9 and Meis1 binding site if it is bound by either Hoxa9 or Meis1in both of the replicate sequencing runs. The sequence tags of nonsignificant peak regions (FDR P < .01) are not displayed. The binding sites are highly conserved as shown by the Phastcon17 conservation track below. No significant binding was detected in the 2 control lanes at any of the regions shown. (B) Confirmation of selected Hoxa9 and Meis1 binding sites by ChIP and quantitative PCR. ChIP experiments were performed with polyclonal anti-HA Abs on HA epitope-tagged Hoxa9-ER/Meis1–transformed myeloblastic cell (HM4) used for ChIP-Seq experiments as described in “Experimental procedures.” Green bars represent PCR signal as a percentage of input for ChIP on cells cultured for 96 hours in the presence of 4-OHT, whereas yellow bars represent ratios for cells cultured for 96 hours in the absence of 4-OHT. These experiments show that Hoxa9 binds at high levels to ChIP-Seq–identified binding sites but not at control peaks and that the Hoxa9 enrichment disappears on 4-OHT withdrawal.