Gfi1b binds directly to the Rag locus and acts through a chromatin modifying mechanism. (A) α-FLAG ChIP-Chip on RAG1-GFP^high or Gfi1b^−/− cells overexpressing FLAG-tagged Gfi1b analyzed using a custom genomic tiling array. Data are shown for the region upstream and including Rag1 and Rag2 as indicated. Arrows denote the location of primer sets used in B. (B) Quantitative PCR of DNA recovered from ChIP using Gfi1b^−/− cells overexpressing FLAG-tagged Gfi1b (left) or Gfi1 (right). IgG is a control IP done in parallel and error bars indicate standard deviation of triplicate assays. (C) Quantitative PCR of H3K9me2 ChIP products in RAG1-GFP^high Gfi1b-ER cells treated for 3 d in the presence (+) or absence (−) of tamoxifen (4-OHT). Error bar represents standard deviation of triplicate PCR assays. Data are representative of two independent experiments. (D) Flow cytometric analysis of RAG1-GFP levels in RAG1-GFP^high cells infected with a retrovirus expressing either a wt or P2A mutant Gfi1b protein compared with uninfected cells. Data are representative of two independent experiments. (E) Quantitative RT-PCR of Rag1 and Rag2 transcripts relative to HPRT in RAG1-GFP^high cells infected with a retroviral Gfi1b-ER construct and treated (+) or not (−) with tamoxifen (4-OHT) for 12 h in the presence (+) or absence (−) of the DNA replication inhibitor aphidicolin. Error bar represents standard deviation of triplicate PCR assays. Numbers indicate fold differences with error bars from triplicate assays.