Expression of mir-196b is dependent on Mll and Menin. (A) Schematic illustration of the murine Hoxa9 locus on chromosomal band 6qB3. Open boxes represent 3 exons and gray boxes indicate CpG islands. Exon II is the homeodomain (HD) containing exon. The canonical Hoxa9 is encoded by exons CD and II. Regions of high homology between species are noted as dark boxes and the region amplified by ChIP primers is indicated by arrows. The location of mir-196b is labeled above exon AB with an arrowhead. (B) Sequence alignment of the conserved mir-196b sequence among different species. The mature mir-196b sequence is highlighted in black. (C) Quantitative RT-PCR of mir-196b levels in WT, Mll^−/−, and Men^−/− MEFs. The experiment was run in triplicate and the results represent average expression plus or minus SD. (D) Quantitative RT-PCR of mir-196b expression in individual clones expressing an empty vector, MLL, or MLLAF4 in Mll^−/− MEF background. Transfection of Mll^−/− MEFs and clone selection was performed as previously described. Mir-196b expression was determined from total RNA. (E) ChIP assay performed in WT and Mll^−/− MEFs. Chromatin was precipitated using indicated antibodies and qPCR performed with primers spanning mir-196b region. All samples were run in triplicate and were normalized to the input chromatin.