Gfi1 physically binds to miR-21 and miR-196B loci. (A) Diagrammatic representation of putative Gfi1 binding sites in the human miR-21 locus relative to the start of the pre-miR transcript on human chromosome 17, along with primer pairs (“A” and “B”; arrows) used for ChIP analyses (top). ChIP analyses using a Gfi1-specific monoclonal antibody (Gfi1) and isotype control mouse IgG (ConIgG; bottom) in human HL60 cells. Primers amplifying a region of the Beta-actin gene (Actin) and primer pair “B” to the miR-21 locus serve as negative controls. (B) EMSA analyses with in vitro transcribed and translated Gfi1 and oligonucleotides encoding wild-type (Probe) or Gfi1-site mutant (Mut probe) miR-21 locus sequences depicted in panel A. (C) Diagrammatic representation of putative Gfi1 binding sites in the human miR-196B locus relative to the start of the pre-miR transcript on chromosome 6, along with primer pairs (“A,” “B,” and “C”) used for ChIP analyses (top). ChIP analyses using a Gfi1-specific monoclonal antibody (Gfi1), isotype control mouse IgG (ConIgG; bottom). Primers amplifying a region of the Beta-actin gene (Actin) and primer pair “C” to the miR-196b locus serve as negative controls. (D) EMSA analyses with in vitro transcribed and translated Gfi1 and oligonucleotides encoding wild-type (probe) or Gfi1-site mutant (Mut probe) miR-196B locus sequences depicted in panel C. P indicates control IVT protein (luciferase); C, cold competition with wild-type miR-21 or miR-196B oligo; NS, neutralizing Gfi1-specific antibody; CS, nonspecific control antibody; and M, cold competition with Gfi1-site mutant oligo. Results shown are representative of at least 3 independent experiments.