NF-Y binding to the HOXB4 promoter in vitro and in situ. (A) The HxRE-1 site and E-box are essential for HOXB4 promoter activity. A luciferase gene, coupled with a wild-type human HOXB4 5′ regulatory sequence or the mutated form of this sequence at either HxRE-1 (HxRE-1^*) or HxRE-2 (HxRE-2^*), was incorporated into the endogenous HOXB4 locus of murine ES cells. Luciferase activities of cell lysates from equal amounts of ES cells in each genetic modification were measured in triplicate as described in “Materials and methods.” The data presented are means ± SD. (B) NF-Y binds to the probe containing HxRE-1 site in EMSA. K562 nuclear extract (NE) was incubated with a ³²P-labeled short probe containing the HxRE-1 site, and the reaction mixture was then run on a 5% nondenaturing PAGE to detect the specific retarded migrating band (arrow, lane 2). In lanes 3 through 7, K562 NE was pretreated with the indicated antibodies before being incubated with the probe. (C) NF-Y binds to HOXB4 promoter in situ. In ChIP assay, the chemically cross-linked and fragmented chromatins were precipitated by antibodies against NF-Ya, USF1, and Yin Yang protein 1 (YY1), as well as by a control IgG. The presence of HOXB4 promoter within those final precipitates was examined by PCR amplification of a specific 363-bp sequence spanning HxRE-1 and HxRE-2. The PCR primers are depicted as arrows in diagram below. (D) NF-Y and USF specifically bind to HxRE-1 and HxRE-2 sites in situ. Chemically linked chromatin from the K562 cells stably transfected with either pGL-3-HOXB4 core promoter (1), pGL-3-HOXB4 core promoter/mutated HxRE-1 (2), mutated HxRE-2 (3) were precipitated with the antibodies as indicated. Transfected HOXB4 promoter DNA bound within the immunoprecipitates were amplified with primers derived from pGL-3 vector sequence surrounding the inserted wild-type or mutated HOXB4 core promoters.