Down-regulation of NF-Ya nuclear protein levels and its regulation on HOXB4 expression with hematopoietic differentiation. (A) NF-Ya protein levels decrease with terminal myeloid differentiation. Western blotting was used to measure 10 μg nuclear protein extracted from primary c-Kit⁺ and c-Kit-Gr-1⁺ BM cells as well as spleen Mac-1⁺ cells. (B) Primitive 32D cells maintained in IL-3 were washed and induced to granulocytic differentiation by 10 ng/mL G-CSF, and NF-Ya levels were measured by Western blotting at indicated time points. The morphology of 32D cells during differentiation is shown (original magnification, × 1000). Note that the level of NF-Ya declines rapidly and precedes the differentiation to mature granulocytes. (C) NF-Ya/HOXB4 ChIP assays in c-Kit⁺ premature BM cells versus mature granulocyte and monocytes/macrophages. Cross-linked chromatin fragments from 5 × 10⁶ indicated cells were precipitated with NF-Ya antibody, and the abundance of HOXB4 promoter sequence in final precipitates was assessed by PCR amplification as described in Figure 1C. One of 2 experiments with similar PCR results is shown. The HOXB4 mRNA levels in indicated cells were examined in parallel by RT-PCR (bottom lane).