The transcription factor nuclear factor Y regulates the proliferation of myeloid progenitor cells.

The transcription factor nuclear factor Y (NF-Y) consists of three subunits; NF-YA, -B and -C, and this complex binds DNA and the basal transcription machinery through binding of TBP. NF-Y targets include cell cycle related genes and hematopoietic (stem) cell genes such as cd34 and hoxb4. Deletion of both nf-ya alleles in mice results in embryonic lethality. Inactivation of NF-YA in mouse embryonic fibroblasts results in a block in proliferation, which is followed by apoptosis. A few studies have been performed addressing the role of NF-Y in hematopoiesis. Mouse bone marrow (BM) cells overexpressing NF-YA are biased towards primitive hematopoiesis and show increased repopulating ability after bone marrow transplantation. During macrophage differentiation NF-Y shows increasing binding activity to several promoters and NF-Y has been shown to mediate the IL-6 response in BM cells, which results in induction of macrophage differentiation. Mantovani et al. constructed a mutant form of NF-YA (YAm29), defective in DNA-binding and shown to have a dominant-negative effect on transcription regulation by wild type NF-Y. To determine the requirement of NF-Y in myelopoiesis, we cloned the YAm29 cDNA in the LZRS vector, containing an IRES-GFP expression cassette. Mouse BM cells were transduced with YAm29 (efficiency of 23±8.9%, 3 independent experiments) as described earlier. As a control we transduced cells with an empty vector (efficiency of 14.5±8.1%). One day after transduction, GFP cells were sorted by flowcytometry and analyzed. Endogenous NF-YA expression was detected by Western blot analysis in the control cells. Clear overexpression of YAm29 was detected in the GFP fraction of the YAm29 transduced cells (Figure 1A). To determine whether expression of YAm29 repressed the expression of NF-Y target genes we measured the levels of Hoxb4 mRNA by qPCR, performed as previously described. Hoxb4 mRNA levels were significantly downregulated in cells transduced with YAm29 compared to cells transduced with empty vector, indicating that YAm29 effectively represses NF-Y function (Figure 1B). To determine the effect of YAm29 expression on colony formation, GFP cells were plated in methylcellulose and colonies were counted after 5-7 days. Transduction with YAm29 resulted in a reduction of granulocytic (CFU-G) and monocytic (CFU-M) colony formation by 70±6% (p<0.01) and 44±19% (p<0.1) respectively, compared to cells transduced with empty vector (Figure 1C). In addition, transduction with YAm29 resulted in a reduction of the size of both colony types (data not shown). The data suggest that NF-Y is required for the proliferation of myeloid progenitor cells. To quantify the inhibitory effect of YAm29 on proliferation, cells were grown in liquid culture. The empty vector transduced population expanded faster than the YAm29 transduced population, resulting in a three-fold difference in cell numbers on day 6 (Figure 2A). To investigate the effect of YAm29 on maturation, the differentiation marker MAC-1 was used. MAC-1 is expressed on maturing myeloid cells, beyond the colony-forming unit (CFU) stage. The percentage of MAC-1 positive cells increased more rapidly within the YAm29 transduced population than in the control population. After eight days, all YAm29 transduced cells were MAC-1 positive (Figure 2B), while in the control population around 17% of the cells were still MAC-1 negative. This suggested that the immature cells in the control population still proliferated and in the YAm29 transduced population proliferation of immature cells was inhibited. The expansion of the MAC-1 negative cell fraction was calculated by multiplying the total amount of cells with the percentage of MAC-1 negative cells. This revealed that proliferation of the YAm29 transduced immature MAC-1 negative population was completely impaired, whereas the MAC-1 negative population in the empty vector transduced cells