The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB, and NF-YC, all of which are required for formation of the NF-Y complex and DNA-binding. NF-YA contains a DNA binding domain in its C-terminal region. We established transgenic fly lines carrying the UAS-HA-dNF-YA or UAS-dNF-YAIR and showed over-expression or knockdown with various GAL4 drivers to be lethal at various developmental stages, suggesting that dNF-YA participate in various gene regulatory pathways during Drosophila development. Expression of dNF-YA with eyeless-GAL4 mainly resulted in lethality with a headless phenotype in pharate-adults. Reduction of the eyeless gene dose enhanced the dNF-YA-induced phenotype, while reduction of the Distal-less gene dose suppressed the phenotype. On the other hand, crossing the dNF-YA over-expressing flies with Notch mutant resulted in no apparent effect on the phenotype. These results suggest that dNF-YA can disturb eye disc specification, but not eye disc growth.

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The CCAAT motif‐binding factor, nuclear factor Y (NF‐Y) consists of three different subunits, NF‐YA, NF‐YB and NF‐YC. Knockdown of Drosophila NF‐YA (dNF‐YA) in the notum compartment of wing discs by a pannir‐GAL4 and UAS‐dNF‐YAIR mainly resulted in a thorax disclosed phenotype. Reduction of the Drosophila c‐Jun N‐terminal kinase (JNK) basket (bsk) gene dose enhanced the knockdown of dNF‐YA‐induced phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)‐LacZ enhancer trap line revealed reduction in the level of the JNK reporter, puc‐LacZ signals, in dNF‐YA RNAi clones. In addition, expression of wild‐type Bsk effectively suppressed the phenotype induced by knockdown of dNF‐YA. The bsk gene promoter contains a CCAAT motif and this motif plays a positive role in the promoter activity. We performed chromatin immunoprecipitation (ChIP) assays in S2 cells with anti‐dNF‐YA IgG and quantitative real‐time PCR. The bsk gene promoter region containing the CCAAT boxes was effectively amplified in the immunoprecipitates by PCR. However, this region was not amplified in the immunoprecipitates from dNF‐YA knockdown cells. Furthermore, the level of endogenous bsk mRNA is reduced in the dNF‐YA knockdown larvae. These results suggest that dNF‐Y is necessary for proper bsk expression and activity of JNK pathway during thorax development.

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