Transcription factor NF‐Y is involved in regulation of the JNK pathway during Drosophila thorax development

Abstract

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.

NF-Y transcriptionally regulates the Drosophila p53 gene

“…F1 transformants were selected on the basis of white eye color rescue (Robertson et al, 1988). The plasmids p5'-dmp53wt-EGFP and p5'-dmp53mut-EGFP were constructed as described below for establishment (Yoshioka et al, 2008) of the transgenic fly strains listed with their chromosomal linkages in Table 1.…”

“…The sonicated cell supernatants were diluted 10-fold in Chip Dilution Buffer and precleared with 60 μl of Salmon Sperm DNA/Protein G agarose-50% slurry for 30 min at 4°C. After brief centrifugation, each supernatant was incubated with 1 μg of the rabbit IgG or anti-dNF-YA polyclonal antibody (Yoshioka et al, 2008) for 16 h at 4°C. Salmon Sperm DNA/Protein A agarose-50% slurry was added and incubated for 1 h at 4°C.…”

“…The fact that knockout of mouse NF-YA results in early embryonic lethality indicates essential roles in early development (Bhattacharya et al, 2003). In the Drosophila model, studies focused on NF-YA have revealed a novel function in regulation of the JNK signal transduction pathway that had not been suggested in mammalian systems (Yoshioka et al, 2008). Moreover, overexpression or knockdown of Drosophila NF-YA (dNF-YA) results in specific phenotypes (Yoshioka et al, 2007(Yoshioka et al, , 2008 providing useful models for studying NF-Y functions in vivo.…”

“…NF-Y specifically binds to multiple CCAAT boxes in many promoters of eukaryotic genes (Mantovani, 1999), many with key roles in the G1/S and G2/M transitions (Elkon et al, 2003). NF-Y modulates the activity of CCAAT box-containing promoters of the E2F1, cyclin A, cyclin B1, topo IIα, and bsk genes during different phases of the cell cycle, in response to DNA damage and activation of the JNK pathway (Huet et al, 1996;Farina et al, 1999;Adachi et al, 2000;Yoshioka et al, 2008). Among the three subunits, only subunit NF-YA contains a DNA binding domain, participates in the cellular responses to DNA damage and is involved in regulation of stress-inducible gene (Jin et al, 2001).…”

DREF is Critical for Drosophila Bristle Development by Regulating Endoreplication in Shaft Cells

“…Knockdown efficiency of these UAS-RNAi lines was evaluated by Western blot analysis and differences in adult wing and eye morphology induced by en-GAL4 and GMR-GAL4, respectively (Yoshida et al, 2004). UAS-DREF (II) (Hirose et al, 2001), UAS-dMyc (II) (a kind gift from Dr. Corinne Benassayag) (Zaffran et al,1998), y hs-flp; Act>y>GAL4, UAS-GFP;+ (a kind gift from Dr. Adachiyamada) (Yoshioka et al, 2008), yw hs-flp122; Act>CD2>GAL4, UAS-GFPnls (a kind gift from Dr. BA Edgar). UAS-RNAi against dMyc (2948), PCNA (108384) were from Vienna Drosophila RNAi Center.…”