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.

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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The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. By genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express DREF in the eye discs, we identified 24 genes that suppressed and 12 genes that enhanced the rough eye phenotype when heterozygous for mutations. Five genes, HP6, pigeon, lace, X box binding protein 1 and guftagu were found to carry replication-related element (DRE) sequences in their 5′-flanking regions. Of these, the HP6 gene carries two sequences that match seven out of eight nucleotides of DRE and two additional sequences that match six out of eight nucleotides of DRE in the 5′-flanking region. Band mobility shift assays using Drosophila Kc cell nuclear extracts demonstrated DREF binding to two of these sites and chromatin immunoprecipitation using anti-DREF antibodies confirmed that this occurs in vivo. Knockdown of DREF in Drosophila S2 cells decreased the HP6 mRNA level. The results, taken together, indicate that DREF directly regulates expression of the HP6 gene. HP6 mRNA was detected throughout development by RT-PCR with highest levels in adult males. In addition, immunostaining analyses revealed colocalization of HP6 and DREF in nuclei at the apical tips in the testes.

BSTRACTIn human cells, appropriate monomethylation of histone H4 lysine 20 by PrSet7 (also known as SET8 and SETD7) is important for the correct transcription of specific genes and timely progression through the cell cycle. Over-methylation appears to be prevented through the interaction of PrSet7 with proliferating cell nuclear antigen (PCNA), which targets PrSet7 for destruction through the pathway mediated by CRL4Cdt2 (the cullin ring finger ligase-4 complex containing Cdt2).However, the factors involved in positive regulation of PrSet7 histone methylation remain undefined. Here, we present biochemical and genetic evidence for a previously undocumented interaction between Drosophila PrSet7 (dPrSet7) and DNA polymerase a in Drosophila. Depletion of the polymerase reduces H4K20 monomethylation suggesting that it is required for dPrSet7 histone methylation activity. We also show that the interaction between PCNA and PrSet7 is conserved in Drosophila, but is only detectable in chromatin fractions. Consistent with this, S2 cells show a significant loss of chromatin-bound dPrSet7 protein as S phase progresses. Based on these data we suggest that interaction with the DNA polymerase represents an important route for stimulation of PrSet7 histone methylase activity that is mediated by allowing loading of dPrSet7 onto chromatin or its subsequent activation.