3.4 Identification of CRCT target genes
CRCT contains a CCT domain at the C-terminal region. The CCT domains of CONSTANS and TOC1 were shown to directly bind to CORE elements (TGTG(X2-3)ATG) and are involved in the regulation of gene expression (Tiwari et al., 2010; Gendron et al., 2012). To clarify whether CRCT can bind to the 5’-flanking regions of starch synthesis-related genes, ChIP-qPCR was conducted using the FLAG-CRCT overexpression line. Primers for qPCR were designed at 300-400 bp intervals on 5’-flanking regions of the starch synthesis-related genes (Figure 5a). The amplicons designed at the position 10 of OsGPT2 , the position 8 of OsAGPL1 , and the position 1 of OsPho1were enriched over 100 fold in ChIP DNA compared to input DNA (Figure 5b, c, f). Additionally, the amplicons designed at the position 6 ofOsBEI and the positions 4 to 7 of OsAGPS1 were enriched several dozen times by ChIP (Figure 5d, e). These results suggest that CRCT can associate with the promoter regions of these genes, especiallyOsAGPL1 , OsGPT2 and OsPho1 in vivo . The CORE element was found in 5’-flanking regions (within 5 kb of the translation initiation) of OsGPT2 , OsAGPL1 , OsAGPS1 andOsBEI (Figure 5a, Table S2). A CORE element was present in the ChIP enriched position 6 of OsBEI . However, other positions of amplicons enriched by ChIP do not overlap the CORE elements. Additionally, the 5’-flanking regions of OsPho1 do not contain a CORE element. Thus, their presence can not explain the results of our ChIP assay. The CCT domain of Ghd7 can bind to more simple DNA elements such as T1ME (CACA) and G-box (CACG) (Nemoto, Nonoue, Yano, & Izawa, 2016). These elements can be found more in the starch synthesis related genes. Thus, it is likely that the CRCT binds to some elements other than the CORE element.