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.