Identification of LEC1 binding sites in Arabidopsis seedlings

LEAFY COTYLEDON1 (LEC1), an atypical subunit of the NF-Y CCAAT binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene network and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. In this part of the study, we identified the genes bound by LEC1 by overexpressing a FLAG-tagged inducible form of LEC1 in Arabidopsis seedlings and inducing LEC1 activity for a short (4 hours) or long (8 days) period of time, to identify the genes bound EARLY and LATE, respectively. LEC1 activity was induced in Arabidopsis 35S:FLAG-LEC1-GR plants, either by treating seedlings for 4h, or germinating seeds and growing seedlings for 8 days in the presence of dexamethasone (DEX). Anti-FLAG antibodies (Sigma F1804) were used to immunoprecipitate LEC1-bound DNA fragments and compared to total fragmented chromatin from the same biological materials. Two biological replicates per conditions were analyzed.