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KNOTTED1-like homeobox (KNOX) genes promote stem cell activity and must be repressed to form determinate lateral organs. StableKNOX gene silencing during organogenesis isknownto involve the predictedDNAbinding proteinsASYMMETRIC LEAVES1 (AS1) and AS2 as well as the chromatin-remodeling factor HIRA. However, the mechanism of silencing is unknown. Here, we show that AS1 and AS2 form a repressor complex that binds directly to the regulatory motifs CWGTTD and KMKTTGAHW present at two sites in the promoters of the KNOX genes BREVIPEDICELLUS (BP) and KNAT2. The two binding sites act nonredundantly, and interaction between AS1-AS2 complexes at these sites is required to repress BP. Promoter deletion analysis further indicates that enhancer elements required for BP expression in the leaf are located between the AS1-AS2 complex binding sites.Wepropose that AS1-AS2 complexes interact to create a loop in theKNOX promoter and, likely through recruitment of HIRA, form a repressive chromatin state that blocks enhancer activity during organogenesis. Our model for AS1-AS2-mediated KNOX gene silencing is conceptually similar to the action of an insulator. This regulatory mechanism may be conserved in simple leafed species of monocot and dicot lineages and constitutes a potential key determinant in the evolution of compound leaves.
INTRODUCTION
The reiterative process of organogenesis characteristic of plants depends on the activity of a population of self-renewing, plurip-otent stem cells present in meristems at the growing tips. Meristem activity in the shoot apex is specified in part by the class I KNOTTED1-like homeobox (KNOX) genes (Long et al., 1996; Vollbrecht et al., 2000; Scofield and Murray, 2006). Lateral organs, such as leaves, initiate on the flank of the shoot apical meristem (SAM), and downregulation of KNOX gene expression is essential to facilitate this process (Jackson et al., 1994; Long et al., 1996). Moreover, acquisition of determinacy in developing organs requires the continued silencing of KNOX genes, as ectopic KNOX expression during organogenesis results in patterning defects and overproliferation of cells (Sinha et al., 1993; Chuck et al., 1996; Kidner et al., 2002). Thus, in plants, the precise balance between stem cell proliferation and differentiation that is critical for development is attained, in part, through the proper regulation of KNOX gene expression.
KNOX repression during organogenesis is mediated by the orthologous MYB domain proteins ROUGH SHEATH2 (RS2) and ASYMMETRIC LEAVES1...