Molecular Mechanism Of Transcription Factors TCPs And Epigenetic Factors CLF And LHP1 In Repression Of Class-Ⅰ KNOX Genes In Arabidopsis Thaliana

Lateral organs such as leaves are generated from cells at the periphery of shoot apical meristems (SAMs) in higher plants. The evolutionarily conserved class-I KNOTTED1-like homeobox (KNOX) genes are involved in maintaining the undifferentiated status of stem cells in SAM, and initiation and subsequent development of a leaf requires stable repression of class-I KNOX genes. In Arabidopsis, class-I KNOX genes comprise SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS (BP), KNAT2 and KNAT6. Two transcription factors, ASYMMETRIC LEAVES1 (AS1) and AS2, are known to form a protein complex to repress BP, KNAT2 and KNAT6. Here we show that AS2 physically interacts with the microRNA319 (miR319)-regulated CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF (TCP) transcription factors in vitro and in vivo. By chromatin immunoprecipitation (ChIP), we demonstrate that AS2 and TCPs bind to similar chromatin regions at the BP and KNAT2 promoters, and that DNA-binding activities of TCP proteins rely on the presence of AS2. The jaw-Dmutant, which overexpresses MIR319a to downregulate several target TCP genes, strongly enhanced the as2 phenotypes and caused more severe ectopic expression of BP, KNAT2 and KNAT6. In addition to transcription factors, epigenetic factors also participate in regulating class-I KNOX gene expression. Repressive epigenetic marker H3K27me3, which is deposited by POLYCOMB REPRESSIVE COMPLEX2 (PRC2), containing a H3K27 methyltransferase such as CLF, and is recognized by LHP1 within the PRCl-like complex, is enriched in the chromatin regions of class-I KNOX genes. We find that TCP3 and CLF, as well as AS1/AS2 and LHP1 exhibit physical interactions in vitro and in vivo. While the H3K27me3 level of class-I KNOX genes is unchanged in asl, as2, jaw-D and as2 jaw-D mutants, interaction between AS/AS2 and LHP1 is critical for LHP1 binding at charomatin of the BP and KNAT2 promoters. Together, our results reveal that repression of class-I KNOX genes involves protein complexes containing different types of transcription factors and epigenetic factors that function together to ensure normal leaf development.