The Cyclophilin AtCYP71 Interacts with CAF-1 and LHP1 and Functions in Multiple Chromatin Remodeling Processes

Hong Li 0 1 Sheng Luan sluan@nature 0 1 0 berkeley.edu , tel. (510) 642-6306, fax (510) 642-4995. a The Author 2011. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE , SIBS, CAS. doi: 10.1093/mp/ssr036, Advance Access publication 18 May 2011 Received 15 February 2011; accepted 25 March 2011 1 Department of Plant and Microbial Biology, University of California , Berkeley, CA 94720 , USA Chromatin is the primary carrier of epigenetic information in higher eukaryotes. AtCYP71 contains both cyclophilin domain and WD40 repeats. Loss of AtCYP71 function causes drastic pleiotropic phenotypic defects. Here, we show that AtCYP71 physically interacts with FAS1 and LHP1, respectively, to modulate their distribution on chromatin. The lhp1 cyp71 double mutant showed more severe phenotypes than the single mutants, suggesting that AtCYP71 and LHP1 synergistically control plant development. Such synergism was in part illustrated by the observation that LHP1 association with its specific target loci requires AtCYP71 function. We also demonstrate that AtCYP71 physically interacts with FAS1 and is indispensable for FAS1 targeting to the KNAT1 locus. Together, our data suggest that AtCYP71 is involved in fundamental processes of chromatin assembly and histone modification in plants. - INTRODUCTION Chromatin is the principal carrier of epigenetic information. It is composed of both histones and DNA. Basic proteins and nucleic acids are assembled into complexes in a reaction that must be facilitated by molecular chaperones in order to prevent protein aggregation and formation of non-specific nucleoprotein complexes. Assembly of H3/H4 requires a number of chaperones to facilitate tethering of complexes for rapid histone deposition (Smith and Stillman, 1991; Akey and Luger, 2003; Natsume et al., 2007; Murzina et al., 2008). Chromatin Assembly Factor-1 (CAF-1) is such a chaperone that is highly conserved in eukaryotes ranging from yeast to human. It is a heterotrimeric complex that facilitates the deposition of histones H3/H4 onto DNA and is the only known histone chaperone involved in DNA replication (Verreault et al., 1996; Kaya et al., 2001; Schaper et al., 2001; Krude, 2002). Three subunits of CAF-1 were identified as FASCIATA1 (FAS1), FASCIATA2 (FAS2), and multicopy suppressor of ira1 (MSI1) in plants (Kaya et al., 2001). Defects in CAF-1-mediated pathway lead to cell death in human cell and mating defects in yeast (Enomoto and Berman, 1998; Nabatiyan and Krude, 2004), whereas mutations in FAS1 or FAS2 result in viable plants with developmental defects (Leyser and Furner, 1992; Game and Kaufman, 1999; Tchenio et al., 2001; Sharp et al., 2002; Hennig et al., 2003; Glowczewski et al., 2004; Linger and Tyler, 2005; Exner et al., 2006; Ramirez-Parra and Gutierrez, 2007; Song et al., 2007). Given the lethal phenotype caused by mutation in CAF-1 in other eukaryotes organisms, it is not well understood why mutations in FAS1/FAS2 are not lethal to plants. Little is known about how CAF1 regulates gene expression and if other unidentified factors are involved in chromatin assembly and function redundantly with CAF-1 in plants. Histones are subject to a wide variety of post-translational covalent modifications that constitute a histone code. A growing body of evidence suggests that the histone codes serve crucial functions in gene activation and silencing by altering accessibility of transcription factors to DNA wrapped in chromatin (Jenuwein and Allis, 2001; Berger, 2007; Li et al., 2007a). Histone acetylation and methylation have been linked to transcriptional control and been extensively studied (Lachner and Jenuwein, 2002; He and Amasino, 2005; Kimura et al., 2005; He, 2009; Strahl and Allis, 2000). The methylation of histones appears to carry more complex information. For example, the methylation of lysine 4 in the N-terminal tail of H3 is associated with actively transcribed genes. On the other hand, methylation of lysine 9 or lysine 27 in H3 (H3K9/27) is associated with gene silencing. Although it is widely acknowledged that histone modification is critical for the regulation of chromatin functions, it remains unclear as to how histone marks are produced and recognized at the molecular level. It is therefore imperative to identify the factors necessary for depositing and recognizing histone marks. Over the past decade, remarkable progress has been made in this area of research. For instance, SU(VAR)39 enzymes are responsible for methylation of the histone H3 at lysine 9, which defines heterochromatin and represses gene activity (Ivanova et al., 1998; Rea et al., 2000; Baumbusch et al., 2001; Cao and Jacobsen, 2002; Gendrel et al., 2002). The Heterochromatin protein 1 (HP1) is a conserved chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. HP1 is proposed to form a three-component complex with the methylated H3K9 and SU(VAR)39 enzymes (Bannister et al., 2001; Maison and Almouzni, 2004). Protein complexes have been identified that are capable of depositing or removing different modifications in insects (Lee et al., 2005). Two well-characterized Polycomb group protein (PcG) complexes, including the PcG repressive complex 1 (PRC1) and 2 (PRC2), have been identified in many organisms. PRC1 functions in the repression of gene expression by recognizing the methylated H3K27me3 marks. The PRC2 complex is required for catalyzing H3K27 methylation and conserved throughout the eukaryotic kingdoms. Multiple PRC2-like components have been identified in Arabidopsis, and different components are proposed to be combined into multiple complexes to have specific function. Plants with a mutation in the PRC2 protein complex display pleiotropic phenotype (Kiyosue et al., 1999; Ohad et al., 1999; Chanvivattana et al., 2004; Calonje et al., 2008; Ahmad et al., 2010). Surprisingly, PRC1-like complexes appear to be missing in plants. Recent studies have made remarkable progresses in unveiling proteins that substitute for PRC1 in plants. One candidate gene, Like Heterochromatin Protein1 (LHP1), has been identified as a subunit of PRC1 because of its association with the H3K27me3. Because the LHP1 mutation causes terminated inflorescence in Arabidopsis, the mutant was also named terminal flower2 (tfl2) (Larsson et al., 1998; Gaudin et al., 2001). In addition, a class of RING finger domain proteins (including AtRING1a and AtRING1b) has also been identified as PRC1 components (Xu and Shen, 2008). However, it is still unclear how these proteins are targeted to specific chromosomal domains and what accessory repressive proteins are required to control euchromatic gene expression. Several PRC components play roles in repressing KNOX genes (Schubert et al., 2006; Xu and Shen, 2008; Hay and Tsiantis, 2009; Shen and Xu, 2009). Class I KNOTTED-like homeobox (KNOX) family genes encode home (...truncated)