In Arabidopsis (Arabidopsis thaliana), the ATP-dependent chromatin remodeler PICKLE (PKL) determines expression of genes associated with developmental identity. PKL promotes the epigenetic mark trimethylation of histone H3 lysine 27 (H3K27me3) that facilitates repression of tissue-specific genes in plants. It has previously been proposed that PKL acts indirectly to promote H3K27me3 by promoting expression of the POLYCOMB REPRESSIVE COMPLEX2 complex that generates H3K27me3. We undertook expression and chromatin immunoprecipitation analyses to further characterize the contribution of PKL to gene expression and developmental identity. Our expression data support a critical and specific role for PKL in expression of H3K27me3-enriched loci but do not support a role for PKL in expression of POLYCOMB REPRESSIVE COMPLEX2. Moreover, our chromatin immunoprecipitation data reveal that PKL protein is present at the promoter region of multiple H3K27me3-enriched loci, indicating that PKL directly acts on these loci. In particular, we find that PKL is present at LEAFY COTYLEDON1 and LEAFY COTYLEDON2 during germination, which is when PKL acts to repress these master regulators of embryonic identity. Surprisingly, we also find that PKL is present at the promoters of actively transcribed genes that are ubiquitously expressed such as ACTIN7 and POLYUBIQUITIN10 that do not exhibit PKL-dependent expression. Taken together, our data contravene the previous model of PKL action and instead support a direct role for PKL in determining levels of H3K27me3 at repressed loci. Our data also raise the possibility that PKL facilitates a common chromatin remodeling process that is not restricted to H3K27me3-enriched regions.

Selective, tissue-specific gene expression is facilitated by the epigenetic modification H3K27me3 (trimethylation of lysine 27 on histone H3) in plants and animals. Much remains to be learned about how H3K27me3-enriched chromatin states are constructed and maintained. Here, we identify a genetic interaction in between the chromodomain helicase DNA binding chromatin remodeler PICKLE (PKL), which promotes H3K27me3 enrichment, and the SWR1-family remodeler PHOTOPERIOD INDEPENDENT EARLY FLOWERING1 (PIE1), which incorporates the histone variant H2A.Z. Chromatin immunoprecipitation-sequencing and RNA-sequencing reveal that, , and the H3K27 methyltransferase CURLY LEAF act in a common gene expression pathway and are required for H3K27me3 levels genome-wide. Additionally, H3K27me3-enriched genes are largely a subset of H2A.Z-enriched genes, further supporting the functional linkage between these marks. We also found that recombinant PKL acts as a prenucleosome maturation factor, indicating that it promotes retention of H3K27me3. These data support the existence of an epigenetic pathway in which PIE1 promotes H2A.Z, which in turn promotes H3K27me3 deposition. After deposition, PKL promotes retention of H3K27me3 after DNA replication and/or transcription. Our analyses thus reveal roles for H2A.Z and ATP-dependent remodelers in construction and maintenance of H3K27me3-enriched chromatin in plants.

Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.

Angiosperm reproduction requires the integrated development of multiple tissues with different genotypes. To achieve successful fertilization, the haploid female gametophytes and diploid ovary must coordinate their development, after which the male gametes must navigate through the maternal sporophytic tissues to reach the female gametes. After fertilization, seed development requires coordinated development of the maternal diploid integuments, the triploid endosperm, and the diploid zygote. Transcription and signaling factors contribute to communication between these tissues, and roles for epigenetic regulation have been described for some of these processes. Here we identify a broad role for CHD3 chromatin remodelers in Arabidopsis thaliana reproductive development. Plants lacking the CHD3 remodeler, PICKLE, exhibit various reproductive defects including abnormal development of the integuments, female gametophyte, and pollen tube, as well as delayed progression of ovule and embryo development. Genetic analyses demonstrate that these phenotypes result from loss of PICKLE in the maternal sporophyte. The paralogous gene PICKLE RELATED 2 is preferentially expressed in the endosperm and acts antagonistically with respect to PICKLE in the seed: loss of PICKLE RELATED 2 suppresses the large seed phenotype of pickle seeds. Surprisingly, the alteration of seed size in pickle plants is sufficient to determine the expression of embryonic traits in the seedling primary root. These findings establish an important role for CHD3 remodelers in plant reproduction and highlight how the epigenetic status of one tissue can impact the development of genetically distinct tissues. KEYWORDS PICKLE; PKR2; ovule; pollen tube; seed size D EVELOPMENT in multicellular organisms requires coordinated morphogenesis and communication between tissues with distinct gene expression profiles and occasionally distinct genotypes. Mutations in epigenetic regulators can perturb mutually dependent cell types and therefore complicate interpretation of the resulting phenotypes. The reproductive system of flowering plants presents an attractive context in which to distinguish autonomous phenotypes from those arising from defects in ancillary tissues. The haploid male and female gametophyte generation is contained within the diploid sporophyte, and development of these genetically distinct tissues is highly interdependent (Ma and Sundaresan 2010;Niklas and Kutschera 2010). The female gametophyte develops within the sporophytic ovary, where it forms into an embryo sac as it is surrounded by the diploid sporophytic integuments of the ovule (Bencivenga et al. 2011;Chevalier et al. 2011). Characterization of ovule-defective mutants in Arabidopsis thaliana has revealed that female gametophyte development is dependent on the sporophyte, particularly integument development, and has also identified a variety of factors that contribute to this relationship including transcription factors, kinases, and components of plant hormone signal transduction pathwa...