ABSTRACT
The chromatin modifying Polycomb group (PcG) and trithorax group (trxG) proteins are central regulators of cell identity that maintain a tightly controlled balance between cell proliferation and cell differentiation. The opposing activities of PcG and trxG proteins ensure the correct expression of specific transcriptional programs at defined developmental stages. Here, we report that the chromatin remodeling factor PICKLE (PKL) and the PcG protein CURLY LEAF (CLF) antagonistically determine root meristem activity. Whereas loss of PKL function caused a decrease in meristematic activity, loss of CLF function increased meristematic activity. Alterations of meristematic activity in pkl and clf mutants were not connected with changes in auxin concentration but correlated with decreased or increased expression of root stem cell and meristem marker genes, respectively. Root stem cell and meristem marker genes are modified by the PcG-mediated trimethylation of histone H3 on lysine 27 (H3K27me3). Decreased expression levels of root stem cell and meristem marker genes in pkl correlated with increased levels of H3K27me3, indicating that root meristem activity is largely controlled by the antagonistic activity of PcG proteins and PKL.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Homeodomain Proteins/metabolism , Meristem/growth & development , Plant Roots/growth & development , Repressor Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/growth & development , Arabidopsis/metabolism , Cell Differentiation , Cell Division , Chromatin Assembly and Disassembly , DNA Helicases , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Histones/metabolism , Meristem/cytology , Meristem/metabolism , Methylation , Mutation , Plant Roots/cytology , Plant Roots/genetics , Plant Roots/metabolism , Polycomb-Group ProteinsABSTRACT
Development of multicellular organisms is based on specialized gene expression programs. Because chromatin establishes the environment for transcription, understanding composition and dynamics of chromatin is an important part of developmental biology. The knowledge about chromatin has been greatly advanced by the chromatin immunoprecipitation (ChIP) technique, because ChIP allows to map the position of proteins as well as modifications of DNA and histones to specific genomic regions. Although ChIP has been applied to a wide range of model organisms, including Arabidopsis, it remains a challenging technique, and a careful experimental setup including appropriate positive and negative controls are required to obtain reliable results. Here, we describe a ChIP protocol adapted for material from Arabidopsis, which we routinely apply in our laboratory, and we discuss required controls and methods for data analysis.
Subject(s)
Arabidopsis/genetics , Chromatin Immunoprecipitation/methods , Chromatin , DNA, Plant/isolation & purification , EpigenomicsABSTRACT
Dynamic regulation of chromatin structure is of fundamental importance for modulating genomic activities in higher eukaryotes. The opposing activities of Polycomb group (PcG) and trithorax group (trxG) proteins are part of a chromatin-based cellular memory system ensuring the correct expression of specific transcriptional programs at defined developmental stages. The default silencing activity of PcG proteins is counteracted by trxG proteins that activate PcG target genes and prevent PcG mediated silencing activities. Therefore, the timely expression and regulation of PcG proteins and counteracting trxG proteins is likely to be of fundamental importance for establishing cell identity. Here, we report that the chromodomain/helicase/DNA-binding domain CHD3 proteins PICKLE (PKL) and PICKLE RELATED2 (PKR2) have trxG-like functions in plants and are required for the expression of many genes that are repressed by PcG proteins. The pkl mutant could partly suppress the leaf and flower phenotype of the PcG mutant curly leaf, supporting the idea that CHD3 proteins and PcG proteins antagonistically determine cell identity in plants. The direct targets of PKL in roots include the PcG genes SWINGER and EMBRYONIC FLOWER2 that encode subunits of Polycomb repressive complexes responsible for trimethylating histone H3 at lysine 27 (H3K27me3). Similar to mutants lacking PcG proteins, lack of PKL and PKR2 caused reduced H3K27me3 levels and, therefore, increased expression of a set of PcG protein target genes in roots. Thus, PKL and PKR2 are directly required for activation of PcG protein target genes and in roots are also indirectly required for repression of PcG protein target genes. Reduced PcG protein activity can lead to cell de-differentiation and callus-like tissue formation in pkl pkr2 mutants. Thus, in contrast to mammals, where PcG proteins are required to maintain pluripotency and to prevent cell differentiation, in plants PcG proteins are required to promote cell differentiation by suppressing embryonic development.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/metabolism , Cell Differentiation , Gene Expression Regulation, Plant , Repressor Proteins/metabolism , Trans-Activators/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Cell Lineage , DNA Helicases , Gene Expression Regulation, Developmental , Histones/metabolism , Plant Roots/cytology , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/metabolism , Polycomb-Group Proteins , Protein Binding , Repressor Proteins/genetics , Trans-Activators/geneticsABSTRACT
Polycomb group (PcG) complexes maintain epigenetically repressed states that need to be reprogrammed when cells become committed to differentiation. In contrast to the previously held belief that PcG complexes regulate only a few selected genes, recent efforts have revealed hundreds of potential PcG targets in mammals, insects and plants. These results have changed our perception about PcG recruitment and function on chromatin. Both in animals and plants, evolutionarily conserved PcG complexes mark the chromatin of their target genes by methylation at histone H3 lysine 27. Surprisingly, however, both the proteins recognizing this mark and the mechanisms causing gene repression differ between both kingdoms. This suggests that different developmental strategies used in plant and animal development entailed the evolution of different repressive maintenance mechanisms.
Subject(s)
Gene Expression Regulation , Repressor Proteins/metabolism , Algorithms , Animals , Cell Differentiation , Chromatin/metabolism , DNA Methylation , Drosophila melanogaster , Gene Expression , Gene Silencing , Histones/chemistry , Humans , Lysine/chemistry , Models, Biological , Plant Proteins/metabolism , Polycomb-Group ProteinsABSTRACT
Genomic imprinting is a phenomenon where only one of the two alleles of a gene is expressed - either the maternally or the paternally inherited allele. Imprinting of the plant gene PHERES1 requires the function of the FERTILIZATION INDEPENDENT SEED (FIS) Polycomb group (PcG) complex for repression of the maternal PHERES1 allele. In this study we investigated the mechanism of PHERES1 imprinting and found that PcG silencing is necessary but not sufficient for imprinting establishment of PHERES1. We provide evidence that silencing of the maternal PHERES1 allele depends on a distantly located region downstream of the PHERES1 locus. This region needs to be methylated to ensure PHERES1 expression but must not be methylated for PHERES1 repression. This mechanism is analogous to the regulation of several imprinted genes in mammals, suggesting the employment of similar evolutionary mechanisms for the regulation of imprinted genes in mammals and flowering plants.