Genic and Global Functions for Paf1C in Chromatin Modification and Gene Expression in Arabidopsis

Citation: Oh S, Park S, van Nocker S ( Genic and Global Functions for Paf1C in Chromatin Modification and Gene Expression in Arabidopsis Sookyung Oh. 0 Sunchung Park. 0 Steven van Nocker 0 Bas van Steensel, Netherlands Cancer Institute, Netherlands 0 Department of Horticulture, Michigan State University , East Lansing, Michigan , United States of America In budding yeast, intragenic histone modification is linked with transcriptional elongation through the conserved regulator Paf1C. To investigate Paf1C-related function in higher eukaryotes, we analyzed the effects of loss of Paf1C on histone H3 density and patterns of H3 methylated at K4, K27, and K36 in Arabidopsis genes, and integrated this with existing gene expression data. Loss of Paf1C did not change global abundance of H3K4me3 or H3K36me2 within chromatin, but instead led to a 39 shift in the distribution of H3K4me3 and a 59 shift in the distribution of H3K36me2 within genes. We found that genes regulated by plant Paf1C showed strong enrichment for both H3K4me3 and H3K27me3 and also showed a high degree of tissue-specific expression. At the Paf1C- and PcG-regulated gene FLC, transcriptional silencing and loss of H3K4me3 and H3K36me2 were accompanied by expansion of H3K27me3 into the promoter and transcriptional start regions and further enrichment of H3K27me3 within the transcribed region. These results highlight both genic and global functions for plant Paf1C in histone modification and gene expression, and link transcriptional activity with cellular memory. - . These authors contributed equally to this work. Post-translational modification of core histones considerably extends the information potential of the genetic code [1,2]. Methylation of specific residues within the amino-terminal tail of nucleosomal histone H3, in particular, has been tied to activation or repression of transcription within the associated gene(s). For example, where studied in budding yeast and human, nucleosomes containing H3 trimethylated at lysine-4 (H3K4me3) are globally enriched near the transcriptional start sites (TSS) and 59 regions of active genes, with the degree of enrichment correlating with gene activity [36]. In yeast, this pattern is thought to be an outcome of cotranscriptional recruitment of the histone methyltransferase SET1 during the early elongation phase [7,8]. SET1 and homologous methyltransferases such as Trithorax (Trx) in fruit fly and mixed lineage leukemia 1 (MLL1) in human target nucleosomal H3K4 for methylation as components of larger protein complexes [911]. However, methylated H3K4 likely serves an instructive and promotive role in transcription as well: methylated H3K4 is required for efficient chromatin remodeling at promoters [12,13], and potentially enhances interaction with the SET1-related complexes [14]. Thus, H3K4me3 may define a mechanism that reinforces the active state of transcription. Di- and trimethylated H3K36 (H3K36me2/me3) is prevalent within transcribed regions in yeast and human, especially near the 39 ends [15,16], reflecting cotranscriptional activity of the H3K36specific SET2 methyltransferase during elongation [17]. Although localized within active genes, H3K36 methylation probably has an overall negative influence on transcription that is mediated at least in part through recruitment of histone deacetylase activity and consequent maintenance of low acetylation levels [1820]. Repressing histone acetylation within transcribed regions is expected to promote internucleosomal interactions and/or chromatin assembly in the wake of PolII, thus minimizing inappropriate intragenic transcriptional initiation at cryptic sites. Methylation of H3K27 is an elaboration seen only in higher eukaryotes, where it has been linked with transcriptional repression. Dimethylated H3K27 (H3K27me2) is abundant within heterochromatin, whereas in human and fruit fly, trimethylated H3K27 (H3K27me3) is found in frequent islands scattered throughout euchromatin, with extended domains surrounding Polycomb-Group (PcG) protein binding sites including the Hox loci [2125]. In plants, H3K27me3 marks weakly expressed and/ or developmentally silenced genes, including known targets of plant PcG proteins [2629]. H3K27 methylation may repress transcription through several mechanisms, including recruitment of PRC1 in metazoans [21], and in plants, direct binding to LHP1, the homolog of Heterochromatin Protein 1 [28]. The conserved PcG protein Enhancer of zeste [E(z)] and associated proteins, designated Polycomb Repressive Complex 2 (PRC2) mediate methylation of H3K27, thus connecting this modification to the maintenance of gene silencing [30]. The PolII-associating factor 1 complex (Paf1C), minimally composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1 has an important role in establishing patterns of methylated H3K4 and H3K36 by promoting ubiquitination of histone H2B [31,32] and linking elongating PolII with SET1 and SET2 [7,8,15]. Paf1C also has transcription-related roles potentially independent of its function in histone modification, related to elongation [33], suppression of intragenic initiation [34], poly(A) site selection [35], mRNA In eukaryotes, DNA is packaged with histones and other proteins into a dynamic fabric called chromatin. Specific modifications of histonesincluding methylation of key lysine residuesprovide genetic information that acts synergistically with the DNA code. In yeast, the conserved transcriptional regulator Paf1C is required for marking histone H3 within active genes by methylation of Lysine-4, a modification thought to promote gene activity. In higher eukaryotes, this mechanism is elaborated through Polycomb-Group (PcG), which maintains transcriptional repression through cell divisions and involves methylation of Lysine-27 of H3. In this study, we mapped these and other key H3 modifications throughout the genome of the plant Arabidopsis thaliana and evaluated the effects of loss of Paf1C on these modifications and gene expression. We found that Paf1C acts globally to maintain histone modification within genes, but is required for appropriate expression of only a handful of genes. These typically showed a high degree of developmental regulation in both Lysine-4 and Lysine-27 methylation. At the flowering regulator FLC, targeted by both Paf1C and PcG, loss of activating (Lysine-4) methylation was accompanied by further accumulation of repressive (Lysine-27) methylation. These results provide a link between transcriptional activity and cellular memory. polyadenylation [36], and 39 end formation on nonpolyadenylated PolII-generated transcripts [37]. Components of Paf1C are also conserved in higher eukaryotes. The product of the human HRPT2 gene, parafibromin, shows moderate homology with Cdc73, and interacts with human homologs of Paf1, Ctr9, and Leo1 as well as elongating (Ser-2/ Ser-5 phosphorylated) PolII in vivo [3840]. The human Paf1C complex (hPAF) also contains hSki8, a protein t (...truncated)