Complementary Activities of TELOMERE REPEAT BINDING Proteins and Polycomb Group Complexes in Transcriptional Regulation of Target Genes

Abstract

In multicellular organisms, Polycomb Repressive Complex 1 (PRC1) and PRC2 repress target genes through histone modification and chromatin compaction. Arabidopsis thaliana mutants strongly compromised in the pathway cannot develop differentiated organs. LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is so far the only known plant PRC1 component that directly binds to H3K27me3, the histone modification set by PRC2, and also associates genome-wide with trimethylation of lysine 27 of histone H3 (H3K27me3). Surprisingly, lhp1 mutants show relatively mild phenotypic alterations. To explain this paradox, we screened for genetic enhancers of lhp1 mutants to identify novel components repressing target genes together with, or in parallel to, LHP1. Two enhancing mutations were mapped to TELOMERE REPEAT BINDING PROTEIN1 (TRB1) and its paralog TRB3. We show that TRB1 binds to thousands of genomic sites containing telobox or related cis-elements with a significant increase of sites and strength of binding in the lhp1 background. Furthermore, in combination with lhp1, but not alone, trb1 mutants show increased transcription of LHP1 targets, such as floral meristem identity genes, which are more likely to be bound by TRB1 in the lhp1 background. By contrast, expression of a subset of LHP1-independent TRB1 target genes, many involved in primary metabolism, is decreased in the absence of TRB1 alone. Thus, TRB1 is a bivalent transcriptional modulator that maintains downregulation of Polycomb Group (PcG) target genes in lhp1 mutants, while it sustains high expression of targets that are regulated independently of PcG.

Figure 1.

trb1 and trb3 Alleles Enhance the lhp1 Mutant Phenotype Independently of Their Role in Telomere Maintenance. (A) Phenotype of Col-0, trb1-1, trb3-1, trb1-1 trb3-1, lhp1-3, trb1-1 lhp1-3, trb3-1 lhp1-3, and trb1-1 trb3-1 lhp1-3 plants 28 d postgermination. Plants were grown at 22°C in long days. Bar = 1 cm. (B) Flowering time of genotypes grown as in (A) scored as number of leaves. Error bars indicate mean ± se (n = 9). Statistical significance was determined by one-way ANOVA with multiple comparison correction by Tukey HSD. Different letters indicate significance groups (P < 0.001). (C) Rosette size of plants as in (B); statistical significance tested as above. (D) Localization of fluorescent TRB1 and TRB3 wild-type and mutant fusion proteins transiently produced in tobacco leaves. Bar = 10 μm. (E) Colocalization of fluorescent TRB1, TRB3, and LHP1 fusion proteins transiently produced in tobacco leaves Bar = 10 μm. (F) Average intensity of TRB1-GFP and LHP1-RFP across regions of interest. Error bars represent Student’s t test confidence intervals (n = 9). (G) Average intensity of TRB3-RFP and LHP1-GFP as in (F). (H) Telomere length analysis. DNA was prepared from Col-0, lhp1-3, trb1-1 lhp1-3, and trb3-1 lhp1-3 pools of (100 to 200) 10-d-old seedlings and 31-d-old individual plants. Material from 10-d-old ku70 and tert (G5) seedlings was included as reference. (I) Phenotype of lhp1-3, ku70 lhp1-3, and tert lhp1-3. Bar = 1 cm.

Figure 2.

Effect of Mutation of TRB1 on Gene Expression in Seedlings. (A) Venn diagram of genes differentially regulated in trb1-1, lhp1-3, and trb1-1 lhp1-3 seedlings compared with wild-type Col based on RNA-seq data (biological replicates n = 3, Baggerley’s test with FDR correction on RPKM values per gene, threshold FDR < 0.05, fold change > 2). Comparison only for upregulated genes indicated in parentheses. (B) Number of genes up- or downregulated in each genotype. Proportion of H3K27me3 target genes indicated in gray. (C) Transcriptional clustering of misregulated genes. K-median (k = 8) clustering after median centering each gene across all samples (arbitrary units). A representative clustering result is shown.

Figure 3.

Analysis of Gene Clusters Affected in the trb1 Mutant Background. (A) Box plot showing median normalized log-transformed expression across all genes per cluster (cl). Thick horizontal lines represent the median, boxes represent the 25th to 75th percentiles, the whiskers represent the 5th and 95th percentiles, and dots indicate outliers. (B) Proportion of H3K27me3 target genes per cluster. Genome average of H3K27me3 is indicated by dashed line. Significant deviation from genome average was tested by Fisher’s exact (*P < 0.05, **P < 0.01, and ***P < 0.005). (C) Gene clusters were further clustered according to the number of shared GO terms (dendrogram). Curved lines indicate positive (+) and negative (−) correlations between expression of the clusters. The most significant shared GO term is indicated below the tree. The pictogram indicates statistical significance of enriched GO term (yellow, lower; orange, intermediate, red, higher significance). An enriched site II sequence motif found in cluster 5 is indicated below.

Figure 4.

Analysis of TRB1 Target Sites in Seedlings. (A) cis-elements enriched in TRB1 target regions. Regions enriched in TRB1:GFP compared with Col were interrogated for centrally enriched cis-elements by MEME-ChIP. Consensus sequences with corresponding E-values (bottom) and probabilities of occurrence around the fragment’s center (top). (B) Proportion of TRB1 targets among the genes encoding ribosomal functions. 1, Ribosomal protein; 2, tRNA; 3, intron-containing tRNA; 4, snoRNA. (C) Proportion of H3K27me3 target (blue) and non-target (red) genes for TRB1 positive (upper pie) and negative (lower pie) genes. (D) and (E) Metagene analysis of TRB1 target genes. (D) Background-corrected TRB1:GFP ChIP-seq reads for all genes (green), all TRB1 target genes (brown), and H3K27me3 negative (purple) or positive (pink) TRB1 target genes. (E) Frequency of RMCCTAR consensus for gene categories as in (D). Genes are represented in relative length from TSS to transcriptional exit site (TES). Sequences 5′ and 3′ of TSS and transcriptional exit site, respectively, are scaled in base pairs.

Figure 5.

Comparison of TRB1 Target Sites in the Wild Type and lhp1. (A) Venn diagram showing the number of regions associated with TRB1:GFP (gray) and TRB1:GFP lhp1 (red). (B) Read depth (corrected for read depth at control precipitation) across all fragments enriched only in TRB1:GFP lhp1 (left panel) or in both genetic backgrounds (right panel). ChIP-seq data are based on two biological replicates of TRB1:GFP (black lines) and TRB1:GFP lhp1 (red lines). Enriched fragments are displayed between gray lines on a fraction of length scale and flanking regions on a base pair scale. (C) Overview of the SEP3 locus. Top panels show gene models with exons and introns illustrated by boxes and lines, respectively. Untranslated regions are depicted by lighter blue fill color; direction of the coding strand is indicated by the arrow. Location of telobox-related, telobox, and celobox motifs are indicated by blue, red, and green boxes, respectively. Middle panels show coverage of TRB1:GFP and TRB1:GFP lhp1 corrected by coverage from Col control precipitation. Values more than 50 reads over background are indicated in black for TRB1:GFP and red for TRB1:GFP lhp1. Black and red boxes indicate location of fragments indicates significantly enriched by SICER (FDR < 0.0001). Two bottom panels show ChIP-chip enrichment of LHP1:HA and H3K27me3 from our previously published data (Dong et al., 2012; Engelhorn et al., 2012). (D) Overview of AG locus; colors and symbols same as for (C). (E) Overview of the AP3 locus; colors and symbols same as for (C).

Figure 6.

Comparison of TRB1 Target Genes within Transcriptional Cluster Groups between the Wild Type and lhp1. (A) Scatterplot of proportion of TRB target genes per transcriptional cluster for the wild type and lhp1 among genes misregulated in trb1-1, lhp1, and trb1-1 lhp1. Genome-wide proportion for the wild type and lhp1 is indicated by a black and a red line, respectively. Statistical difference from expected indicated by asterisks (Fisher’s exact test, *P < 0.05, **P < 0.01, and ***P < 0.005). (B) Proportion of genes that are both TRB1 and H3K27me3 positive in the wild type and lhp1. Genome-wide proportion indicated by a black and a red line for the wild type and lhp1, respectively. Significant deviation from genome average was tested by Fisher’s exact (*P < 0.05, **P < 0.01, and ***P < 0.005).

Figure 7.

Working Model for Transcriptional Regulation by TRB1. (A) and (B) Model for highly expressed genes in cluster 5. (A) TRB1 binds to telobox-like motifs, thereby facilitating the binding of TCP factors to site II motifs. Genes are highly expressed in the absence of TRB (ON), while the presence of both factors helps to express genes at very high levels (ON+), but the presence of TCP is a dominant requirement for expression (OFF). In principle, the mechanisms could also apply to coregulation by other cis-elements and cofactors, which could also be repressors. (B) As in (A) but TRB1 recruits chromatin remodelers to assist binding of TCP factors or support their downstream effect on transcription activation. (C) and (D) TRB1 action at PcG target genes, showing enhanced upregulation in the double mutant. (C) TRB1 basically functions as in (A) but facilitates the action of a repressor that participates in downregulation but has no dominant effect. In the wild type, there is an equilibrium between LHP1-dependent and TRB1-dependent repression and targets are always repressed (OFF). In lhp1 plants, a repressor can bind with the help of TRB1 and attenuate upregulation (ON). In trb1 lhp1 double mutants, attenuation is lost, leading to enhanced expression (ON+). (D) TRB1 acts similar as in (B) by recruiting a chromatin remodeler. The remodeler maintains a more closed chromatin conformation but can only partially compensate for the lack of LHP1. Genes are not expressed in the wild type and trb1 single mutants (OFF), are induced in lhp1 mutants (ON), and are hyperinduced in trb1 lhp1 double mutants (ON+).