Distinct role of subunits of the Arabidopsis RNA polymerase II elongation factor PAF1C in transcriptional reprogramming

Abstract

Transcript elongation by RNA polymerase II (RNAPII) is dynamic and highly regulated, thereby contributing to the implementation of gene expression programs during plant development or in response to environmental cues. The heterohexameric polymerase-associated factor 1 complex (PAF1C) stabilizes the RNAPII elongation complex promoting efficient transcript synthesis. In addition, PAF1C links transcriptional elongation with various post-translational histone modifications at transcribed loci. We have exposed Arabidopsis mutants deficient in the PAF1C subunits ELF7 or CDC73 to elevated NaCl concentrations to provoke a transcriptional response. The growth of elf7 plants was reduced relative to that of wildtype under these challenging conditions, whereas cdc73 plants exhibited rather enhanced tolerance. Profiling of the transcriptional changes upon NaCl exposure revealed that cdc73 responded similar to wildtype. Relative to wildtype and cdc73, the transcriptional response of elf7 plants was severely reduced in accord with their greater susceptibility to NaCl. The data also imply that CDC73 is more relevant for the transcription of longer genes. Despite the fact that both ELF7 and CDC73 are part of PAF1C the strikingly different transcriptional response of the mutants upon NaCl exposure suggests that the subunits have (partially) specific functions.

Keywords: Arabidopsis thaliana; PAF1C; RNA polymerase II; chromatin; histone modifications; transcript elongation.

Figure 1

Growth of PAF1C subunit mutants is differentially affected by exposure to elevated NaCl concentrations. (A) Seeds of the indicated genotypes were sown on solid MS medium containing 100 mM NaCl or control medium (ctrl), and the resulting plants were documented at 14 days after stratification (14 DAS). (B) After growth on solid MS medium for 7 DAS, plants of the indicated genotypes were transferred to control medium or to medium containing 100 mM NaCl. Plants were documented after additional 18d (ctrl) or 21d (100 mM NaCl) of growth. (C, D) 7 DAS plants were grown in absence or presence of 100 mM NaCl for another 11d (ctrl, left) or 14d (100 mM NaCl, right), before rosette diameter [cm] (C) or fresh weight [mg] (D) was determined (n = 15). (E) Plants were grown on vertical MS plates for 7 DAS, before they were transferred to control medium or medium containing 100 mM NaCl. Length of the primary roots was recorded directly after transfer (0h) and 96h after transfer (n = 8). Growth after 96h in presence of NaCl is depicted as percentage of growth of the same genotype on control medium. The relative growth of each genotype was compared to Col-0 using Wilcoxon signed-rank test (ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001).

Figure 2

Changes in transcript levels upon exposure to NaCl. After growth on solid MS medium for 7 DAS, Col-0 plants were transferred to control medium (ctrl) or to medium containing 100 mM NaCl (NaCl). After different periods of the indicated treatment, RNA was isolated and the transcript levels of LTP4 and reference genes (EF1A, GAPC2, ACT2) quantified by RT-qPCR. The depicted relative expression was normalised to the level of ACT2 transcript in Col-0. The bars represent the mean relative expression of three technical replicates and error bars represent SD. The relative expression of LTP4 was compared between control and NaCl treated condition of the same timepoints using Wilcoxon´s signed-rank test indicated by brackets (**, p ≤ 0.01).

Figure 3

Differential gene expression analysis of elf7-3 and cdc73-2 relative to Col-0 upon NaCl treatment. (A–C) differential gene expression between treatment and control groups for each genotype separately. Highlighted in red are genes that are significantly upregulated after salt stress (log-fold change, LFC≥ 1 and padjusted ≤ 0.05), while in blue are genes that are significantly downregulated upon NaCl exposure (LFC≤ −1 and padjusted ≤ 0.05). NS = not significant. Numbers in the legends represent the gene counts in that group. (D, E) Venn diagrams summarising the number of differentially expressed genes in the different genotypes (as in A–C) upon NaCl treatment with total number of differentially expressed genes (D), upregulated (E) or downregulated (F) genes. ns, not significant.

Figure 4

Heatmap and hierarchical clustering of the 500 most variable genes identified by transcript profiling of elf7-3 and cdc73-2 relative to Col-0 upon NaCl treatment. These genes were selected from the scaled counts of the count table using the subread package “featureCounts” and the count table includes the counts for all replicates, conditions and genotypes. Relative transcript abundance is shown as heatmap using the indicated scale (LFC of scaled counts). Genotypes and condition (ctrl: control; NaCl: 3h 100 mM NaCl) are colour-coded on top.

Figure 5

Changes in transcript levels in the different genotypes at individual loci dependent on the presence of NaCl. Coverage of merged RNA-seq samples was normalised to the effective Arabidopsis genome size and visualised using the Integrative Genomics Viewer. (A) coverage of WSD1 (At5g37300), a gene induced by elevated salt concentrations. (B) coverage of DRN1 (At2g45180), a gene repressed by elevated salt concentrations. (C) coverage of ICL (At3g21720), a gene, whose expression enhances salt tolerance.

Figure 6

Transcript length and number of exons of genes uniquely differentially expressed in Col-0 or cdc73-2. (A) average transcript length of genes differentially expressed either in Col-0 or cdc73-2. (B) number of exons of genes differentially expressed either in Col-0 or cdc73-2. Data was evaluated using Wilcoxon signed-rank test (**, p ≤ 0.01; ****, p ≤ 0.0001; ns, not significant).

Figure 7

RNAPII coverage over genes up- and downregulated in elf7-3. Metagene plots of ChIP-seq analysis of Col-0 and elf7-3 using an antibody directed against RNAPII-S2P over DEGs elf7-3/Col-0 under standard condtions (A) or upon exposure to NaCl (B). Mean signals of the three biological replicates were averaged (line) and the shaded area represents the SEM for the three replicates at each position. The tracks were scaled over transcribed regions from transcriptional start site (TSS) to transcriptional end site (TES).