Polycomb Repressive Complex 2 attenuates the very high expression of the Arabidopsis gene NRT2.1

Abstract

PRC2 is a major regulator of gene expression in eukaryotes. It catalyzes the repressive chromatin mark H3K27me3, which leads to very low expression of target genes. NRT2.1, which encodes a key root nitrate transporter in Arabidopsis, is targeted by H3K27me3, but the function of PRC2 on NRT2.1 remains unclear. Here, we demonstrate that PRC2 directly targets and down-regulates NRT2.1, but in a context of very high transcription, in nutritional conditions where this gene is one of the most highly expressed genes in the transcriptome. Indeed, the mutation of CLF, which encodes a PRC2 subunit, leads to a loss of H3K27me3 at NRT2.1 and results, exclusively under permissive conditions for NRT2.1, in a further increase in NRT2.1 expression, and specifically in tissues where NRT2.1 is normally expressed. Therefore, our data indicates that PRC2 tempers the hyperactivity of NRT2.1 in a context of very strong transcription. This reveals an original function of PRC2 in the control of the expression of a highly expressed gene in Arabidopsis.

Figure 1

CLF controls H3K27me3 enrichment at the NRT2.1 locus under both repressive and active conditions for expression. ChIP analysis of H3K27me3 in WT, clf-29, and swn-3 roots of 7 days-old plants grown under (A) high nitrogen (10 mM NH₄NO₃) or (B) low nitrate (0.3 mM NO₃⁻) conditions. LEC2 and ACT2 served as positive or negative control for H3K27me3, respectively. Positions of primers used in qRT-PCR are available in Fig. S1. (C) ChIP analysis of H3K27me3 in WT and clf-29 covering the NRT2.1 locus. (D) ChIP analysis of H3K27me3 at the ProNRT2.1:GUS locus in WT and clf-29 roots of 7 days-old plants grown under low nitrate (0.3 mM NO₃⁻) condition. Quantification by qRT-PCR is shown as the percentage of H3. Error bars represent standard errors of the mean based on 3 biological replicates. Statistical significance was computed using a two-tailed Student’s t-test. Significance cutoff: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Reduction of H3K27me3 in clf-29 increases the expression of NRT2.1 in a context of very high expression. (A) Relative expression of NRT2.1 by qRT-PCR in roots of 7-days old of WT, clf-29, and swn-3 plants grown under high nitrogen (10 mM NH₄NO₃) or low nitrate (0.3 mM NO₃⁻) conditions. Quantification by qRT-PCR is shown as the percentage of ACT2 transcript levels. (B) Relative GUS expression by qRT-PCR in roots of 7-days old ProNRT2.1:GUS WT and clf-29 plants grown under low nitrate (0.3 mM NO₃⁻) condition. Quantification by qRT-PCR is shown as the percentage of ACT2 transcript levels. Error bars represent standard errors of the mean based on 3 biological replicates. Statistical significance was computed using a two-tailed Student’s t-test. Significance cutoff: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Reduction of H3K27me3 in clf-29 in the context of active transcription does not lead to an increase in H3K4me3, H3K36me3 or H3K9ac at the NRT2.1 locus. ChIP analysis of (A) H3K4me3, (B) H3K36me3, (C) H3K9ac in WT and clf-29 roots of 7 days-old plants grown under low nitrate (0.3 mM NO₃⁻) condition. Quantification by qRT-PCR is shown as the percentage of H3. ACT7 served as positive for H3K4me3 and H3K9ac, ACT2 served as positive for H3K36me3, LEC2 served as negative control for H3K4me3, H3K36me3 and H3K9ac. Error bars represent standard errors of the mean based on at least 3 biological replicates. Statistical significance was computed using a two-tailed Student’s t-test. Significance cutoff: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

CLF and H3K27me3 control NRT2.1 expression in NRT2.1-expressing root tissues. Histochemical localization of GUS expression on root transversal sections of 7 days-old Arabidopsis WT (A) and clf-29 (B) lines containing ProNRT21:GUS, and grown under low nitrate (0.3 mM NO₃⁻) condition. Bar = 25 µm.

Figure 5

Comparison of genes with very low or high expression showing H3K27me3 enrichment and regulation by CLF. Venn diagram representing a comparison of the proportion of genes marked by H3K27me3 and regulated by CLF among the very low expressed or very highly expressed genes. Very low expressed genes correspond to the 10 percent of genes the most poorly expressed in the transcriptome; very highly expressed genes correspond to the 10 percent of genes the most highly expressed in the transcriptome. Data of H3K27me3-marked genes in Arabidopsis roots are from, expression data are from, CLF-regulated genes are from.