The characterization of Mediator 12 and 13 as conditional positive gene regulators in Arabidopsis

Abstract

Mediator 12 (MED12) and MED13 are components of the Mediator multi-protein complex, that facilitates the initial steps of gene transcription. Here, in an Arabidopsis mutant screen, we identify MED12 and MED13 as positive gene regulators, both of which contribute broadly to morc1 de-repressed gene expression. Both MED12 and MED13 are preferentially required for the expression of genes depleted in active chromatin marks, a chromatin signature shared with morc1 re-activated loci. We further discover that MED12 tends to interact with genes that are responsive to environmental stimuli, including light and radiation. We demonstrate that light-induced transient gene expression depends on MED12, and is accompanied by a concomitant increase in MED12 enrichment during induction. In contrast, the steady-state expression level of these genes show little dependence on MED12, suggesting that MED12 is primarily required to aid the expression of genes in transition from less-active to more active states.

Fig. 1. Identification of med12 and med13 mutations as morc1 suppressors.

a Schematic diagram of SDC gene structure and its transcription status in different genetic backgrounds. Open rectangle represents the SDC 5’-untranslated region (5’-UTR). Filled rectangle represents the SDC coding sequence (CDS). b Schematic diagram of the Arabidopsis MED12 and MED13 gene structures with the position of each EMS mutation illustrated. Data display conventions as in a. c GFP fluorescence of plants in different genetic backgrounds. GFP-wt and GFP-morc1 are plants carrying the SDC:GFP transgene in wild-type and morc1 mutant backgrounds, respectively. S243, S213, and S486 are med12 and med13 EMS alleles in the GFP-morc1 background. GFP-morc1/med12 is the med12 CRISPR-CAS9 allele re-created in GFP-morc1 background. GFP-morc1/med13 is the med13 T-DNA allele re-created in GFP-morc1 background. S243-F1 is the F1 resulting from the cross between the CRISPR-CAS9 re-created GFP-morc1/med12 and S243; S213-F1 and S486-F1 are the F1s resulting from the crosses between the re-created GFP-morc1/med13 and S213, S486, respectively. Images show 8-day-old seedlings. The auto-fluorescence (gray) from chloroplasts are shown at the top-right corner of each image. d Real-time PCR quantification of SDC:GFP expression compared between different genetic backgrounds. Each vertical bar represents one biological replicate. A minimum of five biological replicates were analyzed for each mutant background. Red asterisk represents statistically significant difference between the groups under comparison (Student’s t test, two-sided). e GFP fluorescence of plants in different genetic backgrounds. GFP-wt and GFP-ddc are plants carrying the SDC:GFP transgene in wild-type and ddc mutant backgrounds, respectively. GFP-ddc/med12 mutant is the med12 CRISPR-CAS9 allele re-created in GFP-ddc background. GFP-ddc/med13 mutant is the med13 CRISPR-CAS9 allele re-created in GFP-ddc background. f Real-time PCR quantification of SDC:GFP expression compared between different genetic backgrounds. Data display conventions as in d. Source data underlying d, f are provided as a Source data file.

Fig. 2. Characterization of med12/13 differentially regulated genes.

a Venn diagram showing the overlap between genes that are downregulated in morc1/med12 and/or morc1/med13 mutants and those that are upregulated in morc1 background compared to wild-type (Col-0). Size is proportional to the number of genes defined for each group. p value indicates a statistical significance of the overlap (hypergeometric test, one sided). b Venn diagram showing the overlap between genes that are upregulated (left) and downregulated (right) in med12 and med13 compared to wild-type plants (fold change >1.5, FDR < 0.05). Size is proportional to the number of genes defined for each group. p value indicates a statistical significance of the overlap (hypergeometric test, one-sided). c Venn diagram showing the overlaps of med12 upregulated (left) and downregulated (right) genes between wild-type and morc1 background (fold change >1.5, FDR < 0.05). Data display conventions as in b. p value indicates a statistical significance of the overlap (hypergeometric test, one sided). d Bar chart showing the number of genes (gray) and TEs (black) that are differentially regulated by MED12 in wild-type (upper) and morc1 (lower) backgrounds. Source data are provided as a Source data file.

Fig. 3. Characterization of epigenetic features of med12 differentially regulated genes.

a Average distribution of DNA methylations over med12 downregulated genes in three different sequence contexts (left to right, CG, CHG, and CHH). Control represents a group of randomly selected MED12 non-DEGs of similar expression levels. −2 kb and +2 kb represent 2 kb upstream of transcription start site (TSS) and 2 kb downstream of transcription termination site (TTS), respectively. b Same as in a, except for med12 upregulated genes. c Average distribution of histone modifications over med12 DEGs, distinguishing genes downregulated (green) and upregulated (purple) in med12 from corresponding controls of similar expression levels (orange and pink, respectively). Top row from left to right, H3, H3K4me3, H3K9me2; Bottom row from left to right, H3K27me3, H3K36me3, H3PanAc. Shaded area represents the standard error (SEM) centered on mean value (dark solid lines). n = 3 biologically independent samples.

Fig. 4. Characterization of the epigenetic features at SDC and other morc1 upregulated loci.

a Screen shot showing the distribution of (from top to bottom) H3, H3K4me3, H3K27me3, H3K36me3, and H3PanAc over the endogenous SDC locus in Col-0 (top track), morc1 (middle track), and ddc (bottom track). Shaded area indicates the region downstream of SDC TSS. b Average distribution of histone modifications over morc1 upregulated genes (green) and corresponding controls of similar expression levels (purple). Shaded area represents the standard error (SEM) centered on mean value (dark solid lines). n = 3 biologically independent samples.

Fig. 5. Characterization of MED12-enriched loci.

a Genome-wide distribution of MED12 on five chromosomes (chr). Each of the five Arabidopsis chromosomes was divided into 500-kb window. The relative enrichments of MED12 within each window was plotted. Y-axis represents the log₂ value of MED12-ChIPseq reads in MED12 complementing plants relative to those in wild-type plants. Orange rectangles indicate the locations of pericentromeric regions. b The distribution of MED12 ChIP-seq peaks overlapping with different genomic features (right). The background distribution of different genomic features is shown on the left. c Average distribution of MED12 over genes of different expression values. All expressed genes were divided evenly into five groups based on their expression levels in wild-type plants. Orange to yellow to green to blue to purple is low-to-high expression tiers. Y-axis represents MED12-FLAG ChIP-seq read counts normalized to sequencing depth. −1 kb and 1 kb represent 1 kb upstream of transcription start site (TSS) and 1 kb downstream of transcription termination site (TTS), respectively.

Fig. 6. Features of MED12-interacting genes.

a Heatmap ordered based on k-means clustering of H3K4me3, H3K27me3, and MED12 ChIP-seq signals at all Arabidopsis genes (cluster n = 5). The number of genes from each cluster is labeled to the right. Color codes are shared with pie chart in b–d. b The percentage of genes derived from each of the five clusters defined in a. The percentage was calculated by dividing the number of genes from each cluster by the total number of Arabidopsis genes. c The percentage of med12 downregulated genes derived from each of the five clusters defined in a. The percentage was calculated by dividing the number of med12 downregulated genes from each cluster by the total number of med12 downregulated genes. p value indicates a statistical significance in the enrichment of med12 downregulated genes in Cluster 5 (hypergeometric test). d The percentage of MED12-interacting genes derived from each of the five clusters defined in a. Similarly calculated as in c, except for MED12-interacting genes. e Statistical significance of GO term analysis of MED12-interacting genes derived from Clusters 2 and 3 as defined in a (Fisher’s exact test). Source data underlying b–e are provided as a Source data file.

Fig. 7. Role of MED12 in mediating light-induced transient gene expression.

a Schematic illustration of the light and control treatments. b Bar plot of light-responsive gene fold induction under light treatments. Left, wild-type plants (Col-0); Right, med12 mutant plants. Asterisks indicate statistical significance in gene expressional changes (Benjamini–Hochberg adjusted p value, two sided). Data are presented as mean values +/− SE. n = 3 biologically independent samples. c Scatter plot showing the fold induction of light-inducible genes in Col-0 and med12 mutant plants upon light treatments. Each dot represents one light-inducible gene with its x-axis position showing fold induction in Col-0 and y-axis position showing fold induction in med12 mutant. The dotted line represents the 45° reference line. d The average distribution of MED12 over light-inducible genes and control genes in the light-treated (purple and red) and non-treated (green and orange) plants. Shaded area represents the standard error (SEM) centered on mean value (dark solid lines). n = 3 biologically independent samples. e Screen shots showing gene expression and MED12 enrichments at HY5 (left) and CGA1 (right) locus under the light-treated and non-treated conditions. Top four tracks, gene expression in Col-0; middle four tracks, gene expression in med12 mutants; bottom four tracks, MED12 enrichments in MED12 complementing plants. Source data underlying b, c are provided as a Source data file.