The Arabidopsis Paf1c complex component CDC73 participates in the modification of FLOWERING LOCUS C chromatin

Abstract

FLOWERING LOCUS C (FLC) is a key repressor of flowering in Arabidopsis (Arabidopsis thaliana) and is regulated, both positively and negatively, by posttranslational histone modifications. For example, vernalization (the promotion of flowering by cold temperatures) epigenetically silences FLC expression through repressive histone modifications such as histone H3 lysine-9 dimethylation (H3K9me2) and H3K27me3. In contrast, an RNA polymerase II-associated complex (Paf1c) activates FLC expression through increased H3K4 and H3K36 methylation. As a result of this regulation, FLC has become a useful model for the study of chromatin structure in Arabidopsis. Here we show that At3g22590 is the Arabidopsis homolog of the yeast (Saccharomyces cerevisiae) Paf1c component CDC73 and is enriched at FLC chromatin. In contrast to other Paf1c component mutants that exhibit pleiotropic developmental phenotypes, the effects of cdc73 mutations are primarily limited to flowering time, suggesting that CDC73 may only be required for Paf1c function at a subset of target genes. In rapid-cycling strains, cdc73 mutants showed reduced FLC mRNA levels and decreased H3K4me3 at the FLC locus. Interestingly, in late-flowering autonomous-pathway mutants, which contain higher levels of FLC, cdc73 mutations only suppressed FLC in a subset of mutants. H3K4me3 was uniformly reduced in all autonomous-pathway cdc73 double mutants tested; however, those showing reduced FLC expression also showed an increase in H3K27me3. Thus, CDC73 is required for high levels of FLC expression in a subset of autonomous-pathway-mutant backgrounds and functions both to promote activating histone modifications (H3K4me3) as well as preventing repressive ones (e.g. H3K27me3).

Figure 1.

Mutations in CDC73 result in early flowering. A, Schematic of the CDC73 locus. Triangles indicate the positions of T-DNA insertions. For cdc73-1, genomic DNA rescued from the left border of the T-DNA mapped to two different locations, suggesting a complex T-DNA insertion. Arrows indicate primers used for RT-PCR in Figure 1E. B and D, Plants grown under short (B) or long days (D). C, Flowering time of cdc73 mutants under short and long days. Bars indicate the total number of leaves formed prior to flowering. Error bars indicate 1 sd. E, Semiquantitative RT-PCR analysis of CDC73 expression. RNA was extracted from 7-d-old seedlings grown under long days. Samples were taken at 3 h (ZT3) and 16 h (ZT16) after relative dawn. Positions of primers are indicated in A. UBIQUITIN10 was used as a control for loading. [See online article for color version of this figure.]

Figure 2.

Analysis of CDC73 expression. A to F, CDC73::GUS expression in seedling (A), root (B), vegetative rosettes (C and D), inflorescence (E), and flower (F; sepals and petals have been removed to expose carpels and stamens). G, Nuclear localization of CDC73::GFP in hypocotyl cells. H and I, Semiquantitative RT-PCR analysis of CDC73 mRNA levels during vernalization (H) and in various late-flowering backgrounds (I). For vernalized samples, V and T indicate the days of cold exposure and subsequent warm temperatures, respectively. FLC was included as a control for vernalization and UBIQUITIN10 was used as a control for loading. J to O, FLC::GUS expression in FRI (J–L) or FRI cdc73 (M–O).

Figure 3.

Effect of cdc73 and Paf1c component mutants on growth and development. A, Loss of cdc73 has less severe effects on plant size than other Paf1c-associated mutants (vip3, vip5, elf7, and elf8). B, Flowering time of cdc73 and Paf1c mutants under long days. Bars indicate the total number of leaves formed prior to flowering. Error bars indicate 1 sd. C, Fully expanded fifth leaves are shown for the indicated genotypes. D, Floral abnormalities present in other Paf1c mutants are absent in cdc73. [See online article for color version of this figure.]

Figure 4.

Effect of cdc73 on the expression of flowering-time genes. A, Quantitative RT-PCR analysis of flowering-time genes. RNA was extracted from 7-d-old seedlings grown under long days. Samples were taken at 3 h (black bars) and 16 h (white bars) after relative dawn. Error bars indicate 1 sd. B, Effect of cdc73 in a FRI-containing background. Plants were grown under long days. C, Flowering time of long-day-grown plants with wild-type CDC73 (black bars) or cdc73-1 (white bars). Error bars indicate 1 sd. D, Quantitative RT-PCR analysis of FLC expression in genotypes containing wild-type CDC73 (black bars) or cdc73-1 (white bars). RNA was extracted from 7-d-old seedlings grown under long days. Error bars indicate 1 sd. Asterisks indicate a statistically significant difference in FLC expression for a given background ± CDC73 (P < 0.05). [See online article for color version of this figure.]

Figure 5.

ChIP analysis of the FLC locus. A, Schematic drawing of the FLC locus. Exons are depicted as thick black lines. The numbered segments indicate the positions of PCR products generated in ChIP analysis. B, ChIP analysis of CDC73::GFP at the FLC locus. C and E, Analysis of H3K4me3 and H3K27me3 using ChIP followed by quantitative RT-PCR. D and F, Percent change in H3K4me3 and H3K27me3 due to loss of cdc73. Asterisks indicate statistically significant differences (P < 0.05).