Vernalization-mediated VIN3 Induction Overcomes the LIKE-HETEROCHROMATIN PROTEIN1/POLYCOMB REPRESSION COMPLEX2-mediated epigenetic repression

Abstract

VERNALIZATION INSENSITIVE3 (VIN3) induction by vernalization is one of the earliest events in the vernalization response of Arabidopsis (Arabidopsis thaliana). However, the mechanism responsible for vernalization-mediated VIN3 induction is poorly understood. Here, we show that the constitutive repression of VIN3 in the absence of the cold is due to multiple repressive components, including a transposable element-derived sequence, LIKE-HETEROCHROMATIN PROTEIN1 and POLYCOMB REPRESSION COMPLEX2. Furthermore, the full extent of VIN3 induction by vernalization requires activating complex components, including EARLY FLOWERING7 and EARLY FLOWERING IN SHORT DAYS. In addition, we observed dynamic changes in the histone modifications present at VIN3 chromatin during the course of vernalization. Our results show that the induction of VIN3 includes dynamic changes at the level of chromatin triggered by long-term cold exposure.

Figure 1.

H3K9me2 enrichment at VIN3 chromatin. A, Schematic diagram showing the gene structure of VIN3. The solid boxes indicate coding regions, and the open boxes indicate untranslated regions (UTRs). Gray box indicates the transposable element-derived sequence (TE). Solid bars (P1 to P5) indicate regions for primer pairs used for ChIP assays. B, ChIP-quantitative PCR to quantify the relative enrichment of H3K9me2. UBQ10, Ta3, and VIL1 regions were used to compare the levels of H3K9me2 enrichments. Primer sequence information used for ChIP assay is shown in Supplemental Table S1.

Figure 2.

Involvement of LHP1 and PRC2 in the repression of VIN3 gene in a nonvernalized condition. A, Real-time reverse transcription (RT)-PCR analysis on VIN3 expression between wild types (Col-0 and Ler) and a series of mutants. B, Schematic diagram showing the gene structures of VIN3 family genes (VIN3, VIL1/VRN5, VIL2/VEL1, and VIL3/VEL2). The solid boxes indicate the coding regions, and the open boxes indicate UTRs. C, Real-time RT-PCR analysis on VIN3 family genes between the wild type (WT; FRI-Col) and lhp1-4 mutants in NV condition. D, Real-time RT-PCR analysis on VIN3 expression in the wild type (Col), clf-29, and clf-29/swn-2 mutants in NV condition. C and D, Relative fold change was determined by normalization with the levels of PP2A gene as previously reported (Czechowski et al., 2005). Primer sequence information used for real-time RT-PCR analysis is shown in Supplemental Table S1.

Figure 3.

ChIP assay using LHP1:GFP and GFP:CLF transgenic plants. A, Schematic diagram showing the gene structure of VIN3. Solid boxes indicate the coding regions, and open boxes indicate UTR. Gray box indicates the transposable element-derived sequence (TE). Solid bars (P1 to P5) indicate regions for primer pairs used for ChIP assays. B, ChIP assay using LHP1:GFP transgenic lines. C, ChIP assay using GFP:CLF transgenic line. AGAMOUS (AG) and UBQ10 were used as controls. As an input control, the preimmunoprecipitated DNA after sonication was used (B and C). D, ChIP assay using LHP1:GFP transgenic lines followed by real-time quantitative PCR. E, ChIP assay using GFP:CLF transgenic line followed by real-time quantitative PCR. For quantifications, the relative enrichments compared to input were calculated, and relative enrichments are shown compared to those of the control, AG (D and E). IP, Immunoprecipitation; NV, nonvernalized; 40V and 40VT0, 40 d of vernalization; 40VT10, 40 d of vernalization followed by 10 d of normal growth temperature; WT, wild type.

Figure 4.

Real-time RT-PCR analysis on VIN3 expression between the wild type (FRI-Col) and lhp1-4 (in FRI-Col) mutants during the course of vernalization. A, Nonvernalized (NV); 40V, 40 d of vernalization; 40VT10, 40 d of vernalization followed by 10 d of normal growth temperature. WT, Wild type. B, Shorter periods of cold exposure. 1V, 1 d of cold; 3V, 3 d of cold; 5V, 5 d of cold; 10V, 10 d of cold; 20V, 20 d of cold. Relative fold change was determined by normalization with the levels of PP2A gene as previously reported (Czechowski et al., 2005). Primer sequence information used for real-time RT-PCR analysis is shown in Supplemental Table S1.

Figure 5.

ChIP assays using anti-H3K27me3, H3K9me2, Pol II, and H3K4me3 antibody during the course of vernalization. A, Schematic diagram showing the gene structure of VIN3. Solid boxes indicate the coding regions, and open boxes indicate UTRs. Gray box indicates the transposable element-derived sequence (TE). Solid bars (P1 to P5) indicate regions for primer pairs used for ChIP assays. B, ChIP assays followed by real-time quantitative PCR using the anti-H3K27me3 antibody during the course of vernalization. For quantifications, the relative enrichments compared to input were calculated, and relative enrichments are shown compared to those of the control, FUS3. A FUS3 region was used as a positive control as previously reported (Makarevich et al., 2006). NV, Nonvernalized. C, ChIP assay followed by real-time quantitative PCR using the anti-H3K4me3 antibody during the course of vernalization. For quantifications, the relative enrichments compared to input were calculated, and relative enrichments are shown compared to those of the control, UBQ10. D, ChIP assay followed by real-time quantitative PCR using the anti-Pol II antibody (8WG16) during the course of vernalization. The Pol II antibody (8WG16) detects both phosphorylated and nonphosphorylated forms of RNA Pol II. For quantifications, the relative enrichments compared to input were calculated, and relative enrichments are shown compared to those of the control, UBQ10. E, ChIP assay followed by real-time quantitative PCR using the anti-H3K9me2 antibody during the course of vernalization. For quantifications, the relative enrichments compared to input were calculated, and relative enrichments are shown compared to those of the control, Ta3. Primer sequence information used for ChIP assay is shown in Supplemental Table S1. NV, Nonvernalized; 40V, 40 d of vernalization; 40VT10, 40 d of vernalization followed by 10 d of normal growth temperature.

Figure 6.

ELF7 and EFS are required for the full induction of VIN3 by vernalization. A, Real-time RT-PCR analysis on VIN3 expression in the wild type (WT; Col-0) and elf7-2 mutants in nonvernalized (NV) and 40 d of vernalization (40VT0) conditions. B, Real-time RT-PCR analysis on VIN3 expression in the wild type, elf7-2, and efs-3 mutants during vernalization. Relative fold change was determined by normalization with the levels of PP2A as previously reported (Czechowski et al., 2005).

Figure 7.

Schematic model on the regulation at VIN3 chromatin by vernalization. In an NV condition, LHP1 and CLF-containing PRC2 complexes are necessary for the repressed state of VIN3. H3K9me2 and H3K27me3 are enriched at VIN3 chromatin prior to vernalization. When plants are exposed to a prolonged cold (i.e. winter), unknown upstream components (X) induce VIN3, although LHP1 and PRC2 complexes are still enriched at VIN3 chromatin. The induction of VIN3 causes the decrease in enrichments of H3K9me2 mark and the increase in enrichments of H3K4me3 mark at the transcription start site of VIN3. The increased enrichments of H3K4me3 are in part due to the activity of activating complexes (i.e. PAF1, EFS, and Trithorax-like proteins). Once plants return to warm growth temperature, H3K4me3 mark decreases and H3K9me2 mark increases again at VIN3 chromatin in the absence of an unknown trigger (X). [See online article for color version of this figure.]