Research ArticleMOLECULAR BIOLOGY

PRC2 recruitment and H3K27me3 deposition at FLC require FCA binding of COOLAIR

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Science Advances  24 Apr 2019:
Vol. 5, no. 4, eaau7246
DOI: 10.1126/sciadv.aau7246
  • Fig. 1 FCA binds COOLAIR in vitro and in vivo.

    (A) Diagram of FCA showing its different domains. (B) Gene structure of FLC, COOLAIR, COLDWRAP, and COLDAIR, indicating exons (boxes), introns (lines), and T-DNA insertions (triangles). The primers used for genotyping are marked with arrows. The locations of the gene regions analyzed by RIP reverse transcription polymerase chain reaction (RT-PCR) are marked. bp, base pair. (C) Beads containing a His tag (His) or His-fused FCAN were tested for their binding of COOLAIR, COLDAIR, COLDWRAP, and FLC RNAs, followed by RT-PCR. (D) Beads containing a His tag (His) or His-fused N terminus of FPA (FPAN) were tested for their binding of COOLAIR RNAs, followed by RT-PCR. (E) Beads containing biotin-fused COOLAIR (+), complementary COOLAIR (−), COLDAIR, COLDWRAP, and FLC RNAs were tested for their binding of FCAN. The GU-rich RNA (GUUGUUUUGUUU) was used as a positive control, and FPA was used as a negative control. (F) Beads containing biotin-fused COOLAIR (+), complementary COOLAIR (−), COLDAIR, COLDWRAP, and FLC RNAs were tested for their FCA binding in vivo. The GU-rich RNA (GUUGUUUUGUUU) was used as a positive control. (G) Thirty-day-old Col-0 and coolair plants under an LD photoperiod. (Photo credit: Y.T., University of Science and Technology of China.) (H) FLC transcripts were tested by RT-PCR in the coolair mutants. Experiments were repeated three or more times, and data from a representative experiment are shown. Data are shown as means ± SE (n = 3 replicates). UBIQUITIN 10 was used as an internal control. (I) The FCA-COOLAIR complex was examined by RIP RT–quantitative PCR in wild-type and coolair mutants. RNA was immunoprecipitated with anti-FCA or anti-immunoglobulin G (IgG), followed by RT-PCR using region 1 primers. The amount of COOLAIR RNA immunoprecipitated with anti-FCA was measured relative to the amount of COOLAIR immunoprecipitated with anti-IgG. Experiments were repeated at least three times, and data from the representative experiments are presented as means ± SE (n = 3 replicates). Data that differ significantly from Col-0 (based on Student’s t test, P < 0.01) are marked with asterisks.

  • Fig. 2 FCA interacts with CLF.

    (A) A yeast two-hybrid study to examine the FCA-CLF interaction. Two dilutions (2 × 10−2 and 2 × 10−3) were placed on SD medium lacking Trp, Leu, His, and adenine. The interaction between FCA and FY was used as a positive control. (B) Beads bound to GST, GST-fused FY, or GST-fused CLF were tested for binding of soluble His-fused FCA and immunoblotted with anti-His. GST-fused FY was used as a positive control in the pull-down assay. (C) Beads bound to a His tag (His) or His-fused FCA were tested for binding of soluble GST-fused FY or GST-fused CLF. The bound proteins were immunoblotted with anti-GST. GST-fused FY was used as a positive control. The asterisk indicates a nonspecific band. (D) Protein fusions containing CLF fused to the N terminus of YFP or the N terminus of YFP were tested for their ability to bind to the C terminus of YFP alone or the C terminus of YFP fused to FCA. Yellow fluorescence and bright-field images were recorded, and the resulting images were merged. FY fused to the N terminus of YFP was used as a positive control, and SET domain protein ATX1 fused to the N terminus of YFP was used as a negative control. Twenty-five cells were examined for each transformation. The nucleus was stained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 10 μm. (E) Co-IP of FCA and CLF. HA-FCA, FLAG-CLF, FLAG-ABI5, and FLAG-ATX1 were cotransformed into Arabidopsis protoplasts. The expressed proteins were immunoprecipitated using an anti-FLAG antibody and then detected with anti-FLAG and anti-HA antibodies. ABI5 was used as a positive control, and ATX1 and IgG were used as negative controls in Co-IP. (F) Co-IP of CLF and FCA in complemented plants. The cell extracts from 10-day-old complemented seedlings with or without ribonuclease (RNase) A treatment were immunoprecipitated using an anti-HA antibody and detected with anti-HA and anti-FCA antibodies. The complemented plants (T10) were used for this assay and are indicated in fig. S5.

  • Fig. 3 FCA and COOLAIR are required for CLF enrichment.

    (A) The profiles of H3K27me3 in different regions of FLC were assessed by ChIP-PCR in wild-type and fca-9 plants. (B) The occupancy of CLF in different regions of FLC was assessed by ChIP-PCR in T10 and ProCLF:HA-CLF clf-29 fca-9 plants. T10 was clf-29 complemented with ProCLF:HA-CLF and indicated in fig. S5. (C) The occupancy of CLF in different regions of FLC was assessed by ChIP-PCR in T10 and ProCLF:HA-CLF clf-29 coolair-1 plants. (D) The profiles of H3K27me3 in different regions of FLC were assessed by ChIP-PCR in wild-type and coolair plants. For (A) to (D), experiments were repeated three or more times, and data from a representative experiment are shown. Data are shown as means ± SE (n = 3 replicates). The regions analyzed by ChIP-PCR are indicated in fig. S1C. ACTIN2 and PP2A were not regulated by FCA or COOLAIR. IgG, fca-9, or coolair-1 was used as a negative control. *P < 0.05; **P < 0.01. Significant difference using Student’s t test.

  • Fig. 4 FCA interacts with SSU72.

    (A) A yeast two-hybrid study to examine the FCA-SSU72 interaction. Two dilutions (2 × 10−2 and 2 × 10−3) were placed on SD medium lacking Trp, Leu, His, and adenine. The interaction between FCA and FY was used as a positive control. (B) Beads containing a GST tag, GST-fused FY, or GST-fused SSU72 were tested for binding of soluble His-fused FCA. The bound proteins were immunoblotted with anti-His. GST-fused FY was used as a positive control in the pull-down assay. (C) Beads containing a His tag (His) or His-fused FCA were tested for binding of soluble GST-fused SSU72 or GST-fused FY. The bound proteins were immunoblotted with anti-GST. Asterisk indicates a nonspecific band. GST-fused FY was used as a positive control in the pull-down assay. (D) SSU72 fused to the N terminus of YFP or the N terminus of YFP alone was tested for the ability of either to bind to the C terminus of YFP fused to FY or the C terminus of YFP fused to FCA. Yellow fluorescence and a bright-field image were recorded, and the resulting images were merged. FY fused to the N terminus of YFP was used as a positive control, and FY fused to the C terminus of YFP was used as a negative control. Twenty-five cells were examined for each transformation. The nucleus was stained by DAPI (blue). Scale bar, 10 μm. (E) Co-IP of FCA and SSU72. HA-FCA, FLAG-ABI5, FLAG-ATX1, and FLAG-SSU72 were cotransformed into Arabidopsis protoplasts. The expressed proteins were immunoprecipitated using an anti-FLAG antibody and then detected with anti-FLAG and anti-HA antibodies. ABI5 was used as a positive control, and ATX1 and IgG were used as negative controls. (F) The RRM1 motif is sufficient for interaction between FCA and SSU72. The growth of two dilutions (2 × 10−2 and 2 × 10−3) of yeast culture on SD medium lacking Trp, Leu, His, and adenine is shown.

  • Fig. 5 Loss of SSU72 function results in early flowering.

    (A) Gene structure of SSU72, indicating exons (boxes), introns (lines), and T-DNA insertions (triangles). (B) Full-length SSU72 transcripts were examined with RT-PCR in the ssu72 mutants. (C) The SSU72 transcripts were quantified by RT-PCR in the ssu72 mutants. UBIQUITIN 10 was used as an internal control. Experiments were repeated three or more times, and data from a representative experiment are shown. Data are shown as means ± SE (n = 3 replicates). (D) Twenty-five–day–old Col-0 and ssu72 mutant plants under an LD photoperiod. (Photo credit: Y.T., University of Science and Technology of China.) (E) Total leaf numbers of Col-0 and ssu72 mutant plants under an LD photoperiod and vernalization (LD + Ver), an SD photoperiod, and GA treatment (SD + GA). Flowering time was measured using the number of leaves (rosette, shown in dark blue; cauline, shown in light blue) at bolting as a proxy. Tests were conducted under LD, LD + Ver, SD, and SD + GA. More than 40 plants were scored for each line. Asterisks indicate a significant difference from total leaf numbers using Student’s t test (P < 0.05 or P < 0.01). (F) Forty-day-old FRI and FRI ssu72 mutant plants grown under an LD photoperiod. (Photo credit: Y.T., University of Science and Technology of China.) (G) Total leaf numbers of FRI and FRI ssu72 mutant plants grown under an LD photoperiod. Flowering time was measured using the number of leaves (rosette, shown in dark blue; cauline, shown in light blue) at bolting as a proxy. More than 40 plants were scored for each line. Asterisks indicate a significant difference from total leaf numbers using Student’s t test (P < 0.01). (H) Twenty-eight–day–old Col-0, ssu72-1, flc-3, and ssu72-1 flc-3 mutant plants under an LD photoperiod. (Photo credit: Y.T., University of Science and Technology of China.) (I) Total leaf numbers of Col-0, ssu72-1, ssu72-2, flc-3, ssu72-1 flc-3, and ssu72-2 flc-3 mutant plants grown under an LD photoperiod. Dark blue indicates rosette leaves, and light blue indicates cauline leaves; more than 40 plants were scored for each line. Asterisks indicate a significant difference from total leaf numbers using Student’s t test (P < 0.05). (J) FLC transcripts were quantified by RT-PCR in ssu72 mutants. UBIQUITIN 10 was used as an internal control. Experiments were repeated three or more times, and data from a representative experiment are shown. Data are shown as means ± SE (n = 3 replicates). Data that differ significantly from Col-0 (based on Student’s t test, P < 0.01) are marked with asterisks.

  • Fig. 6 SSU72 antagonizes FCA binding of COOLAIR.

    (A) Beads containing the His-fused FCAN were tested for binding of COOLAIR RNA with increasing dosage of SSU72, followed by RT-PCR. (B to D) Beads containing biotin-fused COOLAIR (class I) (B), COOLAIR [class II (i)] (C), and COOLAIR [class II (ii)] RNAs (D) were tested for binding of FCA in wild-type and ssu72 plants. (E) The FCA-COOLAIR complex was determined by RIP RT-PCR in wild-type and ssu72 mutants. RNA was immunoprecipitated with anti-FCA and anti-IgG, followed by RT-PCR, using region 1 primers. The amount of COOLAIR RNA immunoprecipitated with anti-FCA was measured relative to the amount of COOLAIR immunoprecipitated with anti-IgG. Experiments were repeated three or more times, and data from a representative experiment are shown. Data are shown as means ± SE (n = 3 replicates). Fca-9 was used as a negative control. Significant difference using Student’s t test, **P < 0.01. The regions analyzed by RIP RT-PCR are indicated in Fig. 1B. (F and G) The deposition of H3K27me3 (F) and H3K4me3 (G) in different regions of FLC was determined by ChIP-PCR in wild-type and ssu72 mutants. (H) The occupancy of CLF in different regions of FLC was determined by ChIP-PCR in T10 and ProCLF:HA-CLF clf-29 ssu72-1 plants. T10 was clf-29 complemented with ProCLF:HA-CLF, as indicated in fig. S5. From (F) to (H), experiments were repeated three or more times, and data from a representative experiment are shown. Data are shown as means ± SE (n = 3 replicates). ACTIN2 and PP2A were used as internal controls. IgG, fca-9, or ssu72-1 was used as a negative control. Significant difference using Student’s t test, *P < 0.05; **P < 0.01. The regions analyzed by ChIP-PCR are indicated in fig. S1C.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaau7246/DC1

    Fig. S1. Specificity of the FCA antibody.

    Fig. S2. Complementation of coolair.

    Fig. S3. Bait proteins for pull-down assay.

    Fig. S4. Co-IP of FCA and CLF.

    Fig. S5. Complementation of clf-29 with ProCLF:HA-CLF.

    Fig. S6. Phenotype of the fca-9 clf-29 double mutant.

    Fig. S7. CLF transcript level and CLF protein level in fca-9 and coolair plants.

    Fig. S8. Co-IP of FCA and SSU72.

    Fig. S9. Complementation of ssu72-1.

    Fig. S10. A model showing that binding of nascent COOLAIR RNA by FCA is required for H3K27me3 and silencing of FLC.

    Data file S1. Plasmids and primers.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Specificity of the FCA antibody.
    • Fig. S2. Complementation of coolair.
    • Fig. S3. Bait proteins for pull-down assay.
    • Fig. S4. Co-IP of FCA and CLF.
    • Fig. S5. Complementation of clf-29 with ProCLF:HA-CLF.
    • Fig. S6. Phenotype of the fca-9 clf-29 double mutant.
    • Fig. S7. CLF transcript level and CLF protein level in fca-9 and coolair plants.
    • Fig. S8. Co-IP of FCA and SSU72.
    • Fig. S9. Complementation of ssu72-1.
    • Fig. S10. A model showing that binding of nascent COOLAIR RNA by FCA is required for H3K27me3 and silencing of FLC.
    • Data file S1. Plasmids and primers.

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