Vernalization-Triggered Intragenic Chromatin Loop Formation by Long Noncoding RNAs

Abstract

Long noncoding RNAs (lncRNAs) affect gene regulation through structural and regulatory interactions with associated proteins. The Polycomb complex often binds to lncRNAs in eukaryotes, and an lncRNA, COLDAIR, associates with Polycomb to mediate silencing of the floral repressor FLOWERING LOCUS C (FLC) during the process of vernalization in Arabidopsis. Here, we identified an additional Polycomb-binding lncRNA, COLDWRAP. COLDWRAP is derived from the repressed promoter of FLC and is necessary for the establishment of the stable repressed state of FLC by vernalization. Both COLDAIR and COLDWRAP are required to form a repressive intragenic chromatin loop at the FLC locus by vernalization. Our results indicate that vernalization-mediated Polycomb silencing is coordinated by lncRNAs in a cooperative manner to form a stable repressive chromatin structure.

Keywords: chromatin loop; flowering; long noncoding RNAs; polycomb repressive complex 2; vernalization.

Figure 1

Identification of COLDWRAP. (A) RIP using polyclonal CLF antibody retrieves COLDWRAP RNA. (+) RT: with reverse transcription, (−) RT: without reverse transcription. (b) Expression patterns of FLC, COOLAIR (proximal and distal), COLDAIR, and COLDWRAP transcripts during the course of vernalization. (A, B) Data (mean ± SD of quantitative PCR; biological replicates n = 3). NV, non-vernalized. 10V, 10 days of vernalization. 20V, 20 days of vernalization. 40V, 40 days of vernalization. 40VT10, 40 days of vernalization followed by 10 days of normal growth temperature. (C) In vitro RNA-binding assays. C-terminal His-tagged CLF recombinant protein binds to in vitro transcribed (IVT)-biotinylated COLDWRAP RNA. (D) RNA-binding assay using IVT-biotinylated RNAs and CLF polyclonal antibody. (E) RNA-binding assay using non-biotinylated competitive RNAs. (C–E) NB: non-biotinylated, B: biotinylated. Also see Supplementary Figures S1 and S2.

Figure 2

COLDWRAP is necessary for vernalization response. (A) Flowering times of flc-2FRI (parental; top), the primary transgenic lines carrying the wild-type FLC transgene in flc-2FRI after vernalization (middle) and the primary transgenic lines carrying the mutant COLDWRAP in flc-2FRI after vernalization (bottom). X-axis; range of rosette leaf numbers. (B) Representative flowering behaviors of flc-2 FRI, flc-2 FRI transformed with the wild-type FLC transgene, and flc-2 FRI transformed with the mutant COLDWRAP after 40 days of vernalization. (C) Flowering times of a representative transgenic line carrying the wild-type FLC transgene, and two representative lines carrying the mutant COLDWRAP. (D) Changes in FLC mRNA during the course of vernalization in the wild type (FRI_Col) and two representative transgenic lines carrying the wild-type FLC transgene (WT FLC #3-4 and #4-1) and two representative transgenic lines carrying the mutant COLDWRAP (Mut FLC #1-6 and #12-2). Data (relative levels; mean ± SD of quantitative RT-PCR; biological replicates n = 3). Also see Supplementary Figures S3 and S4.

Figure 3

COLDWRAP is necessary for vernalization-mediated H3K27me3 enrichment at FLC chromatin. (A) Changes in occupancy of CLF at FLC chromatin during the course of vernalization. (B) Changes in enrichment of H3K27me3 at FLC chromatin during the course of vernalization. (C) Relative fold changes of RNA retrieved by RIP using anti-CLF antibody followed by quantitative RT-PCR in wild type (FRI_Col), a representative transgenic line carrying the wild-type FLC transgene (WT_FLC #3-5), and a representative transgenic line carrying the mutant COLDWRAP (Mut_FLC #1–6). (A–C) Data (mean ± SD of quantitative PCR; biological replicates n=3). Also see Supplementary Figures S3 and S4.

Figure 4

Restoration of vernalization response of transgenic lines carrying the mutant COLDWRAP mutant by COLDWRAP. (A) Representative flowering behaviors of transgenic lines carrying the mutant COLDWRAP and the mutant COLDWRAP, complemented with 35S::COLDWRAP without (NV) and with (40V) vernalization. (B) Flowering times of a representative transgenic line carrying the mutant COLDWRAP (#1-6) without (NV) and with (40V) vernalization (n = 19). (C) Flowering times of the primary transgenic lines carrying the mutant COLDWRAP complemented with the 35S::COLDWRAP (n = 18). (D) Levels of FLC mRNA during the course of vernalization between Mut_FLC gDNA in flc-2FRI (#1-6) and Mut_FLC gDNA in flc-2FRI+35S:COLDWRAP T1 primary transgenic lines. Data (relative levels; mean ± SD of quantitative RT-PCR; n =3). Also see Supplementary Figure S4.

Figure 5

Chromatin conformation capture (3C) assays (A) Relative positions of primer sets used for 3C assays. (B) Relative Interaction Frequency (RIF) in 3C assay between F4 and a series of F primer regions of FLC in the wild type (FRI) and the COLDWRAP RNAi line (#3-2). (C) RIF in 3C assay between F4 and a series of R primer regions of FLC in the wild type (FRI) and the COLDWRAP RNAi line (#3-2). (D) RIF in 3C assay between F4 and R17 regions of FLC in the wild type (FRI) and the COLDWRAP RNAi line (#3-2) during the course of vernalization. (E) RIF in 3C assay between F4 and a series of F primer regions of FLC in the wild type (FRI), the transgenic line carrying the wild-type FLC transgene (WT_FLC #3-4) and the transgenic line carrying the mutant COLDWRAP (Mut_FLC #1-6). (B~E) Maximum interaction frequency is set as 1 and relative fold changes are shown. −V, without vernalization. +V, with vernalization. NV, non-vernalized. 10V, 10 days of vernalization. 20V, 20 days of vernalization. 40V, 40 days of vernalization. 40VT10, 40 days of vernalization followed by 10 days of normal growth temperature. Data (mean ± SD of quantitative 3C; biological replicates n=3). Also see Supplementary Figure S5.

Figure 6

The formation of a repressive chromatin loop at the FLC locus. (A) RIF in 3C assay between F4 and a series of F primer regions of FLC in the wild type (FRI) and vin3 mutant (vin3-1FRI). (B) RIF in 3C assay between F4 and a series of F primer regions of FLC in the wild type (FRI) and vil1 mutant (vil1-1FRI). (C) RIF in 3C assay between F4 and a series of F primer regions of FLC in the wild type (WT) and a representative COLDAIR promoter deletion line (COLDAIR proΔ gFLC). (A~C) Maximum interaction frequency is set as 1 and relative fold changes are shown. −V, without vernalization. +V, with vernalization. (D) A proposed model for Polycomb-mediated FLC repression by two ncRNAs during the course of vernalization. During early cold, COLDAIR functions to recruit PRC2 at the first intron region of FLC. Subsequently, COLDWRAP helps PRC2 spread to the promoter region, which results in spreading of repressive histone mark, H3K27me3. This spreading is achieved at least in part by the formation of an intragenic chromatin loop between the COLDAIR-transcribed region and the promoter of FLC. Also see Supplementary Figure S6.