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Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb repressive complex 2 components.

Jiang D, Wang Y, Wang Y, He Y - PLoS ONE (2008)

Bottom Line: In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci.Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin.Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore.

ABSTRACT
Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.

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Interaction of the CLF-dependent H3K27 trimethylation with H3K4 trimethylation in its target-gene chromatin.(A) Sequential ChIP analysis of FLC and FT chromatin. The chromatin from wild-type Ws seedlings was immunoprecipitated first with anti-trimethyl H3K4 and second with anti-trimethyl H3K27. Examined regions are as illustrated in Figure 4A. “Input” is the total DNA prior to the first immunoprecipitation (diluted 800 times); Ta3, a heterochromatic locus lacking of H3K4me3 and ACT2, a constitutively expressed locus lacking of H3K27me3, served as negative controls. “(-)” is the negative control for immunoprecipitation, residual DNA from the rabbit IgG immunoprecipitation. (B) Levels of trimethyl H3K4 in the FLC, MAF4 and MAF5 chromatin in clf seedlings relative to Col determined by real-time quantitative PCR. Amounts of DNA fragments from Col and clf seedlings after ChIP were quantified and subsequently normalized to an internal control (TUB2). The fold enrichments of clf over Col are shown, and the values shown are means±SD. (C) Levels of trimethyl H3K4 in FT chromatin in clf seedlings relative to Col determined by real-time quantitative PCR. The fold enrichments of clf over Col are shown, and the values shown are means±SD.
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pone-0003404-g007: Interaction of the CLF-dependent H3K27 trimethylation with H3K4 trimethylation in its target-gene chromatin.(A) Sequential ChIP analysis of FLC and FT chromatin. The chromatin from wild-type Ws seedlings was immunoprecipitated first with anti-trimethyl H3K4 and second with anti-trimethyl H3K27. Examined regions are as illustrated in Figure 4A. “Input” is the total DNA prior to the first immunoprecipitation (diluted 800 times); Ta3, a heterochromatic locus lacking of H3K4me3 and ACT2, a constitutively expressed locus lacking of H3K27me3, served as negative controls. “(-)” is the negative control for immunoprecipitation, residual DNA from the rabbit IgG immunoprecipitation. (B) Levels of trimethyl H3K4 in the FLC, MAF4 and MAF5 chromatin in clf seedlings relative to Col determined by real-time quantitative PCR. Amounts of DNA fragments from Col and clf seedlings after ChIP were quantified and subsequently normalized to an internal control (TUB2). The fold enrichments of clf over Col are shown, and the values shown are means±SD. (C) Levels of trimethyl H3K4 in FT chromatin in clf seedlings relative to Col determined by real-time quantitative PCR. The fold enrichments of clf over Col are shown, and the values shown are means±SD.

Mentions: As noted above, PRC2 subunits repress but do not fully silence FLC and FT expression because both genes are still expressed at low levels in wild-type seedlings. It has been shown that active H3K4me3 is associated with FLC chromatin in Arabidopsis accessions which lack of FRI such as Col and Wassileskija (Ws) in which FLC expression is repressed [14], [64], and repressive H3K27me3 is also associated with FLC chromatin in these accessions in the absence of vernalization treatment [39], [64] (also see Figure 6A). However, it remains unknown whether FLC chromatin can simultaneously carry these two modifications as it is formally possible that these modifications could occur in two subpopulations of FLC chromatin and not in the same physical region of FLC. To examine whether FLC chromatin concomitantly carries both H3K4me3 and H3K27me3, we performed a sequential ChIP in which FLC chromatin from seedlings was immunoprecipitated first with anti-trimethyl H3K4 and second with anti-trimethyl H3K27. Both the region around TSS (FLC-P2) and 5′ part of Intron I of FLC (FLC-I) in part of the FLC chromatin concomitantly harbor H3K4me3 and H3K27me3 (Figure 7A). Similarly, using sequential ChIP we also found that the 5′ transcribed region (FT-E) and the middle of FT (FT-I) in part of the FT chromatin simultaneously harbor H3K4me3 and H3K27me3 (Figure 7A). In addition, we did not detect any DNA fragments from a heterochromatic locus Ta3 [65] that lacks of H3K4me3 or from a constitutive expressed house-keeping gene ACTIN 2 (ACT2) carrying abundant H3K4me3 (data not shown) but lacking of H3K27me3 (Figure 7A). Together, these data show that part of the FLC and FT chromatin simultaneously possesses the bivalent chromatin marks of active H3K4me3 and repressive H3K27me3.


Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb repressive complex 2 components.

Jiang D, Wang Y, Wang Y, He Y - PLoS ONE (2008)

Interaction of the CLF-dependent H3K27 trimethylation with H3K4 trimethylation in its target-gene chromatin.(A) Sequential ChIP analysis of FLC and FT chromatin. The chromatin from wild-type Ws seedlings was immunoprecipitated first with anti-trimethyl H3K4 and second with anti-trimethyl H3K27. Examined regions are as illustrated in Figure 4A. “Input” is the total DNA prior to the first immunoprecipitation (diluted 800 times); Ta3, a heterochromatic locus lacking of H3K4me3 and ACT2, a constitutively expressed locus lacking of H3K27me3, served as negative controls. “(-)” is the negative control for immunoprecipitation, residual DNA from the rabbit IgG immunoprecipitation. (B) Levels of trimethyl H3K4 in the FLC, MAF4 and MAF5 chromatin in clf seedlings relative to Col determined by real-time quantitative PCR. Amounts of DNA fragments from Col and clf seedlings after ChIP were quantified and subsequently normalized to an internal control (TUB2). The fold enrichments of clf over Col are shown, and the values shown are means±SD. (C) Levels of trimethyl H3K4 in FT chromatin in clf seedlings relative to Col determined by real-time quantitative PCR. The fold enrichments of clf over Col are shown, and the values shown are means±SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2561057&req=5

pone-0003404-g007: Interaction of the CLF-dependent H3K27 trimethylation with H3K4 trimethylation in its target-gene chromatin.(A) Sequential ChIP analysis of FLC and FT chromatin. The chromatin from wild-type Ws seedlings was immunoprecipitated first with anti-trimethyl H3K4 and second with anti-trimethyl H3K27. Examined regions are as illustrated in Figure 4A. “Input” is the total DNA prior to the first immunoprecipitation (diluted 800 times); Ta3, a heterochromatic locus lacking of H3K4me3 and ACT2, a constitutively expressed locus lacking of H3K27me3, served as negative controls. “(-)” is the negative control for immunoprecipitation, residual DNA from the rabbit IgG immunoprecipitation. (B) Levels of trimethyl H3K4 in the FLC, MAF4 and MAF5 chromatin in clf seedlings relative to Col determined by real-time quantitative PCR. Amounts of DNA fragments from Col and clf seedlings after ChIP were quantified and subsequently normalized to an internal control (TUB2). The fold enrichments of clf over Col are shown, and the values shown are means±SD. (C) Levels of trimethyl H3K4 in FT chromatin in clf seedlings relative to Col determined by real-time quantitative PCR. The fold enrichments of clf over Col are shown, and the values shown are means±SD.
Mentions: As noted above, PRC2 subunits repress but do not fully silence FLC and FT expression because both genes are still expressed at low levels in wild-type seedlings. It has been shown that active H3K4me3 is associated with FLC chromatin in Arabidopsis accessions which lack of FRI such as Col and Wassileskija (Ws) in which FLC expression is repressed [14], [64], and repressive H3K27me3 is also associated with FLC chromatin in these accessions in the absence of vernalization treatment [39], [64] (also see Figure 6A). However, it remains unknown whether FLC chromatin can simultaneously carry these two modifications as it is formally possible that these modifications could occur in two subpopulations of FLC chromatin and not in the same physical region of FLC. To examine whether FLC chromatin concomitantly carries both H3K4me3 and H3K27me3, we performed a sequential ChIP in which FLC chromatin from seedlings was immunoprecipitated first with anti-trimethyl H3K4 and second with anti-trimethyl H3K27. Both the region around TSS (FLC-P2) and 5′ part of Intron I of FLC (FLC-I) in part of the FLC chromatin concomitantly harbor H3K4me3 and H3K27me3 (Figure 7A). Similarly, using sequential ChIP we also found that the 5′ transcribed region (FT-E) and the middle of FT (FT-I) in part of the FT chromatin simultaneously harbor H3K4me3 and H3K27me3 (Figure 7A). In addition, we did not detect any DNA fragments from a heterochromatic locus Ta3 [65] that lacks of H3K4me3 or from a constitutive expressed house-keeping gene ACTIN 2 (ACT2) carrying abundant H3K4me3 (data not shown) but lacking of H3K27me3 (Figure 7A). Together, these data show that part of the FLC and FT chromatin simultaneously possesses the bivalent chromatin marks of active H3K4me3 and repressive H3K27me3.

Bottom Line: In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci.Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin.Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore.

ABSTRACT
Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.

Show MeSH
Related in: MedlinePlus