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. 2007 Jun;3(6):e86.
doi: 10.1371/journal.pgen.0030086. Epub 2007 Apr 17.

Arabidopsis TFL2/LHP1 specifically associates with genes marked by trimethylation of histone H3 lysine 27

Franziska Turck  1 , François Roudier, Sara Farrona, Marie-Laure Martin-Magniette, Elodie Guillaume, Nicolas Buisine, Séverine Gagnot, Robert A Martienssen, George Coupland, Vincent Colot
Affiliations

Arabidopsis TFL2/LHP1 specifically associates with genes marked by trimethylation of histone H3 lysine 27

Franziska Turck et al. PLoS Genet. 2007 Jun.

Abstract

TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) is the only Arabidopsis protein with overall sequence similarity to the HETEROCHROMATIN PROTEIN 1 (HP1) family of metazoans and S. pombe. TFL2/LHP1 represses transcription of numerous genes, including the flowering-time genes FLOWERING LOCUS T (FT) and FLOWERING LOCUS C (FLC), as well as the floral organ identity genes AGAMOUS (AG) and APETALA 3 (AP3). These genes are also regulated by proteins of the Polycomb repressive complex 2 (PRC2), and it has been proposed that TFL2/LHP1 represents a potential stabilizing factor of PRC2 activity. Here we show by chromatin immunoprecipitation and hybridization to an Arabidopsis Chromosome 4 tiling array (ChIP-chip) that TFL2/LHP1 associates with hundreds of small domains, almost all of which correspond to genes located within euchromatin. We investigated the chromatin marks to which TFL2/LHP1 binds and show that, in vitro, TFL2/LHP1 binds to histone H3 di- or tri-methylated at lysine 9 (H3K9me2 or H3K9me3), the marks recognized by HP1, and to histone H3 trimethylated at lysine 27 (H3K27me3), the mark deposited by PRC2. However, in vivo TFL2/LHP1 association with chromatin occurs almost exclusively and co-extensively with domains marked by H3K27me3, but not H3K9me2 or -3. Moreover, the distribution of H3K27me3 is unaffected in lhp1 mutant plants, indicating that unlike PRC2 components, TFL2/LHP1 is not involved in the deposition of this mark. Rather, our data suggest that TFL2/LHP1 recognizes specifically H3K27me3 in vivo as part of a mechanism that represses the expression of many genes targeted by PRC2.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ChIP Assays with TFL2/LHP1:HA
(A) ChIP was carried out with chromatin prepared from TFL2/LHP1:HA-expressing plants using anti-rat IgG control antibodies (1), or anti-HA antibodies developed in rabbit (2–4). Precipitated chromatin was eluted from the beads using low-pH buffer (1 and 2) or different amounts of HA-peptide (3 and 4). Precipitated DNA was tested via semi-quantitative PCR for presence of FT (At1g65480) proximal promoter, with PCR primers centered around position −388 from the transcriptional start (white bars) or WRKY33 (At2g38470) promoter with PCR primers centered around position −226 from the translational start (black bars). (B) ChIP samples prepared from TFL2/LHP1:HA (black bars) or FLC:HA-expressing plants (white bars) and immunoprecipitated with anti-HA antibodies were analyzed by quantitative PCR. PCR amplicons are indicated by triangles below the FT gene model. Data are based on three independent quantitative PCR experiments.
Figure 2
Figure 2. TFL2/LHP1 Associates with Small Euchromatic Gene Domains
(A) Left panel: Size distribution of domains associated with TFL2/LHP1. Right panel: Size distribution of annotated genes present on Chromosome 4 (black dotted line) and of genes bound by TFL2/LHP1 (blue line). Note the absence of any skewing of the latter set compared to the whole chromosome set. (B) Genome browser view of a 1.7-Mb region of the short arm of Chromosome 4 (position 0.5 Mb to 2.2 Mb) centered on the distal boundary of the heterochromatic knob and showing the distribution of TFL2/LHP1 targets. Euchromatin and heterochromatin are depicted above the browser view as orange and brown cylinders, respectively. IP/INPUT ratios (log2) reporting significant TFL2/LHP1 association are marked in dark blue. (C) Genome browser representation of ChIP-chip data over several TFL2/LHP1 target loci. (D) Average value of IP/INPUT ratios along all TFL2/LHP1 target genes and 800 bp of flanking sequences. To accommodate for different gene lengths, positions within genes are indicated as percentage of total length. Highest mean ratios are located around the 5′ end of genes.
Figure 3
Figure 3. Analysis of TFL2/LHP1 Target Genes
(A) Tandemly repeated genes are frequent targets of TFL2/LHP1. Percentage of TFL2/LHP1 targets among all Chromosome 4 genes (white bars), among tandemly repeated genes (black bars), and among genes that are part of segmental duplications (gray bars). To accommodate for possible cross-hybridization issues, the analysis was carried out considering all gene tiles (1), tiles with a single high-score BLAST hit (2), and tiles with one or two high-score BLAST hits. (B) Detailed analysis of TFL2/LHP1 association within a large array of tandemly repeated genes. Upper panel: Genome browser view of a 60-kb large region of Chromosome 4 encompassing the nine chitinase/glycosylase-18 genes (At4g19720–At4g19820), two LTR retrotransposon insertions (At4g19780 and At4g19790), and genes flanking the tandem array. Middle panel: ChIP-chip data presented as IP/INPUT ratios (log2). Lower panel: ChIP-PCR data using gene-specific primers presented as percentage of INPUT (black bars). (C) Gene ontology analysis of TFL2/LHP1 targets. Distribution of TFL2/LHP1 target genes (black bars) and all genes present on Chromosome 4 (white bars) into the different “biological process” categories as defined by TAIR. Note that the slight enrichment observed for “electron transport or energy pathways” is caused by the association of TFL2/LHP1 with the two large clusters of cytochrome P450 genes that are present on Chromosome 4. Asterisks indicate statistically significant differences (p < 0.01) between the two sets. (D) Global expression analysis of TFL2/LHP1 target genes. Median expression levels (horizontal axis) of TFL2/LHP1 target genes (white bars) and genes present of Chromosome 4 (black bars) were estimated from microarray data compiled at The Botany Beowulf Cluster (http://bbc.botany.utoronto.ca). The data were normalized using the standard MAS5.0 algorithm with a target value of 500. (E) Cluster representation [73] of the developmental series of expression data obtained from AtGenExpress (http://www.arabidopsis.org/servlets/TairObject?type=expression_set&id=1006710873) for 417 TFL2/LHP1 target genes (horizontal axis). Expression levels (vertical axis) are visualized as a heat map. (F) AtGenExpress data obtained for the nine tandemly repeated chitinase/glycosylase-18 genes and the two flanking genes. Genes are ordered as on the chromosome on the vertical axis. Expression profiles are indicated on the horizontal axis.
Figure 4
Figure 4. In Vitro Binding of TFL2/LHP1 to H3K27me3, H3K9me3, and H3K9me2
In vitro–translated and 35S-labeled TFL2/LHP1 was pulled down with either H3K9me2-, H3K9me3-, H3K27me3-, or H3-biotinylated peptides, or without peptide (beads). Purified protein was separated on SDS-PAGE and analyzed by autoradiography. Fractions of the input are shown as indicated. A representative autoradiogram of three independent experimental repetitions is shown.
Figure 5
Figure 5. TFL2/LHP1 Associates Specifically with H3K27me3
(A) Genome browser view of a 500-kb region of Chromosome 4 (positions 10.25 Mb to 10.75 Mb) showing the co-extensive association of TFL2/LHP1 with H3K27me3, and the lack of overlap between H3K9me2, H3K9me3, and H3K27me3. IP/INPUT ratios (log2) reporting significant association are marked in dark colors. (B) Venn diagram showing the extent of overlap among genes that are associated with TFL2/LHP1, H3K27me3, or H3K9me3 (see also Table S9). (C) Scanning ChIP-PCR analysis of H3K27me3 (white bars) and TFL2/LHP1 (black bars) over the AG locus (At4g18960), and genome browser view of the corresponding epigenomic maps. (D) Scanning ChIP-PCR analysis of H3K27me3 (white bars) and TFL2/LHP1 (black bars) over the FT locus (At1g65480). In C and D, amplicons are indicated as triangles and numbered consecutively along the scanned regions. Data are based on three independent quantitative PCR experiments.
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References

    1. Birve A, Sengupta AK, Beuchle D, Larsson J, Kennison JA, et al. Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants. Development. 2001;128:3371–3379. - PubMed
    1. Kinoshita T, Harada JJ, Goldberg RB, Fischer RL. Polycomb repression of flowering during early plant development. Proc Natl Acad Sci U S A. 2001;98:14156–14161. - PMC - PubMed
    1. Yoshida N, Yanai Y, Chen L, Kato Y, Hiratsuka J, et al. EMBRYONIC FLOWER2, a novel polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis . Plant Cell. 2001;13:2471–2481. - PMC - PubMed
    1. Pineiro M, Gomez-Mena C, Schaffer R, Martinez-Zapater JM, Coupland G. EARLY BOLTING IN SHORT DAYS is related to chromatin remodeling factors and regulates flowering in Arabidopsis by repressing FT . Plant Cell. 2003;15:1552–1562. - PMC - PubMed
    1. Gaudin V, Libault M, Pouteau S, Juul T, Zhao G, et al. Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis . Development. 2001;128:4847–4858. - PubMed

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