Article Figures & Data
Figures
- Figure 1.
Structure and expression of Arabidopsis PHP. A, Region of chromosome III containing the PHP locus. Exons are shown as black (translated region) or gray (untranslated region) boxes. The positions of the insertions corresponding to the php-1 and php-2 alleles are indicated. Annealing positions of oligonucleotide primers are shown. B, RNA gel-blot analysis of PHP RNAs in wild-type (WT), php-1, and php-2 seedlings. The entire transcribed region of PHP (F1/R2) was used as probe.
- Figure 2.
Characterization of PHP and PHP-associated proteins. A, Nuclear extracts prepared from nontransgenic wild-type (NT) and php-1:PHP-FLAG plants were subjected to immunoprecipitation using anti-FLAG antibody. Immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotting with antisera generated against the indicated proteins. B, Total cellular proteins were prepared from wild type (Col-0 or Col:FRI as indicated), php mutants, or mutants for the plant Paf1C components ELF7, VIP3, VIP4, VIP5, and VIP6 as indicated, and were analyzed by SDS-PAGE and immunoblotting using antisera raised against the indicated proteins. An unrelated, immunoreactive protein species detected by anti-VIP5 antisera is indicated by an asterisk (*). C, Nuclear extracts were subjected to gel filtration chromatography using a Superose 6 column with an effective fractionation range of 5 to 5,000 kD, and fractions were analyzed by SDS-PAGE and immunoblotting using antisera raised against the indicated proteins. Elution positions of molecular mass standards (kD) and chromatographic fraction numbers are indicated at the top and bottom, respectively. In the sections at right, the peak PHP fraction (no. 45) was analyzed together with total cellular extracts from wild type (Col-0) and php, vip3, or vip6 mutants to demonstrate specificity of the antibodies. [See online article for color version of this figure.]
- Figure 3.
Flowering phenotype of php plants. A, Wild-type ecotype Col-0 (left), php-1 (center), and php-1 expressing a PHP-FLAG transgene (right) after 4 weeks of growth. B, Summary of total leaves formed on the main shoot for wild-type ecotype Col-0, Col-0 carrying an introgressed, dominant FRI allele (Col:FRI), and php mutants. Plants were grown under flowering-inductive (16 h light/8 h dark; I) or noninductive (8 h light, 16 h dark; NI) photoperiods. To evaluate the vernalization response, germinating seeds were maintained in the cold for 12, 21, 35, or 70 d. Values represent the mean and sd for at least 12 plants. C, Analysis of flowering in double mutants. Plants were grown under inductive photoperiods. [See online article for color version of this figure.]
- Figure 4.
Gene expression profiling of php mutants. A, Scatter plot of wild type (WT; Col:FRI) versus php-2:FRI datasets composed with log-transformed signal intensities. Genes with P value <0.01 are shown in red, and 2-fold increase or decrease is indicated with a dotted line. Signal positions for FLC and PHP are circled. B, Venn diagram indicating the number and overlap of up-regulated or down-regulated genes in php-2:FRI and vip3:FRI mutants. C, Genes that were determined to be misexpressed in php-2:FRI mutants relative to wild-type plants, based on a criteria of >2-fold change and P < 0.01, were analyzed for misregulation in vip3:FRI mutants, as previously determined (left section; Oh et al., 2008). Genes misexpressed in the vip3:FRI mutant were analyzed for misregulation in the php-2:FRI mutant (right section). In each section, the P-value statistic for expression change is indicated as shown in the key. The x and y axes indicate expression change in each mutant relative to wild type. Mutants and wild type were in the Col:FRI background. Microarray data were derived from two independent biological replicates.
- Figure 5.
Histone methylation profiles within PHP-targeted genes. Mean genic positional signals for H3K4me3, H3K36me2, and H3K27me3 within the subsets of genes up-regulated (solid red) or down-regulated (solid green) in the php-2 mutant were calculated from the data set reported previously (Oh et al., 2008) and are shown relative to the genomic average (black). Signals are depicted across the promoter, transcribed, and 3′ regions as indicated on the x axes. The 95th percentile confidence intervals (dashed black lines) were determined for the mean positional signals within 1,000 randomly resampled gene sets (background gray lines). Mean positional signals for those genes up-regulated or down-regulated in the vip3 mutant are indicated as dashed red or dashed green lines, respectively, as previously determined (Oh et al., 2008).
- Figure 6.
ChIP analysis of histone H3 methylations within FLC chromatin. ChIP was carried out using antibodies recognizing H3K4me3, H3K36me2, or H3K27me3 within a promoter segment (I), intronic region near the 5′ end (II), or 3′ region (III) of the FLC gene from wild-type (WT) Col-0 or php-2 plants. Band intensities from gel images were quantified and normalized based on those for ACTIN7 (lower band in each gel image marked with asterisk). ChIP analysis was performed twice using biologically independent samples and yielded essentially identical results. A depiction of the FLC gene, indicating the position of amplified segments and with exons depicted as black boxes, is shown above. No ab, No antibody control. Error bars show the sd from the mean.
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Supplemental Data
Supplemental Table and Figures
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- Supplemental Data - Supplemental Table and Supplemental Figures 1 and 2
