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. 2010 Jul;30(13):3286-98.
doi: 10.1128/MCB.01520-09. Epub 2010 Apr 26.

PHF8 targets histone methylation and RNA polymerase II to activate transcription

Klaus Fortschegger  1 , Petra de Graaf, Nikolay S Outchkourov, Frederik M A van Schaik, H T Marc Timmers, Ramin Shiekhattar
Affiliations

PHF8 targets histone methylation and RNA polymerase II to activate transcription

Klaus Fortschegger et al. Mol Cell Biol. 2010 Jul.

Abstract

Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. PHF8 contains a plant homeodomain (PHD) in its N terminus and is a member of a family of JmjC domain-containing proteins. While PHDs can act as methyl lysine recognition motifs, JmjC domains can catalyze lysine demethylation. Here, we show that PHF8 is a histone demethylase that removes repressive histone H3 dimethyl lysine 9 marks. Our biochemical analysis revealed specific association of the PHF8 PHD with histone H3 trimethylated at lysine 4 (H3K4me3). Chromatin immunoprecipitation followed by high-throughput sequencing indicated that PHF8 is enriched at the transcription start sites of many active or poised genes, mirroring the presence of RNA polymerase II (RNAPII) and of H3K4me3-bearing nucleosomes. We show that PHF8 can act as a transcriptional coactivator and that its activation function largely depends on binding of the PHD to H3K4me3. Furthermore, we present evidence for direct interaction of PHF8 with the C-terminal domain of RNAPII. Importantly, a PHF8 disease mutant was defective in demethylation and in coactivation. This is the first demonstration of a chromatin-modifying enzyme that is globally recruited to promoters through its association with H3K4me3 and RNAPII.

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Figures

FIG. 1.
FIG. 1.
PHF8 is an H3K9me2 demethylase. (A) Schematic representation of PHF8 with its PHD (red), JmjC domain (cyan), and nuclear localization signals (magenta). Below, conserved amino acids crucial for metal ion chelation (red) and for binding to H3K4me3 (green) and α-ketoglutarate (cyan) are depicted. F279 (blue) is mutated to serine in some patients with XLMR. (B) Recombinant full-length PHF8 was produced in insect cells and quantified by comparison to a BSA standard after Coomassie staining. The individual lanes were spliced from one gel. (C) Bulk histones were incubated without (−) or with (+) recombinant PHF8 and analyzed by immunoblotting using the given specific histone antibodies. (D to K) Indirect-immunofluorescence analyses of 293T cells transfected for 24 h with Flag-PHF8(1-489)NLS wild type or the H247A/D249A, F279S, or D28A/W29A mutant. Flag and the indicated histone H3 methyl lysine antibodies were used. Transfected Flag-positive cells are indicated by the arrowheads. The image field dimension is 68 μm. Quantifications of mean fluorescence signals are given in Table S1 in the supplemental material.
FIG. 2.
FIG. 2.
The PHD of PHF8 binds H3K4me3. (A) Bacterial lysates containing GST-PHF8(2-66) (top) or GST-Taf3(857-924) (bottom) were incubated with streptavidin beads coated with H3 peptides carrying the indicated modifications and a C-terminal lysyl biotin. Bound proteins were analyzed by SDS-PAGE and Coomassie staining. The arrowheads indicate the positions of the GST fusion protein. (B) Wild-type (top) and D28A/W29A mutant (bottom) GST-PHF8-PHD were analyzed as described for panel A for binding to methylated and phosphorylated H3 peptides (1 to 17) as indicated above. (C) Lysates of transfected 293T cells expressing wild-type or mutant Flag-PHF8(1-352) proteins were incubated with streptavidin beads coated with the indicated H3 peptides. Bound proteins were analyzed by immunoblotting using Flag antibodies. The arrowheads show the positions of the indicated proteins, and the asterisks show background binding to H3 peptides. (D) Glutathione beads were coated with GST, GST-PHF8-PHD, GST-PHF8-PHD mutant (D28A/W29A), and GST-Taf3-PHD as a positive control and incubated with native HeLa mononucleosomes. The bound material was analyzed by immunoblotting using the given antibodies or Ponceau S staining as indicated.
FIG. 3.
FIG. 3.
PHF8 reads H3K4me3 at TSSs, and occupancy correlates with transcript levels. ChIP-seq reads of PHF8 in 293T (A), HeLa (B), and serum-stimulated Hs68 (C) cells; of H3K4me3 in serum-starved Hs68 cells (D); of negative-control IgG in HeLa cells (E); and of input in serum-stimulated Hs68 cells (F) were strand specifically correlated with annotated TSSs in a range from bp −1000 to +1000. Normalized count densities of 5′ (red), 3′ (green), and combined raw (black) reads are shown. The putative positions of phased nucleosomes are numbered and depicted as blue ovals. (G) Venn diagram depicting the overlap of PHF8-occupied genes in HeLa (blue), 293T (red), and Hs68 (green) cells. (H) Correlation of H3K4me3 and PHF8 occupancies in Hs68 cells. Genes with H3K4me3 peaks were sorted by read counts and grouped into bins of 500, and the percentage of genes in the bin that were also occupied by PHF8 was determined. Bins with decreasing H3K4me3 levels are shown from left to right; the corresponding percentages of PHF8 cooccupancy are given on the y axis. (I) Correlation of PHF8 and transcript levels in HeLa cells. Genes were sorted by normalized expression levels and grouped into bins of 500, and the percentage of genes in the bin that were occupied by PHF8 was determined. Bins with decreasing transcript levels are shown from left to right; the corresponding percentages of PHF8 occupancy are shown on the y axis.
FIG. 4.
FIG. 4.
PHF8 coactivates reporter gene expression. (A) U2OS cells were transfected in triplicate with the 5XGal4MLP-Luc reporter plasmid and pcDNA3 empty vector or pFlag-CMV2 expression plasmid for full-length PHF8 or its truncated forms, 1-352 and 1-489, in the presence or absence of Gal4-Ash2, Gal4-myc, Gal4-p53, or Gal4-E2F as indicated. The experiment shown is representative of three biological replicates, with error bars indicating standard deviations (SD) within the experiment. The graphs represent the fold activation relative to transfection with the 5XGal4MLP-Luc reporter plasmid alone. Below, Flag-PHF8 expression was examined by immunoblotting. wt, wild type. (B) U2OS cells were transfected as described for panel A with the pcDNA3 empty vector or pFlag-CMV2 expression plasmid of PHF8(1-489) or its D28A/W29A (PHDmut), H247A/D249A (JmjCmut), or F279S mutant in the presence or absence of Gal4-E2F as indicated. (C) U2OS cells were transfected as described for panel A with pcDNA3 empty vector or pFlag-CMV2 expression plasmid of full-length PHF8 or its D28A/W29A (PHDmut), H247A/D249A (JmjCmut), or F279S mutant in the presence or absence of Gal4-Ash2 or Gal4-p53 as indicated.
FIG. 5.
FIG. 5.
PHF8 interacts with the CTD of RNAPII. (A) 293T cells were either mock transfected with pcDNA3 or transfected with pFlag-CMV2 containing full-length PHF8. Cell lysates were collected and subjected to immunoprecipitation using anti-Flag beads (IP M2). Precipitated proteins were analyzed by immunoblotting with the indicated antibodies. 5%inp., 5% input. (B) 293T cells were either mock transfected with pcDNA3 or transfected with full-length, 1-489, or 1-352 PHF8. Immunoprecipitation and detection of precipitated proteins was performed as described for panel A. The RPB1 antibodies 8WG16 and H-224 were directed against the CTD and an N-terminal epitope, respectively. (C) 293T cells were either mock transfected with pcDNA3 or transfected with pFlag-CMV2 containing full-length PHF8, PHDmut (D28A/W29A), JmjCmut (H247A/D249A), or F279S mutant. Immunoprecipitation and detection of precipitated proteins were performed as described for panel A. (D) Bacterial lysates containing GST fusions with the CTD of the RPB1 subunit of RNAPII or with the PHD of murine Taf3 (as a control) were bound to glutathione-agarose beads. “Empty” indicates that no bacterial lysate was added. The beads were washed and incubated with 293T cell lysates transfected with full-length PHF8, PHF8(1-489), or PHF8(1-352). Bound proteins were analyzed by immunoblotting using Flag antibodies. The arrowheads indicate the positions of the PHF8 proteins. (E) GST pulldown experiment with GST fused to the CTD repeats or to UbcH5B (as a control) using 293T cell lysates transfected with PHF8-pFlag-CMV2 or the PHD (D28A/W29A), JmjC (H247A/D249A), and F279S mutants as indicated above the lanes. Binding reactions were processed as for panel D. (F) Bacterial lysates containing GST fusions with the CTD repeats of the RPB1 subunit of RNAPII were bound to glutathione-agarose beads. After being washed, the GST-CTD-coated beads were incubated with or without CDK7/cyclin H/Mat1 or CDK9/cyclin T as indicated above the blots and ATP as indicated below. The beads were washed and incubated with 293T cell lysates transfected with PHF8-pFlag-CMV2. Bound proteins were analyzed by immunoblotting using Flag (top) or GST (bottom) antibodies. The positions of PHF8, GST-CTD, and its phosphorylated form are indicated.
FIG. 6.
FIG. 6.
Knockdown of PHF8 in HeLa cells using shRNA affects specific transcripts. (A) PHF8-shA reduced PHF8 mRNA levels by about 90% compared with control NT-sh, as determined by quantitative RT-PCR. PHF8 transcript levels were normalized to ACTB and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) housekeeping genes using the 2−ΔΔCT method. The values correspond to means ± SD (n = 3). (B) PHF8-shA reduced PHF8 protein levels compared with NT-sh. HeLa whole-cell lysates were used for immunoblots using goat (g) and rabbit (r) PHF8 and mouse (m) β-actin antibodies as indicated. (C) Overview of microarray results. Log2 values of averaged normalized fluorescence ratios (PHF8-shA to NT-sh) were plotted according to their ranks for all 41,000 probes. Probes that were more than 2-fold changed are indicated by arrows (combined, 1,344 probes, or 3.3% of the total). (D) Box-and-whisker plot depicting PHF8 read counts at promoters in untransfected HeLa cells for upregulated and downregulated gene sets. The numbers of genes that were PHF8 occupied and their percentages of the annotated genes in the set are given below. (E) Box-and-whisker plot depicting transcript levels of untransfected HeLa cells (normalized robust multiarray average [RMA] values derived from the work of Cuddapah et al. [10]) for the gene sets that were up- and downregulated upon PHF8 knockdown. The numbers of genes for which expression data were available and their percentages of the annotated genes in the set are given below. In panels C and D, the central lines represent medians, the hinges represent quartiles, and the whiskers represent minimum and maximum values. N. S., no significant difference (according to Student's t test). Experiments were performed 72 h after shRNA vector transfection.
FIG. 7.
FIG. 7.
Model for PHF8 coactivator function. (A) Inactive chromatin bears repressive chromatin marks, like methylated H3K9 or H3K27 (nucleosomes are represented by gray boxes and H3K9me2 by red symbols). (B) Upon induction, transcription factors (TF) bind to target sites close to the transcription start site and bring in H3K4 methylation complexes (MLL; H3K4me3 is indicated by green symbols). (C) PHF8 binds to emerging H3K4me3 marks, removes nearby H3K9me2, helps basal transcription factors to recruit polymerase (RNAPII), and consequently coactivates transcription. However, other binders compete for H3K4me3-binding sites, and the activity of PHF8 itself may be regulated.
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