PHF8 Targets Histone Methylation and RNA Polymerase II To Activate Transcription

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.Posttranslational modifications of histone tails play an important role in chromatin structure and function (27). While the presence of several modifications correlates with gene activation and transcription, others have opposing repressive functions and are enriched in transcriptionally inactive or heterochromatic regions. A well-studied type of histone modification is methylation of lysine residues, which is conferred by histone methyltransferases (KMTs). There are three possible states of lysine methylation, namely, mono-, di-, and trimethylation. As expected for reversible marks involved in dynamic gene regulation, the methyl groups can be removed by histone demethylases (KDMs) of either the LSD1 or the Jumonji C-terminal-containing (JmjC) family of proteins (43, 48).The JmjC family of histone demethylases consists of approximately 30 members in humans (9, 25). Plant homeodomain (PHD) finger-containing proteins 2 and 8 (PHF2 and PHF8, respectively) and KIAA1718 constitute a subgroup containing a single N-terminal PHD followed by the catalytic JmjC domain. Several protein domains (including the PHD; the MBT, WD40, and Tudor domains; and chromodomains) have been shown to bind to peptides containing either an unmodified or a methylated lysine residue, and some PHDs specifically interact with histone H3 trimethylated at lysine 4 (H3K4me3) (42, 51, 53). H3K4me3 is considered to be an activating chromatin mark because it is enriched at active RNA polymerase II (RNAPII) transcription start sites (TSSs) (4, 17). In mammalian cells, H3K4me3 is mainly established by the SET1/KMT2 family, which includes the mixed-lineage leukemia 1 to 4 (MLL1 to -4) proteins. These chromatin modifiers can be recruited to promoters upon gene activation (16, 52), and certain MLL complexes also contain KDMs that concomitantly remove the repressive H3K27me3 mark and enhance expression (7, 21, 30).On the other hand, H3K4me3 can be erased by KDMs of the JARID1/KDM5 family that are part of polycomb-repressive complexes (PRC1/2) (29, 35). These complexes also contain KMTs that are able to methylate histone H3 at lysine 9 or 27. Methylated H3K9 and H3K27 are repressive marks, which are read by PRC1 via chromodomain-containing proteins, a mechanism that is thought to be responsible for enforcement and inheritance of silenced chromatin (5, 18). This obvious coupling of writers (KMTs), readers (like proteins containing PHDs), and erasers (KDMs) enables cross talk between different chromatin modifications (45). Furthermore, many SET and JmjC domain proteins contain additional domains or putative DNA-binding modules, increasing their specificity and affinity for modified chromatin.PHF8 is a ubiquitously expressed nuclear protein whose dysfunction is implicated in disease (28). Mutations in the PHF8 locus on the X chromosome have been linked to Siderius-Hamel syndrome, an X-linked mental retardation (XLMR) that is often accompanied by cleft lip and/or cleft palate (44). The described mutations result mostly in early truncations of the protein before or in the JmjC domain (1, 28). Moreover, a single point mutation (F279S) in the JmjC domain and a microdeletion of the whole locus were also reported to cause the disease phenotype (26, 36).In the present study, we delineate crucial biochemical properties of PHF8. We report that PHF8 is a demethylase specific for H3K9me2 and a binder of H3K4me3-marked nucleosomes. Genome localizations show that PHF8, H3K4me3, and RNAPII cooccupy thousands of promoters. Association studies suggest that PHF8 interacts directly with the C-terminal domain (CTD) of the RPB1 subunit of RNAPII. The F279S mutant of PHF8 displays cellular mislocalization, defective histone demethylation, and aberrant coactivation function.