The key findings of this study are as follows. First, PHF8 was delineated as a novel histone demethylase of the JmjC family specific for dimethyl H3K9 and, to a lesser extent, for the monomethyl state. Second, the PHD of PHF8 was identified as an important determinant in recognition of trimethyl H3K4. Third, a critical function for the PHD in histone demethylation and transcriptional coactivation was revealed. Fourth, it was shown that a single point mutant of PHF8 associated with X-linked mental retardation in humans is defective for histone demethylation and transcriptional coactivation. Fifth, by genome-wide location analysis of PHF8, a pattern of promoter occupancy similar to that of trimethyl H3K4 was revealed. Finally, a direct association between PHF8 and the CTD of the large subunit of RNA polymerase II was demonstrated.
We showed that PHF8 is an active H3K9me2 demethylase similar to the JHDM2/KDM3 family (54
). Loenarz et al. recently additionally reported in vitro
demethylation of H3K27me2 and H3K36me2 using a construct lacking the PHD and the C-terminal domain of PHF8 (31
). However, in our study, full-length PHF8 was unable to demethylate H3K27me2 or H3K36me2 and was quite specific for demethylation of di- and monomethyl H3K9. Moreover, overexpression of PHF8(1-489) did not result in a decrease of any of the investigated histone modifications but H3K9me2 and H3K9me1 (see Table S1 in the supplemental material). Very recently, Feng et al. also reported that PHF8(1-690) has only marginal activity on H3K27- or H3K36-methylated peptides (15
). H3K9me2 is an important repressive chromatin modification whose removal signals for coactivation of gene expression. Indeed the importance of the murine H3K9me2 demethylase Jhdm2a for proper activation of target genes by removing this repressive mark at the respective promoters has already been demonstrated (34
). This demethylation may result in the release of repressive factors, or it may facilitate the recruitment of transcription factors and coactivators.
We found that the PHD of PHF8 plays a critical role in its demethylation activity in vivo
, as well as its transcriptional coactivation function. Our results indicate that the PHD of PHF8 serves as a specific reader of H3K4 tri- and dimethyl modifications and may act as an important determinant in anchoring PHF8 at transcription start sites of PHF8-occupied promoters. Moreover, PHF8 occupies a wide spectrum of H3K4-trimethylated promoters, suggesting that it might contribute to the activating effect of this histone modification on transcription. This contention is further supported by its ability to function as a coactivator for a number of transcriptional activators that we have examined, including Ash2, p53, c-myc, and E2F. These activators have been linked to recruitment of the MLL histone methyltransferase complex to mediate H3K4 methylation at promoters (32
). Therefore, we propose a model by which increased H3K4me3 levels, through the action of an MLL-related family of enzymes, would lead to further recruitment of PHF8 to the promoter of responsive genes. Once at the promoter, PHF8, through its association with RNAPII, may stabilize the preinitiation complex formation, leading to enhanced transcription (Fig. ).
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 (more ...)
We found that the disease-causing mutant of PHF8 (F279S) displays aberrant cellular localization and is devoid of demethylation activity. Furthermore, this mutant exhibited reduced activity in our coactivation assays. While we did not observe a general defect in transcription or increased H3K9me2 levels upon knockdown of PHF8, a specific transcriptional defect in neuronal cells may underlie the disease phenotype of this mutant PHF8. Indeed, XLMR patients display specific defects in the development of neurons and the midline, arguing against a global role for PHF8 in transcription. Possible reasons for this lack of global effects on methylation levels or transcription following PHF8 mutations could be that in most tissues, the loss of PHF8 is compensated for by redundant proteins like JHDM2, PHF2, or KIAA1718, the last of which is induced by PHF8 knockdown, or that the activity of endogenous PHF8 is strictly regulated by signaling pathways and switched on in a tissue-, time-, and/or locus-specific manner.
Our data suggested that the interaction with H3K4me3 was crucial for PHF8 function, since mutation of aromatic-cage residues in the PHD, as well as removal of H3K4me3 by JARID1A, significantly reduced demethylation and coactivation. It has been shown that activation of neuron-specific genes occurs by recruitment of the H3K4 methyltransferase MLL1 during in vitro
differentiation of P19 cells (52
). On the other hand, deletions in the H3K9 methyltransferase EHMT1 cause the 9q34.3 subtelomeric deletion syndrome, one feature of which is mental retardation (24
). Additionally, mutations in other PHD protein-coding genes, like ATRX
, are implicated in X-linked neurological disorders (3
). Several H3K4me3 binders, like the inhibitors of growth protein family (ING1 to -5), the Taf3 subunit of TFIID, or the NURF subunit BPTF, have been demonstrated to be involved in gene transcription and chromatin remodeling (42
). These proteins display different affinities for the H3K4me3 mark, and their recruitment to promoters could be stimulated via interaction with other transcription factors, DNA, or chromatin. Another JmjC protein, SMCX/KDM5C, is also implicated in XLMR, but in contrast to PHF8, one of its two PHDs binds H3K9me3. SMCX has been shown to act as a demethylase specific for H3K4me3 and a transcriptional repressor (22
). This argues for the importance of a balanced H3K4 and H3K9 methylation and readout in neuronal development and brain function (2
). Writers, erasers, or readers exhibit synergistic or opposing roles by interaction at or competition for genomic binding sites in order to produce the desired transcriptional outcome. Disturbances in the fine tuning of this delicate equilibrium brought about by mutations in genes like PHF8
can result in diseases such as hereditary disorders or cancer.