XLMR affects 1–4 out of 2,000 males, causing intellectual disability (Intelligence Quotient (IQ) <70)1
. Approximately 1/3 of them are syndromic patients in that they also have additional clinical symptoms including defects in craniofacial, muscular and hematopoietic development 1
. About 25% of the XLMR genes identified by human genetic studies are predicted to encode nuclear proteins including transcriptional regulators and chromatin remodeling proteins 2,4
. However, molecular mechanisms by which these proteins regulate biological processes relevant to the disease remain incompletely understood.
PHF8 belongs to a subfamily of JmjC domain-containing proteins that also includes KIAA1718 and PHF2. All three proteins share a similar domain structure with an N-terminus PHD finger adjacent to the catalytic JmjC domain (), predicting that they are candidate histone demethylases. We used nucleosomes as substrates and identified a novel H4K20me1-specific demethylase activity for PHF8 and KIAA1718, with additional activities towards H3K9me1/2 and H3K27me2, but not H4K20me2/3 or trimethylated H3K9, 27 and 36 (). However, when histone octomers were used as substrates, we were unable to detect the H4K20me1 demethylase activity (Supplemental Figure 2 and Supplemental Table 1
), consistent with a number of recent reports that identified PHF8 as an H3K9me1/2 and H3K27me2 specific demethylase5–9
. This discrepancy could be due to the use of nucleosomes versus histone octmers as substrates, and, as such, is not unprecedented. For instance, a number of histone methyltransferases have been shown to only methylate nucleosomal substrates or to methylate a specific lysine residue only in the nucleosomal context10,11
. Importantly, the above in vitro
demethylation reactions were also analyzed by mass spectrometry, which confirmed the Western blot results ( and Supplemental Figure 1
). Furthermore, a point mutation in the catalytic domain (phenylalanine to serine, F279S) abolished PHF8 demethylase activities (), suggesting that the reduction of the H3K9me1/2 and H4K20me1 signals by the wild type PHF8 was due to its demethylase activity. The F279S mutation has been found in several male XLMR patients from a Finnish family12
, indicating the importance of the demethylase activity in PHF8-associated mental retardation.
PHF8 and KIA1718 demethylate multiple lysines of histone 3 and 4 in vitro
We next investigated the impact of PHF8 on the global H4K20 and H3K9 methylation states in the cells. PHF8 over-expression in U2OS but not HeLa cells, which express a high level of PHF8 (Supplemental Figure 3
), resulted in a significant reduction of H3K9me2 (but not H3K9me1), and only a slight reduction of the global level of H4K20me1 (~10%) (Supplemental Figure 4, and Table 2
). Although over-expression or RNAi of PHF8 in HeLa cells did not affect the global levels of H4K20me1 and H3K9me1 assayed by Western blotting (Supplemental Figure 5a
), we did observe an increase in the overall H4K20me1 level in the late G2/M and early G1 stages of the cell cycle when we inhibited PHF8 expression by RNAi (Supplemental Figure 5b
). This is consistent with the previous reports that H4K20me1 is cell cycle-regulated13,14
and provides further support that PHF8 regulates H4K20me1 in vivo
. Interestingly, inhibition of PHF8 also reduced cell proliferation (Supplemental Figure 6
), suggesting a possible role for PHF8 in regulation of cell growth via histone demethylation.
To identify the genomic locations of PHF8, we performed ChIP-seq (chromatin immunoprecipitation followed by DNA deep sequencing). We obtained 17,293 peaks in HeLa cells after IgG normalization. Of those, 8,077 peaks representing 7,469 refseq genes are mapped to within 2.5 kb of TSS (Supplemental Table 3
) as well as 9,824 potential PHF8 binding events in the gene bodies (23.5%) and intergenic regions (33.3%) (). This pattern of PHF8 genomic distribution was corroborated by ChIP-chip analysis. Importantly, majority of the PHF8 binding signals identified by ChIP-chip were erased by PHF8 RNAi, supporting the specificity of the PHF8 binding events detected by the PHF8 antibody (Supplemental Figures 3, 7
and Table 4
). Interestingly, the pattern of PHF8 binding around TSS () resembles that of H3K4me315,16
, consistent with the fact that the PHD domain of PHF8 binds H3K4me35,17
. Point mutations (Y7A, W29A, Y7A/Y14A, W29A/Y14A) that abolished binding of PHF8 to H3K4me3 in vitro
disrupted its localization in vivo
(Supplemental Figure 8
), suggesting that PHF8 TSS localization may be dependent on its PHD domain recognizing H3K4me3. Importantly, analysis of the ChIP-seq and gene expression microarray data (Supplemental Table 6
) shows that PHF8 binding events are positively correlated with gene expression (), suggesting that PHF8 binds H3K4me3 and functions to promote gene transcription.
PHF8 differently regulates H4K20me1, H3K9me1 and H3K9me2
We next used ChIP-seq to investigate histone methylation regulation by PHF8 at its direct loci using HeLa cells stably expressing either control or PHF8 shRNA. We found that PHF8 depletion caused a modest albeit significant increase in H4K20me1 and H3K9me1 at the PHF8 TSS target loci (), compared with the regions not bound by PHF8 (p-value < 0.01), suggesting that PHF8 regulates transcription by demethylating H4K20me1 and H3K9me1. Unlike H4K20me1 and H3K9me1, H3K9me2 level remained unchanged (). Interestingly, for the 9824 non-TSS PHF8 sites, we found that reduction of PHF8 is instead correlated with an increase in H3K9me2 (p-value < 0.01) (), but not H3K9me1 or H4K20me1 (), indicating that PHF8 may regulate non-TSS regions via demethylation of H3K9me2. Consistently, a recent study reported PHF8 regulation of rDNA transcription via demethylation of H3K9me25
. The finding that PHF8 may exhibit differential substrate specificities at different genomic locations (TSS versus non-TSS) implies that additional factors are involved in the regulation of PHF8 demethylase specificity in vivo
To confirm the ChIP-seq histone modification data, we initially focused on three TSS-bound PHF8 target genes, FBXO7, TFAP2C and NCOA3, and first confirmed PHF8 binding near TSS (amplicon 2) by conventional ChIP assays (). Inhibition of PHF8 resulted in up-regulation of H4K20me1 and H3K9me1 at amplicon 2 of the target genes (). In some instances, we also observed an increase in H4K20me1 and H3K9me1 in amplicons #1 and 3 for reasons that are currently unclear. In contrast, we found no appreciable changes in H3K9me2, H3K27me1/2 or H3K36me1/2 (Supplemental Figure 9
), many of which are also substrates of PHF8 in vitro
(refer to and Supplemental Figures 1 and 2
). We also did not observe changes in histone H3, suggesting that the increase in H4K20me1/H3K9me1 is not due to an increase in nucleosome density (Supplemental Figure 9
). We further investigated an additional seven PHF8 target genes and found H4K20me1 and H3K9me1 increase for some if not all of these targets (Supplemental Figure 11
). We next investigated the impact of PHF8 inhibition on the expression of these three target genes. As shown in , Consistent with the bioinformatics analysis () implicating PHF8 as a positive regulator of transcription, their expression was down-regulated in PHF8 RNAi cells, restored by wild-type PHF8 but not by the patient F279 mutation or the H3K4me3-binding defective PHD finger mutant (Y7A, W29A). The expression of the addition seven genes was similarly reduced upon PHF8 inhibition (Supplemental Figure 11
). Taken together, these data indicate that PHF8 demethylates H4K20me1 and H3K9me1 both in vitro
and in vivo
, and that both H3K4me3-binding and catalytic functions of PHF8 are important for its ability to positively regulate gene expression.
Depletion of PHF8 increases H3K9me1, H4K20me1, L3MBTL1 at TSS-bound PHF8 target genes
Although H4K20me1 has been found downstream of TSS on active genes16,18
, it has also been associated with repression and recruitment of L3MBTL1, which preferentially binds mono- and dimethylated lysines including H4K20me1 and H3K9me119
and induces chromatin compaction to further negatively regulate gene expression20
. Upon PHF8 knockdown, we also observed a corresponding increase of L3MBTL1 at amplicon 2 but not amplicons 1 and 3 ( and Supplemental Figure 10
). This is interesting in light of the fact that the H4K20me1 increase in PHF8 RNAi cells extends beyond amplicon 2, suggesting that factors, in addition to H4K20me1, are involved in determining L3MBTL1 recruitment. Additionally, we observed a reduction of H3K4me3, a mark that is associated with active genes, consistent with these genes being in a transcriptionally repressed state ( and Supplemental Figure 10
). Taken together, these findings support the model that PHF8, when bound to TSS of the target genes, positively regulates transcription by demethylating H3K9me1 and H4K20me1.
To address the biological function of PHF8, we turned to zebrafish where PHF8 is evolutionarily conserved (Supplemental Figure 11
). zPHF8 expression was first detected at 14 hours post-fertilization (hpf) in the head and tail regions (Supplemental Figure 12
), but starting at 1 day post-fertilization (dpf), zPHF8 was found mostly in the head region. zPHF8 expression was also detectable in the jaw of the embryo at 3 dpf. Injection of a zPHF8 morpholino caused delay in brain development at 24 hpf (, compare panel b with a), and apoptosis in the developing brain and the neural tube at 30 hpf (, compare panel b with a). Significantly, this apoptosis can be rescued by reintroduction of wild type but not catalytically inactive zPHF8 (, compare panels c, d with a, b). However, whether PHF8 regulates cell death by directly modulating the apoptotic machinery or indirectly through regulation of cell proliferation remains to be investigated. Furthermore, it remains to be seen whether PHF8 also plays a role in post-mitotic mature neurons. In addition to apoptosis in the developing brain, we also detected significant differences in the craniofacial development between control and zPHF8 morpholino injected embryos at 3, 4 and 7 dpf, with the most pronounced defect being the absence of a lower jaw (, compare panel a with b, Supplemental Figure 13
). To better understand the craniofacial developmental abnormality, we used Alcian blue to visualize the extent of cartilage development in these embryos. We found that the 1st through 5th pharyngeal arches were all either developmentally affected or were absent upon zPHF8 inhibition (, compare panel d,f with c,e). Specifically, the 1st pharyngeal arch had not migrated to the full extent of the lower jaw and the 2nd pharyngeal was inverted. The 3rd through 5th pharyngeal arches were also significantly under-developed. Importantly, wild type but not catalytically inactive zPHF8 showed significant rescue of the craniofacial defects induced by the zPHF8 morpholino (, Supplemental Table 5
). Collectively, these findings identified a critical role of zPHF8 in zebrafish brain and craniofacial development, in a manner that is dependent on its demethylase activity.
zPHF8 regulates brain and craniofacial development in part through regulation of MSXB
The msh/Msx gene family encodes homeodomain transcription factors that act downstream of the TGFβ, BMP and WNT signaling pathways and play important roles in many developmental processes including craniofacial and neural development3
. Zebrafish has five homologs, MSXA through MSXE, that constitute the zebrafish orthologs of the tetrapod MSX1, MSX2, and MSX3 genes21
, of which, MSX1 was identified as a PHF8 direct target (Supplemental Figures 14a and b
). Interestingly, a MSXB morpholino produced craniofacial deformities similar to those seen in a zPHF8 morphant22
. We therefore investigated whether MSXB was regulated by zPHF8 and whether it functions to mediate the role of zPHF8 in zebrafish craniofacial development. We found that the msx
B transcript level was reduced to 40% of wild-type level in embryos injected with a zPHF8 morpholino (Supplemental Figure 14c
). The msx
B transcript was largely restored to wild type level in the animals rescued with zPHF8 mRNA encoding wild type but not with a catalytically inactive form of the zPHF8 protein (Supplemental Figure 14c
). Importantly, injection of msxB mRNA also significantly corrected the craniofacial defects induced by the zPHF8 morpholino (, compare panels k, l with a, c and g, h). Although the rescue was not complete, the lower jaw developed better and the 2nd pharyngeal arch was no longer inverted (, compare panels l with d and h, and data not shown). Similarly, the apoptosis seen in the developing brain and neural tube was also suppressed by forced expression of MSXB (, panel e). These findings raise the possibility that zPHF8 may control neuronal cell survival and craniofacial development at least in part by regulating MSXB expression. However, it is likely that MSXB is not the only PHF8 target gene that mediates PHF8 biology. Consistent with this idea, bioinformatics analysis of the expression microarray data from HeLa cells identified additional potential PHF8 target genes in a number of pathways including RA (Retinoic Acid) and Notch signaling pathways (Supplemental Table 6
), both of which have been previously shown to play a role in neural and craniofacial development23,24
and therefore may also be involved in PHF8 biological functions. In addition, the PHF8 direct target genes (FBXO7, NCOA3 and TFAP2C) discussed earlier () also function in neural development25–27
. Lastly, ChIP-seq/ChIP-chip identified many XLMR genes as potential PHF8 direct targets (Supplemental Figure 15
), consistent with a recent report7
. This finding raises the interesting possibility of a connection between PHF8 and other XLMR gene products in regulating cognitive functions. However, given that the ChIP-seq data were obtained from heterologous cells, whether PHF8 regulation of other XLMR genes is physiologically and pathologically relevant remains to be investigated.
In conclusion, we have identified the XLMR gene PHF8 to encode a novel H4K20me1 and H3K9me1/2 demethylase, and provided insights into molecular mechanisms by which PHF8 regulates histone methylation and gene transcription. The identification of a role for PHF8 in zebrafish brain apoptosis and craniofacial development suggests a potential biological basis for the involvement of PHF8 mutations in mental retardation and craniofacial deformities. Importantly, our finding of PHF8 as a histone demethylase whose mutations are correlated with XLMR supports the emerging theme of a critical link between histone methylation dynamics in X-linked mental retardation.