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. Author manuscript; available in PMC: 2015 Dec 9.
Published in final edited form as: Hum Mol Genet. 2009 Oct 19;19(2):217–222. doi: 10.1093/hmg/ddp480

PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an Nε-dimethyl lysine demethylase

Christoph Loenarz 1,5, Wei Ge 1,5, Mathew L Coleman 2,6, Nathan R Rose 1,6, Christopher D O Cooper 3, Robert J Klose 4, Peter J Ratcliffe 2, Christopher J Schofield 1,*
PMCID: PMC4673897  EMSID: EMS66201  PMID: 19843542

Abstract

Mutations of human PHF8 cluster within its JmjC encoding exons and are linked to mental retardation (MR) and a cleft lip/palate phenotype. Sequence comparisons, employing structural insights, suggest that PHF8 contains the double stranded β-helix fold and ferrous iron binding residues that are present in 2-oxoglutarate dependent oxygenases. We report that recombinant PHF8 is an Fe(II) and 2-oxoglutarate dependent Nε-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for Nε-di- and mono-methylated lysine residues and does not accept trimethyl substrates. Clinically observed mutations to the PHF8 gene cluster in exons encoding for the double stranded β-helix fold and will therefore disrupt catalytic activity. The PHF8 missense mutation c.836C>T is associated with mild MR, mild dysmorphic features, and either unilateral or bilateral cleft lip and cleft palate in two male siblings. In the c.836C>T encoded F279S variant of PHF8, a conserved hydrophobic pocket is modified; assays with both peptides and intact histones reveal this variant to be catalytically inactive. The dependence of PHF8 activity on oxygen availability is interesting because the occurrence of fetal cleft lip has been demonstrated to increase with maternal hypoxia in mice studies. Cleft lip and other congenital anomalies are also linked indirectly to maternal hypoxia in humans, including from maternal smoking and maternal hyptertension treatment. Our results will enable further studies aimed at defining the molecular links between developmental changes in histone methylation status, congenital disorders, and mental retardation.

INTRODUCTION

Mutations to the human PHF8 gene are associated with Siderius X-linked mental retardation (XLMR) syndrome (OMIM 300263). The symptoms of Siderius XLMR include, in addition to mild MR, facial dysmorphism and cleft lip/palate, implying a role for PHF8 (JHDM1F) in midline formation and cognitive functions (1, 2). The prevalence of cleft lip with or without cleft palate is amongst the most common of all birth defects, averaging at 10.5 per 10,000 live births in the United States; the estimated lifetime costs of treating children born with this disorder each year are ~$700 million (3). Recently, a microdeletion encompassing all of the PHF8 and FAM120C genes and parts of WNK3 was reported in two brothers with autism spectrum disorders, causing hypertelorism and broad halluces in addition to the Siderius XLMR features (4). Episodes of maternal respiratory hypoxia in mice also correlate with increased incidence of cleft lip/palate (gestational day 10-11, 10% O2), while hyperoxia rescued mouse strains that are genetically susceptible for cleft lip/palate (gestational day 10-11, 50% O2) (5), suggesting that gestational oxygen levels mediate genetic and environmental factors.

PHF8 is predicted to be one of a sequence-related group of human proteins, the PHF subfamily, which also includes JHDM1D and PHF2 (JHDM1E), the function(s) of which have not been identified. Predicted amino acid sequence analyses imply that the PHF proteins are part of the JmjC subfamily of human Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases (6) that also depend on cellular oxygen levels for catalytic activity, some of which are histone lysine demethylases. All of the studied 2OG-dependent demethylases appear to be sequence-specific (6). All four types of identified mutations/deletions to PHF8 cluster in the exons encoding for the predicted JmjC (or double-stranded β-helix) domain (Figure 2A), which is common to all identified 2OG oxygenases (2, 6-8).

Figure 2. The effect of clinically observed variants on PHF8 activity.

Figure 2

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(A) PHF8 domain analysis, indicating clinically identified variants (2, 7, 8). Abbreviation: NLS, nuclear localization signal. (B) Mapping of clinical variants (yellow residues) onto a PHF8 structure model suggests PHF8 Δ308-315 inactivity due to a missing β-strand and explains PHF8 F279S inactivity (see Figure S6). (C) The PHF879-447 H247A and F279S variants (5-50 μg) are inactive against H3K9me2 and H3K36me2 on calf histones. The apparent increase in histone staining is due to PHF8 F279S degradation products. (D) PHF879-447 F279S does not demethylate H3K9me1, while PHF879-447 is active. Impurities in the control lane originate from the synthesis of long 19mer peptides. (E) Wild-type HA-PHF8 was localized to the nucleus, while the clinical F279S variant displayed cytoplasmic staining; transfected cells are indicated by arrows.

Post-translational modifications to histones are important in epigenetic regulation, and the enzymes responsible for their placement and removal are currently the focus of intense research. Several JmjC-domain containing histone demethylases have been implicated in diseases including prostate cancer (the JMJD2 family of histone H3K9/36 demethylases) (9) and XLMR (JARID1C, a histone H3K4 demethylase) (10-13). Here we report that PHF8 is an iron and 2OG dependent demethylase specific for di- and mono-Nε-methylated lysine residues; compared to other studied 2OG dependent demethylases, PHF8 appears in vitro to have relatively lax sequence selectivity.

RESULTS

Identification of PHF8 as a 2OG oxygenase by activity assays

To investigate whether, as predicted, PHF8 is a 2OG oxygenase, we prepared recombinant forms of full length PHF8, as well as N- and C-terminal deletions of PHF8; PHF81-447 and PHF879-447 were purified to near homogeneity. Because many 2OG oxygenases can oxidize 2OG in the absence of their ‘prime’ substrate, we then employed an assay for PHF8 measuring the turnover of 1-[14C]-2OG to succinate and [14C]-CO2. We observed PHF8-dependent stimulation of 2OG turnover that was inhibited by lack of Fe(II), as well as by pyridine-2,4-dicarboxylate (2,4-PDCA), a known 2OG oxygenase inhibitor (Figure 1A). Mutation of a predicted Fe(II) binding residue (H247A) ablated activity (Figure S1). These results reveal that PHF8 is a 2OG dependent oxygenase.

Figure 1. PHF8 is a histone lysine demethylase selective for the di- and monomethyl states.

Figure 1

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(A) PHF879-447 stimulated 2OG turnover is dependent on cofactors and is inhibited by 1 mM pyridine-2,4-dicarboxylate. Addition of Nε-methylated histone peptides increased 2OG turnover only for dimethylated substrates. Numbers shown with standard deviation are from one assay performed in triplicates. (B) PHF879-447 reduces H3K9me2 and H3K36me2 levels on calf histones. (C, D) PHF8 is similar to K36me1/2-selective FBXL11 (PDB ID: 2YU2) (32) (Figure S4). Comparison with trimethyl-selective JMJD2A (PDB ID: 2OQ6) (21) suggests structural constraints prevent FBXL11 from accommodating trimethylated lysine (see Figure S3). (E) PHF879-447 demethylates H3K9me2, H3K9me1, H3K27me2 and H3K36me2 on calf histones.

In an effort to identify a substrate for PHF8, we then screened a set of peptides corresponding to fragments of potential target proteins, including Nε-lysine tri- and dimethylated histone fragments, for stimulation of 2OG turnover by PHF8. Increased 2OG turnover was only observed for dimethylated lysine-9 of histone 3 (H3K9me2; analogous abbreviations are used throughout) and H3K36me2 peptides. Notably, H3K9me3 did not show stimulation of 2OG turnover above control levels (Figure 1A), suggesting dimethylated lysine residues are substrates of PHF8.

Establishing PHF8 substrate selectivity by mass spectrometric based screening

We then investigated the sequence selectivity of PHF8 by mass spectrometry using peptide fragments of H3 and H4 histones containing methylated lysine residues. PHF8 catalyzed demethylation of H3K9me2/me1, H3K27me2, and H3K36me2, but not of H4K20 peptides (Figure S1). Under current assay conditions, we did not observe demethylation of H3K36me1, but observed demethylation of H3K36me2 to give both H3K36me1 and some H3K36me0. It is possible that this reflects incomplete release of the H3K36me1 derived from H3K36me2 during catalysis to H3K36me0. These results suggest that PHF8 may have an unusually broad sequence selectivity, at least in vitro, compared to other JmjC histone demethylases (e.g. FBXL11 is reported to be selective for H3K36) (14). Interestingly, both the 2OG oxygenases factor inhibiting HIF (FIH) and JMJD6, which although hydroxylases are sequence-related to the JmjC demethylases, have broad substrate selectivity (15-19), with FIH being shown to have large numbers of substrates in vitro and in vivo. We thus do not exclude the possibility of non-histone substrates for PHF8.

Because some JmjC-domain containing oxygenases function as hydroxylases, rather than demethylases, we tested substrates of FIH (asparaginyl hydroxylation of hypoxia-inducible transcription factor (HIFα) and ankyrins) (17, 20) and JMJD6 (lysyl hydroxylation of splicing-related (SR) proteins) (18) as PHF8 substrates; however, no hydroxylase activity was observed (Figure S2).

PHF8 removes methylation marks on calf histones

Because the selectivity of 2OG oxygenases observed with peptide fragments does not always reflect their selectivity with regards to proteins (18), we then carried out assays with intact calf histones as substrates (Figure 1B), using the known histone demethylase JMJD2A for comparison. As reported (21), JMJD2A reduced H3K9me3 and H3K36me3/me2/me1 methylation levels. PHF8-mediated demethylation was observed for H3K9me2, H3K9me1, H3K36me2, as well as for H3K27me2 (Figure 1E). In contrast to JMJD2A, with PHF8 demethylation was neither observed for trimethylated H3K9, nor for H3K36, nor for any of the investigated H3K4 and H3K79 methylation states. Consistent with the mass spectrometric assays, H3K36me1 was also not found to be a substrate for PHF8.

Structural predictions for PHF8 based on deposited coordinates for FBXL11 (PDB: 2YU2 (22)) and structures of JMJD2A (21) indicate that the methyl lysine pocket for PHF8 is similar to that of FBXL11, which is also selective for di- and mono-methyl lysine residues (14), and smaller than the methyllysine binding pocket of JMJD2A, which is selective for di- and tri-methyl lysine residues (Figures 1C and 1D) (21).

The methylation state selectivity of PHF8 was also confirmed by side-by-side analysis of mono-, di-, and trimethylated H3K9 peptides with PHF8 and JMJD2A in vitro using mass spectrometry (Figure S5): PHF8 was selective for di- and mono-methylated forms whereas JMJD2A was selective for tri- and di-methylated forms.

Clinically observed PHF8 mutants associated with cleft lip abrogate PHF8 activity

We then investigated whether clinically observed PHF8 mutations associated with Siderius XLMR affect PHF8 catalysis (Figure 2A). Analysis of a structural model for PHF8, based on the reported crystallographic data for JMJD2A (21) and FBXL11, reveals that three of the four identified types of PHF8 mutations will lead to major modifications to the double-stranded β-helix/JmjC domain (2, 7, 8), almost certainly causing loss of function (see Figures 2B and S6 for detailed analyses). However, one point mutation, F279S (8), replaces a hydrophobic phenylalanine with a more polar serine residue, within a hydrophobic pocket close to the active site and which is apparently conserved in all members of the FBXL/PHF subfamily (Figure S6). The functional effect of the PHF8 F279S mutation was unclear from the structural model. In affected individuals, this mutation caused symptoms including mild MR, dysmorphic features, and either unilateral or bilateral cleft lip/palate. Notably, PHF879-447 F279S was prepared and found to be completely catalytically inactive, both by 2OG turnover and mass spectrometric assays (Figures 2D and S6). Similarly, when using calf histones as substrates, both H3K9me2 and H3K36me2 levels were reduced with wild-type PHF8, but neither by the F279S nor H247A variants, even when using a 10-fold increase in PHF8 F279S levels (Figure 2C).

To investigate the effect of the F279S variation on cellular localization of PHF8, we transfected HeLa cells with human full-length PHF8 bearing an N-terminal HA epitope tag (HA-PHF8). The HA-PHF8 overexpressing cells were fixed after 24 hours; immunofluorescence assays demonstrated that wild-type PHF8 localizes to the nucleus, consistent with its function as a histone demethylase (Figure 2E). Significant changes in global methylation levels were not observed under these conditions (data not shown), suggesting that PHF8 is either specifically targeted to certain chromosomal regions or that it requires other protein partners. In contrast to the wild-type HA-PHF8, analogous studies revealed that the clinically observed F279S variant did not show clear nuclear localization, with apparent cytoplasmic localization. We conclude that the single site mutation F279S abolishes PHF8 activity, thus linking the pathology of PHF8 mutations causing XLMR with cleft lip/palate to its function as a histone demethylase.

DISCUSSION

In summary, our results reveal that PHF8 is a 2OG oxygenase with selectivity for H3K9me2/me1, H3K27me2 and H3K36me2 residues. We provide evidence that the pathology of PHF8 mutations causing XLMR with cleft lip/palate is linked to its function as a histone demethylase; efforts can now be directed towards determining how lysine methylation status is linked to the pathophysiology of Siderius XLMR. Given that the 2OG oxygenases have an absolute requirement for oxygen (6, 23), and that the HIFα hydroxylases, including FIH, act as sensors in the physiological response to hypoxia (20), it is notable that maternal hypoxia has been linked to increased incidence of cleft lip/palate not only in mice (5), but also in the acardiac human fetus (24). In the human fetus, hypoxia is also proposed to arise as a result of maternal hypertension treatment (25) and maternal smoking (26). The molecular mechanism of how oxygen and histone modifying enzymes such as PHF8 regulate the development and fusion of facial prominences can now be addressed; it is possible that it involves chromatin regulated gene expression including of homeobox genes, which are commonly associated with congenital abnormalities (27).

Finally, because of their role in epigenetics and oncology, histone modifying enzymes are being extensively investigated, both in academia and industry, with histone deacetylase inhibitors already in clinical use (we note that some histone deacetylase inhibitors also inhibit demethylases) (28). Although PHF8 may have different roles in development compared to adult organisms, our work suggests that when targeting histone demethylases or deacetylases, caution should be used in studies on compounds that inhibit PHF8 activity.

MATERIALS & METHODS

Expression plasmids and protein purification

For expression in E. coli, human PHF8 was cloned into the pNIC28-Bsa4 expression vector (residues 79-447 and 1-447) with an N-terminal His6-thioredoxin tag. For mammalian expression, human PHF8 (residues 1-447 and full-length) was cloned into the pEF6 vector with an N-terminal HA tag. Recombinant JMJD2A was prepared as reported (21). All point mutations were generated using thermal cycling; for primer sequences see Table S1. For large-scale expression, plasmids were transformed into E. coli BL21 cells, induced with 0.5 mM IPTG at OD 0.6 and grown for ~4 hours at 30°C. Enzymes were purified by immobilized Ni(II) affinity chromatography and subsequent gel filtration chromatography.

Calf histone incubations

Bulk calf thymus type II-A histones (Sigma H9250) were incubated with purified PHF879-447 in 20 mM Tris-HCl pH 8, 150 mM NaCl, 50 μM FeSO4·7H20, 1 mM 2-oxoglutarate, and 2 mM ascorbate. Wildtype PHF8 (5 μg), PHF8 H247A (5 μg) or PHF8 F279S (5-50 μg) were incubated with 25 μg histones in a total reaction volume of 100 μl. Reactions were performed at 37 °C for 4-6 hours before addition of Laemmli buffer, separation on 15% SDS-PAGE gels and western blot analysis. Rabbit anti-H3K9me1 (07-450), -H3K9me2 (07-441), -H3K9me3 (07-442), -H3K4me1 (07-436), -H3K27me1 (07-447), -H3K27me2 (07-452), and - H3K79me2 (05-835) were from Millipore. Rabbit anti-H3K36me1 (ab9048), H3K36me3 (ab9050), H3K4me2 (ab7766), and -Histone H3 (ab1791) were from AbCam. Rabbit anti-H3K36me2 (9758) was from Cell Signaling.

Immunofluorescence studies

HeLa cells were grown on coverslips in 6-well plates, using DMEM supplemented with foetal bovine serum (10%) and penicillin/streptomycin. Cells were transfected with 1-2 μg PHF81-1024, PHF81-1024 F279S or PHF81-1024 H247A, using FuGene HD (Roche) as transfection reagent. After 24 hours, cells were fixed in paraformaldehyde (4% in PBS), permeabilized (0.5% Triton X-100 in PBS) and blocked (3% BSA in PBS) before staining with primary antibody (HA-FITC, Sigma, H7411) for 4 hours. After primary antibody incubation, cells were incubated with FITC conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 1 hour, prior to staining with DAPI (5 min) and mounting onto microscope slides in fluorescent mounting medium (Dako). Slides were analyzed using an AxioSkop fluorescent microscope (Zeiss).

Structural modeling of PHF8 and FBXL11

The FBXL11 sequence was mapped by pair fitting onto the JMJD2A.H3K9me3 structure using PyMOL (PDB IDs: 2YU2, 2OQ6). The high sequence similarity (61% identity) between the JmjC domains of PHF8 and FBXL11 (amino acids 234-334 and 199-299, respectively) was used to prepare a structure homology model for PHF8 based on an available structure of the FBXL11 protein (PDB ID: 2YU2) (22) using the Phyre server (E-value: 6.95e-17) (29). The metal ion substituting for Fe(II) for crystallographic purposes was replaced by Fe(II) in Figures 1 and S3.

Cosubstrate turnover experiments

Recombinant enzymes and substrates were tested for their ability to stimulate PHF8-dependent decarboxylation of 1-[14C]-labeled 2OG, as described for other 2OG oxygenases (30, 31). Conversion of 2OG was calculated from the percentage of 1-[14C]-2OG that had been converted to 14CO2 gas; data was represented as pmol 2OG turnover per minute per μM of enzyme. Numbers shown are from one assay, performed in triplicates; error bars give the standard deviation.

Mass spectrometric analyses

Demethylation of peptides was analyzed using a MALDI-TOF microMX mass spectrometer in the positive ion reflectron mode. Assay conditions utilized similar 2OG turnover assay conditions except without the radioactive 2OG. Reactions were quenched with an equal volume of 0.1 % formic acid/MeOH at 4 °C. All samples for MS analyses were mixed with α-cyano-4-hydroxy-cinnamic acid (CHCA) as MALDI matrix (1:1) and spotted onto the target plate.

Peptide synthesis

Peptides H3K9me1 (ARTKQTAR-Kme1-STGGKAPRKQ), H3K9me2 (ARTKQTAR-Kme2-STGGKAPRKQ), H3K9me3 (ARTKQTAR-Kme3-STGGKA), H3K36me1 (RKSAPATGGV-Kme1-KPHRYRPG), and H3K36me2 (RKSAPATGGV-Kme2-KPHRYRPG) were prepared using a solid phase peptide synthesizer (CS Bio CS336) employing Rink amide linker, PL-AMS resin (Polymer Laboratories) and standard Fmoc/DIC/HOBt strategy. Final cleavage (CF3CO2H: triisopropylsilane 97.5:2.5) yielded the peptides as C-terminal amides which were purified by preparative reversed-phase HPLC using Vydac 218TP C18 10-15u column (Grace Davison Discovery Sciences) and lyophilized. The mass of the peptide product was confirmed using a Micromass MALDI-TOF (Waters) mass spectrometer. Peptides H3K9me2 24mer (ARTKQTAR-Kme2-STGGKAPRKQLATKA), H3K27me2 (LATKAAR-Kme2-SAPSTGGVKK), H3K36me2 18mer (TGGV-Kme2-KPHRYRPGTVALR), H3K36me3 18mer (TGGV-Kme3-KPHRYRPGTVALR) and H4K20me2 (KGGAKRHR-Kme2-VLRDNIQ) were prepared using an Intavis Multipep automated peptide synthesizer, using Tentagel-S-RAM resin (Rapp-Polymere) and otherwise as above.

Supplementary Material

Supplementary

ACKNOWLEDGEMENTS

We thank the Biotechnology and Biological Sciences Research Council and the Wellcome Trust for the support of this research. We acknowledge support by the Rhodes Trust (C.L.) and the Commonwealth Scholarship Commission (N.R.R.) and thank Udo Oppermann (SGC Oxford) for expression construct design.

REFERENCES

  • 1.Siderius LE, Hamel BCJ, van Bokhoven H, de Jager F, van den Helm B, Kremer H, Heineman-de Boer JA, Ropers H-H, Mariman ECM. X-linked mental retardation associated with cleft lip/palate maps to Xp11.3-q21.3. Am. J. Med. Genet. 1999;85:216–220. [PubMed] [Google Scholar]
  • 2.Laumonnier F, Holbert S, Ronce N, Faravelli F, Lenzner S, Schwartz CE, Lespinasse J, Van Esch H, Lacombe D, Goizet C, et al. Mutations in PHF8 are associated with X linked mental retardation and cleft lip/cleft palate. J. Med. Genet. 2005;42:780–786. doi: 10.1136/jmg.2004.029439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.1999 to 2001 cleft lip and palate data collected from 11 states (Alabama, Arkansas, California, Georgia, Hawaii, Iowa, Massachusetts, North Carolina, Oklahoma, Texas, and Utah), adjusted for race-specific distribution of US live births. The Centers for Diseases Control and Prevention (CDC) National Birth Defects Prevention Network (NBDPN); [Google Scholar]
  • 4.Qiao Y, Liu X, Harvard C, Hildebrand MJ, Rajcan-Separovic E, Holden JJA, Lewis MES. Autism-associated familial microdeletion of Xp11.22. Clin. Genet. 2008;74:134–144. doi: 10.1111/j.1399-0004.2008.01028.x. [DOI] [PubMed] [Google Scholar]
  • 5.Millicovsky G, Johnston MC. Hyperoxia and hypoxia in pregnancy: simple experimental manipulation alters the incidence of cleft lip and palate in CL/Fr mice. Proc. Natl. Acad. Sci. U. S. A. 1981;78:5722–5723. doi: 10.1073/pnas.78.9.5722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Loenarz C, Schofield CJ. Expanding chemical biology of 2-oxoglutarate oxygenases. Nat. Chem. Biol. 2008;4:152–156. doi: 10.1038/nchembio0308-152. [DOI] [PubMed] [Google Scholar]
  • 7.Abidi FE, Miano MG, Murray JC, Schwartz CE. A novel mutation in the PHF8 gene is associated with X-linked mental retardation with cleft lip/cleft palate. Clin. Genet. 2007;72:19–22. doi: 10.1111/j.1399-0004.2007.00817.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Koivisto AM, Ala-Mello S, Lemmelä S, Komu HA, Rautio J, Järvelä I. Screening of mutations in the PHF8 gene and identification of a novel mutation in a Finnish family with XLMR and cleft lip/cleft palate. Clin. Genet. 2007;72:145–149. doi: 10.1111/j.1399-0004.2007.00836.x. [DOI] [PubMed] [Google Scholar]
  • 9.Cloos PAC, Christensen J, Agger K, Maiolica A, Rappsilber J, Antal T, Hansen KH, Helin K. The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3. Nature. 2006;442:307–311. doi: 10.1038/nature04837. [DOI] [PubMed] [Google Scholar]
  • 10.Jensen LR, Amende M, Gurok U, Moser B, Gimmel V, Tzschach A, Janecke AR, Tariverdian G, Chelly J, Fryns J-P, et al. Mutations in the JARID1C gene, which is involved in transcriptional regulation and chromatin remodeling, cause X-linked mental retardation. Am. J. Hum. Genet. 2005;76:227–236. doi: 10.1086/427563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Iwase S, Lan F, Bayliss P, de la Torre-Ubieta L, Huarte M, Qi HH, Whetstine JR, Bonni A, Roberts TM, Shi Y. The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell. 2007;128:1077–1088. doi: 10.1016/j.cell.2007.02.017. [DOI] [PubMed] [Google Scholar]
  • 12.Tahiliani M, Mei P, Fang R, Leonor T, Rutenberg M, Shimizu F, Li J, Rao A, Shi Y. The histone H3K4 demethylase SMCX links REST target genes to X-linked mental retardation. Nature. 2007;447:601–605. doi: 10.1038/nature05823. [DOI] [PubMed] [Google Scholar]
  • 13.Tzschach A, Lenzner S, Moser B, Reinhardt R, Chelly J, Fryns J-P, Kleefstra T, Raynaud M, Turner G, Ropers H-H, et al. Novel JARID1C/SMCX mutations in patients with X-linked mental retardation. Hum. Mutat. 2006;27:389. doi: 10.1002/humu.9420. [DOI] [PubMed] [Google Scholar]
  • 14.Tsukada Y.-i., Fang J, Erdjument-Bromage H, Warren ME, Borchers CH, Tempst P, Zhang Y. Histone demethylation by a family of JmjC domain-containing proteins. Nature. 2006;439:811–816. doi: 10.1038/nature04433. [DOI] [PubMed] [Google Scholar]
  • 15.Kelly L, McDonough MA, Coleman ML, Ratcliffe PJ, Schofield CJ. Asparagine β-hydroxylation stabilizes the ankyrin repeat domain fold. Mol. BioSyst. 2009;5:52–58. doi: 10.1039/b815271c. [DOI] [PubMed] [Google Scholar]
  • 16.Coleman ML, McDonough MA, Hewitson KS, Coles C, Mecinovic J, Edelmann M, Cook KM, Cockman ME, Lancaster DE, Kessler BM, et al. Asparaginyl hydroxylation of the notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor. J. Biol. Chem. 2007;282:24027–24038. doi: 10.1074/jbc.M704102200. [DOI] [PubMed] [Google Scholar]
  • 17.Cockman ME, Lancaster DE, Stolze IP, Hewitson KS, McDonough MA, Coleman ML, Coles CH, Yu X, Hay RT, Ley SC, et al. Posttranslational hydroxylation of ankyrin repeats in I{kappa}B proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH) Proc. Natl. Acad. Sci. U. S. A. 2006;103:14767–14772. doi: 10.1073/pnas.0606877103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Webby CJ, Wolf A, Gromak N, Dreger M, Kramer H, Kessler B, Nielsen ML, Schmitz C, Butler DS, Yates JR, III, et al. Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing. Science. 2009;325:90–93. doi: 10.1126/science.1175865. [DOI] [PubMed] [Google Scholar]
  • 19.Cockman ME, Webb JD, Kramer HB, Kessler BM, Ratcliffe PJ. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins. Mol. Cell. Proteomics. 2009;8:535–546. doi: 10.1074/mcp.M800340-MCP200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat. Rev. Mol. Cell. Biol. 2004;5:343–354. doi: 10.1038/nrm1366. [DOI] [PubMed] [Google Scholar]
  • 21.Ng SS, Kavanagh KL, McDonough MA, Butler D, Pilka ES, Lienard BMR, Bray JE, Savitsky P, Gileadi O, von Delft F, et al. Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity. Nature. 2007;448:87–91. doi: 10.1038/nature05971. [DOI] [PubMed] [Google Scholar]
  • 22.Han Z, Liu P, Gu L, Zhang Y, Li H, Chen S, Chai J. Structural basis for histone demethylation by JHDM1. Frontier Science. 2007;1:52–61. [Google Scholar]
  • 23.Welford RW, Kirkpatrick JM, McNeill LA, Puri M, Oldham NJ, Schofield CJ. Incorporation of oxygen into the succinate co-product of iron(II) and 2-oxoglutarate dependent oxygenases from bacteria, plants and humans. FEBS Lett. 2005;579:5170–4. doi: 10.1016/j.febslet.2005.08.033. [DOI] [PubMed] [Google Scholar]
  • 24.Jones KL, Webster WS, Vaux KK, Benirschke K. Acardiac fetus: evidence in support of a vascular/hypoxia pathogenesis for isolated oral clefting. Birth Defects Res., Part A. 2008;82:597–600. doi: 10.1002/bdra.20473. [DOI] [PubMed] [Google Scholar]
  • 25.Hurst JA, Houlston RS, Roberts A, Gould SJ, Tingey WG. Transverse limb deficiency, facial clefting and hypoxic renal damage: an association with treatment of maternal hypertension? Clin. Dysmorphol. 1995;4:359–363. doi: 10.1097/00019605-199510000-00013. [DOI] [PubMed] [Google Scholar]
  • 26.Shi M, Wehby GL, Murray JC. Review on genetic variants and maternal smoking in the etiology of oral clefts and other birth defects. Birth Defects Res., Part C. 2008;84:16–29. doi: 10.1002/bdrc.20117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Cox TC. Taking it to the max: The genetic and developmental mechanisms coordinating midfacial morphogenesis and dysmorphology. Clin. Genet. 2004;65:163–176. doi: 10.1111/j.0009-9163.2004.00225.x. [DOI] [PubMed] [Google Scholar]
  • 28.Rose NR, Ng SS, Mecinovic J, Lienard BMR, Bello SH, Sun Z, McDonough MA, Oppermann U, Schofield CJ. Inhibitor scaffolds for 2-oxoglutarate-dependent histone lysine demethylases. J. Med. Chem. 2008;51:7053–7056. doi: 10.1021/jm800936s. [DOI] [PubMed] [Google Scholar]
  • 29.Kelley LA, Sternberg MJE. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protocols. 2009;4:363–371. doi: 10.1038/nprot.2009.2. [DOI] [PubMed] [Google Scholar]
  • 30.Hewitson KS, McNeill LA, Riordan MV, Tian YM, Bullock AN, Welford RW, Elkins JM, Oldham NJ, Bhattacharya S, Gleadle JM, et al. Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J. Biol. Chem. 2002;277:26351–26355. doi: 10.1074/jbc.C200273200. [DOI] [PubMed] [Google Scholar]
  • 31.Sabourin PJ, Bieber LL. Purification and characterization of an α-ketoisocaproate oxygenase of rat-liver. J. Biol. Chem. 1982;257:7460–7467. [PubMed] [Google Scholar]
  • 32.Han Z, Liu P, Gu L, Zhang Y, Li H, Chen S, Chai J. Structural basis for histone demethylation by JHDM1. doi: 10.2210/pdb2yu2/pdb. [Google Scholar]

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Supplementary Materials

Supplementary
NIHMS66201-supplement-Supplementary.pdf (587.5KB, pdf)

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