Human Molecular Genetics Advance Access [Accepted Manuscript] published online on October 19, 2009
Human Molecular Genetics, doi:10.1093/hmg/ddp480
PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an N
-dimethyl lysine demethylase
1 Chemistry Research Laboratory and The Oxford Centre for Integrative Systems Biology, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, U.K. 2 Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, U.K. 3 Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford, OX3 7DQ, U.K. 4 The Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.
* Address for correspondence: christopher.schofield{at}chem.ox.ac.uk, fax: +44 1865 275674, phone: +44 1865 275625
Received September 15, 2009; Revised October 15, 2009; Accepted October 15, 2009
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 N
-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for N-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.
5 These authors contributed equally to this work.