Gene regulation mechanisms in cellular events ranging from development to tumorigenesis target the fundamental unit of chromatin structure, the nucleosome, 146 bp of DNA wrapped around a histone octamer. Covalent modifications of histones play key roles in the function of the genome. Histones are methylated on lysine (K) and arginine (R) residues. Methylation has been shown to occur on five K residues of histone H3 (K4, K9, K27, K36 and K79), and on four R residues (R2, R8, R17 and R26). H4K20 and H4R3 are also methylated [1, 2]. Methylation is a reversible modification regulated by site-specific methyltransferases and demethylases [1]. Demethylases are classified into three groups: peptidylarginine deiminase 4 (PADI 4), lysine-specific demethylase (LSD), and Jumonji C (JmjC)-domain-containing proteins. LSD has two members (LSD1 and LSD2), and removes mono-(me) or di-methyl (me2) groups from H3K4 or H3K9 in an amine oxidase reaction [3]. To date, 98 JmjC-domain-containing proteins have been isolated from fission yeast to humans, and classified into seven groups. They catalyze lysine demethylation through an oxidative reaction that requires iron Fe (II) and α-ketoglutarate (αKG) as cofactors [4]. JMJD6 (Jumonji domain containing 6 protein) is iron and 2-oxoglutarate demethylase of ariginine [5]. Demethylase functions have an impact on gene regulation through the specific histone substrate preferences of individual demethylases. The plant homeodomain finger (PHF2/PHF8) proteins (PHF2, PHF8, KIAA1718, spt3, and 4F429) are found in worms, mice and humans, and contain a plant homeodomain (PHD), in addition to a JmjC domain [1]. Importantly, mutations in PHF8 cause inherited X-linked mental retardation (XLMR) and cleft lip/palate [6]. These observations led to questions about how the demethylase activity of PHF8 is regulated, and how PHF8 functions in biological processes that are involved in the development of XLMR. Recently, Yu et al. demonstrated the catalytic specificity of human PHF8 by determining its crystal structure [7]. Working with wild type (WT) PHF8 and a PHD deletion mutant (c-PHF8) discussed below, they showed that human c-PHF8 consists of 16 α helices and 12 β-strands, arranged in a similar manner to a known JmjC-domain-containing histone demethylase (JHDM1a)[7]. In the presence of Fe2+ and αKG, Fe2+ sits in the center of the c-PHF8 catalytic core, which consists of nine hydrophobic residues. The binding of αKG to Fe2+ does not drastically change the conformation, however it causes the side chain of Y257 to turn towards the substrate binding channel, to be in a position to contact the methyl group of K9me2. With αKG and Fe2+, the active center is too narrow to receive the trimethyl group. Recombinant human PHF8 was able to demethylate H3K9me2 and

H3K9me1, as measured by mass spectrometry. A comparison of the c-PHF8 and JHDM1a structures suggests that the c-PHF8 and JHDM1a, which have different lengths and low sequence homology, are responsible for the different binding affinities to H3K36. JHDM1a binds methyl H3K36, while PHF8 does not. PHD domain adjacent to the catalytic domain in PHF8 may recognize methyl H3K9 and contribute to its demethylation. By contrast, the PHD domain of JHDM1a which is distal to its catalytic domain may recognize a different methyl lysine (K36). Yang, et al. demonstrated a role for the PHD by analyzing the crystal