Article Mutations in the KDM5C ARID Domain and Their Plausible Association with Syndromic Claes-Jensen-Type Disease Yunhui Peng 1, Jimmy Suryadi 2, Ye Yang 2, Tugba G. Kucukkal 1, Weiguo Cao 2,* and Emil Alexov 1,* Received: 31 August 2015 ; Accepted: 4 November 2015 ; Published: 13 November 2015 Academic Editor: Stephen A. Bustin 1 Computational Biophysics and Bioinformatics, Department of Physics, Clemson University, Clemson, SC 29634, USA; [email protected] (Y.P.); [email protected] (T.G.K.) 2 Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA; [email protected] (J.S.); [email protected] (Y.Y.) * Correspondence: [email protected] (W.C.); [email protected] (E.A.); Tel.: +864-908-4796 (E.A.); Fax: +864-656-0805 (E.A.) Abstract: Mutations in KDM5C gene are linked to X-linked mental retardation, the syndromic Claes-Jensen-type disease. This study focuses on non-synonymous mutations in the KDM5C ARID domain and evaluates the effects of two disease-associated missense mutations (A77T and D87G) and three not-yet-classified missense mutations (R108W, N142S, and R179H). We predict the ARID domain’s folding and binding free energy changes due to mutations, and also study the effects of mutations on protein dynamics. Our computational results indicate that A77T and D87G mutants have minimal effect on the KDM5C ARID domain stability and DNA binding. In parallel, the change in the free energy unfolding caused by the mutants A77T and D87G were experimentally measured by urea-induced unfolding experiments and were shown to be similar to the in silico predictions. The evolutionary conservation analysis shows that the disease-associated mutations are located in a highly-conserved part of the ARID structure (N-terminal domain), indicating their importance for the KDM5C function. N-terminal residues’ high conservation suggests that either the ARID domain utilizes the N-terminal to interact with other KDM5C domains or the N-terminal is involved in some yet unknown function. The analysis indicates that, among the non-classified mutations, R108W is possibly a disease-associated mutation, while N142S and R179H are probably harmless. Keywords: X-linked syndromic Claes-Jensen type disease; sequence variants; folding free energy changes; binding free energy changes; molecular dynamics; free energy perturbation 1. Introduction Epigenetic processes regulate gene expression and are essential for development and differentiation of cells [1]. Histone proteins are the major components of chromatin, acting as spools around which DNA winds. Particularly, histone lysine methylation is an important epigenetic process which regulates chromatin structure and gene transcription [2,3]. Due to this, loss of balance of histone lysine methylation has been found to have a profound effect on the diverse biological processes and to be involved in many diseases, including cancer development [4–6]. This work focuses on a particular histone protein, the KDM5C protein of 1560 aa, which is a member of the SMCY homolog family. The KDM5C protein specifically reverses tri- and di-methylation of Lys4 of histone H3 (H3K4), helps maintain the dynamic balance of histone H3K4 methylation states, and also plays a crucial role in functional discrimination between enhancers and Int. J. Mol. Sci. 2015, 16, 27270–27287; doi:10.3390/ijms161126022 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2015, 16, 27270–27287 core promoters [7–9]. It is a multi-functional protein, which contains highly-conserved domains, including ARID/Bright, JmjN, JmjC, C5HC2 zinc finger, and two PHD zinc finger domains (Figure1). These domainsInt. J. Mol. were Sci. 2015 shown, 16, page–page to have specific functions alone or to function in concert with the other KDM5C domains.including ARID/Bright, Thus, the ARIDJmjN, JmjC, (A–T C5HC2 rich zinc interaction finger, and domain)two PHD zinc is afing helix–turn–helixer domains (Figure 1). motif-based DNA-bindingThese domain, domains were which shown is to highly have specific conserved function ins alone all eukaryoticor to function proteinsin concert with and the plays other important KDM5C domains. Thus, the ARID (A–T rich interaction domain) is a helix–turn–helix motif-based roles in development, tissue-specific gene expression, and cell growth regulation [10,11]. The DNA DNA-binding domain, which is highly conserved in all eukaryotic proteins and plays important sequence bindingroles in development, preference istissue-speci still unclearfic gene for expression, the ARID and domain cell grow ofth KDM5C.regulation [10,11]. The other The DNA domain, JmjC, catalyzes demethylationsequence binding of preference H3K4me3 is still to unclear H3K4me1 for the [7 ARID]. The domain JmjN of domain KDM5C. and The itsother interaction domain, with the JmjC catalyticJmjC, domain catalyzes are demethylation important of for H3K4me3 the KDM5C to H3K4me1 function [7]. The [ 12JmjN]. Thedomain N-terminal and its interaction PHD zinc finger with the JmjC catalytic domain are important for the KDM5C function [12]. The N-terminal PHD is a histonezinc methyl-lysine finger is a histone binding methyl-lysine motif binding and wasmotif shownand was shown to have to have a preferential a preferential binding binding to to histone H3K9me3 [7histone,13]. H3K9me3 [7,13]. Figure 1. KDM5C protein domains. The numbers indicate approximate domain boundaries. The Figure 1. KDM5Cknown disease-associated protein domains. missense The mutations numbers are provided indicate as well. approximate domain boundaries. The known disease-associated missense mutations are provided as well. Previous studies have shown that many mutations in the KDM5C gene cause X-linked mental retardation (XLMR), the syndromic Claes-Jensen-type disease [7,14,15]. Mental retardation (MR) Previousgenerally studies causes have significant shown limitations that many both mutations in intellectual in functioning the KDM5C and genein adaptive cause behavior, X-linked mental retardationcovering (XLMR), the social the syndromicand practical skills Claes-Jensen-type that originate before disease the age [of7 ,1814 ,years15]. [16]. Mental The estimated retardation (MR) generally causesprevalence significant of MR among limitations the general both population in intellectual is around functioning1%–3% [15,17]. and The infrequency adaptive of behavior, mutations in the KDM5C gene approximately accounts for 2.8% to 3.3% of families with XLMR [18]. covering the social and practical skills that originate before the age of 18 years [16]. The estimated Thirteen missense mutations associated with XLMR, the syndromic Claes-Jensen-type disease in prevalence ofKDM5C MR amonghave been the reported general to population date and affected is around individuals 1%–3% with [15 KDM5C,17]. The mutations frequency show ofa mutations in the KDM5Cmild-to-severegene approximately range of intellectual accounts disability. for Most 2.8% of to mutations 3.3% of are families located in with JmjC XLMR domain, [ 18ZF]. Thirteen missense mutationsdomain (C5HC2 associated zinc finger with domain), XLMR, and theinter-doma syndromicin regions Claes-Jensen-type and affect the demethylation disease activity in KDM5C have [7,8]. The severity of associated XLMR is roughly related to the cellular demethylase activities of been reportedKDM5C to date mutants and [19]. affected In this individualsstudy, we focus with on KDM5Cthe mutationsmutations in the ARID show domain. a mild-to-severe Two MR range of intellectualassociated disability. mutations Most (A77T of mutationsand D87G) are are reported located inin the JmjC ARID domain, domain [14,20]. ZF domain The D87G (C5HC2 zinc finger domain),mutation and causes inter-domain mild to moderate regions MR including and affect aggressive the demethylation behavior, epileptic activity seizures, [ 7and,8]. speech The severity of impairment, while the A77T results in severe MR including speech impairment, short stature, associated XLMR is roughly related to the cellular demethylase activities of KDM5C mutants [19]. seizures, microcephaly, hyper reflexia, and aggressive behavior [14,20]. However, recent work has In this study,shown we that focus the onD87G the has mutations a minimal effect in the on ARIDKDM5C domain. demethylase Two activity MR associatedin vivo [19] indicating mutations (A77T and D87G)that are the reported disease-associated in the ARID effect domainis not demethyl [14,20ation.]. The Combined D87G mutationwith the lack causes of data mild for the to moderate MR includingmolecular aggressive effect of behavior, A77T mutation, epileptic it can seizures, be concluded and that speech the disease-associated impairment, whileeffects of the both A77T results A77T and D87G mutations are unknown. In this work, we extend the list of mutations, which will be in severe MRinvestigated, including to include speech three impairment, currently non-classi shortfied stature, missense seizures,mutations in microcephaly, the ARID domain. hyper The reflexia, and aggressivenon-classified behavior mutations [14,20 ].are However, R108W(rs146232504), recent workN142S(rs377166019), has shown and that R178H(rs201805773), the D87G has a minimal effect on KDM5Ctaken from demethylase the NCBI dbSNP activity databasein vivo [21].[ 19They] indicating were identified that from the disease-associatedpopulation cohorts effect is participating in the NHLBI Exome Sequencing Project [22]. This project is designed to identify not demethylation. Combined with the lack of data for the molecular effect of A77T mutation,