Cellular DNA in eukaryotes is organized in chromatin, which contains a multitude of proteins. Histone proteins comprise a major structural component of chromatin. Histones carry diverse post-translational modifications, particularly on the lysines of their N-terminus. These modifications include methylation, acetylation, ubiquitylation and phosphorylation [1–4]. Histone modifications are involved in crucial cellular processes, including transcription, DNA replication, and the DNA-damage response [1, 3, 5]. As might be expected, chromatin and histones undergo major reorganization during DNA replication. After replication, the appropriate chromatin structure has to be restored in order to maintain the passage of information from mother to daughter cells.
One of the major histone modifications is methylation of the lysine 4 residue of histone H3 (H3K4). Trimethylation of H3K4 is a hallmark of active transcription, and conversely, a loss of this modification represses transcription. Histone methylation is a very stable modification compared with, for example, acetylation. It had been long thought that this modification was irreversible . This was supported by the fact that histone methyltransferases were identified, but their counterparts, histone demethylases, were not yet known. This situation changed with the first discovery of a histone demethylase, termed lysine-specific demethylase (LSD)1 . LSD1 prefers double-methylated H3K4 as its substrate . Following this discovery, many other histone demethylases were discovered, with various preferences for specific methylated histone residues. One of the newly discovered demethylases is SMCX (Smcy homolog, X-linked (mouse); also termed lysine-specific demethylase 5C (KDM5C) and jumonji, AT rich interactive domain 1C (JARID1C)) .
SMCX is a ubiquitously expressed protein, which has been linked to mental retardation [9–14]. In addition, a novel mutation in the SMCX gene was found in a patient with autism spectrum disorder (ASD) . SMCX contains a JmjC domain, and belongs to a family of demethylases, which includes the Y-linked homolog SMCY, retinoblastoma binding protein (RBP)2, and; lysine-specific demethylase 5B (KDM5B; also termed PLU-1) [8, 16]. In addition to JmjC, SMCX also contains the N-terminal plant homeodomain (PHD) finger, which binds to the trimethylated H3K4 (H3K4me3). SMCX reverses H3K4me3 to di- and mono-, but not unmethylated products . SMCX was reported to function as a transcriptional repressor, and its loss impairs neuronal gene regulation mediated by regulatory element-1-silencing transcription factor (REST) [17, 18]. However, little is known about how the SMCX-mediated demethylase activity is regulated during the cell cycle and other crucial cellular processes.
In this study, we found that SMCX-mediated H3K4 demethylation is regulated by proliferating cell nuclear antigen (PCNA). We show that association of SMCX with chromatin and H3K4me3 demethylation depends on PCNA. Further, we identified a aconserved sequence motif, [NQ]xx(L/I/M/V)xx (F/Y/W), called the PCNA-interaction protein motif (PIP box) in the SMCX protein. Finally, site-directed mutagenesis demonstrates that the PIP box is important for SMCX association with PCNA.