Abstract
Histone methylation is regulated to shape the epigenome by modulating DNA compaction, thus playing central roles in fundamental chromatin-based processes including transcriptional regulation, DNA repair and cell proliferation. Histone methylation is erased by demethylases including the well-established KDM4 subfamily members, however, little is known about their dimerization capacity and its impact on their demethylase activity. Using the powerful bimolecular fluorescence complementation technique, we herein show the in situ formation of human KDM4A and KDM4C homodimers and heterodimers in nuclei of live transfectant cells and evaluate their H3K9me3 demethylation activity. Using size exclusion HPLC as well as Western blot analysis, we show that endogenous KDM4C undergoes dimerization under physiological conditions. Importantly, we identify the JmjN domain as the KDM4C dimerization interface and pin-point specific charged residues therein to be essential for this dimerization. We further demonstrate that KDM4A/C dimerization is absolutely required for their demethylase activity which was abolished by the expression of free JmjN peptides. In contrast, KDM4B does not dimerize and functions as a monomer, and hence was not affected by free JmjN expression. KDM4 proteins are overexpressed in numerous malignancies and their pharmacological inhibition or depletion in cancer cells was shown to impair tumor cell proliferation, invasion and metastasis. Thus, the KDM4 dimer-interactome emerging from the present study bears potential implications for cancer therapeutics via selective inhibition of KDM4A/C demethylase activity using JmjN-based peptidomimetics.
Highlights
Histone methylation is a key post-translational modification modulating chromatin compaction and regulating gene expression, alternative splicing and other nucleic acid-associated processes [1,2,3,4,5]
We further demonstrate that KDM4A/C dimerization is absolutely required for their demethylase activity which was abolished by the expression of free JmjN peptides
While KDM4B and KDM4C retained full demethylase activity, as demonstrated by the complete loss of H3K9me3 staining in YFP positive cells (Figure 1B, indicated by white arrows), KDM4AYFP displayed reduced H3K9me3 demethylation activity compared to the original HA-KDM4A (Figure 1, compare 1B-b to 1A-b); very high KDM4A-YFP expression levels allowed the demethylation of H3K9me3, whereas low to moderate expression levels were not sufficient to exert detectable demethylase activity (Figure 1B-b, compare white-filled arrows to outlined arrows), suggesting that the large YFP tag interferes with the demethylase activity of KDM4A
Summary
Histone methylation is a key post-translational modification modulating chromatin compaction and regulating gene expression, alternative splicing and other nucleic acid-associated processes [1,2,3,4,5]. Histones can be methylated on specific lysine residues by different lysine methyl transferases (KMTs), resulting in mono-, di- or trimethylated states [6]. This methylation is erased by specific histone lysine demethylases (KDMs) [6]. There are two known molecular mechanisms of lysine demethylation; the first is an FAD-dependent amine oxidation, catalyzed by the lysine specific demethylases LSD1/2 [7, 8]. The second involves a dioxygenase reaction which requires Fe2 + , O2, and α-ketoglutarate cofactors, and is catalyzed by Jumonji C domain-containing (JMJD) proteins [9, 10]. H3K9 methylation is involved in chromatin condensation necessary for cell division [15] and mediates heterochromatin formation during DNA double strand break response [16]
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