The Vaccinia-Related Kinase I (VRK1) in epigenetic regulation of chromatin

Abstract

[Introduction]: The differences in chromatin compaction are determined by the interaction of DNA with histones and other non histone chromosomal proteins. Chromatin needs to relax or condense to regulate gene transcription or DNA damage response, among other processes. These key reversible mechanisms depend on histone post translational modifications, such as lysine acetylation or methylation, which are regulated by ATP-dependent enzymes: histone acetyltransferases (HAT) and methyltransferases (KMT), called “writers”, and histone deacetylases (HDACs) and demethylases (KDMT), called “erasers”. Epigenetics deregulation is gradually gaining importance as potential player in aging and neurodegeneration. VRK1 is a nuclear Ser-Thr kinase implicated in many cellular processes, such as proliferation, cell cycle or DNA Damage Repair (DDR). Previously we observed that VRK1 is present in the chromatin fraction and is able to interact and phosphorylate H2AX in Ser139 after DNA Damage induced by irradiation. Besides, it is necessary for H4 acetylation in an ATM-independent manner. [Objectives]: We aim to study if VRK1regulates epigenetic mechanisms and has a role in chromatin condensation/relaxation by regulating histone writers or erasers. [Methods]: We used pharmacological inhibitors of HATs (C646 and MG149); HDACs (Entinostat and SAHA); KMTs (Chaetocin); and KDMTs (JMJD2 inhibitor). Some of them are advancing in preclinical regulatory studies. Serum-deprived U2OS cells were silenced for VRK1 and immunofluorescence experiments for lysine-specific H3 methylation and H4 acetylation were performed. [Results]: First, as we knew that VRK1 was necessary for 53BP1 and γ H2AX foci formation after irradiation and to confirm that VRK1 has a role in relaxing chromatin after DNA damage, we tested the effect of histone acetylation/deacetylation and methylation/demethylation inhibition on foci formation. The depletion of VRK1 by siRNA in U2OS, A549 and ATM-/- cells affected the acetylation of H3 in Lys14 and Lys9 and also the H4 acetylation in Lys16, important markers of transcriptional activation. In contrast, siVRK1 enhanced the H3 methylation in Lys9 (a gene repressor marker) but not in Lys4 (transcriptional activator residue), confirming the role of VRK1 in positively mediating gene transcription. As expected, the pharmacological treatment of C646 and of MG149 reduced H3K9 and H4K16 acetylation, while the inhibition of HATs and VRK1 depletion didn’t rescue both acetylations, showing that siVRK1 works as a HATs inhibitor. Moreover, the H3K9 trimethylation enhanced by siVRK1 or JMJD2 inhibitor had a further increase with the presence of the both inhibitions, suggesting that siVRK1 is also a strong KDMT inhibitor. [Conclusions]: In conclusion, we suppose that VRK1 regulates epigenetic mechanisms and supports the chromatin relaxation through two ways: activating HATs and also activating KDMTs. These results could be useful to further studies that will expand the roles of VRK1 in chromatin remodeling and also in the number of target proteins regulated by VRK1. Futurperspectives could be focus on the activation of HATs p300 and Tip60 or some of the members of the Jumonji family by VRK1.