The role of VRK1 in chromatin remodelingregulation of histone post-translational modifications and epigenetic enzymes

Laburpena

DNA organization is essential for proper chromatin packaging and necessary to facilitate different processes that require dynamic chromatin remodeling. Modulation of chromatin structure is critical for the regulation of gene expression, since it determines which genes are accessible for transcription and the sequential recruitment of regulatory factors to the underlying DNA. Thus, to deal with inaccessible chromatin, eukaryotic cells have developed mechanisms that facilitate the opening of chromatin. Epigenetic alterations are defined as mechanisms that control DNA accessibility for the regulation of gene expression patterns and normal development. The epigenetic transcriptional control can occur through DNA methylation, histone post-translational modifications (PTMs), the reading of these modifications by epigenetic enzymes, histone-variants exchange, and noncoding RNA. Errors in the epigenetic regulation can alter the control of chromatin-based processes, ultimately leading to abnormal gene expression. Pathological conditions such as cancers, metabolic disorders, and inflammatory and neurodegenerative diseases have been found to be related to epigenetic errors. Post-translational modifications (PTMs) of the N-terminal tail of histones regulate DNA access. Importantly, histone PTMs are reversible, and their coordination requires a tight regulation of multiple epigenetic enzymes, known as writers (enzymes that add a mark) and erasers (enzymes that remove a mark)111. Among the different PTMs, acetylation and methylation are especially important. They have been extensively investigated in a context of cancer and therapy responses. The balance between de- and acetylation is controlled by deacetylases (HDACs) and acetyltransferases (HATs), while de- and methylation are regulated by demethylases (KDMs) and methyltransferases (KMTs). An abnormal activity of the epigenetic enzymes and, subsequently, a disturbed histone PTM landscape can alter different cellular processes such as proliferation, DNA repair, gene transcription, and DNA replication, together with the expression of tumor-suppressor or cancer-associated genes. Thus, it is crucial to unveil the molecular mechanisms that modulate these changes and the histone-modifying enzymes involved in such regulation. VRK1 (Vaccinia-related kinase 1) is a nuclear kinase implicated in different cellular processes, such as modulation of transcription factors (TFs) or proteins implicated in the DNA damage response (DDR). The location of VRK1 as a nucleus-resident kinase makes it a suitable candidate to participate in chromatin remodeling and, thus, a potential therapeutic target. Therefore, this thesis aims to broaden the knowledge of the role of the human kinase VRK1 in chromatin remodeling, deciphering the regulation of histone PTMs patterns and characterizing new possible epigenetic enzymes substrates of VRK1. Another goal of this work is characterizing a novel VRK1 inhibitor, VRK-IN-1, to understand its mechanism of action and propose VRK1 inhibition as a potential cancer therapy. In this work, we demonstrate that the absence of VRK1 alters the histone PTM landscape, mimicking the effect of some epigenetic enzyme inhibitors. VRK1 depletion causes a decrease of H3K4me3, H3K9ac, H3K27ac, H3K79me2 and H4K16ac levels, and an increase of H3K9me3 and H3K27me3 levels in lung adenocarcinoma and osteosarcoma cellular models. Furthermore, VRK1 can form a stable protein complex with HDAC1, SIRT1, SIRT2, SETDB1, LSD1, JMJD1A and JMJD2A, suggesting that VRK1 controls the activity of these epigenetic enzymes. In addition, we observed that the inhibition of VRK1 kinase activity by using VRK-IN-1 resembles the VRK1 depletion, thus blocking cell proliferation, impairing histone PTM patterns and altering the DDR upon DNA damage induction. Altogether, these findings reveal VRK1 as an orchestrator of chromatin remodeling, capable of interacting with different epigenetic enzymes and maintaining histone PTMs associated with relaxed chromatin. Moreover, the present data provides a resource for investigating novel VRK1 inhibitors, as well as exploring new target therapies through the biochemical mechanisms here uncovered.