Search

[Background]: Dynamic chromatin remodeling is associated with changes in the epigenetic pattern of histone acetylations and methylations required for processes based on dynamic chromatin remodeling and implicated in different nuclear functions. These histone epigenetic modifications need to be coordinated, a role that may be mediated by chromatin kinases such as VRK1, which phosphorylates histones H3 and H2A., [Methods]: The effect of VRK1 depletion and VRK1 inhibitor, VRK-IN-1, on the acetylation and methylation of histone H3 in K4, K9 and K27 was determined under different conditions, arrested or proliferating cells, in A549 lung adenocarcinoma and U2OS osteosarcoma cells., [Results]: Chromatin organization is determined by the phosphorylation pattern of histones mediated by different types of enzymes. We have studied how the VRK1 chromatin kinase can alter the epigenetic posttranslational modifications of histones by using siRNA, a specific inhibitor of this kinase (VRK-IN-1), and of histone acetyl and methyl transferases, as well as histone deacetylase and demethylase. Loss of VRK1 implicated a switch in the state of H3K9 posttranslational modifications. VRK1 depletion/inhibition causes a loss of H3K9 acetylation and facilitates its methylation. This effect is similar to that of the KAT inhibitor C646, and to KDM inhibitors as iadademstat (ORY-1001) or JMJD2 inhibitor. Alternatively, HDAC inhibitors (selisistat, panobinostat, vorinostat) and KMT inhibitors (tazemetostat, chaetocin) have the opposite effect of VRK1 depletion or inhibition, and cause increase of H3K9ac and a decrease of H3K9me3. VRK1 stably interacts with members of these four enzyme families. However, VRK1 can only play a role on these epigenetic modifications by indirect mechanisms in which these epigenetic enzymes are likely targets to be regulated and coordinated by VRK1, [Conclusions]: The chromatin kinase VRK1 regulates the epigenetic patterns of histone H3 acetylation and methylation in lysines 4, 9 and 27. VRK1 is a master regulator of chromatin organization associated with its specific functions, such as transcription or DNA repair.

[Background]: Dynamic chromatin remodeling is associated with changes in the epigenetic pattern of histone acetylations and methylations required for processes based on dynamic chromatin remodeling and implicated in different nuclear functions. These histone epigenetic modifications need to be coordinated, a role that may be mediated by chromatin kinases such as VRK1, which phosphorylates histones H3 and H2A., [Methods]: The effect of VRK1 depletion and VRK1 inhibitor, VRK-IN-1, on the acetylation and methylation of histone H3 in K4, K9 and K27 was determined under different conditions, arrested or proliferating cells, in A549 lung adenocarcinoma and U2OS osteosarcoma cells., [Results]: Chromatin organization is determined by the phosphorylation pattern of histones mediated by different types of enzymes. We have studied how the VRK1 chromatin kinase can alter the epigenetic posttranslational modifications of histones by using siRNA, a specific inhibitor of this kinase (VRK-IN-1), and of histone acetyl and methyl transferases, as well as histone deacetylase and demethylase. Loss of VRK1 implicated a switch in the state of H3K9 posttranslational modifications. VRK1 depletion/inhibition causes a loss of H3K9 acetylation and facilitates its methylation. This effect is similar to that of the KAT inhibitor C646, and to KDM inhibitors as iadademstat (ORY-1001) or JMJD2 inhibitor. Alternatively, HDAC inhibitors (selisistat, panobinostat, vorinostat) and KMT inhibitors (tazemetostat, chaetocin) have the opposite effect of VRK1 depletion or inhibition, and cause increase of H3K9ac and a decrease of H3K9me3. VRK1 stably interacts with members of these four enzyme families. However, VRK1 can only play a role on these epigenetic modifications by indirect mechanisms in which these epigenetic enzymes are likely targets to be regulated and coordinated by VRK1., [Conclusions]: The chromatin kinase VRK1 regulates the epigenetic patterns of histone H3 acetylation and methylation in lysines 4, 9 and 27. VRK1 is a master regulator of chromatin organization associated with its specific functions, such as transcription or DNA repair.

Distal hereditary neuropathies and neuro motor diseases are complex neurological phenotypes associated with pathogenic variants in a large number of genes, but in some the origin is unknown. Recently, rare pathogenic variants of the human VRK1 gene have been associated with these neurological phenotypes. All VRK1 pathogenic variants are recessive, and their clinical presentation occurs in either homozygous or compound heterozygous patients. The pathogenic VRK1 gene pathogenic variants are located in three clusters within the protein sequence. The main, and initial, shared clinical phenotype among VRK1 pathogenic variants is a distal progressive loss of motor and/or sensory function, which includes diseases such as spinal muscular atrophy, Charcot-Marie-Tooth, amyotrophic lateral sclerosis and hereditary spastic paraplegia. In most cases, symptoms start early in infancy, or in utero, and are slowly progressive. Additional neurological symptoms vary among non-related patients, probably because of their different VRK1 variants and their genetic background. The underlying common pathogenic mechanism, by its functional impairment, is a likely consequence of the roles that the VRK1 protein plays in the regulation on the stability and assembly of Cajal bodies, which affect RNA maturation and processing, neuronal migration of RNPs along axons, and DNA-damage responses. Alterations of these processes are associated with several neuro sensory or motor syndromes. The clinical heterogeneity of the neurological phenotypes associated with VRK1 is a likely consequence of the protein complexes in which VRK1 is integrated, which include several proteins known to be associated with Cajal bodies and DNA damage responses. Several hereditary distal neurological diseases are a consequence of pathogenic variants in genes that alter these cellular functions. We conclude that VRK1-related distal hereditary neuropathies and motor neuron diseases represent a novel subgroup of Cajal body relat

The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of chromatin associated to histone H4 acetylation in K16, mediated by Tip60/KAT5. In this work, we have studied the role that the VRK1 chromatin kinase plays on the activation of Tip60 during this process. In the DNA damage response induced by doxorubicin, VRK1 directly phosphorylates Tip60. However, the phosphorylated Tip60 residues and their functional roles are unknown. In DDR, we have identified these two Tip60 phosphorylated residues and the cooperation of the participating kinases. The T158 phosphorylation, mediated by VRK1, is early and transient, preceding that of S199, which is more sustained in time, and mediated by DNA-PK. The role of each phosphorylated residues was determined by using phosphomimetic and phosphonull mutants and their combination. T158 phosphorylation protects Tip60 from ubiquitin-mediated degradation, promotes its recruitment to chromatin from the nucleoplasm, and is necessary for its full trans-acetylase activity. The phosphorylation in S199 by DNA-PK directly facilitates Tip60 autoacetylation, but it is not enough for trans-acetylation of two of its targets, histone H4 and ATM, which requires a double phosphorylation of Tip60 in T158 and S199. DNA-PK inhibitors block the phosphorylation of S199. We propose a model in which the cooperation between VRK1 and DNA-PK mediates the sequential phosphorylation of Tip60/KAT5, and contributes to the recruitment of this protein to initiate the sequential remodeling of chromatin in DDR. Both proteins are candidates for novel synthetic lethality strategies in cancer treatment.

[Background and Objectives]: To conduct a genetic and molecular functional study of a family with members affected of hereditary spastic paraplegia (HSP) of unknown origin and carrying a novel pathogenic vaccinia-related kinase 1 (VRK1) variant., [Methods]: Whole-exome sequencing was performed in 2 patients, and their parents diagnosed with HSP. The novel VRK1 variant was detected by whole-exome sequencing, molecularly modeled and biochemically characterized in kinase assays. Functionally, we studied the role of this VRK1 variant in DNA damage response and its effect on the assembly of Cajal bodies (CBs)., [Results]: We have identified a very rare homozygous variant VRK1-D263G with a neurologic phenotype associated with HSP and moderate intellectual disability. The molecular modeling of this VRK1 variant protein predicted an alteration in the folding of a loop that interferes with the access to the kinase catalytic site. The VRK1-D263G variant is kinase inactive and does not phosphorylate histones H2AX and H3, transcription factors activating transcription factor 2 and p53, coilin needed for assembly of CBs, and p53 binding protein 1, a DNA repair protein. Functionally, this VRK1 variant protein impairs CB formation and the DNA damage response., [Discussion]: This report expands the neurologic spectrum of neuromotor syndromes associated with a new and rare VRK1 variant, representing a novel pathogenic participant in complicated HSP and demonstrates that CBs and the DNA damage response are impaired in these patients.

Dynamic remodeling of chromatin requires acetylation and methylation of histones, frequently affecting the same lysine residue. These alternative epigenetic modifications require the coordination of enzymes, writers and erasers, mediating them such as acetylases and deacetylases. In cells in G0/G1, DNA damage induced by doxorubicin causes an increase in histone H4K16ac, a marker of chromatin relaxation. In this context, we studied the role that VRK1, a chromatin kinase activated by DNA damage, plays in this early step. VRK1 depletion or MG149, a Tip60/KAT5 inhibitor, cause a loss of H4K16ac. DNA damage induces the phosphorylation of Tip60 mediated by VRK1 in the chromatin fraction. VRK1 directly interacts with and phosphorylates Tip60. Furthermore, the phosphorylation of Tip60 induced by doxorubicin is lost by depletion of VRK1 in both ATM +/+ and ATM−/− cells. Kinase-active VRK1, but not kinase-dead VRK1, rescues Tip60 phosphorylation induced by DNA damage independently of ATM. The Tip60 phosphorylation by VRK1 is necessary for the activating acetylation of ATM, and subsequent ATM autophosphorylation, and both are lost by VRK1 depletion. These results support that the VRK1 chromatin kinase is an upstream regulator of the initial acetylation of histones, and an early step in DNA damage responses (DDR).

[Background]: Distal motor neuropathies with a genetic origin have a heterogeneous clinical presentation with overlapping features affecting distal nerves and including spinal muscular atrophies and amyotrophic lateral sclerosis. This indicates that their genetic background is heterogeneous., [Patient and methods]: In this work, we have identified and characterized the genetic and molecular base of a patient with a distal sensorimotor neuropathy of unknown origin. For this study, we performed whole‐exome sequencing, molecular modelling, cloning and expression of mutant gene, and biochemical and cell biology analysis of the mutant protein., [Results]: A novel homozygous recessive mutation in the human VRK1 gene, coding for a chromatin kinase, causing a substitution (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since childhood a progressive distal sensorimotor neuropathy and spinal muscular atrophy syndrome, with normal intellectual development. Molecular modelling predicted this mutant VRK1 has altered the kinase activation loop by disrupting its interaction with the C‐terminal regulatory region. The p.Y213H mutant protein has a reduced kinase activity with different substrates, including histones H3 and H2AX, proteins involved in DNA damage responses, such as p53 and 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) protein is unable to rescue the formation of Cajal bodies assembled on coilin, in the absence of wild‐type VRK1., [Conclusion]: The VRK1(Y213H) mutant protein alters the activation loop, impairs the kinase activity of VRK1 causing a functional insufficiency that impairs the formation of Cajal bodies assembled on coilin, a protein that regulates SMN1 and Cajal body formation.

[ES]: Las atrofias musculares espinales (SMA) son un conjunto de enfermedades neurodegenerativas para las que no existe actualmente tratamiento, que representan la principal causa genética de muerte infantil. Se deben a defectos en proteínas que llevan a cabo diferentes funciones en la célula, pero aún no está claro el mecanismo molecular exacto por el que las neuronas degeneran. Se han descrito mutaciones en los genes VRK1, SMN y VCP asociadas tanto a esta patología neuromuscular como a ataxia, degeneración pontocerebelar y esclerosis lateral amiotrófica. En este trabajo nos hemos propuesto caracterizar las relaciones entre las proteínas SMN, VCP y VRK1 en líneas celulares tumorales, para entender la base molecular común de estas patologías neurodegenerativas. Hemos visto mediante ensayos de inmunoprecipitación, que estas tres proteínas interaccionan entre sí en alguna fase del ciclo celular formando un complejo proteico. Lo siguiente a evaluar será en qué compartimento celular se producen dichas interacciones y cómo la alteración de cada uno de estos componentes afecta al conjunto y causa estas patologías neurodegenerativas., [EN]: Spinal muscular atrophy (SMA), a hereditary neurodegenerative disease, is the leading genetic cause of infant mortality. It is caused because of the loss of important proteins which carry different functions in the cells. However, the molecular mechanism by neurons degenerate is not yet clear. Mutations in the VRK1, SMN and VCP genes have been observed in SMA, ataxia, pontocerebellar hipoplasia, amyotrophic lateral sclerosis. In this work, we study the relationship between SMN, VCP and VRK1, using tumor cell lines, in order to understand the common molecular basis of these neurodegenerative diseases. We have seen that these three proteins interact forming a complex in the cell. The next to investigate will be in which cellular compartment such interactions occur and how the alterations of these components affect the whole and cause neurodegenerative diseases.