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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&minus, /&minus, 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).

VRK1 is a nuclear chromatin Ser-Thr kinase functionally associated with different processes that require chromatin remodeling. The VRK1 activity is regulated by entry in cell cycle, and this kinase is also required for nuclear envelope dynamics, chromatin condensation, and Golgi fragmentation. Among its substrates, there are several transcription factors: p53, c-Jun, ATF2, CREB1, and SOX2. VRK1 probably acts cooperating with other signaling pathways that also phosphorylate these transcription factors. VRK1 stabilizes p53 by a specific phosphorylation in Thr18 and after induction of p53 responses, there is a VRK1 downregulation in the lysosome, in which the autophagic pathway participates in a p53-dependent manner. VRK1 also contributes to chromatin condensation, nuclear envelop kinetics by phosphorylation of histone H3 and BAF, and the regulation of Cajal bodies by phosphorylation of coilin. In DNA-damage responses, VRK1 actively participates in chromatin remodeling and regulates H2AX, NBS1, and 53BP1. VRK1 is downstream of MEK1 and Plk3 in the induction of Golgi fragmentation during mitosis. Furthermore, VRK1 is expressed in most tissues, is necessary in the early G1 phase for entry in cell cycle, and is associated with proliferation markers.

Resumen del póster presentado a la EMBO Conference: "Chromatin and Epigenetics", celebrada en Heidelberg (Alemania) del 3 al 6 de mayo de 2017., Genome integrity is continuously challenged by endogenous and exogenous agents causing DNA damage. To counteract the adverse consequences associated to DNA lesions, eukaryotic cells have developed DNA damage signaling and repair machineries specifically adapted to the type of damage. Defects in DNA damage response contribute to aging and several disorders, including cancer and neurodegenerative diseases, which highlights their critical importance for cell viability. Double-strand breaks (DSBs), the most deleterious form of DNA damage, occur in the context of a highly organized chromatin environment. The dynamics of DNA repair proteins, which in many cases form detectable foci, are dictated by chromatin organization and transcriptional activity. Because of that, all eukaryotic DNA repair pathways need to unravel the compacted chromatin structure to facilitate access of the repair machinery and restoration of the original chromatin state afterwards. However, how accessibility and transcriptional silencing are orchestrated and interpreted at DSB sites is still unclear. In order to study the connection between chromatin structure alterations and DSB repair foci formation, pharmacological inhibitors of histone deacetylases, acetylases, methylases or demethylases were used in tumor cell lines before and after inducing DNA damage by both ionizing radiation or Olaparib treatment, widely employed in cancer therapies. Their effect on the assembly of DSB repair foci were assessed later, focusing on sensor and mediator proteins, such as γH2AX, Nbs1, MDC1 or 53BP1. Our results seem to indicate that some HAT and KMT inhibitors impair the formation of 53BP1 foci, not the γH2AX ones, at damage sites. This supports the idea that specific histone acetylations and methylations are necessary for the assembly or stability of 53BP1 foci in resting cells. Further experiments are being performed to determine the molecular mechanism and the modifications of histones implicated in this process.

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., [Introduction]: Genome integrity is continuously challenged by exogenous and endogenous agents causing DNA damage, both in euchromatin and heterocromatin. The repair of these lesions involves changes in the histone organization, such as recruitment and/or incorporation of histone variants or covalent modifications of histones, in order to promote the formation of open, relaxed chromatin structues. Recently, a growing number of chromatin components, remodelers and modifications has been identificated as key players in DNA repair, emphasizing the complex role of chromatin in this process. On the other hand, defects in DNA repair pathways enable cancer cells to survive DNA damage. For this reason, the combination of DNA-damaging agents with specific inhibitors of these pathways could be used in cancer treatment. Currently, specific inhibitors against chromatin remodelers have been developed, but it is still unclear how they act in tumor cells. Our aim ls to detemine the effect of histone acetylation/deacetylation and methylation/demethylation inhibition on DNA repair foci. [Results and conclusion]: We use pharmacological inhibitors of HAIs (C646 and MGl49); HDACs (Entinostat and SAHA); KMTs (Chaetocin); and KDMs (JMJD2 inhibitor). Some of them are being employed in preclinical regulatory studies. In this analysis, we can observe that closed chromatin induced by HAIs inhibitors seems to affect the formation of yH2AX and 53BP1 foci, and the dynamics offoci formation corresponds with the increase of fluorescence level of H4 acetylation after inducing DNA damage. Based on these results, we conclude that chromatin relaxation is an essential early step in DNA repair, which can be blocked by specific inhibitors against HAIs, sensibilizing cells to treatments

Hashimoto's thyroiditis (HT) is an autoimmune disorder characterized by the presence of specific antibodies and by a lymphocytic infiltration of the thyroid secreting inflammatory cytokines. Macrophages, lymphocytes and cytokines play a pivotal role in both development and progression of Th1-mediated autoimmune diseases, and a direct role in the destruction of thyroid follicles and follicular cell function in autoimmune thyroiditis. Integrins are integral membrane receptors involved in cell-extra-cellular matrix (ECM) interaction with both structural and signaling functions. The integrin-ECM interaction is necessary for the correct function and survival of thyroid follicular cells. The purpose of this study was to determine the effect of cytokine stimulation on integrin expression and signaling in the thyroid cell. Primary cultures from normal thyroids were treated with interferon-γ (IFN-γ), INF-α, tumor necrosis factor-α, interlukin 1a or these cytokines all together. Integrin expression, cell adhesion to fibronectin (FN) and FN-stimulated ERK phosphorylation were determined after cytokine treatment. IFN-γ and IFN-α were the most effective, reducing the expression of the integrin αvβ3 and slightly increasing the α3β1. Cell treatment with IFN-γ strongly impaired cell adhesion to FN. At the same time, the treatment with IFN-γ dramatically inhibited the stimulation of ERK phosphorylation induced by cell adhesion to FN. In conclusion, IFN-γ inhibits the expression of the integrin αvβ3, reducing the cell adhesion to FN and the following intracellular signaling in thyroid cells in culture. These results suggest that integrins may be a target of the infiltrating lymphocytes and have a role in the pathogenesis of autoimmune thyroiditis.

Resumen del póster presentado a la European Association for Cancer Research (EACR) Conference on Basic Epigenetic Mechanisms in Cancer, celebrada en Berlin (Alemania) del 8 al 11 de noviembre de 2015., Chromatin organization is regulated by covalent modifications of histones such as acetylation or methylation in specific residues, which can be regulated by phosphorylations. VRK1 is a nuclear Ser-Thr kinase implicated in many nuclear processes, such as proliferation, cell cycle or DNA Damage Repair (DDR). VRK1 is present in the chromatin fraction and is able to interact and phosphorylate H2AX in Ser139 after DNA Damage induced by irradiation, and also phosphorylates histone H3 in Thr3. In this work we have studied the role of human VRK1 Ser-Thr in the control of DNA damage response to IR and the role of specific acetylation and methylation of histones. We have shown that VRK1 is necessary for a proper formation of 53BP1 and γH2AX foci formation at double strand-breaks induced by ionizing radiation in and ATM-independent manner. DSB imply a local relaxation of chromatin and therefore it is likely that it might alter the modifications of histones. Fist we tested if acetylation is required for formation of DSB foci, and whether these acetylations require VRK1. The depletion of VRK1 by siRNA i 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. Also we have determined if manipulation of VRK1 levels can alter the tumour cell sensitivity to drugs currently used in clinical trials that target histone acetylation or methylation. Pharmacological inhibition of histone acetyl transferases (HATs) or VRK1 depletion did not rescue H3K9 and H4K16 acetylations, indicating that VRK1 is a regulator of HATs. We conclude that VRK1 regulates epigenetic mechanisms and has a role in chromatin condensation.

Resumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015., [Introduction]: In basal conditions, cells are exposed to exogenous and endogenous agents, such as ionizing radiation (IR) or oxidative stress, among others, which are responsible for double- and single-strand breaks (DSBs and SSBs, respectively) in the DNA. As a consequence of this, cells can activate different DNA repair mechanisms based on the type of DNA lesions. All these mechanisms take part in a global process called DNA Damage Response (DDR), essential to maintain the integrity and stability of genetic information. It is widely known that oxidative stress induces both SSBs and DSBs, particularly in cells with high metabolic rates, like cancer or neural cells. For this reason, it is particularly relevant to know the proteins which detect and repair the damage, returning to homeostatic equilibrium afterwards. Recently, VRK1 has been described to be required for the assembly of 53BP1 foci and participates in the recruitment and formation of γ H2AX foci in response to ionizing radiation. Furthermore, VRK1 interacts with p53 and is activated by UV-induced DNA damage. Overall, these processes could be an indicator of the crucial role this kinase may play in several steps of DDR. [Objectives]: Our aim is to determine whether VRK1 is activated and participates in the assembly of 53BP1 foci after inducing oxidative stress in human cancer cell lines., [Methods]: To study VRK1 activation by oxidative stress, cell lines were starved and treated with 10 mM of hydrogen peroxide (H2O2). Next, endogenous p53 was immunoprecipitated at different points in time and the level of threonine 18 phosphorylation, which depends on VRK1 activation, was determined with a phosphospecific antibody. Moreover, in vitro assays were performed, using purified protein GST-p53 (1-85) as substrate and analyzing its phosphorylation in threonine 18. Along this same line, the assembly of 53BP1 foci was assessed by immunofluorescence in the presence or absence of VRK1. [Results]: In this work analysis, we can observe that VRK1 is activated by oxidative stress in the absence of serum, which reduces VRK1 activation mediated by growth factors. Exclusively under H2O2 exposition, this kinase is able to activate and phosphorylate p53. In turn, what it is shown is that VRK1 plays an essential role in DNA repair after oxidative stress induction. When cell lines were treated with H2O2, the number of 53BP1 foci increased considerably for the following 30-60 minutes, beginning to decrease at that point. On the contrary, VRK1 depletion was accompanied by a huge reduction of the number of 53BP1 foci, despite the fact that cells had been previously treated with hydrogen peroxide. [Conclusions]: Based on these results, we conclude that VRK1 participates in DNA repair process after H2O2 exposure, because it is activated and required for the assembly of 53BP1 foci under these conditions. Furthermore, it could also be possible that this kinase plays a crucial role in the early steps of DDR, mainly indicated by its hypothetical involvement in the recruitment and formation of γ H2AX foc.

Resumen del póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada (España) del 9 al 12 de septiembre de 2014., El daño local al ADN altera la estructura de la cromatina y el detectar y el responder a esta alteración es probablemente el inicio de la Respuesta al Daño del ADN (RDA). En este contexto, las cromatina quinasas son unos buenos candidatos por participar en la detección de la alteración local de la cromatina por daño al ADN y en sus respuestas celulares. VRK1 es una Ser-Thr quinasa nucleosomal identificada en el nuestro laboratorio como un nuevo sensor de la RDA. Está descrito que VRK1 regula muchos procesos celulares como por ejemplo el ciclo celular, condensación de la cromatina y protección de las células al daño génico por regulación positiva de p53. Nuestros resultados demuestran que su depleción por “knock-down” causa una pérdida global de la acetilación de las histonas independientemente de ATM. También hemos demostrado que VRK1 interacciona directamente con H2AX fosforilándola en Ser139 inmediatamente tras el daño al ADN inducido por radiación ionizante. El efecto sobre la fosforilación de H2AX y la formación de los focos es prevenido por el knock-out de VRK1 y rescatado por la quinasa activa, pero no con la inactiva, demostrando que es necesaria la actividad de VRK1 para la respuesta al daño del ADN. Además, el pretratamiento con los inhibidores de ATM demostró una cooperación de ATM y VRK1 sobre la formación de los focos de reparación. Concluimos que VRK1 es un nuevo componente de la cromatina, que reacciona a sus alteraciones y que participa a la respuesta muy temprana al daño génico, funcionando ella sola o en colaboración de ATM.

The calcium-calmodulin dependent kinase II (CaMKII) is an ubiquitous serine/threonine protein kinase involved in multiple signalings and biological functions. It has been demonstrated that in epithelial and mesenchimal cells CaMKII participates with Ras to Raf-1 activation and that it is necessary for ERK activation by diverse factors. Raf-1 activation is complex. Maximal Raf-1 activation is reached by phosphorylation at Y341 by Src and at S338. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 is not definitively identified. Aim of my thesis is to go more insight into the molecular mechanisms of CaMKII/Raf-1 interaction and to verify the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To this purpose, I investigated the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors, in COS-7 and NIH3T3 cells. Serum, SrcY527 and RasV12 activated CaMKII. CaMKII was necessary for Raf-1 and ERK activation by all these factors. CaMKII was necessary to the phosphorylation of S338 Raf-1 by serum, fibronectin or oncogenic Ras. Conversely, the inhibition of phosphatidylinositol 3-kinase, which in turn activates Pak3, was ineffective. The direct kinase activity of CaMKII on the serine 338 residue, was demonstrated in vitro by interaction of purified kinases. These results demonstrate that CaMKII phosphorylates Raf-1 at S338 and partecipates to ERK activation upon different physiologic and pathologic stimuli in the MAPK cascade. This kinase, might have a role in cancers harbouring oncogenic Ras and could represent a new therapeutic target for pharmacological intervention in these tumors.

The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src (Y527) and Ras (V12) activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol 3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli.

Hashimoto's thyroiditis is the most frequent autoimmune disorder, characterized by the presence of a large lymphocytic infiltration and secretion of inflammatory cytokines in the thyroid. Infiltrating lymphocytes and cytokines play a pivotal role in the progression of HT, characterized by the progressive destruction of the normal follicular architecture of the gland and death of follicular cells, ending with loss of thyroid function. Integrins are plasma membrane receptors for the cell–extra-cellular matrix components, with both structural and signaling functions. Integrin-mediated fibronectin (FN) binding is necessary for the correct function and survival of thyroid follicular cells. The purpose of this study was to determine the effect of interferon-γ (IFN-γ) stimulation on integrin expression and signaling in the thyroid cell. Cytotoxicity, integrin expression, cell adhesion to FN, and FN-stimulated ERK and AKT phosphorylation were determined in a normal human thyroid cell line treated with IFN-γ. IFN-γ induced apoptosis and reduced the expression of the integrin αvβ3. Integrin-mediated cell adhesion to FN was strongly impaired. Similarly, FN-stimulated ERK and AKT phosphorylation were inhibited. In conclusion, our study in a thyroid cell model demonstrates that IFN-γ induces apoptosis and inhibits the expression of the integrin αvβ3, reducing cell adhesion to FN and the succeeding outside-in signaling. These results suggest that integrins mediate the cytotoxic effect of IFN-γ and are involved in the destructive mechanism of autoimmune thyroiditis.