Malignant melanoma is an aggressive cancer known for its notorious resistance to most current therapies. The basic helix-loop-helix microphthalmia transcription factor (MITF) is the master regulator determining the identity and properties of the melanocyte lineage, and is regarded as a lineage-specific 'oncogene' that has a critical role in the pathogenesis of melanoma. MITF promotes melanoma cell proliferation, whereas sustained supression of MITF expression leads to senescence. By combining chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) and RNA sequencing analyses, we show that MITF directly regulates a set of genes required for DNA replication, repair and mitosis. Our results reveal how loss of MITF regulates mitotic fidelity, and through defective replication and repair induces DNA damage, ultimately ending in cellular senescence. These findings reveal a lineage-specific control of DNA replication and mitosis by MITF, providing new avenues for therapeutic intervention in melanoma. The identification of MITF-binding sites and gene-regulatory networks establish a framework for understanding oncogenic basic helix-loop-helix factors such as N-myc or TFE3 in other cancers.

We identified a novel loss-of-function mutation of Nav1.4 that leads to a recessive phenotype combining clinical symptoms and signs of congenital myasthenic syndrome and periodic paralysis, probably by decreasing channel availability for muscle action potential genesis at the neuromuscular junction and propagation along the sarcolemma.

Background and PurposeFor decades, inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been used as tools to investigate the role and function of CFTR conductance in cystic fibrosis research. In the early 2000s, two new and potent inhibitors of CFTR, CFTRinh-172 and GlyH-101, were described and are now widely used to inhibit specifically CFTR. However, despite some evidence, the effects of both drugs on other types of Cl−-conductance have been overlooked. In this context, we explore the specificity and the cellular toxicity of both inhibitors in CFTR-expressing and non–CFTR-expressing cells.Experimental ApproachUsing patch-clamp technique, we tested the effects of CFTRinh-172 and GlyH-101 inhibitors on three distinct types of Cl− currents: the CFTR-like conductance, the volume-sensitive outwardly rectifying Cl− conductance (VSORC) and finally the Ca2+-dependent Cl− conductance (CaCC). We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines.Key ResultsWe confirmed that these two compounds were potent inhibitors of the CFTR-mediated Cl− conductance. However,GlyH-101 also inhibited the VSORC conductance and the CaCC at concentrations used to inhibit CFTR. The CFTRinh-172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM. Neither inhibitor (20 µM; 24 h exposure) affected cell viability, but both were cytotoxic at higher concentrations.Conclusions and ImplicationsBoth inhibitors affected Cl− conductances apart from CFTR. Our results provided insights into their use in mouse models.

DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.

Andersen's syndrome is a rare disorder affecting muscle, heart, and bone that is associated with mutations leading to a loss of function of the inwardly rectifying K channel Kir2.1. Although the Kir2.1 function can be anticipated in excitable cells by controlling the electrical activity, its role in non-excitable cells remains to be investigated. Using Andersen's syndrome-induced pluripotent stem cells, we investigated the cellular and molecular events during the osteoblastic and chondrogenic differentiation that are affected by the loss of the Ik1 current. We show that loss of Kir2.1 channel function impairs both osteoblastic and chondrogenic processes through the downregulation of master gene expression. This downregulation is the result of an impairment of the bone morphogenetic proteins signaling pathway through dephosphorylation of the Smad proteins. Restoring Kir2.1 channel function in Andersen's syndrome cells rescued master genes expression and restored normal osteoblast and chondrocyte behavior. Our results show that Kir2.1-mediated activity controls endochondral and intramembranous ossification signaling pathways. © 2018 American Society for Bone and Mineral Research.

Mutations in NaV1.4, the skeletal muscle voltage-gated Na+ channel, underlie several skeletal muscle channelopathies. We report here the functional characterization of two substitutions targeting the R1451 residue and resulting in 3 distinct clinical phenotypes. The R1451L is a novel pathogenic substitution found in two unrelated individuals. The first individual was diagnosed with non-dystrophic myotonia, whereas the second suffered from an unusual phenotype combining hyperkalemic and hypokalemic episodes of periodic paralysis (PP). The R1451C substitution was found in one individual with a single attack of hypoPP induced by glucocorticoids. To elucidate the biophysical mechanism underlying the phenotypes, we used the patch-clamp technique to study tsA201 cells expressing WT or R1451C/L channels. Our results showed that both substitutions shifted the inactivation to hyperpolarized potentials, slowed the kinetics of inactivation, slowed the recovery from slow inactivation and reduced the current density. Cooling further enhanced these abnormalities. Homology modeling revealed a disruption of hydrogen bonds in the voltage sensor domain caused by R1451C/L. We concluded that the altered biophysical properties of R1451C/L well account for the PMC-hyperPP cluster and that additional factors likely play a critical role in the inter-individual differences of clinical expression resulting from R1451C/L.

Nasu-Hakola disease (NHD) is a recessively inherited rare disorder characterized by a combination of neuropsychiatric and bone symptoms which, while being unique to this disease, do not provide a rationale for the unambiguous identification of patients. These individuals, in fact, are likely to go unrecognized either because they are considered to be affected by other kinds of dementia or by fibrous dysplasia of bone. Given that dementia in NHD has much in common with Alzheimer’s disease and other neurodegenerative disorders, it cannot be expected to achieve the differential diagnosis of this disease without performing a genetic analysis. Under this scenario, the availability of protein biomarkers would indeed provide a novel context to facilitate interpretation of symptoms and to make the precise identification of this disease possible. The work here reported was designed to generate, for the first time, protein profiles of lymphoblastoid cells from NHD patients. Two-dimensional electrophoresis (2-DE) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) have been applied to all components of an Italian family (seven subjects) and to five healthy subjects included as controls. Comparative analyses revealed differences in the expression profile of 21 proteins involved in glucose metabolism and information pathways as well as in stress responses.

Andersen's syndrome (AS) is a rare and dominantly inherited pathology, linked to the inwardly rectifying potassium channel Kir2.1. AS patients exhibit a triad of symptoms that include periodic paralysis, cardiac dysrhythmia and bone malformations. Some progress has been made in understanding the contribution of the Kir2.1 channel to skeletal and cardiac muscle dysfunctions, but its role in bone morphogenesis remains unclear. We isolated myoblast precursors from muscle biopsies of healthy individuals and typical AS patients with dysmorphic features. Myoblast cultures underwent osteogenic differentiation that led to extracellular matrix mineralization. Osteoblastogenesis was monitored through the activity of alkaline phosphatase, and through the hydroxyapatite formation using Alizarin Red and Von Kossa staining techniques. Patch-clamp recordings revealed the presence of an inwardly rectifying current in healthy cells that was absent in AS osteoblasts, showing the dominant-negative effect of the Kir2.1 mutant allele in osteoblasts. We also found that while control cells actively synthesize hydroxyapatite, AS osteoblasts are unable to efficiently form any extracellular matrix. To further demonstrate the role of the Kir2.1 channels during the osteogenesis, we inhibited Kir2.1 channel activity in healthy patient cells by applying extracellular Ba(2+) or using adenoviruses carrying mutant Kir2.1 channels. In both cases, cells were no longer able to produce extracellular matrixes. Moreover, osteogenic activity of AS osteoblasts was restored by rescue experiments, via wild-type Kir2.1 channel overexpression. These observations provide a proof that normal Kir2.1 channel function is essential during osteoblastogenesis.