Control of neuronal differentiation by sumoylation of BRAF35, a subunit of the LSD1-CoREST histone demethylase complex.

The LSD1-CoREST histone demethylase complex is required to repress neuronal genes in nonneuronal tissues. Here we show that sumoylation of Braf35, one of the subunits of the complex, is required to maintain full repression of neuron-specific genes and for occupancy of the LSD1-CoREST complex at its gene targets. Interestingly, expression of Braf35 was sufficient to prevent neuronal differentiation induced by bHLH neurogenic transcription factors in P19 cells and in neuronal progenitors of the chicken embryo neural tube. Sumoylation of Braf35 is required for this antineurogenic activity. We also show that iBraf, a paralogue of Braf35, forms heterodimers with Braf35. Braf35-iBraf heterodimerization impairs Braf35 interaction with the LSD1-CoREST complex and inhibits Braf35 sumoylation. Consistent with these results, iBraf prevents the antineurogenic activity of Braf35 in vivo. Our data uncover a mechanism of regulation of the LSD1-CoREST complex and provide a molecular explanation for the antagonism between Braf35 and iBraf in neuronal differentiation.

Microtubule nucleation at the cis-side of the Golgi apparatus requires AKAP450 and GM130.

We report that microtubule (MT) nucleation at the Golgi apparatus requires AKAP450, a centrosomal gamma-TuRC-interacting protein that also forms a distinct network associated with the Golgi. Depletion of AKAP450 abolished MT nucleation at the Golgi, whereas depletion of the cis-Golgi protein GM130 led to the disorganisation of AKAP450 network and impairment of MT nucleation. Brefeldin-A treatment induced relocalisation of AKAP450 to ER exit sites and concomitant redistribution of MT nucleation capacity to the ER. AKAP450 specifically binds the cis-side of the Golgi in an MT-independent, GM130-dependent manner. Short AKAP450-dependent growing MTs are covered by CLASP2. Like for centrosome, dynein/dynactin complexes are necessary to anchor MTs growing from the Golgi. We further show that Golgi-associated AKAP450 has a role in cell migration rather than in cell polarisation of the centrosome-Golgi apparatus. We propose that the recruitment of AKAP450 on the Golgi membranes through GM130 allows centrosome-associated nucleating activity to extend to the Golgi, to control the assembly of subsets of MTs ensuring specific functions within the Golgi or for transporting specific cargos to the cell periphery.

Cell consequences of loss of function of the epigenetic factor EHMT1.

EHMT1 is an epigenetic factor with histone methyltransferase activity that appears mutated in Kleefstra syndrome, a neurodevelopmental genetic disorder characterized by developmental delay, intellectual disability, and autistic-like features. Despite recent progress in the study of the function of this gene and the molecular etiology of the disease, our knowledge of how EHMT1 haploinsufficiency causes Kleefstra syndrome is still very limited. Here, we show that EHMT1 depletion in RPE1 cells leads to alterations in the morphology and distribution of different subcellular structures, such as the Golgi apparatus, the lysosomes and different cell adhesion components. EHMT1 downregulation also increases centriolar satellites detection, which may indicate a role for EHMT1 in centrosome functioning. Furthermore, the migration process is also altered in EHMT1 depleted cells, which show reduced migration capacity. We consider that the described phenotypes could open new possibilities for understanding the functional impact of EHMT1 haploinsufficiency in Kleefstra syndrome, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in this neurodevelopmental disorder. This knowledge could be relevant not only for the treatment of this syndrome, but also for other neurodevelopmental conditions that could share similar deregulated cellular pathways.

MRGBP, a member of the NuA4 complex, inhibits DNA double-strand break repair.

The repair of DNA breaks takes place in the context of chromatin and thus involves the activity of chromatin remodelers. The nucleosome acetyltransferase of H4 (NuA4) remodeler complex enables DNA break repair by relaxing flanking chromatin. Here, we show that MRG domain binding protein (MRGBP), a member of this complex, acts as a general inhibitor of DNA double-strand break repair. Upon its downregulation, repair is generally increased. This is particularly evident for the stimulation of early events of homologous recombination. Thus, MRGBP has an opposing role to the main catalytic subunits of the NuA4 complex. Our data suggest that MRGBP acts by limiting the activity of this complex in DNA repair, specifically by narrowing the extent of DNA-end resection.

Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms.

BACKGROUND: The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes in vitro has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind in vivo and how this contributes to GMAP210 localisation and function remains to be investigated. RESULTS: By using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi in vivo. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER) exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein. CONCLUSION: We conclude that the Golgi localisation of GMAP210 is the result of the combined action of the two N- and C-terminal domains that recognise different sub-regions of the cis-GA. Based on present and previous data, we propose a model in which GMAP210 would participate in homotypic fusion of cis-cisternae by anchoring the surface of cisternae via its C-terminus and projecting its distal N-terminus to bind the rims or to stabilise tubular structures connecting neighbouring cis-cisternae.

TBL1 is required for the mesenchymal phenotype of transformed breast cancer cells.

The epithelial-to-mesenchymal transition (EMT) and its reversion (MET) are related to tumor cell dissemination and migration, tumor circulating cell generation, cancer stem cells, chemoresistance, and metastasis formation. To identify chromatin and epigenetic factors possibly involved in the process of EMT, we compare the levels of expression of epigenetic genes in a transformed human breast epithelial cell line (HMEC-RAS) versus a stable clone of the same cell line expressing the EMT master regulator ZEB1 (HMEC-RAS-ZEB1). One of the factors strongly induced in the HMEC-RAS-ZEB1 cells was Transducin beta-like 1 (TBL1), a component of the NCoR complex, which has both corepressor and coactivator activities. We show that TBL1 interacts with ZEB1 and that both factors cooperate to repress the promoter of the epithelial gene E-cadherin (CDH1) and to autoactivate the ZEB1 promoter. Consistent with its central role, TBL1 is required for mesenchymal phenotypes of transformed breast epithelial and breast cancer cell lines of the claudin-low subtype. Importantly, a high expression of the TBL1 gene correlates with poor prognosis and increased proportion of metastasis in breast cancer patients, indicating that the level of TBL1 expression can be used as a prognostic marker.

Percepciones sobre la gestión, exposición, bioseguridad y manipulación de citostáticos en el personal de enfermería de una institución de salud privada de la Ciudad Autónoma de Buenos Aires / Perceptions on the management, exposure, biosafety and handling of cytostatics in the nursing staff of a private health institution in the Autonomous City of Buenos Aires

Introducción: El profesional de enfermería oncológica es formado con conocimientos sobre el cáncer, modalidades de tratamiento, escenarios de cuidado del paciente. El riesgo a la exposición resultante de la manipulación de los citostáticos puede afectar la salud de los agentes sanitarios. Objetivo: describir las medidas de bioseguridad para la manipulación de citostáticos y los signos clínicos y síntomas producto de la exposición a estos medicamentos en el personal de enfermería de una institución de salud privada de la Ciudad Autónoma de Buenos Aires. Métodos: Se realizó un estudio observacional, descriptivo, de corte transversal, retrospectivo. La muestra estuvo conformada por 31 profesionales de enfermería. Como instrumento recolección de datos se utilizó el propuesto por Villa y Varela-Díaz (2020), este instrumento registra datos sociodemográficos, laborales, de salud y sobre medidas de bioseguridad. Resultados: La muestra estuvo confirmada por un 74,2% del sexo femenino, la edad promedio fue de 37,67±6,79, el 58,1% era Licenciado/a en Enfermería, con una experiencia promedio en oncología de 4,06±4,09. El 96,8 % de los participantes administraba citostáticos y el 51,6 % participaba en el desecho. Los principales síntomas reportados fueron la cefalea y el dolor abdominal con 64,5% y 45,2% respectivamente. El 41,9% refiere la realización de exámenes paraclínicos y control por parte de la institución. Conclusiones: El personal de enfermería está expuesto a altos riesgos laborales cuando se trata de citostáticos. Se requiere el cumplimiento de los protocolos el manejo y descarte, con la finalidad de elevar los estándares de seguridad del paciente y seguridad laboral (AU)