Estudio de la regulación de los genes Six3 en vertebrados

Teis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular Bioquíca. Fecha de lectura:19-05-201

A trans-Regulatory code for the forebrain expression of Six3.2 in the Medaka fish

A well integrated and hierarchically organized gene regulatory network is responsible for the progressive specification of the forebrain. The transcription factor Six3 is one of the central components of this network. As such, Six3 regulates several components of the network, but its upstream regulators are still poorly characterized. Here we have systematically identified such regulators, taking advantage of the detailed functional characterization of the regulatory region of the medaka fish Six3.2 ortholog and of a time/cost-effective trans-regulatory screening, which complemented and overcame the limitations of in silico prediction approaches. The candidates resulting from this search were validated with dose-response luciferase assays and expression pattern criteria. Reconfirmed candidates with a matching expression pattern were also tested with chromatin immunoprecipitation and functional studies. Our results confirm the previously proposed direct regulation of Pax6 and further demonstrate that Msx2 and Pbx1 are bona fide direct regulators of early Six3.2 distribution in distinct domains of the medaka fish forebrain. They also point to other transcription factors, including Tcf3, as additional regulators of different spatial-temporal domains of Six3.2 expression. The activity of these regulators is discussed in the context of the gene regulatory network proposed for the specification of the forebrain.Spanish Ministerio de Economía y Competitividad (MINECO) Grants BFU2010-16031 and BFU2013-43213-P, cofounded by FEDER Funds; Comunidad Autónoma de Madrid (CAM) Grant CELL-DD S2010/BMD-2315; Fundaluce; Fundación ONCE; the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) del Instituto Carlos III (ISCIII); and an Institutional Grant from the Fundación Ramón Areces.Peer Reviewe

Genetic testing for cerebral cavernous malformations

Abstract Cavernous cerebral malformations (CCM) are vascular malformations of the brain and spinal cord. CCM affect up to 0.5% of the general population, predisposing to headaches, seizures, cerebral hemorrhage and focal neurological deficit. CCM may be familial or sporadic. Familial forms have autosomal dominant inheritance. This Utility Gene Test was prepared on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution

Phosphorylated filamin A regulates actin-linked caveolae dynamics

Caveolae are relatively stable membrane invaginations that compartmentalize signaling, regulate lipid metabolism and mediate viral entry. Caveolae are closely associated with actin fibers and internalize in response to diverse stimuli. Loss of cell adhesion is known to induce rapid and robust caveolae internalization and trafficking toward a Rab11-positive recycling endosome; however, pathways governing this process are poorly understood. Here, we report that filamin A is required to maintain the F-actin-dependent linear distribution of caveolin-1. High spatiotemporal resolution particle tracking of caveolin-1-GFP vesicles by total internal reflection fluorescence (TIRF) microscopy revealed that FLNa is required for the F-actin-dependent arrest of caveolin-1 vesicles in a confined area and their stable anchorage to the plasma membrane. The linear distribution and anchorage of caveolin-1 vesicles are both required for proper caveolin-1 inwards trafficking. De-adhesion-triggered caveolae inward trafficking towards a recycling endosome is impaired in FLNa-depleted HeLa and FLNa-deficient M2-melanoma cells. Inwards trafficking of caveolin-1 requires both the ability of FLNa to bind actin and cycling PKCα-dependent phosphorylation of FLNa on Ser2152 after cell detachment. © 2011. Published by The Company of Biologists Ltd.Peer Reviewe