Role of enhancer activity and chromatin architecture on the regulation and evolution of developmental gene expression: The HoxD cluster paradigm.

Los genes Hox, también conocidos como genes arquitecto por su papel en la especificación del bauplan de vertebrados e invertebrados, se expresan en distintas combinaciones en varias estructuras embrionarias. Estudiando la regulación de estos genes hemos podido comprender mejor como la actividad de secuencias “enhancer” y la organización 3D de la cromatina contribuyen al control de la expresión génica y a su evolución.Gracias a la ayuda para conferenciantes de los fondos propios del Departamento de Biología Molecular y Bioquímica Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

The population of young low-mass stars in Trumpler 14

Massive star-forming regions are thought to be the most common birth environments in the Galaxy and the only birth places of very massive stars. Their presence in the stellar cluster alters the conditions within the cluster impacting at the same time the evolution of other cluster members. In principle, copious amounts of ultraviolet radiation produced by massive stars can remove material from outer parts of the protoplanetary disks around low- and intermediate-mass stars in the process of external photoevaporation, effectively reducing the planet-formation capabilities of those disks. Here, we present deep VLT/MUSE observations of low-mass stars in Trumpler 14, one of the most massive, young, and compact clusters in the Carina Nebula Complex. We provide spectral and stellar properties of 717 sources and based on the distribution of stellar ages derive the cluster age of $\sim$1~Myr. The majority of the stars in our sample have masses $\leqslant$1~$M_\odot$, what makes our spectroscopic catalogue the most deep to date in term of masses, and proves that detailed investigations of low-mass stars are possible in the massive but distant regions. Spectroscopic studies of low-mass members of the whole Carina Nebula Complex are missing. Our work provides an important step forward towards filling this gap and set the stage for follow-up investigation of accretion properties in Trumpler 14.Comment: Accepted for publication in A&A, 27 pages, 28 figure

The HoxD cluster is a dynamic and resilient TAD boundary controlling the segregation of antagonistic regulatory landscapes

The mammalian HoxD cluster lies between two topologically associating domains (TADs) matching distinct enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of Hoxd genes in forearm cells, whereas the centromeric TAD subsequently regulates more posterior Hoxd genes in digit cells. Therefore, the TAD boundary prevents the terminal Hoxd13 gene from responding to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryos, we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts of this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient, and only a 400-kb deletion, including the whole-gene cluster, was eventually able to merge the neighboring TADs into a single structure. In this unified TAD, both proximal and distal limb enhancers nevertheless continued to work independently over a targeted transgenic reporter construct. We propose that the whole HoxD cluster is a dynamic TAD border and that the exact boundary position varies depending on both the transcriptional status and the developmental context. Press

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

The logic of gene regulatory networks in early vertebrate forebrain patterning

The vertebrate forebrain or prosencephalon is patterned at the beginning of neurulation into four major domains: the telencephalic, hypothalamic, retinal and diencephalic anlagen. These domains will then give rise to the majority of the brain structures involved in sensory integration and the control of higher intellectual and homeostatic functions. Understanding how forebrain pattering arises has thus attracted the interest of developmental neurobiologists for decades. As a result, most of its regulators have been identified and their hierarchical relationship is now the object of active investigation. Here, we summarize the main morphogenetic pathways and transcription factors involved in forebrain specification and propose the backbone of a possible gene regulatory network (GRN) governing its specification, taking advantage of the GRN principles elaborated by pioneer studies in simpler organisms. We will also discuss this GRN and its operational logic in the context of the remarkable morphological and functional diversification that the forebrain has undergone during evolution. © 2012 Elsevier Ireland Ltd.the Spanish MICINN (BFU2010-16031); Comunidad Autonoma de Madrid (CAM, CELL-DD S2010/BMD-2315); Fundaluce; Fundación ONCE; CIBERER.Peer Reviewe