Regulatory landscape fusion in rhabdomyosarcoma through interactions between the PAX3 promoter and FOXO1 regulatory elements.

BACKGROUND: The organisation of vertebrate genomes into topologically associating domains (TADs) is believed to facilitate the regulation of the genes located within them. A remaining question is whether TAD organisation is achieved through the interactions of the regulatory elements within them or if these interactions are favoured by the pre-existence of TADs. If the latter is true, the fusion of two independent TADs should result in the rewiring of the transcriptional landscape and the generation of ectopic contacts. RESULTS: We show that interactions within the PAX3 and FOXO1 domains are restricted to their respective TADs in normal conditions, while in a patient-derived alveolar rhabdomyosarcoma cell line, harbouring the diagnostic t(2;13)(q35;q14) translocation that brings together the PAX3 and FOXO1 genes, the PAX3 promoter interacts ectopically with FOXO1 sequences. Using a combination of 4C-seq datasets, we have modelled the three-dimensional organisation of the fused landscape in alveolar rhabdomyosarcoma. CONCLUSIONS: The chromosomal translocation that leads to alveolar rhabdomyosarcoma development generates a novel TAD that is likely to favour ectopic PAX3:FOXO1 oncogene activation in non-PAX3 territories. Rhabdomyosarcomas may therefore arise from cells which do not normally express PAX3. The borders of this novel TAD correspond to the original 5'- and 3'- borders of the PAX3 and FOXO1 TADs, respectively, suggesting that TAD organisation precedes the formation of regulatory long-range interactions. Our results demonstrate that, upon translocation, novel regulatory landscapes are formed allowing new intra-TAD interactions between the original loci involved.

Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells.

The dermal Panniculus carnosus (PC) muscle is important for wound contraction in lower mammals and represents an interesting model of muscle regeneration due to its high cell turnover. The resident satellite cells (the bona fide muscle stem cells) remain poorly characterized. Here we analyzed PC satellite cells with regard to developmental origin and purported function. Lineage tracing shows that they originate in Myf5(+), Pax3/Pax7(+) cell populations. Skin and muscle wounding increased PC myofiber turnover, with the satellite cell progeny being involved in muscle regeneration but with no detectable contribution to the wound-bed myofibroblasts. Since hematopoietic stem cells fuse to PC myofibers in the absence of injury, we also studied the contribution of bone marrow-derived cells to the PC satellite cell compartment, demonstrating that cells of donor origin are capable of repopulating the PC muscle stem cell niche after irradiation and bone marrow transplantation but may not fully acquire the relevant myogenic commitment.

MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity.

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.

Estructura y tipología familiar en pacientes con dependencia o abuso de sustancias psicoactivas en un centro de rehabilitación de adicciones en el Municipio de Chía, Cundinamarca / Structure and Family Type in Patients With Substance Abuse or Dependence Psychoactive Rehabilitation Center of Addiction in the Municipality of Chia Cundinamarca

Objetivo: Determinar la estructura y la tipología familiar de un grupo de pacientes con dependencia o abuso de sustancias que se encontraban en un centro de rehabilitación de adicciones durante el periodo comprendido entre agosto y octubre de 2009. Métodos: A través de una metodología descriptiva cualitativa-interpretativa, se estudió a 10 pacientes y sus familias que cumplían los criterios de inclusión por dependencia o abuso de sustancias; el trabajo de campo y las transcripciones se realizaron durante 3 meses mediante observación no participante, entrevista no estructurada y revisión de la historia clínica del paciente. Resultados: De las familias entrevistadas, siete eran monoparentales, con una organización no convencional respecto a «roles de género¼. La familia monoparental favorece la soledad, la dificultad para poner reglas, la desidealización del lugar del padre en la estructura familiar y la búsqueda de una complicidad constante. En el análisis por categorías, se concluyó que en las 10 familias del estudio de personas con adicciones son frecuentes características de la estructura familiar como la comunicación inadecuada, la ausencia de autoridad, reglas y límites, la presencia de triangulaciones, la falta de cohesión dada por la existencia de un patrón de relación desligado y el cambio de roles convencionales con respecto a género. La búsqueda del afecto de la madre ante su ausencia emocional por la sobrecarga en los roles y la falta del padre, suscitada por la separación de la pareja, se encontró como un aspecto esencial subyacente al comportamiento adictivo. Se configura un patrón de abandono parental. Conclusiones: Se confirma lo mencionado por diversos autores acerca de las características de la tipología y la estructura familiar encontradas en pacientes con adicciones, además de su necesidad de afecto junto con la premura por una figura maternal. La tipología familiar no determina por sí misma el abuso de sustancias psicoactivas, sino la influencia de otros factores como la estructura familiar, especialmente las interacciones afectivas deficientes, lo cual debe considerarse en el desarrollo de las estrategias terapéuticas.

Objective: To determine the family type, family structure in a group of patients with a diagnosis of substance abuse or dependence who were at a rehabilitation center for addiction during the period between August and October 2009. Methods: Through a descriptive qualitative-interpretative methodology 10 patients who met inclusion criteria for substance dependence or abuse were studied. The fieldwork and transcripts were made for three months by non-participant observation, non-structured interviews and examination of patients' clinical history. Results: Seven of the families interviewed were single-parent families with an unconventional organization on "gender roles". Single-parent families favored loneliness, difficulty in rule-setting, de-idealization of the place of the father in the family structure and a constant search for complicity. In the analysis by categories, we found that in 10 families in the study of individuals with addictions it is common to find family structure characteristics such as inadequate communication, lack of authority rules and limits, presence of triangulations, the lack of cohesion due to the existence of a disconnected relationship pattern and changed roles compared to conventional gender. The search for the affection of the mother at her emotional overload absence of roles and lack of father, raised by the separation of the couple, was found as an essential aspect underlying the addictive behavior. A pattern of parental abandonment is configured. Conclusion: The findings confirmed what has been mentioned by various authors regarding the characteristics of the family typology structure and personal factors in patients with addictions, in addition to their need for affection combined with the desire for the mother's presence. The family typology does not determine for itself the abuse of psychoactive substances, but the influence of other factors such as family structure, especially deficient affective interactions, which should be considered in the development of therapeutic strategies.

Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in mouse myoblasts using CRISPR-Cas9 nuclease.

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.

A polymorphic enhancer near GREM1 influences bowel cancer risk through differential CDX2 and TCF7L2 binding.

A rare germline duplication upstream of the bone morphogenetic protein antagonist GREM1 causes a Mendelian-dominant predisposition to colorectal cancer (CRC). The underlying disease mechanism is strong, ectopic GREM1 overexpression in the intestinal epithelium. Here, we confirm that a common GREM1 polymorphism, rs16969681, is also associated with CRC susceptibility, conferring ∼20% differential risk in the general population. We hypothesized the underlying cause to be moderate differences in GREM1 expression. We showed that rs16969681 lies in a region of active chromatin with allele- and tissue-specific enhancer activity. The CRC high-risk allele was associated with stronger gene expression, and higher Grem1 mRNA levels increased the intestinal tumor burden in Apc(Min) mice. The intestine-specific transcription factor CDX2 and Wnt effector TCF7L2 bound near rs16969681, with significantly higher affinity for the risk allele, and CDX2 overexpression in CDX2/GREM1-negative cells caused re-expression of GREM1. rs16969681 influences CRC risk through effects on Wnt-driven GREM1 expression in colorectal tumors.

Genetic tracing via DNGR-1 expression history defines dendritic cells as a hematopoietic lineage.

Mononuclear phagocytes are classified as macrophages or dendritic cells (DCs) based on cell morphology, phenotype, or select functional properties. However, these attributes are not absolute and often overlap, leading to difficulties in cell-type identification. To circumvent this issue, we describe a mouse model to define DCs based on their ontogenetic descendence from a committed precursor. We show that precursors of mouse conventional DCs, but not other leukocytes, are marked by expression of DNGR-1. Genetic tracing of DNGR-1 expression history specifically marks cells traditionally ascribed to the DC lineage, and this restriction is maintained after inflammation. Notably, in some tissues, cells previously thought to be monocytes/macrophages are in fact descendants from DC precursors. These studies provide an in vivo model for fate mapping of DCs, distinguishing them from other leukocyte lineages, and thus help to unravel the functional complexity of the mononuclear phagocyte system.

Expression pattern of the FoxO1 gene during mouse embryonic development.

In order to fully describe the expression pattern of the transcription factor FoxO1, we have screened the ES cell genetrap repository databases and obtained a clone that contains the ß-geo reporter gene inserted within intron 1 of FoxO1. We then used the ES cell clone to generate a new mouse strain (B6;129P2- Foxo1(Gt(AD0086)Wtsi/JJC)), which expresses ß-geo according to the endogenous FoxO1 pattern, and collected embryo stages from 7.0dpc to 18.5dpc. We show that the expression of FoxO1 is highly dynamic, starting in the neuroepithelium and then extending into the developing vasculature, including all early stages of heart formation. There is a dramatic switch of expression at 11.5dpc in which most vascular expression is abolished and replaced by skeletal muscle expression. In addition FoxO1 is also expressed in several epithelial structures including the olfactory and otic systems, the cornea and at different levels of the gut depending on developmental stage. At later foetal stages, FoxO1 is upregulated again in the same tissues were it is active during early development, including skeletal muscle, vascular system and neuroepithelium.

Peripheral nerve sheath tumors of the foot and ankle.

BACKGROUND: Peripheral nerve sheath tumors (PNSTs) are soft tissue neoplasms found in intimate association with a peripheral nerve. They are rarely seen in the foot and ankle where they have an innocuous appearance, but these tumors have the potential to become malignant. This study reports a large series of foot and ankle PNSTs surgically treated at a single institution. MATERIALS AND METHODS: Retrospectively, all cases of PNSTs confirmed by biopsy and surgically treated from 1992 to 2008 were included in the study. Preoperative, perioperative and postoperative variables were collected. Foot and ankle tumors were compared with the overall group of PNSTs. Fisher's test and Student's t-test were implemented to address the significance of the findings (p ≤ 0.05). RESULTS: From all PNSTs identified (n = 137), there were 14 (10.2%) foot and ankle. A painful mass was the most common presenting symptom. Schwannoma represented the most common histologic type. Two cases of malignant foot and ankle PNSTs were identified. Foot and ankle PNSTs were significantly smaller in size and showed fewer postoperative neurologic deficits than the overall group of PNSTs. No differences were found in terms of histologic type distribution, age at diagnosis, duration of symptoms and postoperative tumor recurrence, but there was a preponderance of females in this series of foot and ankle tumors. CONCLUSION: PNSTs of the foot and ankle usually present as innocuous, slow-growing masses leading to misdiagnosis and suboptimal surgical treatment of a potentially malignant lesion. A comprehensive diagnostic evaluation, preoperative planning and meticulous surgical excision are required to prevent tumor recurrence and minimize the incidence of postoperative sequelae, thus improving postoperative functional outcomes.

A novel Gli3 enhancer controls the Gli3 spatiotemporal expression pattern through a TALE homeodomain protein binding site.

The zinc finger transcription factor Gli3 is an essential mediator of hedgehog signaling. Gli3 has a dynamic expression pattern during embryonic development. In the neural tube, Gli3 transcripts are patterned along the anteroposterior and dorsoventral axes such that the initial broad expression in the posterior neural tube becomes dorsally restricted as neurogenesis takes place. Little is known about the molecular mechanisms that regulate this dynamic expression. Here, we report on a phylogenetic analysis of the Gli3 locus that uncovered a novel regulatory element, HCNE1. HCNE1 contains a compound Pbx/Meis binding site that binds Pbx and Meis/Prep proteins in vitro and in vivo. We show that HCNE1 recapitulates Gli3 expression in the developing neural tube and that mutations in the Pbx/Meis binding site affect the spatiotemporal control of HCNE1 transcriptional activity. Ectopic expression or loss of function of Pbx and Meis/Prep proteins in the chick and mouse embryo results in aberrant expression of endogenous Gli3 transcripts. We propose a novel role for TALE proteins in establishing the correct spatiotemporal expression pattern of Gli3 in the vertebrate spinal cord, thus implicating TALE transcription factors in early embryonic patterning events controlled by Sonic hedgehog signaling.

Estudio Comparativo sobre técnicas quirúrgicas en la luxación recidivante anterior de hombro

Este es un equilibrio descriptivo retrospectivo-prospectivo, sobre el tratamiento quirúrgico de la Luxación Recidivante Anterior de Hombro, en el cual se compararon las técnicas de Magnuson, Bankart-Magnuson modificado (Hermida) y Dutoir-Roux con la cirugía de Bankart-Putti-Platt modificado (Rockwood), las cuales tienen en común que sólo reparan tejidos blandos, lo que hace estos 2 grupos comparables y no vicia los resultados. Los hallazgos demostraron que el procedimiento de Rockwood ofrece ventajas técnicas clínicas, económicas y de pronóstico sobre los anteriores. La Técnica de Bankart-Putti-Platt modificado (Rockwood), en conclusión, es buena opción para el tratamiento de la Luxación Recidivante Anterior de Hombro en nuestro medio