Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping

Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity.

Shying away: An observer-based microprocess analysis of experiential avoidance and resource activation in outpatients with generalized anxiety spectrum disorder

Despite long-standing calls for patient-focused research on individuals with generalized anxiety spectrum disorder there is little systematized knowledge about the in-session behaviors of these patients. The primary objective of this study was to describe of in-session trajectories of the patients' level of explication (as an indicator of an elaborated exposure of negative emotionality) and the patients' focus on their own resources and how these trajectories are associated with post-treatment outcome. In respect to GAD patients, a high level of explication might be seen as an indicator of successful exposure of avoided negative emotionality during therapy sessions. Observers made minute-by-minute ratings of 1100 minutes of video of 20 patients-therapists dyads. The results indicated that a higher level of explication generally observed at a later stage during the therapy sessions and the patients' focus on competencies at an early stage was highly associated with positive therapy outcome at assessment at post treatment, independent of pretreatment distress, rapid response of well-being and symptom reduction, as well as the therapists' professional experience and therapy lengths. These results will be discussed under the perspective of emotion regulation of patients and therapist's counterregulation. It is assumed that GAD-Patients are especially skilled in masking difficult emotions. Explication level and emotion regulation are important variables for this patient group but there's relation to outcome is different.

Functional analysis of histones H3 and H4 in Tup1 repression and in GCN5 activation

Tup1 forms a complex with Ssn6 in yeast. Ssn6-Tup1 complex is recruited via direct interactions with specific DNA binding proteins to a specific promoter region and mediates repression of several sets of genes including a-cell specific genes (asg) in $\alpha$ cells. It has been shown that repression of asgs also requires histone H4 and that Tup1 can directly interact with H3 and H4 in vitro. To address whether histone H3 is required for the repression of asgs, I have examined the effect of H3 and H4 mutations on the expression of a $\alpha$2-controlled LacZ reporter. Assay of $\beta$-glactosidase shows that mutations in either H3 or H4 cause a weak derepression of the reporter gene. Some double mutations result in a stronger derepression, while others do not. The H3 N-terminal deletion also leads to a slightly decreased expression of the reporter gene in $\alpha$ cells. Our data suggest that the N-termini of both H3 and H4 are cooperatively involved in the repression of a-cell specific genes in $\alpha$ cells, possibly through their interaction with Tup1.^ GCN5 was originally identified as a transcriptional regulator required to activate a subset of genes in yeast. Recently, it has been shown that GCN5 encodes the catalytic subunit of a nuclear histone acetyltransferase, providing the first direct link between histone acetylation and gene transcription. Recombinant Gcn5p (rGcn5p) exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. In order to define the sites of histone acetylation mediated by Gcn5p in vivo and assess the significance of histone acetylation, more than 30 yeast strains have been constructed to bear specific H3 and/or H4 mutations in the presence or absence of GCN5 function. Our genetic data suggest that Gcn5p may have additional targets in vivo that are not identified as the targets of rGcn5p by previous studies. Western analysis using antibodies specifically recognizing particular acetylated isoforms of H3 and H4 led us to conclude that Gcn5p is necessary for full acetylation of multiple sites in both H3 and H4 in vivo. Consistent with these observations, rGcn5p still acetylates histones H3 and H4 bearing mutations either in H3 K14 or H4 K8,16, sites previously identified as the targets of acetylation by rGcn5p in H3 and H4. Our data also demonstrated that Gcn5p-mediated acetylation events are important for normal progression of the cell cycle and for transcriptional activation. Furthermore, a critical overall level of acetylation is essential for cell viability. ^

The human vestibular cortex revealed by coordinate-based activation likelihood estimation meta-analysis.

The vestibular system contributes to the control of posture and eye movements and is also involved in various cognitive functions including spatial navigation and memory. These functions are subtended by projections to a vestibular cortex, whose exact location in the human brain is still a matter of debate (Lopez and Blanke, 2011). The vestibular cortex can be defined as the network of all cortical areas receiving inputs from the vestibular system, including areas where vestibular signals influence the processing of other sensory (e.g. somatosensory and visual) and motor signals. Previous neuroimaging studies used caloric vestibular stimulation (CVS), galvanic vestibular stimulation (GVS), and auditory stimulation (clicks and short-tone bursts) to activate the vestibular receptors and localize the vestibular cortex. However, these three methods differ regarding the receptors stimulated (otoliths, semicircular canals) and the concurrent activation of the tactile, thermal, nociceptive and auditory systems. To evaluate the convergence between these methods and provide a statistical analysis of the localization of the human vestibular cortex, we performed an activation likelihood estimation (ALE) meta-analysis of neuroimaging studies using CVS, GVS, and auditory stimuli. We analyzed a total of 352 activation foci reported in 16 studies carried out in a total of 192 healthy participants. The results reveal that the main regions activated by CVS, GVS, or auditory stimuli were located in the Sylvian fissure, insula, retroinsular cortex, fronto-parietal operculum, superior temporal gyrus, and cingulate cortex. Conjunction analysis indicated that regions showing convergence between two stimulation methods were located in the median (short gyrus III) and posterior (long gyrus IV) insula, parietal operculum and retroinsular cortex (Ri). The only area of convergence between all three methods of stimulation was located in Ri. The data indicate that Ri, parietal operculum and posterior insula are vestibular regions where afferents converge from otoliths and semicircular canals, and may thus be involved in the processing of signals informing about body rotations, translations and tilts. Results from the meta-analysis are in agreement with electrophysiological recordings in monkeys showing main vestibular projections in the transitional zone between Ri, the insular granular field (Ig), and SII.

Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

A Comparison of different Uncertainty Activation Cross-Section Data Libraries and collapsed values for different neutron spectra: ADS, FISSION and FUSION

PART I:Cross-section uncertainties under differentneutron spectra. PART II: Processing uncertainty libraries

Analysis of Neutron Thermal Scattering Data Uncertainties in PWRs

Analysis of Neutron Thermal Scattering Data Uncertainties in PWRs

Application of a Bayesian/Generalised Least-Squares method to generate correlations between independent neutron fission yield data

Fission product yields are fundamental parameters for several nuclear engineering calculations and in particular for burn-up/activation problems. The impact of their uncertainties was widely studied in the past and valuations were released, although still incomplete. Recently, the nuclear community expressed the need for full fission yield covariance matrices to produce inventory calculation results that take into account the complete uncertainty data. In this work, we studied and applied a Bayesian/generalised least-squares method for covariance generation, and compared the generated uncertainties to the original data stored in the JEFF-3.1.2 library. Then, we focused on the effect of fission yield covariance information on fission pulse decay heat results for thermal fission of 235U. Calculations were carried out using different codes (ACAB and ALEPH-2) after introducing the new covariance values. Results were compared with those obtained with the uncertainty data currently provided by the library. The uncertainty quantification was performed with the Monte Carlo sampling technique. Indeed, correlations between fission yields strongly affect the statistics of decay heat. Introduction Nowadays, any engineering calculation performed in the nuclear field should be accompanied by an uncertainty analysis. In such an analysis, different sources of uncertainties are taken into account. Works such as those performed under the UAM project (Ivanov, et al., 2013) treat nuclear data as a source of uncertainty, in particular cross-section data for which uncertainties given in the form of covariance matrices are already provided in the major nuclear data libraries. Meanwhile, fission yield uncertainties were often neglected or treated shallowly, because their effects were considered of second order compared to cross-sections (Garcia-Herranz, et al., 2010). However, the Working Party on International Nuclear Data Evaluation Co-operation (WPEC)

High power beam dump project for the accelerator prototype LIPAc: Cooling design and analysis

En el campo de la fusión nuclear y desarrollándose en paralelo a ITER (International Thermonuclear Experimental Reactor), el proyecto IFMIF (International Fusion Material Irradiation Facility) se enmarca dentro de las actividades complementarias encaminadas a solucionar las barreras tecnológicas que aún plantea la fusión. En concreto IFMIF es una instalación de irradiación cuya misión es caracterizar materiales resistentes a condiciones extremas como las esperadas en los futuros reactores de fusión como DEMO (DEMOnstration power plant). Consiste de dos aceleradores de deuterones que proporcionan un haz de 125 mA y 40 MeV cada uno, que al colisionar con un blanco de litio producen un flujo neutrónico intenso (1017 neutrones/s) con un espectro similar al de los neutrones de fusión [1], [2]. Dicho flujo neutrónico es empleado para irradiar los diferentes materiales candidatos a ser empleados en reactores de fusión, y las muestras son posteriormente examinadas en la llamada instalación de post-irradiación. Como primer paso en tan ambicioso proyecto, una fase de validación y diseño llamada IFMIFEVEDA (Engineering Validation and Engineering Design Activities) se encuentra actualmente en desarrollo. Una de las actividades contempladas en esta fase es la construcción y operación de una acelarador prototipo llamado LIPAc (Linear IFMIF Prototype Accelerator). Se trata de un acelerador de deuterones de alta intensidad idéntico a la parte de baja energía de los aceleradores de IFMIF. Los componentes del LIPAc, que será instalado en Japón, son suministrados por diferentes países europeos. El acelerador proporcionará un haz continuo de deuterones de 9 MeV con una potencia de 1.125 MW que tras ser caracterizado con diversos instrumentos deberá pararse de forma segura. Para ello se requiere un sistema denominado bloque de parada (Beam Dump en inglés) que absorba la energía del haz y la transfiera a un sumidero de calor. España tiene el compromiso de suministrar este componente y CIEMAT (Centro de Investigaciones Energéticas Medioambientales y Tecnológicas) es responsable de dicha tarea. La pieza central del bloque de parada, donde se para el haz de iones, es un cono de cobre con un ángulo de 3.5o, 2.5 m de longitud y 5 mm de espesor. Dicha pieza está refrigerada por agua que fluye en su superficie externa por el canal que se forma entre el cono de cobre y otra pieza concéntrica con éste. Este es el marco en que se desarrolla la presente tesis, cuyo objeto es el diseño del sistema de refrigeración del bloque de parada del LIPAc. El diseño se ha realizado utilizando un modelo simplificado unidimensional. Se han obtenido los parámetros del agua (presión, caudal, pérdida de carga) y la geometría requerida en el canal de refrigeración (anchura, rugosidad) para garantizar la correcta refrigeración del bloque de parada. Se ha comprobado que el diseño permite variaciones del haz respecto a la situación nominal siendo el flujo crítico calorífico al menos 2 veces superior al nominal. Se han realizado asimismo simulaciones fluidodinámicas 3D con ANSYS-CFX en aquellas zonas del canal de refrigeración que lo requieren. El bloque de parada se activará como consecuencia de la interacción del haz de partículas lo que impide cualquier cambio o reparación una vez comenzada la operación del acelerador. Por ello el diseño ha de ser muy robusto y todas las hipótesis utilizadas en la realización de éste deben ser cuidadosamente comprobadas. Gran parte del esfuerzo de la tesis se centra en la estimación del coeficiente de transferencia de calor que es determinante en los resultados obtenidos, y que se emplea además como condición de contorno en los cálculos mecánicos. Para ello por un lado se han buscado correlaciones cuyo rango de aplicabilidad sea adecuado para las condiciones del bloque de parada (canal anular, diferencias de temperatura agua-pared de decenas de grados). En un segundo paso se han comparado los coeficientes de película obtenidos a partir de la correlación seleccionada (Petukhov-Gnielinski) con los que se deducen de simulaciones fluidodinámicas, obteniendo resultados satisfactorios. Por último se ha realizado una validación experimental utilizando un prototipo y un circuito hidráulico que proporciona un flujo de agua con los parámetros requeridos en el bloque de parada. Tras varios intentos y mejoras en el experimento se han obtenido los coeficientes de película para distintos caudales y potencias de calentamiento. Teniendo en cuenta la incertidumbre de las medidas, los valores experimentales concuerdan razonablemente bien (en el rango de 15%) con los deducidos de las correlaciones. Por motivos radiológicos es necesario controlar la calidad del agua de refrigeración y minimizar la corrosión del cobre. Tras un estudio bibliográfico se identificaron los parámetros del agua más adecuados (conductividad, pH y concentración de oxígeno disuelto). Como parte de la tesis se ha realizado asimismo un estudio de la corrosión del circuito de refrigeración del bloque de parada con el doble fin de determinar si puede poner en riesgo la integridad del componente, y de obtener una estimación de la velocidad de corrosión para dimensionar el sistema de purificación del agua. Se ha utilizado el código TRACT (TRansport and ACTivation code) adaptándalo al caso del bloque de parada, para lo cual se trabajó con el responsable (Panos Karditsas) del código en Culham (UKAEA). Los resultados confirman que la corrosión del cobre en las condiciones seleccionadas no supone un problema. La Tesis se encuentra estructurada de la siguiente manera: En el primer capítulo se realiza una introducción de los proyectos IFMIF y LIPAc dentro de los cuales se enmarca esta Tesis. Además se describe el bloque de parada, siendo el diseño del sistema de rerigeración de éste el principal objetivo de la Tesis. En el segundo y tercer capítulo se realiza un resumen de la base teórica así como de las diferentes herramientas empleadas en el diseño del sistema de refrigeración. El capítulo cuarto presenta los resultados del relativos al sistema de refrigeración. Tanto los obtenidos del estudio unidimensional, como los obtenidos de las simulaciones fluidodinámicas 3D mediante el empleo del código ANSYS-CFX. En el quinto capítulo se presentan los resultados referentes al análisis de corrosión del circuito de refrigeración del bloque de parada. El capítulo seis se centra en la descripción del montaje experimental para la obtención de los valores de pérdida de carga y coeficiente de transferencia del calor. Asimismo se presentan los resultados obtenidos en dichos experimentos. Finalmente encontramos un capítulo de apéndices en el que se describen una serie de experimentos llevados a cabo como pasos intermedios en la obtención del resultado experimental del coeficiente de película. También se presenta el código informático empleado para el análisis unidimensional del sistema de refrigeración del bloque de parada llamado CHICA (Cooling and Heating Interaction and Corrosion Analysis). ABSTRACT In the nuclear fusion field running in parallel to ITER (International Thermonuclear Experimental Reactor) as one of the complementary activities headed towards solving the technological barriers, IFMIF (International Fusion Material Irradiation Facility) project aims to provide an irradiation facility to qualify advanced materials resistant to extreme conditions like the ones expected in future fusion reactors like DEMO (DEMOnstration Power Plant). IFMIF consists of two constant wave deuteron accelerators delivering a 125 mA and 40 MeV beam each that will collide on a lithium target producing an intense neutron fluence (1017 neutrons/s) with a similar spectra to that of fusion neutrons [1], [2]. This neutron flux is employed to irradiate the different material candidates to be employed in the future fusion reactors, and the samples examined after irradiation at the so called post-irradiative facilities. As a first step in such an ambitious project, an engineering validation and engineering design activity phase called IFMIF-EVEDA (Engineering Validation and Engineering Design Activities) is presently going on. One of the activities consists on the construction and operation of an accelerator prototype named LIPAc (Linear IFMIF Prototype Accelerator). It is a high intensity deuteron accelerator identical to the low energy part of the IFMIF accelerators. The LIPAc components, which will be installed in Japan, are delivered by different european countries. The accelerator supplies a 9 MeV constant wave beam of deuterons with a power of 1.125 MW, which after being characterized by different instruments has to be stopped safely. For such task a beam dump to absorb the beam energy and take it to a heat sink is needed. Spain has the compromise of delivering such device and CIEMAT (Centro de Investigaciones Energéticas Medioambientales y Tecnológicas) is responsible for such task. The central piece of the beam dump, where the ion beam is stopped, is a copper cone with an angle of 3.5o, 2.5 m long and 5 mm width. This part is cooled by water flowing on its external surface through the channel formed between the copper cone and a concentric piece with the latter. The thesis is developed in this realm, and its objective is designing the LIPAc beam dump cooling system. The design has been performed employing a simplified one dimensional model. The water parameters (pressure, flow, pressure loss) and the required annular channel geometry (width, rugoisty) have been obtained guaranteeing the correct cooling of the beam dump. It has been checked that the cooling design allows variations of the the beam with respect to the nominal position, being the CHF (Critical Heat Flux) at least twice times higher than the nominal deposited heat flux. 3D fluid dynamic simulations employing ANSYS-CFX code in the beam dump cooling channel sections which require a more thorough study have also been performed. The beam dump will activateasaconsequenceofthe deuteron beam interaction, making impossible any change or maintenance task once the accelerator operation has started. Hence the design has to be very robust and all the hypotheses employed in the design mustbecarefully checked. Most of the work in the thesis is concentrated in estimating the heat transfer coefficient which is decisive in the obtained results, and is also employed as boundary condition in the mechanical analysis. For such task, correlations which applicability range is the adequate for the beam dump conditions (annular channel, water-surface temperature differences of tens of degrees) have been compiled. In a second step the heat transfer coefficients obtained from the selected correlation (Petukhov- Gnielinski) have been compared with the ones deduced from the 3D fluid dynamic simulations, obtaining satisfactory results. Finally an experimental validation has been performed employing a prototype and a hydraulic circuit that supplies a flow with the requested parameters in the beam dump. After several tries and improvements in the experiment, the heat transfer coefficients for different flows and heating powers have been obtained. Considering the uncertainty in the measurements the experimental values agree reasonably well (in the order of 15%) with the ones obtained from the correlations. Due to radiological reasons the quality of the cooling water must be controlled, hence minimizing the copper corrosion. After performing a bibligraphic study the most adequate water parameters were identified (conductivity, pH and dissolved oxygen concentration). As part of this thesis a corrosion study of the beam dump cooling circuit has been performed with the double aim of determining if corrosion can pose a risk for the copper beam dump , and obtaining an estimation of the corrosion velocitytodimension the water purification system. TRACT code(TRansport and ACTivation) has been employed for such study adapting the code for the beam dump case. For such study a collaboration with the code responsible (Panos Karditsas) at Culham (UKAEA) was established. The work developed in this thesis has supposed the publication of three articles in JCR journals (”Journal of Nuclear Materials” y ”Fusion Engineering and Design”), as well as presentations in more than four conferences and relevant meetings.

A logical analysis of T cell activation and anergy

Interaction of the antigen-specific receptor of T lymphocytes with its antigenic ligand can lead either to cell activation or to a state of profound unresponsiveness (anergy). Although subtle changes in the nature of the ligand or of the antigen-presenting cell have been shown to affect the outcome of T cell receptor ligation, the mechanism by which the same receptor can induce alternative cellular responses is not completely understood. A model for explaining both positive (cell proliferation and cytokine production) and negative (anergy induction) signaling of T lymphocytes is described herein. This model relies on the autophosphorylative properties of the tyrosine kinases associated with the T cell receptor. One of its basic assumptions is that the kinase activity of these receptor-associated enzymes remains above background level after ligand removal and is responsible for cellular unresponsiveness. Using a simple Boolean formalism, we show how the timing of the binding and intracellular signal-transduction events can affect the properties of receptor signaling and determine the type of cellular response. The present approach integrates into a common framework a large body of experimental observations and allows specification of conditions leading to cellular activation or to anergy.

Analysis of exocytosis mutants indicates close coupling between regulated secretion and transcription activation in Tetrahymena

Stimulation of regulated secretory cells promotes protein release via the fusion of cytoplasmic storage vesicles with the plasma membrane. In Tetrahymena thermophila, brief exposure to secretagogue results in synchronous fusion of the entire set of docked dense-core granules with the plasma membrane. We show that stimulation is followed by rapid new dense-core granule synthesis involving gene induction. Two genes encoding granule matrix proteins, GRL1 and GRL4, are shown to undergo induction following stimulation, resulting in ≈10-fold message accumulation within 1 h. The mechanism of induction involves transcriptional regulation, and the upstream region of GRL1 functions in vivo as an inducible promoter in a heterologous reporter construct using the gene encoding green fluorescent protein. Taking advantage of the characterized exocytosis (exo−) mutants available in this system, we asked whether the signals for regranulation were generated directly by the initial stimulation, or whether downstream events were required for transcription activation. Three mutants, with defects at three distinct stages in the regulated secretory pathway, failed to show induction of GRL1 and GRL4 after exposure to secretagogue. These results argue that regranulation depends upon signals generated by the final steps in exocytosis.

Activation and Functional Analysis of Janus Kinase 2 in BA/F3 Cells Using the Coumermycin/Gyrase B System

Janus kinase 2 (Jak2) protein tyrosine kinase plays an important role in interleukin-3– or granulocyte–macrophage colony-stimulating factor–mediated signal transduction pathways leading to cell proliferation, activation of early response genes, and inhibition of apoptosis. However, it is unclear whether Jak2 can activate these signaling pathways directly without the involvement of cytokine receptor phosphorylation. To investigate the specific role of Jak2 in the regulation of signal transduction pathways, we generated gyrase B (GyrB)–Jak2 fusion proteins, dimerized through the addition of coumermycin. Coumermycin induced autophosphorylation of GyrB–Jak2 fusion proteins, thus bypassing receptor activation. Using different types of chimeric Jak2 molecules, we observed that although the kinase domain of Jak2 is sufficient for autophosphorylation, the N-terminal regions are essential for the phosphorylation of Stat5 and for the induction of short-term cell proliferation. Moreover, coumermycin-induced activation of Jak2 can also lead to increased levels of c-myc and CIS mRNAs in BA/F3 cells stably expressing the Jak2 fusion protein with the intact N-terminal region. Conversely, activation of the chimeric Jak2 induced neither phosphorylation of Shc or SHP-2 nor activation of the c-fos promoter. Here, we showed that the GyrB–Jak2 system can serve as an excellent model to dissect signals of receptor-dependent and -independent events. We also obtained evidence indicating a role for the N-terminal region of Jak2 in downstream signaling events.

Analysis of the Xenopus laevis CCAAT-enhancer binding protein α gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPα genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPα genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPα gene (xC/EBPα). The –1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPα or xC/EBPβ. Deletion analysis showed that the –321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPα promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPα promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPα expression, with the factor able to repress the murine promoter but activate the Xenopus gene.