Progesterone Receptor induces bcl-x expression through intragenic binding sites favoring RNA Polymerase II elongation

Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of pro- gestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions sur- rounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-dis- tribution of the active Pol II toward the 30-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene ex- pression by facilitating the proper passage of the polymerase along hormone-dependent genes.Fil: Bertucci, Paola Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Nacht, Ana Silvina. Universitat Pompeu Fabra; España. Centro de Regulación Genómica; EspañaFil: Alló, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Rocha Viegas, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Ballaré, Cecilia. Universitat Pompeu Fabra; España. Centro de Regulación Genómica; EspañaFil: Soronellas, Daniel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Castellano, Giancarlo. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Zaurin, Roser. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Beato, Miguel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Vicent, Guillermo. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Pecci, Adali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro

BACKGROUND: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. METHODS: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. RESULTS: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. CONCLUSION: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action

Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells

p38α, p38β, p38γ, and p38δ are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38α and p38γ transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38δ mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38β is minimally expressed in early CD34(+) hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38α, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38α, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38α does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38α and p38δ isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor α, an inflammatory cytokine that is modulated by p38α, is expressed by differentiating erythroblasts and inhibition of p38α or tumor necrosis factor α results in reduction in differentiation. Taken together, our data demonstrate that both p38α and δ isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation

Quantifying Wavelengths Constrained by Simulted SWOT Observations in a SubMesoscale Resolving Ocean Analysis/Forecasting System

Using a suite of Observing System Simulation Experiments (OSSEs), the utility of simulated Surface Water Ocean Topography (SWOT) observations is estimated in a high-resolution (1 km) ocean analysis/forecasting system. Sampling a Nature Run provides observations for the OSSEs and the realism of the Nature Run is established by comparison to climatological data and an independent ocean analysis/forecast system. Each OSSE experiment assimilated different sets of simulated observations including traditional nadir altimeters, satellite sea surface temperature (SST), in situ profile data, and SWOT. OSSE evaluation metrics include area-averaged errors and wavenumber spectra with the latter providing much finer differentiation between experiments. 100 m temperature, sea surface height (SSH), and mixed layer depth (MLD) errors across the observed wavenumber spectra were reduced by up to 20% for OSSEs assimilating the simulated SWOT observations. The minimum constrained wavelength was found to be 130 km when both nadir altimetry and SWOT observations were used. The experiment using only nadir altimetry produced a value of 161 km. This 31 km gain in skill of predictable scales suggests that ocean forecasts can expect substantial gains in capability when utilizing the forthcoming SWOT data. Experimentation with the analysis decorrelation length scale suggests that emerging multi-scale assimilation methodologies will provide additional advancements in predictive skill