The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (Inc) RNAs from EFNA3 locus and these IncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output.

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension.We found that the changes in transcription rates induced by hypoxia are the major determinant of RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half-life in normoxia of 8.7 hours, to 5.7 hours in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect was mimicked by forced activation of the Hypoxia Inducible Factor pathway and prevented by its interference. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter..

Aims Atrial fibrillation (AF) is a progressive cardiac arrhythmia that increases the risk of hospitalization and adverse cardiovascular events. There is a clear demand for more inclusive and large-scale approaches to understand the molecular drivers responsible for AF, as well as the fundamental mechanisms governing the transition from paroxysmal to persistent and permanent forms. In this study, we aimed to create a molecular map of AF and find the distinct molecular programs underlying cell type-specific atrial remodelling and AF progression. Methods and Results We used a sheep model of long-standing, tachypacing-induced AF, sampled right and left atrial tissue and isolated cardiomyocytes from control, intermediate (transition) and late time points during AF progression, and performed transcriptomic and proteome profiling. We have merged all these layers of information into a meaningful 3-component space in which we explored the genes and proteins detected and their common patterns of expression. Our data-driven analysis points at extracellular matrix remodelling, inflammation, ion channel, myofibril structure, mitochondrial complexes, chromatin remodelling, and genes related to neural function, as well as critical regulators of cell proliferation as hallmarks of AF progression. Most important, we prove that these changes occur at early transitional stages of the disease, but not at later stages, and that the left atrium undergoes significantly more profound changes than the right atrium in its expression program. The pattern of dynamic changes in gene and protein expression replicate the electrical and structural remodelling demonstrated previously in the sheep and in humans, and uncover novel mechanisms potentially relevant for disease treatment. Conclusions Transcriptomic and proteomic analysis of AF progression in a large animal model shows that significant changes occur at early stages, and that among others involve previously undescribed increase in mitochondria, changes to the chromatin of atrial cardiomyocytes, and genes related to neural function and cell proliferation. Translational Perspective We have generated a detailed molecular map of AF progression in a clinically relevant large-animal model. Such data would be very difficult if not impossible to obtain from patients. Our results provide a framework for a comprehensive molecular analysis of the disease, pointing to novel avenues of research toward identifying early events that can lead to therapeutically targets to prevent AF-induced atrial remodelling.

Pluripotent cells are a transient population present in the early mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while simultaneously suppressing lineage specification. Interestingly, these factors are not exclusive to uncommitted cells, but are also expressed during early phases of differentiation. However, their role in the transition from pluripotency to lineage specification is largely unknown. Using genetic models for controlled Oct4 or Nanog expression during postimplantation stages, we found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that the HoxB cluster is coordinately regulated in this way by OCT4 binding sites located at the 3' end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the pre-committed epiblast, but are also necessary for the proper deployment of subsequent developmental programs. Dual regulation of Hox genes by pluripotency factors Lopez-Jimenez et al. 3

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma.

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