A broad spectrum of biological activities has been proposed for transforming growth factor-beta 3 (TGF-beta 3). To study TGF-beta 3 function in development, TGF-beta 3 null mutant mice were generated by gene-targeting. Within 20 hours of birth, homozygous TGF-beta 3-/- mice die with unique and consistent phenotypic features including delayed pulmonary development and defective palatogenesis. Unlike other null mutants with cleft palate, TGF-beta 3-/- mice lack other concomitant craniofacial abnormalities. This study demonstrates an essential function for TGF-beta 3 in the normal morphogenesis of palate and lung, and directly implicates this cytokine in mechanisms of epithelial-mesenchymal interaction.

Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3β (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.

Hypoxia has recently been shown to activate the endoplasmic reticulum kinase PERK, leading to phosphorylation of eIF2a and inhibition of mRNA translation initiation. Using a quantitative assay, we show that this inhibition exhibits a biphasic response mediated through two distinct pathways. The first occurs rapidly, reaching a maximum at 1-2 h and is due to phosphorylation of eIF2a. Continued hypoxic exposure activates a second, eIF2a-independent pathway that maintains repression of translation. This phase is characterized by disruption of eIF4F and sequestration of eIF4E by its inhibitor 4E-BP1 and transporter 4E-T. Quantitative RT-PCR analysis of polysomal RNA indicates that the translation efficiency of individual genes varies widely during hypoxia. Furthermore, the translation efficiency of individual genes is dynamic, changing dramatically during hypoxic exposure due to the initial phosphorylation and subsequent dephosphorylation of eIF2a. Together, our data indicate that acute and prolonged hypoxia regulates mRNA translation through distinct mechanisms, each with important contributions to hypoxic gene expression.

The highly homologous Rnf2 (Ring1b) and Ring1 (Ring1a) proteins were identified as in vivo interactors of the Polycomb Group (PcG) protein Bmi1. Functional ablation of Rnf2 results in gastrulation arrest, in contrast to relatively mild phenotypes in most other PcG gene null mutants belonging to the same functional group, among which is Ring1. Developmental defects occur in both embryonic and extraembryonic tissues during gastrulation. The early lethal phenotype is reminiscent of that of the PcG-gene knockouts Eed and Ezh2, which belong to a separate functional PcG group and PcG protein complex. This finding indicates that these biochemically distinct PcG complexes are both required during early mouse development. In contrast to the strong skeletal transformation in Ring1 hemizygous mice, hemizygocity for Rnf2 does not affect vertebral identity. However, it does aggravate the cerebellar phenotype in a Bmi1 nullmutant background. Together, these results suggest that Rnf2 or Ring1-containing PcG complexes have minimal functional redundancy in specific tissues, despite overlap in expression patterns. We show that the early developmental arrest in Rnf2-null embryos is partially bypassed by genetic inactivation of the Cdkn2a (Ink4a͞ARF) locus. Importantly, this finding implicates Polycomb-mediated repression of the Cdkn2a locus in early murine development. P olycomb Group (PcG) proteins and their genetic counterparts, the trithorax Group proteins (trxG), maintain Hox gene expression boundaries (1-4), which are critical for regional patterning along the antero-posterior (AP) axis (5-7). Based on biochemical characteristics, mammalian PcG proteins are currently grouped into at least two distinct functional groups: the first comprises Eed, Ezh1, and Ezh2 in the mouse (8, 9); the second consists of the highly related protein pairs Cbx2 (M33)͞Cbx4 (MPc2), Bmi͞Zfp144 (Mel18), and Edr1 and 2 (Rae28͞Mph1 and 2), respectively (10, 11). For ease of this discussion we refer to them as groups I and II, respectively. Group I and II homologs are evolutionarily conserved from Drosophila to humans, only group I homologs are found in plants and Caenorhabditis elegans as well, supporting the concept of separate function (12, 13). In addition, complex composition differs throughout development in a temporal and cell-type-specific manner (14, 15). Interaction of Eed with histone deacetylases and the intrinsic histone methyltransferase activity of Ezh proteins suggest mechanisms for repression by group I complexes (16)(17)(18)(19). Although a mammalian hPRC-H (group II) complex harbors an intrinsic capacity to stabilize a repressive chromatin structure and counteract SWI͞SNF chromatin remodeling complexes in vitro, its in vivo mode of action is not well understood (20). Association with histone methyl transferase activity may in part help explain the repressive action of some group II complexes (21).Rnf2 and Ring1 have been identified as in vivo interactors of the group II PcG protein Bmi1 by us and others (22). These Ring finger proteins...

MicroRNAs are small regulatory RNAs that post-transcriptionally control gene expression. Reduced expression of DICER, the enzyme involved in microRNA processing, is frequently observed in cancer and is associated with poor clinical outcome in various malignancies. Yet the underlying mechanisms are not well understood. Here, we identify tumor hypoxia as a regulator of DICER expression in large cohorts of breast cancer patients. We show that DICER expression is suppressed by hypoxia through an epigenetic mechanism that involves inhibition of oxygen-dependent H3K27me3 demethylases KDM6A/B and results in silencing of the DICER promoter. Subsequently, reduced miRNA processing leads to derepression of the miR-200 target ZEB1, stimulates the epithelial to mesenchymal transition and ultimately results in the acquisition of stem cell phenotypes in human mammary epithelial cells. Our study uncovers a previously unknown relationship between oxygen-sensitive epigenetic regulators, miRNA biogenesis and tumor stem cell phenotypes that may underlie poor outcome in breast cancer.

Diseases caused by mutations in lamins A and C (laminopathies) suggest a crucial role for A-type lamins in different cellular processes. Laminopathies mostly affect tissues of mesenchymal origin. As transforming growth factor-beta1 (TGF-beta1) signalling impinges on the retinoblastoma protein (pRB) and SMADs, we tested the hypothesis that lamins modulate cellular responses to TGF-beta1 signalling, via the regulation of these transcription factors in mesenchymal cells. Here, we report that A-type lamins are essential for the inhibition of fibroblast proliferation by TGF-beta1. TGF-beta1 dephosphorylated pRB through PP2A, both of which, we show, are associated with lamin A/C. In addition, lamin A/C modulates the effect of TGF-beta1 on collagen production, a marker of mesenchymal differentiation. Our findings implicate lamin A/C in control of gene activity downstream of TGF-beta1, via nuclear phosphatases such as PP2A. This biological function provides a novel explanation for the observed mesenchymal dysfunction in laminopathies.

ARF , which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcGmediated repression as a dynamically controlled process.

Philadelphia (Ph)-positive leukemias invariably contain a chromosomal translocation fusing BCR to ABL. The BCR-ABL protein is responsible for leukemogenesis. Here we show that exposure of bcr-null mutant mice to gram-negative endotoxin led to severe septic shock and increased tissue injury by neutrophils. Neutrophils of bcr (-/-) mice showed a pronounced increase in reactive oxygen metabolite production upon activation and were more sensitive to priming stimuli. Activated (-/-) neutrophils displayed a 3-fold increased p21rac2 membrane translocation compared with (+/+) neutrophils. These results connect Bcr in vivo with the regulation of Rac-mediated superoxide production by the NADPH-oxidase system of leukocytes and suggest a link between Bcr function and the cell type affected in Ph-positive leukemia.