Mek is a dual-specificity kinase that activates the extracellular-signal-regulated (Erk) mitogen-activated protein (MAP) kinases upon agonist binding to receptors. The Erk MAP kinase cascade is involved in cell-fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single mek gene is present in Caenorhabditis elegans, Drosophila and Xenopus, two mek homologs, Mek1 and Mek2, are present in the mammalian cascade. In the present study, we describe a mutant mouse line in which the mek1 gene has been disrupted by insertional mutagenesis. The null mutation was recessive lethal, as the homozygous mutant embryos died at 10.5 days of gestation. Histopathological analyses revealed a reduction in vascularization of the placenta that was due to a marked decrease of vascular endothelial cells in the labyrinthine region. The failure to establish a functional placenta probably explains the death of the mek1 -/embryos. Cell-migration assays indicated that mek1 -/fibroblasts could not be induced to migrate by fibronectin, although the levels of Mek2 protein and Erk activation were normal. Re-expression of Mek1 in the mutant mouse embryonic fibroblasts (MEFs) restored their ability to migrate. Our findings provide genetic evidence that establishes the unique role played by Mek1 in signal transduction. They also suggest that mek1 function is required for normal response to angiogenic signals that might promote vascularization of the labyrinthine region of the placenta.

To uncover roles for the Hoxa-5 gene during embryogenesis, we have focused on identifying structural and functional defects in organ systems underlying the perinatal lethality in Hoxa-5 homozygous mutants. Analysis of the mutant phenotype shows that Hoxa-5 is essential for normal organogenesis and function of the respiratory tract. In homozygous newborn mutants, improper tracheal and lung morphogenesis can lead to tracheal occlusion, and to respiratory distress associated with a marked decrease in the production of surfactant proteins. Collectively, these defects likely underlie the pronounced mortality of homozygous mutant pups. Furthermore, the loss of Hoxa-5 function results in altered TTF-1, HNF-3 beta, and N-myc gene expression in the pulmonary epithelium. Since expression of Hoxa-5 is confined to the mesenchymal component of the developing trachea and lung, the effects observed in epithelial cells may result from a disruption of normal epithelial-mesenchymal interactions.

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogenactivated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.The mitogen-activated protein (MAP) kinase signaling pathways consist of protein kinase cascades linking extracellular stimuli to various targets scattered in the cytoplasm, the cytoskeleton, the membrane, and the nucleus (38). There are at least three distinct MAP kinase signaling pathways in mammals, including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases, and the p38 MAP kinase (12). These kinases are activated in cascades by phosphorylation on both threonine and tyrosine residues in the regulatory TXY loop present in all MAP kinases. This phosphorylation is carried out via distinct upstream dual-specificity MAP kinase kinases (MAPKKs). The classical pathway, which appears to be the major one in growth factor signaling, uses MAP kinaseor ERK-activating kinases (MEK and MAPKK) and ERK isoforms (MAP kinase) and is named the ERK/MAP kinasesignaling pathway. In mammals, MAPKK constitutes a small family of related proteins, but only MEK1 and MEK2 are known participants in the ERK/MAP kinase cascade (38). Directly downstream of MEK1 and MEK2, ERK1 and ERK2 phosphorylate a large number of substrates located in the cytoplasm and the nucleus (13,25,27,38,45).The ERK/MAP kinase pathway is also involved in cell fate determination in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis (21,28,41,44). While two different MEK proteins are present in the ERK/MAP kinase cascade in mammals, a single Mek gene fulfills this role in these species. Sequence analysis revealed that the murine MEK1 protein is more related to the Xenopus MEK than to the mouse MEK2.Indeed, MEK2 protein is only 80% identical and 90% similar to MEK1, whereas Xenopus MEK is 91% i...

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).

Targeting of activated plasma membrane receptors to endocytic pathways is important in determining the outcome of growth factor signaling. However, the molecular mechanisms are still poorly understood. Here, we show that the synaptotagmin-related membrane protein E-Syt2 is essential for rapid endocytosis of the activated FGF receptor and for functional signal transduction during Xenopus development. E-Syt2 depletion prevents an early phase of activated FGF receptor endocytosis that we show is required for ERK activation and the induction of the mesoderm. E-Syt2 interacts selectively with the activated FGF receptor and with Adaptin-2, and is required upstream of Ras activation and of receptor autophosphorylation for ERK activation and the induction of the mesodermal marker Xbra. The data identify E-Syt2 as an endocytic adaptor for the clathrin-mediated pathway whose function is conserved in human and suggest a broader role for the E-Syt subfamily in growth factor signaling.

The combination of Chromatin Immunoprecipitation and Massively Parallel Sequencing, or ChIP-Seq, has greatly advanced our genome-wide understanding of chromatin and enhancer structures. However, its resolution at any given genetic locus is limited by several factors. In applying ChIP-Seq to the study of the ribosomal RNA genes, we found that a major limitation to resolution was imposed by the underlying variability in sequence coverage that very often dominates the protein–DNA interaction profiles. Here, we describe a simple numerical deconvolution approach that, in large part, corrects for this variability, and significantly improves both the resolution and quantitation of protein–DNA interaction maps deduced from ChIP-Seq data. This approach has allowed us to determine the in vivo organization of the RNA polymerase I preinitiation complexes that form at the promoters and enhancers of the mouse (Mus musculus) and human (Homo sapiens) ribosomal RNA genes, and to reveal a phased binding of the HMG-box factor UBF across the rDNA. The data identify and map a “Spacer Promoter” and associated stalled polymerase in the intergenic spacer of the human ribosomal RNA genes, and reveal a very similar enhancer structure to that found in rodents and lower vertebrates.

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