Stephen A. Murray

The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response

p53 is activated in response to various genotoxic stresses resulting in cell cycle arrest or apoptosis. It is well documented that DNA damage leads to phosphorylation and activation of p53 (refs 1–3), yet how p53 is activated is still not fully understood. Here we report that DNA damage specifically induces p53 phosphorylation on Ser/Thr-Pro motifs, which facilitates its interaction with Pin1, a member of peptidyl-prolyl isomerase. Furthermore, the interaction of Pin1 with p53 is dependent on the phosphorylation that is induced by DNA damage. Consequently, Pin1 stimulates the DNA-binding activity and transactivation function of p53. The Pin1-mediated p53 activation requires the WW domain, a phosphorylated Ser/Thr-Pro motif interaction module, and the isomerase activity of Pin1. Moreover, Pin1-deficient cells are defective in p53 activation and timely accumulation of p53 protein, and exhibit an impaired checkpoint control in response to DNA damage. Together, these data suggest a mechanism for p53 regulation in cellular response to genotoxic stress.

Repression of PTEN Phosphatase by Snail1 Transcriptional Factor during Gamma Radiation-Induced Apoptosis

ABSTRACT The product of the Snail1 gene is a transcriptional repressor required for triggering the epithelial-to-mesenchymal transition. Furthermore, ectopic expression of Snail1 in epithelial cells promotes resistance to apoptosis. In this study, we demonstrate that this resistance to γ radiation-induced apoptosis caused by Snail1 is associated with the inhibition of PTEN phosphatase. In MDCK cells, mRNA levels of the p53 target gene PTEN are induced after γ radiation; the transfection of Snail1 prevents this up-regulation. Decreased mRNA levels of PTEN were also detected in RWP-1 cells after the ectopic expression of this transcriptional factor. Snail1 represses and associates to the PTEN promoter as detected both by the electrophoretic mobility shift assay and chromatin immunoprecipitation experiments performed with either endogenous or ectopic Snail1. The binding of Snail1 to the PTEN promoter increases after γ radiation, correlating with the stabilization of Snail1 protein, and prevents the association of p53 to the PTEN promoter. These results stress the critical role of Snail1 in the control of apoptosis and demonstrate the regulation of PTEN phosphatase by this transcriptional repressor.

High-throughput discovery of novel developmental phenotypes.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource

Full realization of the value of the loxP-flanked alleles generated by the International Knockout Mouse Consortium will require a large set of well-characterized cre-driver lines. However, many cre driver lines display excision activity beyond the intended tissue or cell type, and these data are frequently unavailable to the potential user. Here we describe a high-throughput pipeline to extend characterization of cre driver lines to document excision activity in a wide range of tissues at multiple time points and disseminate these data to the scientific community. Our results show that the majority of cre strains exhibit some degree of unreported recombinase activity. In addition, we observe frequent mosaicism, inconsistent activity and parent-of-origin effects. Together, these results highlight the importance of deep characterization of cre strains, and provide the scientific community with a critical resource for cre strain information.

Multiple functions of Snail family genes during palate development in mice

Palate development requires precise regulation of gene expression changes, morphogenetic movements and alterations in cell physiology. Defects in any of these processes can result in cleft palate, a common human birth defect. The Snail gene family encodes transcriptional repressors that play essential roles in the growth and patterning of vertebrate embryos. Here we report the functions of Snail (Snai1) and Slug (Snai2) genes during palate development in mice. Snai2-/- mice exhibit cleft palate, which is completely penetrant on a Snai1 heterozygous genetic background. Cleft palate in Snai1+/- Snai2-/- embryos is due to a failure of the elevated palatal shelves to fuse. Furthermore, while tissue-specific deletion of the Snai1 gene in neural crest cells does not cause any obvious defects, neural-crest-specific Snai1 deletion on a Snai2-/- genetic background results in multiple craniofacial defects, including a cleft palate phenotype distinct from that observed in Snai1+/- Snai2-/- embryos. In embryos with neural-crest-specific Snai1 deletion on a Snai2-/- background, palatal clefting results from a failure of Meckels cartilage to extend the mandible and thereby allow the palatal shelves to elevate, defects similar to those seen in the Pierre Robin Sequence in humans.

Snail family genes are required for left–right asymmetry determination, but not neural crest formation, in mice

Snail family genes encode zinc finger transcriptional repressors that are key regulators of epithelial–mesenchymal transitions in vertebrates, including the transitions that generate the mesoderm and neural crest. Here, we show that, contrary to observations in frog and avian embryos, the Snail family genes Snail (Snai1) and Slug (Snai2) are not required for formation and delamination of the neural crest in mice. However, embryos with conditional inactivation of Snai1 function exhibit defects in left–right asymmetry determination. This work demonstrates that although some aspects of Snail family gene function, such as a role in left–right asymmetry determination, appear to be evolutionarily conserved, their role in neural crest cell formation and delamination is not.

Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.

Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: Effects of TGF-β1 and serum deprivation

Transforming growth factor‐β1 (TGF‐β1) markedly reduced cell proliferation and elevated steady state lysyl oxidase (LO) mRNA 3‐fold in neonatal rat aorta smooth muscle cells cultured in medium containing 10% fetal bovine serum. The increase in LO mRNA was prevented by the presence of cycloheximide, indicative of controlling events at the level of protein synthesis. The basal level of mRNA in cells proliferating in 10% fetal bovine serum in the absence of TGF‐β1 was enhanced 7‐fold upon decreasing growth by shifting to medium containing 0.5% serum. Changes in LO activity paralleled those in LO mRNA. Nuclear run‐on assays revealed that the stimulation of expression in 0.5% serum involved increased gene transcription whereas that caused by TGF‐β1 was mostly post‐transcriptional in origin. LO mRNA was quite labile (t½ approximately 3 h) in 10% serum but was markedly stabilized (t½ > 12 h) by the presence of TGF‐β1 in the 10% serum medium. LO mRNA was also considerably more stable under retarded growth conditions (0.5% serum) in the absence of TGF‐β1. LO promoter activity in luciferase reporter constructs transfected into these cells was low and not significantly affected by the addition of TGF‐β1 to the 10% serum medium but was markedly elevated by shifting from 10 to 0.5% serum in the absence of TGF‐β1. Thus, LO expression is inversely correlated with cell proliferation, and is subject to control at transcriptional and post‐transcriptional levels. TGF‐β1 enhances LO expression in these cells by dramatically stabilizing LO mRNA. J. Cell. Biochem. 65:395–407.

The FaceBase Consortium: a comprehensive program to facilitate craniofacial research.

The FaceBase Consortium consists of ten interlinked research and technology projects whose goal is to generate craniofacial research data and technology for use by the research community through a central data management and integrated bioinformatics hub. Funded by the National Institute of Dental and Craniofacial Research (NIDCR) and currently focused on studying the development of the middle region of the face, the Consortium will produce comprehensive datasets of global gene expression patterns, regulatory elements and sequencing; will generate anatomical and molecular atlases; will provide human normative facial data and other phenotypes; conduct follow up studies of a completed genome-wide association study; generate independent data on the genetics of craniofacial development, build repositories of animal models and of human samples and data for community access and analysis; and will develop software tools and animal models for analyzing and functionally testing and integrating these data. The FaceBase website (http://www.facebase.org) will serve as a web home for these efforts, providing interactive tools for exploring these datasets, together with discussion forums and other services to support and foster collaboration within the craniofacial research community.

Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening

Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data.