Markus Morkel

c-Met is essential for wound healing in the skin.

Wound healing of the skin is a crucial regenerative process in adult mammals. We examined wound healing in conditional mutant mice, in which the c-Met gene that encodes the receptor of hepatocyte growth factor/scatter factor was mutated in the epidermis by cre recombinase. c-Met–deficient keratinocytes were unable to contribute to the reepithelialization of skin wounds. In conditional c-Met mutant mice, wound closure was slightly attenuated, but occurred exclusively by a few (5%) keratinocytes that had escaped recombination. This demonstrates that the wound process selected and amplified residual cells that express a functional c-Met receptor. We also cultured primary keratinocytes from the skin of conditional c-Met mutant mice and examined them in scratch wound assays. Again, closure of scratch wounds occurred by the few remaining c-Met–positive cells. Our data show that c-Met signaling not only controls cell growth and migration during embryogenesis but is also essential for the generation of the hyperproliferative epithelium in skin wounds, and thus for a fundamental regenerative process in the adult.

MEDIPS: genome-wide differential coverage analysis of sequencing data derived from DNA enrichment experiments

Motivation: DNA enrichment followed by sequencing is a versatile tool in molecular biology, with a wide variety of applications including genome-wide analysis of epigenetic marks and mechanisms. A common requirement of these diverse applications is a comparison of read coverage between experimental conditions. The amount of samples generated for such comparisons ranges from few replicates to hundreds of samples per condition for epigenome-wide association studies. Consequently, there is an urgent need for software that allows for fast and simple processing and comparison of sequencing data derived from enriched DNA. Results: Here, we present a major update of the R/Bioconductor package MEDIPS, which allows for an arbitrary number of replicates per group and integrates sophisticated statistical methods for the detection of differential coverage between experimental conditions. Our approach can be applied to a diversity of quantitative sequencing data. In addition, our update adds novel functionality to MEDIPS, including correlation analysis between samples, and takes advantage of Bioconductor’s annotation databases to facilitate annotation of specific genomic regions. Availability and implementation: The latest version of MEDIPS is available as version 1.12.0 and part of Bioconductor 2.13. The package comes with a manual containing detailed description of its functionality and is available at http://www.bioconductor.org. Contact: lienhard@molgen.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.

An E2F-like repressor of transcription

The activity of a variety of genes whose products are involved in DNA replication and cell-cycle progression are regulated by E2F transcription factors, which bind to E2F sites on DNA in cooperation with members of the DP family of transcription factors. E2F sites can act as both positive and negative control elements. Transcriptional activation from E2F sites is mediated by ‘free’ E2F, whereas repression conventionally requires the formation of a complex with a pocket protein (retinoblastoma protein (RB), p107 or p130). Here we describe an E2F-like protein, termed EMA (for E2F-binding site modulating activity), which can act as a repressor of transcription without a pocket protein.

A Colorectal Cancer Expression Profile That Includes Transforming Growth Factor β Inhibitor BAMBI Predicts Metastatic Potential

BACKGROUND & AIMS Much is known about the genes and mutations that cause colorectal cancer (CRC), yet only a few have been associated with CRC metastasis. We performed expression-profiling experiments to identify genetic markers of risk and to elucidate the molecular mechanisms of CRC metastasis. METHODS We compared gene expression patterns between metastatic and nonmetastatic stage-matched human colorectal carcinomas by microarray analysis. Correlations between BAMBI and metastasis-free survival were examined by quantitative real-time polymerase chain reaction (PCR) using an independent set of human colon carcinomas. Human colon cancer cell lines were analyzed for BAMBI regulation, cell motility, and experimental metastasis. RESULTS We established a signature of 115 genes that differentiated metastatic from nonmetastatic primary tumors. Among these, the transforming growth factor (TGF) beta inhibitor BAMBI was highly expressed in approximately half of metastatic primary tumors and metastases but not in nonmetastatic tumors. BAMBI is a target of canonical Wnt signaling that involves the beta-catenin coactivator BCL9-2. We observed an inverse correlation between level of BAMBI expression and metastasis-free survival time of patients. BAMBI inhibits TGF-beta signaling and increases migration in colon cancer cells. In mice, overexpression of BAMBI caused colon cancer cells to form tumors that metastasized more frequently to liver and lymph nodes than control cancer cells. CONCLUSIONS BAMBI regulates CRC metastasis by connecting the Wnt/beta-catenin and TGF-beta-signaling pathways. The metastatic expression signature we describe, along with BAMBI levels, can be used in prognosis. Developmental signaling pathways appear to act in hierarchies and cooperate in tumor cell migration, invasion, and metastasis.

Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway dependent gene signatures in colorectal cancer cells.

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

A Role for E2F6 in the Restriction of Male-Germ-Cell-Specific Gene Expression

E2F transcription factors play a pivotal role in the regulation of cellular proliferation and can be subdivided into activating and repressing family members [1]. Like other E2Fs, E2F6 binds to E2F consensus sites, but in contrast to E2F1-5, it lacks an Rb binding domain and functions as an Rb-independent transcriptional repressor [2, 3, 4 and 5]. Instead, E2F6 has been shown to complex with Polycomb (PcG) group proteins [6 and 7], which have a well-established role in gene silencing. Here, we show that E2F6 plays an unexpected and essential role in the tissue specificity of gene expression. E2F6-deficient mice ubiquitously express the alpha-tubulin 3 and 7 genes, which are expressed strictly testis-specifically in control mice. Like an additional E2F6 target gene, Tex12, that we identified, tubulin 3 and 7 are normally expressed in male germ cells only. The promoters of the alpha-tubulin and Tex12 genes share a perfectly conserved E2F site, which E2F6 binds to. Mechanistically, E2F6-mediated repression involves CpG hypermethylation locking target promoters in an inactive state. Thus, E2F6 is essential for the long-term somatic silencing of certain male-germ-cell-specific genes, but it is dispensable for cell-cycle regulation.

DNA–Methylome Analysis of Mouse Intestinal Adenoma Identifies a Tumour-Specific Signature That Is Partly Conserved in Human Colon Cancer

Aberrant CpG methylation is a universal epigenetic trait of cancer cell genomes. However, human cancer samples or cell lines preclude the investigation of epigenetic changes occurring early during tumour development. Here, we have used MeDIP-seq to analyse the DNA methylome of APCMin adenoma as a model for intestinal cancer initiation, and we present a list of more than 13,000 recurring differentially methylated regions (DMRs) characterizing intestinal adenoma of the mouse. We show that Polycomb Repressive Complex (PRC) targets are strongly enriched among hypermethylated DMRs, and several PRC2 components and DNA methyltransferases were up-regulated in adenoma. We further demonstrate by bisulfite pyrosequencing of purified cell populations that the DMR signature arises de novo in adenoma cells rather than by expansion of a pre-existing pattern in intestinal stem cells or undifferentiated crypt cells. We found that epigenetic silencing of tumour suppressors, which occurs frequently in colon cancer, was rare in adenoma. Quite strikingly, we identified a core set of DMRs, which is conserved between mouse adenoma and human colon cancer, thus possibly revealing a global panel of epigenetically modified genes for intestinal tumours. Our data allow a distinction between early conserved epigenetic alterations occurring in intestinal adenoma and late stochastic events promoting colon cancer progression, and may facilitate the selection of more specific clinical epigenetic biomarkers.

Genomic organization of the human excitatory amino acid transporter gene GLT-1.

WE present the genomic structure of the human glutamate transporter GLT-1 coding region, the intronic sequences adjacent to the exons, and oligonucleotide primer sequences for single strand conformational analysis. The exon–intron boundaries were determined using long-distance PCR and direct sequencing. The human GLT-1 coding region is composed of 10 exons spanning > 50 kb of genomic DNA. The exons range from 127 to 251 bp in length. The intron lengths vary considerably from 2.2 kb to > 15 kb. These data provide the basis for implementing a comprehensive screen for genetic alterations in the human GLT-1 gene using genomic DNA as a template.

An inducible RNA interference system for the functional dissection of mouse embryogenesis

Functional analysis of multiple genes is key to understanding gene regulatory networks controlling embryonic development. We have developed an integrated vector system for inducible gene silencing by shRNAmir-mediated RNA interference in mouse embryos, as a fast method for dissecting mammalian gene function. For validation of the vector system, we generated mutant phenotypes for Brachyury, Foxa2 and Noto, transcription factors which play pivotal roles in embryonic development. Using a series of Brachyury shRNAmir vectors of various strengths we generated hypomorphic and loss of function phenotypes allowing the identification of Brachyury target genes involved in trunk development. We also demonstrate temporal control of gene silencing, thus bypassing early embryonic lethality. Importantly, off-target effects of shRNAmir expression were not detectable. Taken together, the system allows the dissection of gene function at unprecedented detail and speed, and provides tight control of the genetic background minimizing intrinsic variation.

In vivo knockdown of Brachyury results in skeletal defects and urorectal malformations resembling caudal regression syndrome

The T-box transcription factor BRACHYURY (T) is a key regulator of mesoderm formation during early development. Complete loss of T has been shown to lead to embryonic lethality around E10.0. Here we characterize an inducible miRNA-based in vivo knockdown mouse model of T, termed KD3-T, which exhibits a hypomorphic phenotype. KD3-T embryos display axial skeletal defects caused by apoptosis of paraxial mesoderm, which is accompanied by urorectal malformations resembling the murine uro-recto-caudal syndrome and human caudal regression syndrome phenotypes. We show that there is a reduction of T in the notochord of KD3-T embryos which results in impaired notochord differentiation and its subsequent loss, whereas levels of T in the tailbud are sufficient for axis extension and patterning. Furthermore, the notochord in KD3-T embryos adopts a neural character and loses its ability to act as a signaling center. Since KD3-T animals survive until birth, they are useful for examining later roles for T in the development of urorectal tissues.