Lars Hummerich

Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins

Psoriasis is a frequent, inflammatory disease of skin and joints with considerable morbidity. Here we report that in psoriatic lesions, epidermal keratinocytes have decreased expression of JunB, a gene localized in the psoriasis susceptibility region PSORS6. Likewise, inducible epidermal deletion of JunB and its functional companion c-Jun in adult mice leads (within two weeks) to a phenotype resembling the histological and molecular hallmarks of psoriasis, including arthritic lesions. In contrast to the skin phenotype, the development of arthritic lesions requires T and B cells and signalling through tumour necrosis factor receptor 1 (TNFR1). Prior to the disease onset, two chemotactic proteins (S100A8 and S100A9) previously mapped to the psoriasis susceptibility region PSORS4, are strongly induced in mutant keratinocytes in vivo and in vitro. We propose that the abrogation of JunB/activator protein 1 (AP-1) in keratinocytes triggers chemokine/cytokine expression, which recruits neutrophils and macrophages to the epidermis thereby contributing to the phenotypic changes observed in psoriasis. Thus, these data support the hypothesis that epidermal alterations are sufficient to initiate both skin lesions and arthritis in psoriasis.

Identification of novel oligodendroglioma-associated candidate tumor suppressor genes in 1p36 and 19q13 using microarray-based expression profiling

Loss of heterozygosity (LOH) on chromosomal arms 1p and 19q is the most common genetic alteration in oligodendroglial tumors and associated with response to radio‐ and chemotherapy as well as favorable prognosis. Using microsatellite analysis, we previously identified the chromosomal regions 1p36.22‐p36.31 and 19q13.3, as candidate tumor suppressor gene regions being commonly deleted in these tumors. To identify genes within these regions that are downregulated in oligodendroglial tumors with LOH 1p/19q, we performed cDNA microarray‐based RNA expression profiling of 35 gliomas with known allelic status on 1p and 19q, including 7 oligodendrogliomas and 8 diffuse astrocytomas of World Health Organization (WHO) grade II, as well as 14 anaplastic oligodendrogliomas and 6 anaplastic oligoastrocytomas of WHO grade III. The microarrays used for expression profiling carried ∼7,000 gene‐specific cDNAs, with complete coverage of the genes located in 1p36.13‐p36.31 and 19q13.2‐q13.33. Microarray analysis identified 8 genes from these regions (MGC4399, SRM, ICMT, RPL18, FTL, ZIN, FLJ10781 and DBP), which all showed significantly lower expression in 1p/19q‐deleted gliomas when compared to gliomas without 1p/19q losses. Quantitative real‐time reverse transcription‐PCR analyses were performed for the MGC4399, ICMT and RPL18 genes and confirmed the microarray findings. In addition, we found that the cytosolic phospholipase A2 (PLA2G4C) gene at 19q13.3 demonstrated significantly lower expression in anaplastic oligodendrogliomas (WHO grade III) when compared to well‐differentiated oligodendrogliomas (WHO grade II). Taken together, our study provides a set of interesting novel candidate genes that may play important roles in the pathogenesis of oligodendroglial tumors.

Identification of novel tumour-associated genes differentially expressed in the process of squamous cell cancer development

Chemically induced mouse skin carcinogenesis represents the most extensively utilized animal model to unravel the multistage nature of tumour development and to design novel therapeutic concepts of human epithelial neoplasia. We combined this tumour model with comprehensive gene expression analysis and could identify a large set of novel tumour-associated genes that have not been associated with epithelial skin cancer development yet. Expression data of selected genes were confirmed by semiquantitative and quantitative RT-PCR as well as in situ hybridization and immunofluorescence analysis on mouse tumour sections. Enhanced expression of genes identified in our screen was also demonstrated in mouse keratinocyte cell lines that form tumours in vivo. Self-organizing map clustering was performed to identify different kinetics of gene expression and coregulation during skin cancer progression. Detailed analysis of differential expressed genes according to their functional annotation confirmed the involvement of several biological processes, such as regulation of cell cycle, apoptosis, extracellular proteolysis and cell adhesion, during skin malignancy. Finally, we detected high transcript levels of ANXA1, LCN2 and S100A8 as well as reduced levels for NDR2 protein in human skin tumour specimens demonstrating that tumour-associated genes identified in the chemically induced tumour model might be of great relevance for the understanding of human epithelial malignancies as well.

Identification of novel AP-1 target genes in fibroblasts regulated during cutaneous wound healing

Mesenchymal–epithelial interactions are increasingly considered to be of vital importance for epithelial homeostasis and regeneration. In skin, the transcription factor AP-1 was shown to be critically involved in the communication between keratinocytes and dermal fibroblasts. After skin injury, the release of IL-1 from keratinocytes induces the activity of the AP-1 subunits c-Jun and JunB in fibroblasts leading to a global change in gene expression. To identify AP-1 target genes in fibroblasts, which are involved in the process of cutaneous repair, we performed gene expression profiling of wild-type, c-jun- and junB-deficient fibroblasts in response to IL-1, mimicking the initial phase of wound healing. Using a 15K cDNA collection, over 1000 genes were found to be Jun-dependent and additional 300 clones showed IL-1 responsiveness. Combinatorial evaluation allowed for the dissection of the specific contribution of either AP-1 subunit to gene regulation. Besides previously identified genes that are involved in cutaneous repair, we have identified novel genes regulated during wound healing in vivo and showed their expression by fibroblasts on wound sections. The identification of novel Jun target genes should provide a basis for understanding the molecular mechanisms underlying mesenchymal–epithelial interactions and the critical contribution of AP-1 to tissue homeostasis and repair.

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry

To investigate protein–protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH–MutL–DNA complex.

FACT – a framework for the functional interpretation of high-throughput experiments

BackgroundInterpreting the results of high-throughput experiments, such as those obtained from DNA-microarrays, is an often time-consuming task due to the high number of data-points that need to be analyzed in parallel. It is usually a matter of extensive testing and unknown beforehand, which of the possible approaches for the functional analysis will be the most informativeResultsTo address this problem, we have developed the Flexible Annotation and Correlation Tool (FACT). FACT allows for detection of important patterns in large data sets by simplifying the integration of heterogeneous data sources and the subsequent application of different algorithms for statistical evaluation or visualization of the annotated data. The system is constantly extended to include additional annotation data and comparison methods.ConclusionFACT serves as a highly flexible framework for the explorative analysis of large genomic and proteomic result sets. The program can be used online; open source code and supplementary information are available at http://www.factweb.de.

Corrigendum: Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins

This corrects the article DOI: 10.1038/nature03963

Erratum: Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins (Nature (2005) 437, (369-375))

This corrects the article DOI: 10.1038/nature03963