Christian Schönbach

Functional annotation of a full-length mouse cDNA collection

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Novel peroxisomal protease Tysnd1 processes PTS1- and PTS2-containing enzymes involved in β-oxidation of fatty acids

Peroxisomes play an important role in β‐oxidation of fatty acids. All peroxisomal matrix proteins are synthesized in the cytosol and post‐translationally sorted to the organelle. Two distinct peroxisomal signal targeting sequences (PTSs), the C‐terminal PTS1 and the N‐terminal PTS2, have been defined. Import of precursor PTS2 proteins into the peroxisomes is accompanied by a proteolytic removal of the N‐terminal targeting sequence. Although the PTS1 signal is preserved upon translocation, many PTS1 proteins undergo a highly selective and limited cleavage. Here, we demonstrate that Tysnd1, a previously uncharacterized protein, is responsible both for the removal of the leader peptide from PTS2 proteins and for the specific processing of PTS1 proteins. All of the identified Tysnd1 substrates catalyze peroxisomal β‐oxidation. Tysnd1 itself undergoes processing through the removal of the presumably inhibitory N‐terminal fragment. Tysnd1 expression is induced by the proliferator‐activated receptor α agonist bezafibrate, along with the increase in its substrates. A model is proposed where the Tysnd1‐mediated processing of the peroxisomal enzymes promotes their assembly into a supramolecular complex to enhance the rate of β‐oxidation.

FIMM, a database of functional molecular immunology

FIMM database (http://sdmc.krdl.org.sg:8080/fimm ) contains data relevant to functional molecular immunology, focusing on cellular immunology. It contains fully referenced data on protein antigens, major histocompatibility complex (MHC) molecules, MHC-associated peptides and relevant disease associations. FIMM has a set of search tools for extraction of information and results are presented as lists or as reports.

Id4, a New Candidate Gene for Senile Osteoporosis, Acts as a Molecular Switch Promoting Osteoblast Differentiation

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Pparγ2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Pparγ2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

Computational promoter analysis of mouse, rat, and human antimicrobial peptide-coding genes

BackgroundMammalian antimicrobial peptides (AMPs) are effectors of the innate immune response. A multitude of signals coming from pathways of mammalian pathogen/pattern recognition receptors and other proteins affect the expression of AMP-coding genes (AMPcgs). For many AMPcgs the promoter elements and transcription factors that control their tissue cell-specific expression have yet to be fully identified and characterized.ResultsBased upon the RIKEN full-length cDNA and public sequence data derived from human, mouse and rat, we identified 178 candidate AMP transcripts derived from 61 genes belonging to 29 AMP families. However, only for 31 mouse genes belonging to 22 AMP families we were able to determine true orthologous relationships with 30 human and 15 rat sequences. We screened the promoter regions of AMPcgs in the three species for motifs by an ab initio motif finding method and analyzed the derived promoter characteristics. Promoter models were developed for alpha-defensins, penk and zap AMP families. The results suggest a core set of transcription factors (TFs) that regulate the transcription of AMPcg families in mouse, rat and human. The three most frequent core TFs groups include liver-, nervous system-specific and nuclear hormone receptors (NHRs). Out of 440 motifs analyzed, we found that three represent potentially novel TF-binding motifs enriched in promoters of AMPcgs, while the other four motifs appear to be species-specific.ConclusionOur large-scale computational analysis of promoters of 22 families of AMPcgs across three mammalian species suggests that their key transcriptional regulators are likely to be TFs of the liver-, nervous system-specific and NHR groups. The computationally inferred promoter elements and potential TF binding motifs provide a rich resource for targeted experimental validation of TF binding and signaling studies that aim at the regulation of mouse, rat or human AMPcgs.

Identification of novel PPARγ target genes by integrated analysis of ChIP-on-chip and microarray expression data during adipocyte differentiation

PPARgamma (peroxisome proliferator-activated receptor gamma) acts as a key molecule of adipocyte differentiation, and transactivates multiple target genes involved in lipid metabolic pathways. Identification of PPARgamma target genes will facilitate to predict the extent to which the drugs can affect and also to understand the molecular basis of lipid metabolism. Here, we have identified five target genes regulated directly by PPARgamma during adipocyte differentiation in 3T3-L1 cells using integrated analyses of ChIP-on-chip and expression microarray. We have confirmed the direct PPARgamma regulation of five genes by luciferase reporter assay in NIH-3T3 cells. Of these five genes Hp, Tmem143 and 1100001G20Rik are novel PPARgamma targets. We have also detected PPREs (PPAR response elements) sequences in the promoter region of the five genes computationally. Unexpectedly, most of the PPREs detected proved to be atypical, suggesting the existence of more atypical PPREs than previously thought in the promoter region of PPARgamma regulated genes.

FIMM, a database of functional molecular immunology: update 2002

FIMM database (http://sdmc.krdl.org.sg:8080/fimm) contains data relevant to functional molecular immunology, focusing on cellular immunology. It contains fully referenced data on protein antigens, major histocompatibility complex (MHC) molecules, MHC-associated peptides and relevant disease associations. FIMM has a set of search tools for extraction of information and results are presented as lists or as reports.

Efficient discovery of immune response targets by cyclical refinement of QSAR models of peptide binding

Peptides that induce and recall T-cell responses are called T-cell epitopes. T-cell epitopes may be useful in a subunit vaccine against malaria. Computer models that simulate peptide binding to MHC are useful for selecting candidate T-cell epitopes since they minimize the number of experiments required for their identification. We applied a combination of computational and immunological strategies to select candidate T-cell epitopes. A total of 86 experimental binding assays were performed in three rounds of identification of HLA-A11 binding peptides from the six preerythrocytic malaria antigens. Thirty-six peptides were experimentally confirmed as binders. We show that the cyclical refinement of the ANN models results in a significant improvement of the efficiency of identifying potential T-cell epitopes.

Calibrating Evolutionary Rates at Major Histocompatibility Complex Loci

Unlike alleles at many other loci, major histocompatibility complex (Mhc) locus alleles often differ by nucleotide substitutions at more than one site, often as many as 88 sites. The substitutions accumulate gradually during evolution by the same process that leads to the divergence of genes in two biological species. The difference between the inter- and intraspecific variation is that in the former, substitutions become fixed in the population (reach a frequency of 1.0), whereas in the latter, they reach polymorphic frequencies (≥ 0.01, < 1.0). Since accumulation of interspecific differences is believed by many geneticists to proceed with a clock-like regularity within certain taxonomic groups, there is no a priori reason why the same should not be true for the accumulation of polymorphic differences. Here we demonstrate the validity of this assumption by comparing alleles at the Mhc-DRB and Mhc-DQB loci of different primate species. We then estimate the evolutionary rates at the DRB and DQB loci; the overall rates of these loci are 0.97 ± 0.17 and 1.2 ± 0.39 (site/billion years), respectively. However, the rate of the sites (both synonymous and nonsynonymous) encoding the peptide (antigen)-binding region (PBR) is 4 to 7 times higher than in the rest of the gene. As previously suggested, the enhanced nonsynonymous rate at the PBR is most likely due to balancing selection, but the PBR as a whole may be a hot spot of nucleotide substitutions.

Conservative evolution of the Mbc-DP region in anthropoid primates

To determine the organization of the DP region in the Mbc of anthropoid primates, we constructed contig maps from cosmid clones of the chimpanzee and orangutan, representatives of the infraorder Catarrhini, as well as of the cotton-top tamarin, a representative of the infraorder Platyrrhini. We found the maps to be remarkably similar to each other and to the previously published map of the human DP region. In each of the four species, the DP region consists of four loci arranged in the same order (DPB2 . . . DPA2 . . . DPB1 . . . DPA1) and in the same transcriptional orientation (tail-to-tail). The regions in the four species are of approximately the same length and many of the restriction sites are shared between species. The inserts of most Alu elements, of a ribosomal protein pseudogene, and of an IgC epsilon-like pseudogene are found in corresponding positions in all four species. The data indicate that the human-type organization of the DP region was established before the divergence of the Catarrhini and Platyrrhini lines more than 37 million years ago and that it has remained principally intact since that time. This conservation of the DP region is in striking contrast to the evolutionary instability of certain other Mbc regions, in particular those occupied by the DRB or C4 and CYP21 loci. We interpret the stability of the DP region as an indication that the region is being phased out functionally.