Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs.

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

IL-4 receptor α blockade prevents sensitization and alters acute and long-lasting effects of allergen-specific immunotherapy of murine allergic asthma.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only causal treatment for allergy. However, success rates vary depending on the type of allergy and disease background of the patient. Hence, strategies targeting an increased therapeutic efficacy are urgently needed. Here, the effects of blockade of IL-4 and IL-13 signaling on different phases of AIT were addressed. METHODS: The impact of the recombinantly produced IL-4 and IL-13 antagonist IL-4 mutein (IL-4M) on allergic sensitization and AIT outcome in experimental allergic asthma were analyzed in a murine model. The effects of IL-4M administration were assessed prior/during sensitization, immediately after AIT under allergen challenge, and two weeks post-treatment. RESULTS: Intervention with IL-4M prior/during sensitization led to strong induction of IgG1, IgG2a, IgG2b, and IgG3, decrease of specific and total IgE, as well as of IL-5 in serum. Similar effects on the serum immunoglobulin levels were observed immediately after IL4M-supplemented AIT during allergen challenge. Additionally, IL4M markedly suppressed type-2 cytokine secretion of splenocytes beyond the effect of AIT alone. These effects were equaled to those of AIT alone two weeks post-treatment. Intriguingly, here, IL-4M induced a sustained decrease of Th2-biased Tregs (ST2+ FOXP3+ GATA3intermediate ). CONCLUSIONS: IL-4 and IL-13 blockade during experimental AIT demonstrates beneficial effects on immunological key parameters such as immunoglobulin and cytokine secretion immediately after AIT. Although two weeks later these effects were dropped to those of AIT alone, the number of potentially disease-triggering Th2-biased Tregs was further significantly decreased by IL-4M treatment. Hence, IL-4/IL13-targeting therapies prime the immune memory in therapy success-favoring manner.

A paternal methyl donor-rich diet altered cognitive and neural functions in offspring mice.

Dietary intake of methyl donors, such as folic acid and methionine, shows considerable intra-individual variation in human populations. While it is recognized that maternal departures from the optimum of dietary methyl donor intake can increase the risk for mental health issues and neurological disorders in offspring, it has not been explored whether paternal dietary methyl donor intake influences behavioral and cognitive functions in the next generation. Here, we report that elevated paternal dietary methyl donor intake in a mouse model, transiently applied prior to mating, resulted in offspring animals (methyl donor-rich diet (MD) F1 mice) with deficits in hippocampus-dependent learning and memory, impaired hippocampal synaptic plasticity and reduced hippocampal theta oscillations. Gene expression analyses revealed altered expression of the methionine adenosyltransferase Mat2a and BK channel subunit Kcnmb2, which was associated with changes in Kcnmb2 promoter methylation in MD F1 mice. Hippocampal overexpression of Kcnmb2 in MD F1 mice ameliorated altered spatial learning and memory, supporting a role of this BK channel subunit in the MD F1 behavioral phenotype. Behavioral and gene expression changes did not extend into the F2 offspring generation. Together, our data indicate that paternal dietary factors influence cognitive and neural functions in the offspring generation.

Targeted disruption of the mouse Npal3 gene leads to deficits in behavior, increased IgE levels, and impaired lung function.

The non-imprinted in Prader-Willi/Angelman syndrome (NIPA) proteins are highly conserved receptors or transporters. Translocation of NIPA genes were found in patients with Prader-Willi syndrome, and loss-of-function of the NIPA1 gene was identified in hereditary spastic paraplegia. The family of NIPA-like domain containing (NPAL) proteins is closely related to the NIPA proteins, but to date nothing is known about their function. Here, we could demonstrate that both human NPAL3 and mouse NPAL3 are ubiquitously expressed and encode highly conserved proteins. To further elucidate the function of the Npal3 gene, knockout (Npal3(-/-)) mice were generated. Intensive phenotypic analyses revealed that disruption of the Npal3 gene results in a pleiotropic phenotype. The function of the nervous system was impaired in both mutant males and females which could be demonstrated in behavioral tests. In addition, in NPAL3 mutants the number of NK cells was decreased and changes in IgM, IgG(2), and IgA were observed, indicating that the immune system is also affected. Interestingly, increased IgE levels as well as impaired lung functions were observed in mutant males but not in mutant females. It should be noted that the human Npal3 gene is located at 1p36.12-->p35.1, and atopic diseases were previously linked to this genomic region. Thus, the Npal3(-/-) mice could serve as a valuable model system for studying atopic diseases.

Analysis of a meiosis-specific URS1 site: sequence requirements and involvement of replication protein A.

URS1 is a transcriptional repressor site found in the promoters of a wide variety of yeast genes that are induced under stress conditions. In the context of meiotic promoters, URS1 sites act as repressor sequences during mitosis and function as activator sites during meiosis. We have investigated the sequence requirements of the URS1 site of the meiosis-specific HOP1 gene (URS1H) and have found differences compared with a URS1 site from a nonmeiotic gene. We have also observed that the sequence specificity for meiotic activation at this site differs from that for mitotic repression. Base pairs flanking the conserved core sequence enhance meiotic induction but are not required for mitotic repression of HOP1. Electrophoretic mobility shift assays of mitotic and meiotic cell extracts show a complex pattern of DNA-protein complexes, suggesting that several different protein factors bind specifically to the site. We have determined that one of the complexes of URS1H is formed by replication protein A (RPA). Although RPA binds to the double-stranded URS1H site in vitro, it has much higher affinity for single-stranded than for double-stranded URS1H, and one-hybrid assays suggest that RPA does not bind to this site at detectable levels in vivo. In addition, conditional-lethal mutations in RPA were found to have no effect on URS1H-mediated repression. These results suggest that although RPA binds to URS1H in vitro, it does not appear to have a functional role in transcriptional repression through this site in vivo.

Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene.

The meiosis-specific gene HOP1, which encodes a component of the synaptonemal complex, is controlled through two regulatory elements, UASH and URS1H. Sites similar to URS1H have been identified in the promoter region of virtually every early meiosis-specific gene, as well as in many promoters of nonmeiotic genes, and it has been shown that the proteins that bind to this site function to regulate meiotic and nonmeiotic transcription. Sites similar to the UASH site have been found in a number of meiotic and nonmeiotic genes as well. Since it has been shown that UASH functions as an activator site in vegetative haploid cells, it seemed likely that the factors binding to this site regulate both meiotic and nonmeiotic transcription. We purified the factor binding to the UASH element of the HOP1 promoter. Sequence analysis identified the protein as Abf1 (autonomously replicating sequence-binding factor 1), a multifunctional protein involved in DNA replication, silencing, and transcriptional regulation. We show by mutational analysis of the UASH site, that positions outside of the proposed UASH consensus sequence (TNTGN[A/T]GT) are required for DNA binding in vitro and transcriptional activation in vivo. A new UASH consensus sequence derived from this mutational analysis closely matches a consensus Abf1 binding site. We also show that an Abf1 site from a nonmeiotic gene can replace the function of the UASH site in the HOP1 promoter. Taken together, these results show that Abf1 functions to regulate meiotic gene expression.

Transcriptional regulation of the SMK1 mitogen-activated protein kinase gene during meiotic development in Saccharomyces cerevisiae.

Meiotic development (sporulation) in Saccharomyces cerevisiae is characterized by an ordered pattern of gene expression, with sporulation-specific genes classified as early, middle, mid-late, or late depending on when they are expressed. SMK1 encodes a mitogen-activated protein kinase required for spore morphogenesis that is expressed as a middle sporulation-specific gene. Here, we identify the cis-acting DNA elements that regulate SMK1 transcription and characterize the phenotypes of mutants with altered expression patterns. The SMK1 promoter contains an upstream activating sequence (UASS) that specifically interacts with the transcriptional activator Abf1p. The Abf1p-binding sites from the early HOP1 and the middle SMK1 promoters are functionally interchangeable, demonstrating that these elements do not play a direct role in their differential transcriptional timing. Timing of SMK1 expression is determined by another cis-acting DNA sequence termed MSE (for middle sporulation element). The MSE is required not only for activation of SMK1 transcription during middle sporulation but also for its repression during vegetative growth and early meiosis. In addition, the SMK1 MSE can repress vegetative expression in the context of the HOP1 promoter and convert HOP1 from an early to a middle gene. SMK1 function is not contingent on its tight transcriptional regulation as a middle sporulation-specific gene. However, promoter mutants with different quantitative defects in SMK1 transcript levels during middle sporulation show distinct sporulation phenotypes.

Smad-interacting protein 1 affects acute and tonic, but not chronic pain.

BACKGROUND: Smad-interacting protein 1 (also named Zeb2 and Zfhx1b) is a transcription factor that plays an important role in neuronal development and, when mutated, causes Mowat-Wilson syndrome (MWS). A corresponding mouse model carrying a heterozygous Zeb2 deletion was comprehensively analysed in the German Mouse Clinic. The most prominent phenotype was the reduced pain sensitivity. In this study, we investigated the role of Zeb2 in inflammatory and neuropathic pain. METHODS: For this, we tested mutant Zeb2 animals in different models of inflammatory pain like abdominal constriction, formalin and carrageenan test. Furthermore, we studied the pain reactivity of the mice after peripheral nerve ligation. To examine the nociceptive transmission of primary sensory dorsal root ganglia (DRG) neurons, we determined the neuronal activity in the spinal dorsal horn after the formalin test using staining of c-Fos. Next, we characterized the neuronal cell population in the DRGs and in the sciatic nerve to study the effect of the Zeb2 mutation on peripheral nerve morphology. RESULTS: The present data show that Zeb2 is involved in the development of primary sensory DRG neurons, especially of C- and Aδ fibres. These alterations contribute to a hypoalgesic phenotype in inflammatory but not in neuropathic pain in these Zeb2(+/-) mice. CONCLUSION: Our data suggest that the under-reaction to pain observed in MWS patients results from a reduced responsivity to nociceptive stimulation rather than an inability to communicate discomfort.

Inbred wild type mouse strains have distinct spontaneous morphological phenotypes.

The mouse is the most commonly used animal for modelling human disease. New approaches for generating genetically manipulated mouse models to represent human disease, as well as target the function of specific genes, has increased the importance of mice in biomedical science. For the correct interpretation of alterations in mouse phenotype the basic morphology of background mouse strains must be known. Despite on-going efforts to create publicly available baseline phenotypic data, the information concerning spontaneous lesions in wild-type mice is incomplete and scattered so far, and further studies are needed. We addressed this problem by screening haematoxylin-eosin stained sections of brain, reproductive organs, urinary bladder, kidney, thyroid, parathyroid, heart, lung, spleen, thymus, lymph nodes, adrenal glands, stomach, intestine, liver, skin and pancreas of six commonly used inbred mouse strains (C57BL6/J, C57BL6/NTac, C3HeB/FeJ, BALB/cByJ, 129P2/OlaHsd and FVB/N) for inherent spontaneous morphological lesions. Interesting spontaneous phenotypes were seen in morphology of the liver, pancreas, adrenal glands, lungs, intestines and heart. In conclusion, care should be taken when choosing the background mouse strain for genetic manipulations, since different mouse strains harbour different inherent lesions that can affect the function of targeted genes, interpretation of results and translation of results to model human disease.

The German Mouse Clinic: a platform for systemic phenotype analysis of mouse models.

The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line--a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, host-pathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

Deletion of glucose transporter GLUT8 in mice increases locomotor activity.

Transport of glucose into neuronal cells is predominantly mediated by the glucose transporters GLUT1 and GLUT3. In addition, GLUT8 is expressed in some regions of the brain. By in situ hybridization we detected GLUT8-mRNA in hippocampus, thalamus, and cortex. However, its cellular and physiological function is still unknown. Thus, GLUT8 knockout (Slc2a8 -/-) mice were used for a screening approach in the modified hole board (mHB) behavioral test to analyze the role of GLUT8 in the central nervous system. Slc2a8 -/- mice showed increased mean velocity, total distance traveled and performed more turns in the mHB test. This hyperactivity of Slc2a8 -/- mice was confirmed by monitoring locomotor activity in the home cage and voluntary activity in a running wheel. In addition, Slc2a8 -/- mice showed increased arousal as indicated by elevated defecation, reduced latency to the first defecation and a tendency to altered grooming. Furthermore, the mHB test gave evidence that Slc2a8 -/- mice exhibit a reduced risk assessment because they performed less rearings in an unprotected area and showed significantly reduced latency to stretched body posture. Our data suggest that behavioral alterations of Slc2a8 -/- mice are due to dysfunctions in neuronal processes presumably as a consequence of defects in the glucose metabolism.

Creatine improves health and survival of mice.

The supplementation of creatine (Cr) has a marked neuroprotective effect in mouse models of neurodegenerative diseases. This has been assigned to the known bioenergetic, anti-apoptotic, anti-excitotoxic, and anti-oxidant properties of Cr. As aging and neurodegeneration share pathophysiological pathways, we investigated the effect of oral Cr supplementation on aging in 162 aged C57Bl/6J mice. Outcome variables included "healthy" life span, neurobehavioral phenotyping, as well as morphology, biochemistry, and expression profiling from brain. The median healthy life span of Cr-fed mice was 9% higher than in control mice, and they performed significantly better in neurobehavioral tests. In brains of Cr-treated mice, there was a trend towards a reduction of reactive oxygen species and significantly lower accumulation of the "aging pigment" lipofuscin. Expression profiling showed an upregulation of genes implicated in neuronal growth, neuroprotection, and learning. These data show that Cr improves health and longevity in mice. Cr may be a promising food supplement to promote healthy human aging.

Experimental data of a single promoter can be used for in silico detection of genes with related regulation in the absence of sequence similarity.

Gene expression is presently a major focus in genome analysis, and the experimental data on regulatory mechanisms and functional transcription factor binding sites are steadily growing. However, the annotation of transcriptional regulation of sequences cannot keep pace with the exponential growth of sequence databases. Employing detailed experimental data of a single promoter or enhancer to predict genes with similar regulation would provide a powerful method to link the literature about transcriptional regulation and sequence databases. To this end, we used information on individual functional transcription factor binding sites to compose in silico promoter and enhancer models of muscle-specific genes and to analyze the rodents section of EMBL with these models. Exhaustive evaluation of all hits revealed every second to third match to be a muscle-associated gene. Moreover, functionally related regulatory regions were detected by our model-based approach even in the absence of sequence similarity. We believe that this new approach is a substanial extension to database analysis by BLAST or FASTA, which are restricted to sequence similarity.

Sum1 and Hst1 repress middle sporulation-specific gene expression during mitosis in Saccharomyces cerevisiae.

Meiotic development in yeast is characterized by the sequential induction of temporally distinct classes of genes. Genes that are induced at the middle stages of the pathway share a promoter element, termed the middle sporulation element (MSE), which interacts with the Ndt80 transcriptional activator. We have found that a subclass of MSEs are strong repressor sites during mitosis. SUM1 and HST1, genes previously associated with transcriptional silencing, are required for MSE-mediated repression. Sum1 binds specifically in vitro to MSEs that function as strong repressor sites in vivo. Repression by Sum1 is gene specific and does not extend to neighboring genes. These results suggest that mechanisms used to silence large regions of chromatin may also be used to regulate the expression of specific genes during development. NDT80 is regulated during mitosis by both the Sum1 and Ume6 repressors. These results suggest that progression through sporulation may be controlled by the regulated competition between the Sum1 repressor and Ndt80 activator at key MSEs.

First pass annotation of promoters on human chromosome 22.

The publication of the first almost complete sequence of a human chromosome (chromosome 22) is a major milestone in human genomics. Together with the sequence, an excellent annotation of genes was published which certainly will serve as an information resource for numerous future projects. We noted that the annotation did not cover regulatory regions; in particular, no promoter annotation has been provided. Here we present an analysis of the complete published chromosome 22 sequence for promoters. A recent breakthrough in specific in silico prediction of promoter regions enabled us to attempt large-scale prediction of promoter regions on chromosome 22. Scanning of sequence databases revealed only 20 experimentally verified promoters, of which 10 were correctly predicted by our approach. Nearly 40% of our 465 predicted promoter regions are supported by the currently available gene annotation. Promoter finding also provides a biologically meaningful method for "chromosomal scaffolding", by which long genomic sequences can be divided into segments starting with a gene. As one example, the combination of promoter region prediction with exon/intron structure predictions greatly enhances the specificity of de novo gene finding. The present study demonstrates that it is possible to identify promoters in silico on the chromosomal level with sufficient reliability for experimental planning and indicates that a wealth of information about regulatory regions can be extracted from current large-scale (megabase) sequencing projects. Results are available on-line at http://genomatix.gsf.de/chr22/.

The Helmholtz Network for Bioinformatics: an integrative web portal for bioinformatics resources.

SUMMARY: The Helmholtz Network for Bioinformatics (HNB) is a joint venture of eleven German bioinformatics research groups that offers convenient access to numerous bioinformatics resources through a single web portal. The 'Guided Solution Finder' which is available through the HNB portal helps users to locate the appropriate resources to answer their queries by employing a detailed, tree-like questionnaire. Furthermore, automated complex tool cascades ('tasks'), involving resources located on different servers, have been implemented, allowing users to perform comprehensive data analyses without the requirement of further manual intervention for data transfer and re-formatting. Currently, automated cascades for the analysis of regulatory DNA segments as well as for the prediction of protein functional properties are provided. AVAILABILITY: The HNB portal is available at http://www.hnbioinfo.de