Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines.

We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.

Loss of miR-10a activates lpo and collaborates with activated Wnt signaling in inducing intestinal neoplasia in female mice.

miRNAs are small regulatory RNAs that, due to their considerable potential to target a wide range of mRNAs, are implicated in essentially all biological process, including cancer. miR-10a is particularly interesting considering its conserved location in the Hox cluster of developmental regulators. A role for this microRNA has been described in developmental regulation as well as for various cancers. However, previous miR-10a studies are exclusively based on transient knockdowns of this miRNA and to extensively study miR-10a loss we have generated a miR-10a knock out mouse. Here we show that, in the Apc(min) mouse model of intestinal neoplasia, female miR-10a deficient mice develop significantly more adenomas than miR-10(+/+) and male controls. We further found that Lpo is extensively upregulated in the intestinal epithelium of mice deprived of miR-10a. Using in vitro assays, we demonstrate that the primary miR-10a target KLF4 can upregulate transcription of Lpo, whereas siRNA knockdown of KLF4 reduces LPO levels in HCT-116 cells. Furthermore, Klf4 is upregulated in the intestines of miR-10a knockout mice. Lpo has previously been shown to have the capacity to oxidize estrogens into potent depurinating mutagens, creating an instable genomic environment that can cause initiation of cancer. Therefore, we postulate that Lpo upregulation in the intestinal epithelium of miR-10a deficient mice together with the predominant abundance of estrogens in female animals mainly accounts for the sex-related cancer phenotype we observed. This suggests that miR-10a could be used as a potent diagnostic marker for discovering groups of women that are at high risk of developing colorectal carcinoma, which today is one of the leading causes of cancer-related deaths.

PRDM11 is dispensable for the maintenance and function of hematopoietic stem and progenitor cells.

Hematopoietic stem cells (HSC)(1) supply organisms with life-long output of mature blood cells. To do so, the HSC pool size has to be maintained by HSC self-renewing divisions. PRDM3 and PRDM16 have been documented to regulate HSC self-renewal, maintenance and function. We found Prdm11 to have similar expression patterns in the hematopoietic stem and progenitor cell (HSPC) compartments as Prdm3 and Prdm16. Therefore, we undertook experiments to test if PRDM11 regulates HSC self-renewal, maintenance and function by investigating the Prdm11(-/-) mice. Our data shows that phenotypic HSPCs are intact in bone marrow (BM) of one-year-old Prdm11(-/-) mice. In addition, Prdm11(-/-) mice were able to fully regenerate the hematopoietic system upon BM transplantation (BMT) into lethally irradiated mice with a mild drop in lymphoid output only. Taken together, this suggests that PRDM11, in contrast to PRDM3 and PRDM16, is not directly involved in regulation of HSPCs in mice.

Chromatin-modifying proteins in cancer.

Chromatin-modifying proteins mold the genome into areas that are accessible for transcriptional activity and areas that are transcriptionally silent. This epigenetic gene regulation allows for different transcriptional programs to be conducted in different cell types at different timepoints-despite the fact that all cells in the organism contain the same genetic information. A large amount of data gathered over the last decades has demonstrated that deregulation of chromatin-modifying proteins is etiologically involved in the development and progression of cancer. Here we discuss how epigenetic alterations influence cancer development and review known cancer-associated alterations in chromatin-modifying proteins.

Novel overlapping coding sequences in Chlamydia trachomatis.

Chlamydia trachomatis is the aetiological agent of trachoma and sexually transmitted infections. The C. trachomatis genome sequence revealed an organism adapted to the intracellular habitat with a high coding ratio and a small genome consisting of 1.042-kilobase (kb) with 895 annotated protein coding genes. Here, we repredict the protein-coding genes of the C. trachomatis genome using the gene-finder EasyGene that was trained specifically for C. trachomatis, and compare it with the primary C. trachomatis annotation. Our work predicts 15 genes not listed in the primary annotation and 853 that are in agreement with the primary annotation. Forty two genes from the primary annotation are not predicted by EasyGene. The majority of these genes are listed as hypothetical in the primary annotation. The 15 novel predicted genes all overlap with genes on the complementary strand. We find homologues of several of the novel genes in C. trachomatis Serovar A and Chlamydia muridarum. Several of the genes have typical gene-like and protein-like features. Furthermore, we confirm transcriptional activity from 10 of the putative genes. The combined evidence suggests that at least seven of the 15 are protein coding genes. The data suggest the presence of overlapping active genes in C. trachomatis.

Cationic liposomes containing mycobacterial lipids: a new powerful Th1 adjuvant system.

The immunostimulation provided by the mycobacterial cell wall has been exploited for many decades, e.g., in Freund's complete adjuvant. Recently, the underlying mechanism behind this adjuvant activity, including Toll receptor signaling, has begun to be unraveled, confirming the potential of mycobacterial constituents to act as adjuvants. In this study, the immunostimulatory properties of a Mycobacterium bovis BCG lipid extract were tested for their adjuvant activity. Administration of the lipids in dimethyl dioctadecyl ammonium bromide-based cationic liposomes induced a powerful Th1 response characterized by markedly elevated antigen-specific immunoglobulin G2a (IgG2a) isotype antibodies and substantial production of gamma interferon. The adjuvant formulation (designated mycosomes) elicited high levels of gamma interferon both in C57BL/6 as well as in Th2-prone BALB/c mice. Furthermore, the mycosomes induced immune responses to protein antigens from several sources including Mycobacterium tuberculosis, Chlamydia muridarum, and tetanus toxoid. In a tuberculosis challenge model, the mycosomes combined with the Ag85B-ESAT-6 fusion protein were demonstrated to have a unique ability to maintain sustained immunological memory at a level superior to live BCG.

Minute virus of mice small non-structural protein NS2 localizes within, but is not required for the formation of, Smn-associated autonomous parvovirus-associated replication bodies.

The non-structural proteins NS1 and NS2 of the parvovirus minute virus of mice (MVM) are required for efficient virus replication. It has previously been shown that NS1 and NS2 interact and colocalize with the survival motor neuron (Smn) gene product in novel nuclear structures that are formed late in infection, termed Smn-associated APAR (autonomous parvovirus-associated replication) bodies (SAABs). It is not clear what molecular viral intermediate(s) contribute to SAAB formation. The current results address the role of NS2 in SAAB formation. In highly synchronized wild-type MVM infection of murine A9(2L) cells, NS2 colocalizes with Smn and other SAAB constituents. An MVM mutant that does not produce NS2 still generates SAABS, albeit with a temporal delay. The lag in SAAB formation seen in the absence of NS2 is probably related to the temporal delay in virus replication, suggesting that, whilst NS2 is required for efficient viral infection, it is dispensable for SAAB formation.

Minute virus of mice small nonstructural protein NS2 interacts and colocalizes with the Smn protein.

The small nonstructural protein NS2 of the minute virus of mice (MVM) is required for efficient viral replication, although its mode of action is unclear. Here we demonstrate that NS2 and survival motor neuron protein (Smn) interact in vitro and in vivo. NS2 and Smn also colocalize in infected nuclei at late times following MVM infection.

Minute virus of mice NS1 interacts with the SMN protein, and they colocalize in novel nuclear bodies induced by parvovirus infection.

The human survival motor neuron (SMN) gene is the spinal muscular atrophy-determining gene, and a knockout of the murine Smn gene results in preembryonic lethality. Here we show that SMN can directly interact in vitro and in vivo with the large nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVM), a protein essential for viral replication and a potent transcriptional activator. Typically, SMN localizes within nuclear Cajal bodies and diffusely in the cytoplasm. Following transient NS1expression, SMN and NS1 colocalize within Cajal bodies. At early time points following parvovirus infection, NS1 fails to colocalize with SMN within Cajal bodies; however, during the course of MVM infection, dramatic nuclear alterations occur. Formerly distinct nuclear bodies such as Cajal bodies, promyelocytic leukemia gene product (PML) oncogenic domains (PODs), speckles, and autonomous parvovirus-associated replication (APAR) bodies are seen aggregating at later points in infection. These newly formed large nuclear bodies (termed SMN-associated APAR bodies) are active sites of viral replication and viral capsid assembly. These results highlight the transient nature of nuclear bodies and their contents and identify a novel nuclear body formed during infection. Furthermore, simple transient expression of the viral nonstructural proteins is insufficient to induce this nuclear reorganization, suggesting that this event is induced specifically by a step in the viral infection process.