The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells

Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterize the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterized extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSC s exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.Key words: genomic imprinting, embryonic stem cells, mesenchymal stem cells, differentiation, methylation, epigenetic stability     

Mutant presenilin‐1 deregulated peripheral immunity exacerbates Alzheimer‐like pathology

Mutations in the presenilin‐1 (PS1) gene are independent causes of familial Alzheimer's disease (AD). AD patients have dysregulated immunity, and PS1 mutant mice exhibit abnormal systemic immune responses. To test whether immune function abnormality caused by a mutant human PS1 gene (mhPS1) could modify AD‐like pathology, we reconstituted immune systems of AD model mice carrying a mutant human amyloid precursor protein gene (mhAPP; Tg2576 mice) or both mhAPP and mhPS1 genes (PSAPP mice) with allo‐geneic bone marrow cells. Here, we report a marked reduction in amyloid‐β (Aβ) levels, β‐amyloid plaques and brain inflammatory responses in PSAPP mice following strain‐matched wild‐type PS1 bone marrow reconstitution. These effects occurred with immune switching from pro‐inflammatory T helper (Th) 1 to anti‐inflammatory Th2 immune responses in the periphery and in the brain, which likely instructed microglia to phagocytose and clear Aβ in an ex vivo assay. Conversely, Tg2576 mice displayed accelerated AD‐like pathology when reconstituted with mhPS1 bone marrow. These data show that haematopoietic cells bearing the mhPS1 transgene exacerbate AD‐like pathology, suggesting a novel therapeutic strategy for AD based on targeting PS1 in peripheral immune cells.     

Mannosidase 2, alpha 1 deficiency is associated with ricin resistance in embryonic stem (ES) cells

Host gene products required for mediating the action of toxins are potential targets for reversing or controlling their pathogenic impact following exposure. To identify such targets libraries of insertional gene-trap mutations generated with a PiggyBac transposon in Blm-deficient embryonic stem cells were exposed to the plant toxin, ricin. Resistant clones were isolated and genetically characterised and one was found to be a homozygous mutant of the mannosidase 2, alpha 1 (Man2α1) locus with a matching defect in the homologous allele. The causality of the molecular lesion was confirmed by removal of the transposon following expression of PB-transposase. Comparative glycomic and lectin binding analysis of the Man2α1 (−/−) ricin resistant cells revealed an increase in the levels of hybrid glycan structures and a reduction in terminal β-galactose moieties, potential target receptors for ricin. Furthermore, naïve ES cells treated with inhibitors of the N-linked glycosylation pathway at the mannosidase 2, alpha 1 step exhibited either full or partial resistance to ricin. Therefore, we conclusively identified mannosidase 2, alpha 1 deficiency to be associated with ricin resistance.     

Loss of glucocorticoid receptor expression by DNA methylation prevents glucocorticoid induced apoptosis in human small cell lung cancer cells

Human small cell lung cancer (SCLC) is highly aggressive, and quickly develops resistance to therapy. SCLC cells are typically insensitive to glucocorticoids due to impaired glucocorticoid receptor (GR) expression. This is important as we have previously shown that expression of a GR transgene induces cell death in-vitro, and inhibits tumor growth in-vivo. However, the underlying mechanism for loss of GR expression is unknown. The SCLC cell line, DMS79, has low GR expression, compared to non-SCLC cell lines and normal bronchial epithelial cells. Retroviral GR expression in DMS79 cells caused activation of the apoptotic pathway as evidenced by marked induction of caspase-3 activity. Methylation analysis of the GR promoter revealed some methylation in the 1D, and 1E promoters of the GR gene, however the ubiquitous constitutively active 1C promoter was heavily methylated. In the 1C promoter there was a highly significant increase in DNA methylation in a panel of 14 human SCLC cell lines compared to a mixed panel of GR expressing, and non-expressing cell lines, and to peripheral blood mononuclear cells. Furthermore, within the panel of SCLC cell lines there was a significant negative correlation seen between methylation of the 1C promoter, and GR protein expression. Reversal of GR gene methylation with DNA methyltransferase inhibition caused increased GR mRNA and protein expression in SCLC but not non-SCLC cells. This resulted in increased Gc sensitivity, decreased Bcl-2 expression and increased caspase-3 activity in SCLC cells. These data suggest that DNA methylation decreases GR gene expression in human SCLC cells, in a similar manner to that for conventional tumor suppressor genes.     

Genome Sequence of Campylobacter jejuni strain 327, a strain isolated from a turkey slaughterhouse

Campylobacter is one of the leading causes of food-borne gastroenteritis and has a high prevalence in poultry. Campylobacter jejuni subsp. jejuni 327 is a subspecies of the genus Campylobacter of the family Campylobacteraceae in the phylum Proteobacteria. The microaerophilic, spiral shaped, catalase positive bacterium obtains energy from the metabolism of amino acids and Krebs cycle intermediates. Strain 327 was isolated from a turkey slaughter production line and is considered environmentally sensitive to food processing (cold, heat, drying) and storage conditions. The 327 whole genome shotgun sequence of 1,618,613 bp long consists of 1,740 protein-coding genes, 46 tRNA genes and 3 rRNA operons. A protein based BLAST analysis places the turkey isolate 327 close to the human clinical strain 81116 (NCTC 11828).     

Chelator-facilitated chemical modification of IMP-1 metallo-β-lactamase and its consequences on metal binding

A method involving the reversible chemical modification of an active site, zinc-binding cysteine residue (Cys221) for the specific removal of one of the two zinc ions in the metallo-β-lactamase IMP-1 was explored. Covalent modification of Cys221 by 5,5′-dithio-bis(2-nitrobenzoic acid) was greatly enhanced by the presence of dipicolinic acid, and subsequent removal of the modifying group was easily achieved by reduction of the disulfide bond. However, mass spectrometric analyses and an assessment of IMP-1’s catalytic competence are consistent with the maintenance of the enzyme’s binuclear status. The consequences arising from chemical modification of Cys221 are thus distinct from those reported for Cys → Ala/Ser mutants of IMP-1 and other metallo-β-lactamases, which are mononuclear.     

Regulatory Evolution of a Duplicated Heterodimer Across Species and Tissues of Allopolyploid Clawed Frogs (<Emphasis Type="Italic">Xenopus</Emphasis>)

Changes in gene expression contribute to reproductive isolation of species, adaptation, and development and may impact the genetic fate of duplicated genes. African clawed frogs (genus Xenopus) offer a useful model for examining regulatory evolution, particularly after gene duplication, because species in this genus are polyploid. Additionally, these species can produce viable hybrids, and expression divergence between coexpressed species-specific alleles in hybrids can be attributed exclusively to cis-acting mechanisms. Here we have explored expression divergence of a duplicated heterodimer composed of the recombination activating genes 1 and 2 (RAG1 and RAG2). Previous work identified a phylogenetically biased pattern of pseudogenization of RAG1 wherein one duplicate—RAG1β—was more likely to become a pseudogene than the other one—RAG1α. In this study we show that ancestral expression divergence between these duplicates could account for this. Using comparative data we demonstrate that regulatory divergence between species and between duplicated genes varies significantly across tissue types. These results have implications for understanding of variables that influence pseudogenization of duplicated genes generated by polyploidization, and for interpretation of the relative contributions of cis versus trans mechanisms to expression divergence at the cellular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cophylogeny of Nosema (Microsporidia: Nosematidae) and bees (Hymenoptera: Apidae) suggests both cospeciation and a host-switch

Some microsporidian parasites belonging to the genus Nosema infect bees. Previous phylogenies of these parasites have produced alternative, conflicting relationships. We analyzed separately, and in combination, large and small subunit ribosomal DNA sequences of Nosema species infecting bees under neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian frameworks. We observed a sister relationship between Nosema ceranae and Nosema bombi, with Nosema apis as a basal member to this group. When compared to their respective hosts (Apis cerana, Bombus spp., and A. mellifera), 2 plausible evolutionary scenarios emerged. The first hypothesis involves a common ancestor of N. bombi host-switching from a historical Bombus lineage to A. cerana. The second suggests an ancestral N. ceranae host-switching to a species of Bombus. The reported events offer insight into the evolutionary history of these organisms and may explain host specificity and virulence of Nosema in these economically important insects.     

Splenic mass of masked shrews, Sorex cinereus, in relation to body mass, sex, age, day of the year, and bladder nematode, Liniscus (=Capillaria) maseri, infection

The spleen is an important organ of vertebrates. Splenic mass can change in response to a variety of factors. We tested whether splenic mass of masked shrews, Sorex cinereus, was related to sex, age, time of the year, or intensity of bladder nematode (Liniscus [=Capillaria] maseri) infection, after controlling for host body mass. For females, body mass was a strong predictor of splenic mass. For males, splenic masses were greater later in the year and in more heavily infected males. The latter appeared to represent a threshold response wherein only the most heavily infected individuals had enlarged spleens.