Project description:DNA methylation is a chromatin modification that is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation have been identified, relatively little is understood about how DNA methylation influences gene expression. Using complementary genetic and biochemical approaches we identified a protein complex that antagonizes the transcriptional gene silencing of two LUCIFERASE (LUC) reporters in a manner that requires DNA methylation. At its core, this complex contains LOW IN LUCIFERASE EXPRESSION (LIL), an α-crystallin domain protein, and METHYL-CpG-BINDING DOMAIN 7 (MBD7), a protein previously associated with DNA methylation. At the LUC reporters, loss of MBD7 or LIL resulted in decreased LUC expression concomitant with modest, but reproducible increases in DNA methylation that can be phenocopied by DNA demethylase mutants. These findings are consistent with other reports and reveal a genetic connection between MBD7, LIL and DNA demethylation. However, we found that the hyper-methylation and gene expression phenotypes at a LUC reporter can be genetically uncoupled, demonstrating that changes in DNA methylation alone are not sufficient to silence LUC expression, and suggesting a role for the MBD7-LIL complex downstream of DNA methylation. Consistent with this hypothesis, our more extensive analysis of DNA methylation in mbd7 and lil mutants revealed only a small number of hyper-methylated loci genome wide. Furthermore, these loci displayed minimal overlap with demethylase targets, suggesting that, in general, the DNA demethylation machinery does not function in a manner dependent on the MBD7-LIL complex. Taken together, our findings place the MBD7-LIL complex amongst a small number of factors that regulate gene expression without causing significant changes in DNA methylation. This complex, however, is unique in that it functions to suppress, rather than enforce the silencing effects of DNA methylation, enabling gene expression of several transgene reporters despite high levels of promoter methylation.

Project description:Reprogramming of differentiated somatic cells into induced pluripotent stem (iPS) cells has potential for derivation of patient-specific cells for therapy as well as for development of models with which to study disease progression. Derivation of iPS cells from human somatic cells has been achieved by viral transduction of human fibroblasts with early developmental genes. Because forced expression of these genes by viral transduction results in transgene integration with unknown and unpredictable potential mutagenic effects, identification of cell culture conditions that can induce endogenous expression of these genes is desirable. Here we show that primary adult human fibroblasts have basal expression of mRNA for OCT4, SOX2, and NANOG. However, translation of these messages into detectable proteins and their subcellular localization depends on cell culture conditions. Manipulation of oxygen concentration and FGF2 supplementation can modulate expression of some pluripotency related genes at the transcriptional, translational, and cellular localization level. Changing cell culture condition parameters led to expression of REX1, potentiation of expression of LIN28, translation of OCT4, SOX2, and NANOG, and translocation of these transcription factors to the cell nucleus. We also show that culture conditions affect the in vitro lifespan of dermal fibroblasts, nearly doubling the number of population doublings before the cells reach replicative senescence. Our results suggest that it is possible to induce and manipulate endogenous expression of stem cell genes in somatic cells without genetic manipulation, but this short-term induction may not be sufficient for acquisition of true pluripotency. Further investigation of the factors involved in inducing this response could lead to discovery of defined culture conditions capable of altering cell fate in vitro. This would alleviate the need for forced expression by transgenesis, thus eliminating the risk of mutagenic effects due to genetic manipulation.

Project description:BackgroundWHO guidelines recommend concurrent iron and antimalarial treatment in children with malaria and iron deficiency, but iron may not be well absorbed or utilized during a malaria episode.ObjectivesWe aimed to determine whether starting iron 28 d after antimalarial treatment in children with severe malaria and iron deficiency would improve iron status and lower malaria risk.MethodsWe conducted a randomized clinical trial on the effect of immediate compared with delayed iron treatment in Ugandan children 18 mo-5 y of age with 2 forms of severe malaria: cerebral malaria (CM; n = 79) or severe malarial anemia (SMA; n = 77). Asymptomatic community children (CC; n = 83) were enrolled as a comparison group. Children with iron deficiency, defined as zinc protoporphyrin (ZPP) ≥ 80 µmol/mol heme, were randomly assigned to receive a 3-mo course of daily oral ferrous sulfate (2 mg · kg-1 · d-1) either concurrently with antimalarial treatment (immediate arm) or 28 d after receiving antimalarial treatment (delayed arm). Children were followed for 12 mo.ResultsAll children with CM or SMA, and 35 (42.2%) CC, were iron-deficient and were randomly assigned to immediate or delayed iron treatment. Immediate compared with delayed iron had no effect in any of the 3 study groups on the primary study outcomes (hemoglobin concentration and prevalence of ZPP ≥ 80 µmol/mol heme at 6 mo, malaria incidence over 12 mo). However, after 12 mo, children with SMA in the delayed compared with the immediate arm had a lower prevalence of iron deficiency defined by ZPP (29.4% compared with 65.6%, P = 0.006), a lower mean concentration of soluble transferrin receptor (6.1 compared with 7.8 mg/L, P = 0.03), and showed a trend toward fewer episodes of severe malaria (incidence rate ratio: 0.39; 95% CI: 0.14, 1.12).ConclusionsIn children with SMA, delayed iron treatment did not increase hemoglobin concentration, but did improve long-term iron status over 12 mo without affecting malaria incidence.This trial was registered at clinicaltrials.gov as NCT01093989.

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The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status.

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The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status.

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