During gestation there is a high demand for the essential nutrient choline. Adult rats supplemented with choline during embryonic days (E) 11-17 have improved memory performance and do not exhibit age-related memory decline, whereas prenatally choline-deficient animals have memory deficits. Choline, via betaine, provides methyl groups for the production of S-adenosylmethionine, a substrate of DNA methyltransferases (DNMTs). We describe an apparently adaptive epigenomic response to varied gestational choline supply in rat fetal liver and brain. S-Adenosylmethionine levels increased in both organs of E17 fetuses whose mothers consumed a choline-supplemented diet. Surprisingly, global DNA methylation increased in choline-deficient animals, and this was accompanied by overexpression of Dnmt1 mRNA. Previous studies showed that the prenatal choline supply affects the expression of multiple genes, including insulin-like growth factor 2 (Igf2), whose expression is regulated in a DNA methylation-dependent manner. The differentially methylated region 2 of Igf2 was hypermethylated in the liver of E17 choline-deficient fetuses, and this as well as Igf2 mRNA levels correlated with the expression of Dnmt1 and with hypomethylation of a regulatory CpG within the Dnmt1 locus. Moreover, mRNA expression of brain and liver Dnmt3a and methyl CpG-binding domain 2 (Mbd2) protein as well as cerebral Dnmt3l was inversely correlated to the intake of choline. Thus, choline deficiency modulates fetal DNA methylation machinery in a complex fashion that includes hypomethylation of the regulatory CpGs within the Dnmt1 gene, leading to its overexpression and the resultant increased global and gene-specific (e.g. Igf2) DNA methylation. These epigenomic responses to gestational choline supply may initiate the long term developmental changes observed in rats exposed to varied choline intake in utero.

The histone demethylase Ubiquitously Transcribed Tetratricopeptide Repeat Protein X-Linked (UTX/KDM6A) demethylates H3K27me2/3 at genes and enhancers and is often inactivated by mutations in urothelial carcinoma (UC). The consequences of its inactivation are however poorly understood. We have investigated the consequences of moderate UTX overexpression across a range of UC cell lines with or without mutations in KDM6A or its interaction partners and in a normal control cell line. Effects on cell proliferation, especially long-term, varied dramatically between the cell lines, ranging from deleterious to beneficial. Similarly, effects on global gene expression determined by RNA-Seq were variable with few overlapping up- or downregulated genes between the cell lines. Our data indicate that UTX does not act in a uniform fashion in UC. Rather, its effect depends on several contingencies including, prominently, the status of KMT2C and KMT2D which interact with UTX in the COMPASS complex. In particular, we provide evidence that these factors determine the amount of nuclear UTX.

Disturbances in histone acetyltransferases (HATs) are common in cancers. In urothelial carcinoma (UC), p300 and CBP are often mutated, whereas the GNAT family HATs GCN5 and PCAF (General Control Nonderepressible 5, p300/CBP-Associated Factor) are often upregulated. Here, we explored the effects of specific siRNA-mediated knockdown of GCN5, PCAF or both in four UC cell lines (UCCs). Expression of various HATs and marker proteins was measured by qRT-PCR and western blot. Cellular effects of knockdowns were analyzed by flow cytometry and ATP-, caspase-, and colony forming-assays. GCN5 was regularly upregulated in UCCs, whereas PCAF was variable. Knockdown of GCN5 or both GNATs, but not of PCAF alone, diminished viability and inhibited clonogenic growth in 2/4 UCCs, inducing cell cycle changes and caspase-3/7 activity. PCAF knockdown elicited GCN5 mRNA upregulation. Double knockdown increased c-MYC and MDM2 (Mouse Double Minute 2) in most cell lines. In conclusion, GCN5 upregulation is especially common in UCCs. GCN5 knockdown impeded growth of specific UCCs, whereas PCAF knockdown elicited minor effects. The limited sensitivity towards GNAT knockdown and its variation between the cell lines might be due to compensatory effects including HAT, c-MYC and MDM2 upregulation. Our results predict that developing drugs targeting individual HATs for UC treatment may be challenging.

Pneumocystis jirovecii pneumonia (PCP) is an opportunistic fungal infection that is associated with a high morbidity and mortality in immunocompromised individuals. In this study, we analysed the microbiome of the lower respiratory tract from critically ill intensive care unit patients with and without pneumocystosis. Broncho-alveolar fluids from 65 intubated and mechanically ventilated intensive care unit patients (34 PCP+ and 31 PCP- patients) were collected. Sequence analysis of bacterial 16S rRNA gene V3/V4 regions was performed to study the composition of the respiratory microbiome using the Illumina MiSeq platform. Differences in the microbial composition detected between PCP+ and PCP- patients were not statistically significant on class, order, family and genus level. In addition, alpha and beta diversity metrics did not reveal significant differences between PCP+ and PCP- patients. The composition of the lung microbiota was highly variable between PCP+ patients and comparable in its variety with the microbiota composition of the heterogeneous collective of PCP- patients. The lower respiratory tract microbiome in patients with pneumocystosis does not appear to be determined by a specific microbial composition or to be dominated by a single bacterial species.