Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs) in mouse embryonic stem cells.

Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation

Increase of Dub3 expression upon UV-induced DNA damage in mESCs.

<p>(A) Schematic representation of the experimental setup. Mouse ESCs were nocodazole arrested and 2 hours post released mock or UV-irradiated (6 J/m²) and collected at indicated time points for RNA extraction and gene expression analysis. (B) FACS analysis of total DNA content (PI staining) of mES cells released from nocodazole collected at indicated time points (hours after release). (C) Mouse ESCs cells released from nocodazole and collected at indicated time points (hours after release) for qPCR quantification of Dub3 and Cyclin E1 mRNA levels. Data was normalized to multiple reference genes and expressed as average of three biological replicates. Error bars indicate standard deviation. (D) qPCR quantification of Nanog and Oct4 mRNA normalised to multiple reference genes from mESCs released from nocodazole and collected at indicated time points (hours after release). expressed as average of three biological replicates. Error bars indicate standard deviation. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093663#pone.0093663.s001" target="_blank">Figure S1</a>).</p

Cell cycle dependent oscillations of Nanog, Dub3 and NCoAs.

<p>(A) qPCR quantification of Cyclin E1, Cyclin A2, Nanog and Oct4 mRNA normalised to multiple reference genes from mESCs released from nocodazole collected during a full cell cycle at indicated time points (hours after release) (B) qPCR quantification of β-TrCP, Cdh1 and Dub3 mRNA (upper panel), NCoA1, NCoA2 and NCoA3 mRNA (middle panel) and Esrrb and Sox2 mRNA (lower panel) normalised to multiple reference genes from mESCs released from nocodazole collected during a full cell cycle at indicated time points (hours after release). (C) qPCR quantification of NCoA1, NCoA2, NCoA3 and Dub3 mRNA normalised to multiple reference genes from mESCs released from nocodazole collected at indicated time points (hours after release). Data is shown as average of three biological replicates and the error bars indicate the standard deviation. (D) Western blot analysis of mESCs released from nocodazole and harvested at indicated time points (hours after release). Antibodies used for immunoblotting of proteins are indicated and * indicates non-specific band. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093663#pone.0093663.s002" target="_blank">Figure S2</a>).</p

Gut Microbiota and Celiac Disease

Recent evidence regarding celiac disease has increasingly shown the role of innate immunity in triggering the immune response by stimulating the adaptive immune response and by mucosal damage. The interaction between the gut microbiota and the mucosal wall is mediated by the same receptors which can activate innate immunity. Thus, changes in gut microbiota may lead to activation of this inflammatory pathway. This paper is a review of the current knowledge regarding the relationship between celiac disease and gut microbiota. In fact, patients with celiac disease have a reduction in beneficial species and an increase in those potentially pathogenic as compared to healthy subjects. This dysbiosis is reduced, but might still remain, after a gluten-free diet. Thus, gut microbiota could play a significant role in the pathogenesis of celiac disease, as described by studies which link dysbiosis with the inflammatory milieu in celiac patients. The use of probiotics seems to reduce the inflammatory response and restore a normal proportion of beneficial bacteria in the gastrointestinal tract. Additional evidence is needed in order to better understand the role of gut microbiota in the pathogenesis of celiac disease, and the clinical impact and therapeutic use of probiotics in this setting