ID2 controls differentiation of enteroendocrine cells in mouse small intestine.

AIMS: The mammalian gut is the largest endocrine organ. Dozens of hormones secreted by enteroendocrine cells regulate a variety of physiological functions of the gut but also of the pancreas and brain. Here, we examined the role of the helix-loop-helix transcription factor ID2 during the differentiation of intestinal stem cells along the enteroendocrine lineage. METHODS: To assess the functions of ID2 in the adult mouse small intestine, we used single-cell RNA sequencing, genetically modified mice, and organoid assays. RESULTS: We found that in the adult intestinal epithelium Id2 is predominantly expressed in enterochromaffin and peptidergic enteroendocrine cells. Consistently, the loss of Id2 leads to the reduction of Chromogranin A-positive enteroendocrine cells. In contrast, the numbers of tuft cells are increased in Id2 mutant small intestine. Moreover, ablation of Id2 elevates the numbers of Serotonin+ enterochromaffin cells and Ghrelin+ X-cells in the posterior part of the small intestine. Finally, ID2 acts downstream of BMP signalling during the differentiation of Glucagon-like peptide-1+ L-cells and Cholecystokinin+ I-cells towards Neurotensin+ PYY+ N-cells. CONCLUSION: ID2 plays an important role in cell fate decisions in the adult small intestine. First, ID2 is essential for establishing a differentiation gradient for enterochromaffin and X-cells along the anterior-posterior axis of the gut. Next, ID2 is necessary for the differentiation of N-cells thus ensuring a differentiation gradient along the crypt-villi axis. Finally, ID2 suppresses the commitment of secretory intestinal epithelial progenitors towards tuft cell lineage and thus controls host immune response to commensal and parasitic microbiota.

Regulation of aerobic and anaerobic D-malate metabolism of Escherichia coli by the LysR-type regulator DmlR (YeaT).

Escherichia coli K-12 is able to grow under aerobic conditions on D-malate using DctA for D-malate uptake and the D-malate dehydrogenase DmlA (formerly YeaU) for converting D-malate to pyruvate. Induction of dmlA encoding DmlA required an intact dmlR (formerly yeaT) gene, which encodes DmlR, a LysR-type transcriptional regulator. Induction of dmlA by DmlR required the presence of D-malate or L- or meso-tartrate, but only D-malate supported aerobic growth. The regulator of general C(4)-dicarboxylate metabolism (DcuS-DcuR two-component system) had some effect on dmlA expression. The anaerobic L-tartrate regulator TtdR or the oxygen sensors ArcB-ArcA and FNR did not have a major effect on dmlA expression. DmlR has a high level of sequence identity (49%) with TtdR, the L- and meso-tartrate-specific regulator of L-tartrate fermentation in E. coli. dmlA was also expressed at high levels under anaerobic conditions, and the bacteria had D-malate dehydrogenase activity. These bacteria, however, were not able to grow on D-malate since the anaerobic pathway for D-malate degradation has a predicted yield of < or = 0 ATP/mol D-malate. Slow anaerobic growth on D-malate was observed when glycerol was also provided as an electron donor, and D-malate was used in fumarate respiration. The expression of dmlR is subject to negative autoregulation. The network for regulation and coordination of the central and peripheral pathways for C(4)-dicarboxylate metabolism by the regulators DcuS-DcuR, DmlR, and TtdR is discussed.

Regulation of tartrate metabolism by TtdR and relation to the DcuS-DcuR-regulated C4-dicarboxylate metabolism of Escherichia coli.

Escherichia coli catabolizes L-tartrate under anaerobic conditions to oxaloacetate by the use of L-tartrate/succinate antiporter TtdT and L-tartrate dehydratase TtdAB. Subsequently, L-malate is channelled into fumarate respiration and degraded to succinate by the use of fumarase FumB and fumarate reductase FrdABCD. The genes encoding the latter pathway (dcuB, fumB and frdABCD) are transcriptionally activated by the DcuS-DcuR two-component system. Expression of the L-tartrate-specific ttdABT operon encoding TtdAB and TtdT was stimulated by the LysR-type gene regulator TtdR in the presence of L- and meso-tartrate, and repressed by O(2) and nitrate. Anaerobic expression required a functional fnr gene, and nitrate repression depended on NarL and NarP. Expression of ttdR, encoding TtdR, was repressed by O(2), nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR specifically bound to the ttdR-ttdA promoter region. TtdR was also required for full expression of the DcuS-DcuR-dependent dcuB gene in the presence of tartrate. Overall, expression of the ttdABT genes is subject to L-/meso-tartrate-dependent induction, and to aerobic and nitrate repression. The control is exerted directly at ttdA and in addition indirectly by regulating TtdR levels. TtdR recognizes a subgroup (L- and meso-tartrate) of the stimuli perceived by the sensor DcuS, which responds to all C(4)-dicarboxylates; both systems apparently communicate by mutual regulation of the regulatory genes.

RppH can faithfully replace TAP to allow cloning of 5'-triphosphate carrying small RNAs.

RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. •We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.