RANK links thymic regulatory T cells to fetal loss and gestational diabetes in pregnancy.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.

Influence of RNA circularity on Target RNA-Directed MicroRNA Degradation.

A subset of circular RNAs (circRNAs) and linear RNAs have been proposed to 'sponge' or block microRNA activity. Additionally, certain RNAs induce microRNA destruction through the process of Target RNA-Directed MicroRNA Degradation (TDMD), but whether both linear and circular transcripts are equivalent in driving TDMD is unknown. Here, we studied whether circular/linear topology of endogenous and artificial RNA targets affects TDMD. Consistent with previous knowledge that Cdr1as (ciRS-7) circular RNA protects miR-7 from Cyrano-mediated TDMD, we demonstrate that depletion of Cdr1as reduces miR-7 abundance. In contrast, overexpression of an artificial linear version of Cdr1as drives miR-7 degradation. Using plasmids that express a circRNA with minimal co-expressed cognate linear RNA, we show differential effects on TDMD that cannot be attributed to the nucleotide sequence, as the TDMD properties of a sequence often differ when in a circular versus linear form. By analysing RNA sequencing data of a neuron differentiation system, we further detect potential effects of circRNAs on microRNA stability. Our results support the view that RNA circularity influences TDMD, either enhancing or inhibiting it on specific microRNAs.

Unsupervised modeling of cell morphology dynamics for time-lapse microscopy.

Analysis of cellular phenotypes in large imaging data sets conventionally involves supervised statistical methods, which require user-annotated training data. This paper introduces an unsupervised learning method, based on temporally constrained combinatorial clustering, for automatic prediction of cell morphology classes in time-resolved images. We applied the unsupervised method to diverse fluorescent markers and screening data and validated accurate classification of human cell phenotypes, demonstrating fully objective data labeling in image-based systems biology.

Nuclear factor (NF)-κB controls expression of the immunoregulatory glycan-binding protein galectin-1.

The inflammatory response is a self-limiting process which involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Galectin-1 (Gal-1), an endogenous lectin found in peripheral lymphoid organs and inflammatory sites, elicits a broad spectrum of biological functions predominantly by acting as a potent anti-inflammatory factor and as a suppressive agent for T-cell responses. However, the molecular pathways underlying Gal-1 expression and function remain poorly understood. Here we identified a regulatory loop linking Gal-1 expression and function to NF-κB activation. NF-κB-activating stimuli increased Gal-1 expression on T cells, an effect which could be selectively prevented by inhibitors of NF-κB signaling. Accordingly, transient transfection of the p65 subunit of NF-κB was sufficient to induce high Gal-1 expression. Using in silico studies and chromatin immunoprecipitation analysis we have identified a functional NF-κB binding site within the first intron of the LGALS1 gene. In addition, our results show that exogenous Gal-1 can attenuate NF-κB activation, as shown by inhibition of IκB-α degradation induced by pro-inflammatory stimuli, higher cytoplasmic retention of p65, lower NF-κB DNA binding activity and impaired transcriptional activation of target genes. The present study suggest a novel regulatory loop by which NF-κB induces expression of Gal-1, which in turn may lead to negative control of NF-κB signaling.

Control of alternative splicing through siRNA-mediated transcriptional gene silencing.

When targeting promoter regions, small interfering RNAs (siRNAs) trigger a previously proposed pathway known as transcriptional gene silencing by promoting heterochromatin formation. Here we show that siRNAs targeting intronic or exonic sequences close to an alternative exon regulate the splicing of that exon. The effect occurred in hepatoma and HeLa cells with siRNA antisense strands designed to enter the silencing pathway, suggesting hybridization with nascent pre-mRNA. Unexpectedly, in HeLa cells the sense strands were also effective, suggesting that an endogenous antisense transcript, detectable in HeLa but not in hepatoma cells, acts as a target. The effect depends on Argonaute-1 and is counterbalanced by factors favoring chromatin opening or transcriptional elongation. The increase in heterochromatin marks (dimethylation at Lys9 and trimethylation at Lys27 of histone H3) at the target site, the need for the heterochromatin-associated protein HP1alpha and the reduction in RNA polymerase II processivity suggest a mechanism involving the kinetic coupling of transcription and alternative splicing.

A polar mechanism coordinates different regions of alternative splicing within a single gene.

Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.

A slow RNA polymerase II affects alternative splicing in vivo.

Changes in promoter structure and occupation have been shown to modify the splicing pattern of several genes, evidencing a coupling between transcription and alternative splicing. It has been proposed that the promoter effect involves modulation of RNA pol II elongation rates. The C4 point mutation of the Drosophila pol II largest subunit confers on the enzyme a lower elongation rate. Here we show that expression of a human equivalent to Drosophila's C4 pol II in human cultured cells affects alternative splicing of the fibronectin EDI exon and adenovirus E1a pre-mRNA. Most importantly, resplicing of the Hox gene Ultrabithorax is stimulated in Drosophila embryos mutant for C4, which demonstrates the transcriptional control of alternative splicing on an endogenous gene. These results provide a direct proof for the elongation control of alternative splicing in vivo.