Mammalian female tissues consist of two mixed cell populations, each with either the maternally or paternally inherited X chromosome marked for inactivation. To overcome this heterogeneity, assessments of human XCI have often been confined to the use of artificial cell systems1 or to samples that have skewed XCI1,2, that is, preferential inactivation of one of the two X chromosomes; this is common in clonal cell lines but rare in karyotypically normal, primary human tissues8 (Extended Data Fig. 1 and Supplementary Note). Others have used bias in DNA methylation3,4,9 or in gene expression5,10 between males and females as a proxy for XCI status. Surveys of XCI are powerful in engineered model organisms, for example, mouse models with completely skewed XCI11, but the degree to which these discoveries are generalizable to human XCI remains unclear given marked differences in XCI initiation and the extent of escape across species7. Here we describe a systematic survey of the landscape of human XCI using three complementary RNA sequencing (RNA-seq)-based approaches (Fig. 1) that together enable the assessment of XCI from individual cells to population level across a diverse range of human tissues.
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Although sex bias on the X chromosome is broadly specific to escape genes, some genes show unexpected patterns. Eight genes with some previous evidence for inactivation show >90% concordance in effect direction and significant sex bias (Fig. 2d and Supplementary Table 3), suggesting that variable escape can also have considerable population-level effects. For example, CHM demonstrates such concordance in sex bias and escape at this gene is confirmed when using single-cell RNA-seq (scRNA-seq; see below). One gene (RP11-706O15.3) without an assigned XCI status shows a similar sex bias pattern to escape genes. RP11-706O15.3 resides between escape and variable escape genes PRKX and NLGN4X (Fig. 2d), consistent with known clustering of escape genes1,2. Some escape genes show more heterogeneous sex bias, for example, ACE2 (Fig. 2a and Supplementary Discussion). Many such genes lie in the evolutionarily older region of the chromosome16, in Xq, where escape genes also show higher tissue-specificity and lower expression levels (Extended Data Fig. 5), characteristics that have been linked with higher protein evolutionary rates17,18.
We assigned each cell to either of two cell populations distinguished by the parental X-chromosome designated for inactivation using genotype phasing. For the YRI samples, where parental genotype data was available, the assignment to the two parental cell populations was unambiguous for all cells where X-chromosomal sites outside PAR1 or frequently biallelic sites were expressed. For 24A, no parental genotype data were available, and we therefore used the correlation structure of the expressed X-chromosomal alleles across the 948 cells to infer the two parental haplotypes using the fact that in individual cells the expressed alleles at the chrX sites subject to full inactivation (that is, the majority chrX ASE sites), are from the X chromosome active in each cell (Supplementary Methods). In other words, while monoallelic expression in scRNA-seq in the autosomes is largely stochastic in origin, in the X chromosome the pattern of monoallelic expression is consistent across cells with the same parental X chromosome active22, unless a gene is expressed also from the inactive X. As such, for the phase inference calculations, we excluded all PAR1 sites and all additional sites that were frequently biallelic, to minimize the contribution of escape genes to the phase estimation. After assigning each informative cell to either of the parental cell populations, the reference and alternate allele reads for each ASE site were mapped to active and inactive allele reads within each sample using the actual or inferred parental haplotypes. The data were first combined per variant by taking the sum of active and inactive counts separately across cells, and further similarly combined per gene, if multiple SNPs per gene were available. For 24A the allele expressed at XIST was assumed the Xi allele, in line with the exclusive Xi expression in the Yoruba samples confirmed using the information on parental haplotypes.
The GTEx (Genotype-Tissue Expression) Consortium has established a reference catalogue and associated tissue biobank for gene-expression levels across individuals for diverse tissues of the human body, with a broad sampling of normal, non-diseased human tissues from postmortem donors. The consortium now presents the deepest survey of gene expression across multiple tissues and individuals to date, encompassing 7,051 samples from 449 donors across 44 human tissues. Barbara Engelhardt and colleagues characterize the relationship between genetic variation and gene expression, and find that most genes are regulated by genetic variation near to the affected gene. In accompanying GTEx studies, Alexis Battle, Stephen Montgomery and colleagues examine the effect of rare genetic variation on gene expression across human tissues, Daniel MacArthur and colleagues systematically survey the landscape of X chromosome inactivation in human tissues, and Jin Billy Li and colleagues provide a comprehensive cross-species analysis of adenosine-to-inosine RNA editing in mammals. In an accompanying News & Views, Michelle Ward and Yoav Gilad put the latest results in context and discuss how these findings are helping to crack the regulatory code of the human genome.
Figure 4 Precision engineering of an A2-CAR into the TRAC locus of human Tregs. (A) Expansion strategy of A2-CAR Tregs, unedited resting Tregs (unEd rTreg) and unedited stimulated Tregs after sorting from peripheral blood of an HLA-A2 positive donor. For homology-directed repair-mediated integration into the TRAC locus, the A2-CAR template was inserted using AAV6 transduction after electroporation of two CRISPR/Cas9 ribonucleoprotein (RNP) complexes targeting the TRAC and HLA-A loci. (B) Representative flow cytometry of the editing efficiency measured 10 days later in 3 independent experiments. CD3, MYC-tag, and HLA-A2 surface expression is shown. (C) Treg fold-expansion and percentage of MYC-tag+ Tregs over time. Fitted line plots are shown. (D) Fourteen days after activation, FOXP3 and HELIOS expression were assessed on edited Tregs and compared to that of unedited T cells and unedited Day 9 stimulated Tregs (unEd sTregs). (E) The same cells were co-cocultured with or without irradiated (4000 rad) HLA-A2+ NALM6 cells. CD71 expression was assessed 48 hours later. (F) In vitro suppression assays were performed using HLA-A2+ stimulated B cells (sBCs) as stimulator cells, A2-CAR+TCRdeficient CD4+ T cells as responder cells (0.05 x 106 cell/96well) and A2-CAR+TCRdeficient Tregs at different ratios. After 3 days, 0.5μCi/well of 3[H] thymidine (Perkin Elmer, Waltham, MA) was added for the final 16 h of culture. Proliferation was assessed by 3[H] thymidine incorporation (counts per minute - cpm). Two-way ANOVA was used to determine the statistical significance of the difference. Data corresponds to the cells infused in Figure 5. Similar results were obtained with 3 independent donors. **p .
Abstract SUR2A is an ATP-binding protein that serves as a regulatory subunit of cardioprotective ATP-sensitive K+ (KATP) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled KATP channels, we have suggested that nicotinamide-rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide-rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild-type mice on nicotinamide-rich diet. The experiments focused on action membrane potential and intracellular Ca2+ concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal KATP channels and steady Ca2+ levels in cardiomyocytes in response to β-adrenergic stimulation. In contrast, the same challenge in the wild-type was characterized by a lack of the channel activation and rise in intracellular Ca2+. Nicotinamide-rich diet was ineffective to increase physical endurance in mice lacking KATP channels. This study has shown that nicotinamide-rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide-rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable. PMID:20731746
ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K+ channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels. PMID:26416970 2ff7e9595c
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