Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of diseas

A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis

Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease

TNFR1 Signaling Is Associated with Backbone Conformational Changes of Receptor Dimers Consistent with Overactivation in the R92Q TRAPS Mutant

The widely accepted model for tumor necrosis factor 1 (TNFR1) signaling is that ligand binding causes receptor trimerization, which triggers a reorganization of cytosolic domains and thus initiates intracellular signaling. This model of stoichiometrically driven receptor activation does not account for the occurrence of ligand independent signaling in overexpressed systems, nor does it explain the constitutive activity of the R92Q mutant associated with TRAPS. More recently, ligand binding has been shown to result in the formation of high molecular weight, oligomeric networks. Although the dimer, shown to be the preligand structure, is thought to remain present within ligand–receptor networks, it is unknown whether network formation or ligand-induced structural change to the dimer itself is the trigger for TNFR1 signaling. In the present study, we investigate the available crystal structures of TNFR1 to explore backbone dynamics and infer conformational transitions associated with ligand binding. Using normal-mode analysis, we characterize the dynamic coupling between the TNFR1 ligand binding and membrane proximal domains and suggest a mechanism for ligand-induced activation. Furthermore, our data are supported experimentally by FRET showing that the constitutively active R92Q mutant adopts an altered conformation compared to wild-type. Collectively, our results suggest that the signaling competent architecture is the receptor dimer and that ligand binding modifies domain mobilities intrinsic to the receptor structure, allowing it to sample a separate, active conformation mediated by network formation

Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

<div><p>Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the <i>x-y</i> imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.</p></div

Qualitative and quantitative comparison of different photodestruction methods.

<p>(A) Photodestruction with NaBH₄ quenching alone results in noticeable cross-talk from the original image. Quantification of residual localizations after photodestruction shows an average cross-talk of ~4.5%, from the distribution shown. (B) Photodestruction with NaBH₄ alone and imaging post-photodestruction done in the presence of low intensity 405 nm excitation shows considerable increase in cross-talk and noticeable residual tubulin structure. Quantitative comparison shows ~13% cross-talk. (C) Photodestruction with both photobleaching and NaBH₄ quenching shows little detectable cross-talk. Subregion quantification shows cross-talk of ~0.2%, representing a ~20-fold improvement over NaBH₄ alone. (D) Photodestruction via both photobleaching and NaBH₄ quenching, and imaging post-photodestruction with 405 nm excitation shows considerable improvement and no residual tubulin structure. Quantitative comparison of residual localizations shows ~0.3% cross-talk, a ~40-fold improvement over NaBH₄ quenching alone. Therefore photobleaching and NaBH₄ quenching provides an ideal method for photodestruction in s-SR. Scale bars in original image (left), 2 μm; scale bars in small subregions (right), 1 μm. Note, the image contrast in all post-photodestruction images (middle column) was increased by 3× in order to make visible the any remaining signal. Cross-talk was estimated by calculating residual localization in small, ~2 x 2 μm, subregions after photo-destruction (see Subregion comparison, right column). Data for each histogram represents ~30 subregions taken from at least five independent cells.</p

Sequential superresolution imaging.

<p>(A) Schematic of sequential imaging, including labeling/imaging, photodestruction, relabeling/imaging, and overlay. Both targets are labeled with the same fluorophore (thus with an identical optical path) and alignment to a reference image is done both prior to and during image acquisition. (B) Sequential imaging of clathrin followed by tubulin, both with AF647, with complete photodestruction between images; including photobleaching and fluorophore quenching (described below). Shown at right is the resulting overlay image of clathrin (magenta) and tubulin (green). (C-D) Zoomed region of sequential imaging of clathrin (left) and tubulin (middle) and the resulting overlay (right—clathrin in magenta, tubulin in green) shown as a diffraction limited image (C) and superresolution reconstruction (D). In the superresolution reconstruction for tubulin, residual localizations from clathrin are notably absent, highlighted by the circles shown. Scale bars 500 nm.</p

Four color s-SR imaging using a single fluorophore.

<p>(A-B) Four color reconstruction overlay showing clathrin (yellow), α-tubulin (green), actin (orange), and EGFR (blue), imaged sequentially in the order listed, with each target imaged with AF647-conjugated primary antibody with the exception of actin, which was imaged with phalloidin-AF647. (B) Zoomed region highlighted in (A). (C-F) The original reconstruction for each individual component—clathrin (C), α-tubulin (D), actin (E), and EGFR (F)—of the region shown in (B) and highlighted in (A), scale bars 500 nm. Note the lack of any measurable cross-talk between images. (G-J) Two-label reconstructions for clathrin and tubulin (G), tubulin and actin (H), actin and EGFR (I), and clathrin and EGFR (J).</p