Parallel reverse genetic screening in mutant human cells using transcriptomics

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.     

Mapping of three unique Ca(2+)-binding sites in human annexin II.

Site-directed mutagenesis was employed to map and characterize Ca(2+)-binding sites in annexin II, a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin II and the various mutant proteins were scored for their affinity towards Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266, 14,732-14,739] that a Ca(2+)-binding site is present in the third of the four repeat segments which comprise the 33-kDa protein core of annexin II. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca(2+)-binding site in solution is very similar, if not identical, to that of Ca2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., R?misch, J. and Paques, E.-P. (1990) FEBS Lett. 275, 15-21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin II. Again, an acidic amino acid which is located 40 residues C-terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for high-affinity Ca2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin II together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca2+ sites. Ca(2+)-binding sites of similar structure are present in repeats 2, 3, and 4 of annexin II while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin II derivative with mutations in all three Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2+ site(s) of presumably different architecture.     

Primary structure of human, chicken, and Xenopus laevis p11, a cellular ligand of the Src-kinase substrate, annexin II.

The p11 protein is a member of the S-100 family of Ca(2+)-binding proteins and serves within the cell as a ligand of the tyrosine kinase substrate, annexin II. To obtain more structural information on this molecule, we have isolated and characterized p11 cDNA clones from several different species. A comparison of the deduced amino acid (aa) sequences reveals that mammalian and avian p11 are highly similar (at least 90% identical at the aa level), whereas p11 from Xenopus laevis shows a considerable degree of sequence variation (the aa sequence identity drops to approx. 60% when compared to mammalian or chicken p11). Interestingly, the C-terminal 18 aa, which are unique to p11 within the S-100 family, show a relatively high conservation among species. This high evolutionary conservation is in line with a structurally and/or functionally important role of this C terminus, e.g., in annexin II binding.     

Incorporation kinetics in a membrane, studied with the pore-forming peptide alamethicin.

The reaction of fluorescence-labeled alamethicin with unilamellar phospholipid vesicles (DOPC and DMPC) has been investigated in a stopped-flow apparatus. Clearly single exponential time functions have been observed at temperatures above the phase transition of the bilayer. This can be interpreted in terms of an essentially one-step incorporation process. The pseudo first-order forward rate is found to be quite fast, falling in a range somewhat below the diffusion controlled upper bound. The data are quantitatively very well described on the basis of a simple mechanism. This comprises diffusion of peptide into the bilayer accompanied by a more or less slower change of the secondary structure. Aggregation of the incorporated molecules at higher concentrations is indicated to be comparatively rapid.     

The p36 substrate of pp60src kinase is located at the cytoplasmic surface of the plasma membrane of fibroblasts; an immunoelectron microscopic analysis

p36, a member of the family of Ca2+/lipid-binding proteins, is a major cellular substrate for the tyrosine kinase encoded by the src oncogene. It occurs in two distinct physical states, as either a monomer or a heterotetramer (protein I), which comprises two copies each of p36 and a p11 polypeptide. Immunofluorescence microscopy and cell fractionation studies suggest that p36 and p11 are located underneath the plasma membrane. To investigate whether p36 is indeed associated with the plasma membrane, we have examined its cellular distribution at the electron microscopic level with gold-labeled antibodies. In human fibroblasts, p36 is clearly associated with the cytoplasmic side of the plasma membrane and shows a uniform and regular distribution. Decoration with monoclonal antibodies against p11 reveals the same distribution, suggesting that the p36(2)p11(2) complex (protein I) occurs in the cell in a strict association with the plasma membrane. Titration experiments show that this association is Ca2+ dependent and still occurs at physiological Ca2+ concentrations (10(-7) M). Fodrin, a non-erythroid spectrin, known to bind p36 in vitro, shows a very similar distribution on the cytoplasmic side of the plasma membrane. The results suggest that in a resting and unstimulated cell p36 and p11 reside as a complex bound to the inner side of the plasma membrane.     

A Variant in the Neuropeptide Receptor npr-1 is a Major Determinant of Caenorhabditis elegans Growth and Physiology

The mechanistic basis for how genetic variants cause differences in phenotypic traits is often elusive. We identified a quantitative trait locus in Caenorhabditis elegans that affects three seemingly unrelated phenotypic traits: lifetime fecundity, adult body size, and susceptibility to the human pathogen Staphyloccus aureus. We found a QTL for all three traits arises from variation in the neuropeptide receptor gene npr-1. Moreover, we found that variation in npr-1 is also responsible for differences in 247 gene expression traits. Variation in npr-1 is known to determine whether animals disperse throughout a bacterial lawn or aggregate at the edges of the lawn. We found that the allele that leads to aggregation is associated with reduced growth and reproductive output. The altered gene expression pattern caused by this allele suggests that the aggregation behavior might cause a weak starvation state, which is known to reduce growth rate and fecundity. Importantly, we show that variation in npr-1 causes each of these phenotypic differences through behavioral avoidance of ambient oxygen concentrations. These results suggest that variation in npr-1 has broad pleiotropic effects mediated by altered exposure to bacterial food.     

Shifts in N and P Budgets of Heathland Ecosystems: Effects of Management and Atmospheric Inputs

In the present study we analyzed the combined effects of management (grazing, mowing, prescribed burning, sod-cutting) and atmospheric deposition on N and P budgets of heathland ecosystems (Lüneburger Heide nature reserve; N Germany). We hypothesize that management measures such as grazing and mowing can accelerate a deposition-induced imbalance of N and P pools as a result of a disproportionally high output of P. We analyzed management and deposition affected input–output flows of N and P and related them to changes in the nutritional status of Calluna vulgaris 5 years after treatment application. We found that grazing and mowing caused the highest net loss of P due to high P concentrations in the aboveground biomass. In contrast, prescribed burning only slightly affected P pools, as P remained in the system due to ash deposition. Management-mediated effects on N and P pools were mirrored in the nutritional status of Calluna vulgaris: at the grazed and mown sites, the P content of current season’s shoots significantly decreased within 5 years after treatments, whereas the N content remained unchanged. We conclude that grazing and mowing can accelerate declining availability of P and, thus, accelerate a deposition-induced shift from N- to P-limited plant growth in the medium term. In the face of ongoing atmospheric N loads management schemes need to combine high- and low-intensity measures to maintain both a diverse structure and balanced nutrient budgets in the long term.