Publications in eLife

Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC – a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, we demonstrate that the rs6740960 SNP is sufficient to confer chondrocyte-specific differences in PKDCC expression. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in the maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features.

During apoptosis, caspases degrade 8 out of ~30 nucleoporins to irreversibly demolish the nuclear pore complex. However, for poorly understood reasons, caspases are also activated during cell differentiation. Here, we show that sublethal activation of caspases during myogenesis results in the transient proteolysis of four peripheral Nups and one transmembrane Nup. ‘Trimmed’ NPCs become nuclear export-defective, and we identified in an unbiased manner several classes of cytoplasmic, plasma membrane, and mitochondrial proteins that rapidly accumulate in the nucleus. NPC trimming by non-apoptotic caspases was also observed in neurogenesis and endoplasmic reticulum stress. Our results suggest that caspases can reversibly modulate nuclear transport activity, which allows them to function as agents of cell differentiation and adaptation at sublethal levels. © 2023, eLife Sciences Publications Ltd. All rights reserved.

Non-membrane-bound biomolecular condensates have been proposed to represent an important mode of subcellular organization in diverse biological settings. However, the fundamental principles governing the spatial organization and dynamics of condensates at the atomistic level remain unclear. The Saccharomyces cerevisiae Lge1 protein is required for histone H2B ubiquitination and its N-terminal intrinsically disordered fragment (Lge11-80) undergoes robust phase separation. This study connects single-and multi-chain all-atom molecular dynamics simulations of Lge11-80 with the in vitro behavior of Lge11-80 condensates. Analysis of modeled protein-protein interactions elucidates the key determinants of Lge11-80 condensate formation and links configurational entropy, valency, and compactness of proteins inside the condensates. A newly derived analytical formalism, related to colloid fractal cluster formation, describes condensate architecture across length scales as a function of protein valency and compactness. In particular, the formalism provides an atomisti-cally resolved model of Lge11-80 condensates on the scale of hundreds of nanometers starting from individual protein conformers captured in simulations. The simulation-derived fractal dimensions of condensates of Lge11-80 and its mutants agree with their in vitro morphologies. The presented framework enables a multiscale description of biomolecular condensates and embeds their study in a wider context of colloid self-organization. © Polyansky, Gallego et al.

The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-he-moglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34 +hemato-poietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies. © 2023, eLife Sciences Publications Ltd. All rights reserved.

The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires’ disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-de-rived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmi-tate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD-and FadL-dependent fatty acid metabolism of intracellular L. pneumophila. © Hüsler et al.

Proteins associated with the spindle apparatus, a cytoskeletal structure that ensures the proper segregation of chromosomes during cell division, experienced an unusual number of amino acid substitutions in modern humans after the split from the ancestors of Neandertals and Denisovans. Here, we analyze the history of these substitutions and show that some of the genes in which they occur may have been targets of positive selection. We also find that the two changes in the kinetochore scaffold 1 (KNL1) protein, previously believed to be specific to modern humans, were present in some Neandertals. We show that the KNL1 gene of these Neandertals shared a common ancestor with present-day Africans about 200,000 years ago due to gene flow from the ancestors (or relatives) of modern humans into Neandertals. Subsequently, some non-Africans inherited this modern human-like gene variant from Neandertals, but none inherited the ancestral gene variants. These results add to the growing evidence of early contacts between modern humans and archaic groups in Eurasia and illustrate the intricate relationships among these groups.

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples — so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample — a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Forager focus on wild cereal plants has been documented in the core zone of domestication in southwestern Asia, while evidence for forager use of wild grass grains remains sporadic elsewhere. In this paper, we present starch grain and phytolith analyses of dental calculus from 60 Mesolithic and Early Neolithic individuals from five sites in the Danube Gorges of the central Balkans. This zone was inhabited by likely complex Holocene foragers for several millennia before the appearance of the first farmers ~6200 cal BC. We also analyzed forager ground stone tools for evidence of plant processing. Our results based on the study of dental calculus show that certain species of Poaceae (species of the genus Aegilops) were used since the Early Mesolithic, while ground stone tools exhibit traces of a developed grass grain processing technology. The adoption of domesticated plants in this region after ~6500 cal BC might have been eased by the existing familiarity with wild cereals.