Juxtaposition of Chemical and Mutation-Induced Developmental Defects in Zebrafish Reveal a Copper-Chelating Activity for Kalihinol F

SummaryA major hurdle in using complex systems for drug screening is the difficulty of defining the mechanistic targets of small molecules. The zebrafish provides an excellent model system for juxtaposing developmental phenotypes with mechanism discovery using organism genetics. We carried out a phenotype-based screen of uncharacterized small molecules in zebrafish that produced a variety of chemically induced phenotypes with potential genetic parallels. Specifically, kalihinol F caused an undulated notochord, defects in pigment formation, hematopoiesis, and neural development. These phenotypes were strikingly similar to the zebrafish mutant, calamity, an established model of copper deficiency. Further studies into the mechanism of action of kalihinol F revealed a copper-chelating activity. Our data support this mechanism of action for kalihinol F and the utility of zebrafish as an effective system for identifying therapeutic and target pathways

Germline variants of ATG7 in familial cholangiocarcinoma alter autophagy and p62

Funding Information: The authors recognize and appreciate the patients and families who contributed to the current study. We acknowledge the Icelandic Cancer Registry for assistance in the ascertainment of the Icelandic cancer patients. We thank deCODE genetics for access to data and facilities, assistance with data analysis and helpful discussions. This work was supported by the National Institutes of Health [P01HG000205 to SUG and HPJ, 1U01CA15192001-A1 to HPJ, 1U01CA176299 to HPJ, HG006137-07 to HPJ, R01 CA116468NIH to DAJ, 5K08CA166512 to LDN]; Intermountain Healthcare to SUG, JC and HPJ; a Research Scholar Grant from the American Cancer Society [RSG-13-297-01-TBG to JC and HPJ]; Clayville Foundation to HPJ; Gastric Cancer Foundation to HPJ and LDN; the Samuel Waxman Cancer Research Foundation to DAJ; Oklahoma Center for Adult Stem Cell Research (OCASCR) to DAJ; Oklahoma Medical Research Foundation (OMRF) to DAJ; the Conquer Cancer Foundation (Young Investigator Award) to LDN; the Carl Kawaja Foundation to LDN; the Research Fund of Iceland [130230-0529 to ES and MHO, 184861-052 to ES, 184727-051 to MHO]; and a grant from the Icelandic Cancer Society Research Fund to MHO. Publisher Copyright: © 2022, The Author(s).Autophagy is a housekeeping mechanism tasked with eliminating misfolded proteins and damaged organelles to maintain cellular homeostasis. Autophagy deficiency results in increased oxidative stress, DNA damage and chronic cellular injury. Among the core genes in the autophagy machinery, ATG7 is required for autophagy initiation and autophagosome formation. Based on the analysis of an extended pedigree of familial cholangiocarcinoma, we determined that all affected family members had a novel germline mutation (c.2000C>T p.Arg659* (p.R659*)) in ATG7. Somatic deletions of ATG7 were identified in the tumors of affected individuals. We applied linked-read sequencing to one tumor sample and demonstrated that the ATG7 somatic deletion and germline mutation were located on distinct alleles, resulting in two hits to ATG7. From a parallel population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G p.Asp522Glu (p.D522E)) associated with increased risk of cholangiocarcinoma. To characterize the impact of these germline ATG7 variants on autophagy activity, we developed an ATG7-null cell line derived from the human bile duct. The mutant p.R659* ATG7 protein lacked the ability to lipidate its LC3 substrate, leading to complete loss of autophagy and increased p62 levels. Our findings indicate that germline ATG7 variants have the potential to impact autophagy function with implications for cholangiocarcinoma development.Peer reviewe

\u3ci\u3eDrosophila\u3c/i\u3e Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu