Author contributions L.M.M. and S.K. conceived the project and designed the experiments. L.M.M., M.L., E.G. and R.M. curated patient samples. S.K. led data production and performed the experiments together with A.S.K., A.M. and L.M.M. G.X.Y.Z. provided healthy bone marrow and peripheral blood CITE-seq data. S.K. analyzed the scADT-seq data with contribution from B.P. M.R.C. performed data analysis. J.M.G. conceived the analytical workflows and performed the data analysis for scATAC-seq and scRNA-seq supervised by H.Y.C. and
DNA double strand breaks are generated by genotoxic agents and by cellular endonucleases as intermediates of several important physiologic processes. The cellular response to genotoxic DNA breaks includes the activation of transcriptional programs known primarily to regulate cell cycle checkpoints and cell survival1–5. DNA double strand breaks are generated in all developing lymphocytes during the assembly of antigen receptor genes, a process that is essential for normal lymphocyte development. Here we demonstrate that these physiologic DNA breaks activate a broad transcriptional program. This program transcends the canonical DNA double strand break response and includes many genes that regulate diverse cellular processes important for lymphocyte development. Moreover, the expression of several of these genes is regulated similarly in response to genotoxic DNA damage. Thus, physiologic DNA double strand breaks provide cues that can regulate cell-type-specific processes not directly involved in maintaining the integrity of the genome, and genotoxic DNA breaks could disrupt normal cellular functions by corrupting these processes.
Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain–containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes1. Although Hif regulates many metabolic pathways2, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.
A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp 683 was the most critical for cell binding and toxicity, causing an ϳ1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr 681 , Asn 682 , and Pro 686 also reduced toxicity significantly, by 10 -100-fold. Of these, only Asn 682 and Pro 686 were also critical for interactions with 14B7. However, residues Lys 684 , Leu 685 , Leu 687 , and Tyr 688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5-and 5-fold, respectively.Bacillus anthracis secretes two toxins: edema toxin and lethal toxin. Each is composed of a common binding component, protective antigen (PA), 1 together with an enzymatic component, edema factor (EF), in the case of edema toxin and lethal factor (LF) in the case of lethal toxin (1-3). The current model for toxin entry into the cell illustrates the centrality of PA for toxin action. PA binds to cellular receptors, recently identified as splice variants of either tumor endothelial marker 8 (TEM8) (4 -6) or the closely related capillary morphogenesis protein 2 (CMG2) (7). Furin cleaves PA, releasing a 20-kDa fragment and leaving behind a 63-kDa portion (PA 63 ) capable of forming a heptamer, which has a newly exposed surface that binds . Heptamer complexes enter the endocytic pathway by receptor-mediated endocytosis (13), and upon acidification of the vesicle, the PA 63 heptamer undergoes a conformational change to form a pore through which EF and LF translocate into the cytoplasm (10, 11, 14 -16). Once in the cytoplasm, EF and LF exert their toxic effects.The PA protein can be divided into four domains based on its crystal structure, and functions can be attributed to the different domains based on mutational and biochemical analyses (16). Domain 1 (residues 1-258) contains the furin cleavage site as well as the hydrophobic portion of PA, which is exposed upon furin cleavage to allow EF and LF to bind (16,17). Several lines of evidence indicate that domain 2 (residues 259 -487) is involved in oligomerization and contains the loop that inserts into the membrane to form the channel through which the LF and EF enter the cytosol (16, 18 -20). Various amino acids in domain 3 (residues 488 -595) are necessary for oligomerization, and this has been the only function attributed to domain 3 to date (21,22). Domain 4 (residues 596 -735) is essential for binding to cellular receptor as indicated by several lines of...
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