Characterization of Collaborative Cross mouse founder strain CAST/EiJ as a novel model for lethal COVID-19.

Mutations in SARS-CoV-2 variants of concern (VOCs) have expanded the viral host range beyond primates, and a limited range of other mammals, to mice, affording the opportunity to exploit genetically diverse mouse panels to model the broad range of responses to infection in patient populations. Here we surveyed responses to VOC infection in genetically diverse Collaborative Cross (CC) founder strains. Infection of wild-derived CC founder strains produced a broad range of viral burden, disease susceptibility and survival, whereas most other strains were resistant to disease despite measurable lung viral titers. In particular, CAST/EiJ, a wild-derived strain, developed high lung viral burdens, more severe lung pathology than seen in other CC strains, and a dysregulated cytokine profile resulting in morbidity and mortality. These inbred mouse strains may serve as a valuable platform to evaluate therapeutic countermeasures against severe COVID-19 and other coronavirus pandemics in the future.

Characterizing somatic hypermutation and gene conversion in the chicken DT40 cell system.

The secondary immunoglobulin gene diversification processes, somatic hypermutation (SHM), immunoglobulin gene conversion (GCV), and class switch recombination, are important for efficient humoral immune responses. They require the action of activation-induced cytidine deaminase, an enzyme that deaminates cytosine in the context of single-stranded DNA. The chicken DT40 B-cell line is an important model system for exploring the mechanisms of SHM and GCV, as both processes occur constitutively without the need for stimulation. In addition, standard gene targeting strategies can be used for defined manipulations of the DT40 genome. Thus, these cells represent an excellent model of choice for genetic studies of SHM and GCV. Problems arising from defects in early B-cell development that are of concern when using genetically engineered mice are avoided in this system. Here, we describe how to perform gene targeting in DT40 cells and how to determine the effects of such modifications on SHM and GCV.

Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus.

Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3' regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus.

Lysine residues in N-terminal and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase.

In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3 and H4 to form nucleosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails (e.g., acetylation and methylation of histones). These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence that biotinidase and holocarboxylase synthetase (HCS) catalyze the covalent binding of biotin to histones has been provided. The primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation and whether biotinidase and HCS localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histones H2A and H2AX are targets for biotinylation and that K125, K127 and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modification-specific antibodies. Antibodies to biotinidase and HCS localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that its biotinylation sites include amino acid residues from the C-terminus.

Biotinylation of K12 in histone H4 decreases in response to DNA double-strand breaks in human JAr choriocarcinoma cells.

We tested the hypothesis that biotinylation of K12 in histone H4 plays a role in the cellular response to double-strand breaks (DSB) of DNA in human cells. DSB were caused by treating choriocarcinoma JAr cells with etoposide. Biotinylation of K12 in histone H4 decreased by 50% as early as 10-20 min after initiation of treatment with etoposide. Biotinylation returned to initial levels 30-40 min after the addition of etoposide to the medium. Temporal patterns of K12-biotinylation were similar for human lymphoma cells. Phosphorylation of S14 of histone H2B and poly(ADP-ribosylation) of glutamate residues on histone H2A are known markers of DSB in DNA; these modifications increased 10-40 min after alterations in K12-biotinylation occurred. Decreased biotinylation of K12 of histone H4 was specific for DSB but was not detectable in response to single-strand breaks or the formation of thymine dimers. Biotin-deficient choriocarcinoma cells exhibited a 40% decrease in rates of survival in response to etoposide compared with biotin-sufficient controls. These studies suggest that the lack of biotinylation of K12 in histone H4 is an early signaling event in response to DSB.

Biological functions of biotinylated histones.

Histones H1, H2A, H2B, H3 and H4 are DNA-binding proteins that mediate the folding of DNA into chromatin. Various posttranslational modifications of histones regulate processes such as transcription, replication and repair of DNA. Recently, a novel posttranslational modification has been identified: covalent binding of the vitamin biotin to lysine residues in histones, mediated by biotinidase and holocarboxylase synthetase. Here we describe a novel peptide-based technique, which was used to identify eight distinct biotinylation sites in histones H2A, H3 and H4. Biotinylation site-specific antibodies were generated to investigate biological functions of histone biotinylation. Evidence was provided that biotinylation of histones plays a role in cell proliferation, gene silencing and cellular response to DNA damage.