A subset of B‐cell lymphoma patients have dominant mutations in the histone H3 lysine 27 (H3K27) methyltransferase EZH2, which change it from a monomethylase to a trimethylase. These mutations occur ...in aromatic resides surrounding the active site and increase growth and alter transcription. We study the N‐terminal trimethylase NRMT1 and the N‐terminal monomethylase NRMT2. They are 50% identical, but differ in key aromatic residues in their active site. Given how these residues affect EZH2 activity, we tested whether they are responsible for the distinct catalytic activities of NRMT1/2. Additionally, NRMT1 acts as a tumor suppressor in breast cancer cells. Its loss promotes oncogenic phenotypes but sensitizes cells to DNA damage. Mutations of NRMT1 naturally occur in human cancers, and we tested a select group for altered activity. While directed mutation of the aromatic residues had minimal catalytic effect, NRMT1 mutants N209I (endometrial cancer) and P211S (lung cancer) displayed decreased trimethylase and increased monomethylase/dimethylase activity. Both mutations are located in the peptide‐binding channel and indicate a second structural region impacting enzyme specificity. The NRMT1 mutants demonstrated a slower rate of trimethylation and a requirement for higher substrate concentration. Expression of the mutants in wild type NRMT backgrounds showed no change in N‐terminal methylation levels or growth rates, demonstrating they are not acting as dominant negatives. Expression of the mutants in cells lacking endogenous NRMT1 resulted in minimal accumulation of N‐terminal trimethylation, indicating homozygosity could help drive oncogenesis or serve as a marker for sensitivity to DNA damaging chemotherapeutics or γ‐irradiation.
Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully ...elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function, and dynamics of the antibody response in disease.
Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically ...dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.
T cell receptors (TCRs) recognize peptide fragments presented by the major histocompatibility complex (MHC) and are critical to T cell-mediated immunity. Recent data have indicated that genetic ...diversity within TCR-encoding gene regions is underexplored, limiting understanding of the impact of TCR loci polymorphisms on TCR function in disease, even though TCR repertoire signatures (1) are heritable and (2) associate with disease phenotypes. To address this, we developed a targeted long-read sequencing approach to generate highly accurate haplotype resolved assemblies of the TCR beta (TRB) and alpha/delta (TRA/D) loci, facilitating the genotyping of all variant types, including structural variants. We validate our approach using two mother-father-child trios and 5 unrelated donors representing multiple populations. This resulted in improved genotyping accuracy and the discovery of 84 undocumented V, D, J, and C alleles, demonstrating the utility of this framework for improving our understanding of TCR diversity and function in disease.
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•Novel framework to characterize genomic diversity in the T cell receptor loci•Benchmarking revealed accurate assemblies, variant calls, and gene annotation sets•Variant detection with long-read sequencing framework outperforms short-read methods•Discovery of large number of previously undocumented T cell receptor gene alleles
Genetic variation within T cell receptor (TCR) genes influences the composition of the TCR repertoire and TCR-peptide-major histocompatibility complex interactions. Yet, diversity within human TCR loci is not well documented. Rodriguez et al. report a novel scalable method for targeted long-read sequencing of the TCR beta, alpha, and delta loci.
A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative ...testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study.
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•Ten assays were evaluated with 12 fecal sources using 11 challenge sample units of measure.•Method performance depended on the classification of detected but not quantifiable samples.•HF183 Taqman® was the most effective marker in this California-based study.•Standardization of protocols is essential for widespread implementation.
Genetic screens are used to identify genes involved in specific biological processes. An EMS mutagenesis screen in
Drosophila melanogaster
identified growth control phenotypes in the developing eye. ...One mutant line from this screen,
H.3.2
, was phenotypically characterized using the FLP/FRT system and genetically mapped by complementation analysis and genomic sequencing by undergraduate students participating in the multi-institution Fly-CURE consortium.
H.3.2
was found to have a nonsense mutation in
short stop
(
shot
), an
ortholog of the mammalian spectraplakin
dystonin
(
DST
).
shot
and
DST
are involved in cytoskeletal organization and play roles during cell growth and proliferation.
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