Biology subjects

Garcia, E. M.

Publications and source records attributed to Garcia, E. M..

5 recordsLinked to original sources

bioRxiv2023

Base Editor Scanning Reveals Activating Mutations of DNMT3A

DNA methyltransferase 3A (DNMT3A) is a de novo cytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/536656v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1de1d3eorg.highwire.dtl.DTLVardef@151f12dorg.highwire.dtl.DTLVardef@b0e2dborg.highwire.dtl.DTLVardef@fdce96_HPS_FORMAT_FIGEXP M_FIG C_FIG Using base editor mutagenesis and a DNA methylation reporter optimized to find activating mutations, we identify novel hyperactivating mutations in DNMT3A that suggest new mechanisms of allosteric control.

biochemistry↗

bioRxiv2023

Genotypic and phenotypic differences among phase-variable colony variants conserved across Gardnerella spp.

The Gardnerella genus, now made up of more than 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested Gardnerella strains, representing at least ten different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops in vivo, we characterized their differences. Beyond increased colony size, large colony variants (Lg) showed reduced vaginolysin secretion and faster growth rate relative to small colony variants (Sm). The ability to inhibit growth of Neisseria gonorrhoeae and commensal lactobacillus species varied by strain and in some instances differed between variants. Proteomics analyses indicate that 127-173 proteins are differentially expressed between variants. Further, whole genome sequencing analyses revealed an abundance of genes associated with variable poly-guanine tracts, implicating slipped strand mispairing in Gardnerella phase variation, and illuminating the potential for previously unrecognized variability within clonal populations. Collectively, these results suggest that colony variants may be primed to serve different roles in BV pathogenesis.

microbiology↗

bioRxiv2022

IL-17C is a driver of damaging inflammation during Neisseria gonorrhoeae infection of human Fallopian tube

The human-restricted pathogen Neisseria gonorrhoeae ascends into the upper female reproductive tract to cause damaging inflammation within the Fallopian tubes (salpingitis) and pelvic inflammatory disease (PID), increasing the risk of infertility and life-threatening ectopic pregnancy. The loss of ciliated cells from the epithelium is thought to be both a consequence of inflammation and a cause of the associated adverse sequelae. However, the links between infection, inflammation, and ciliated cell extrusion remain unresolved. With the use of ex vivo cultures of human Fallopian tube paired with RNA sequencing we defined the tissue response to gonococcal challenge, identifying cytokine, chemokine, cell adhesion, and apoptosis related transcripts not previously recognized as potentiators of gonococcal PID. Unexpectedly, the cytokine IL-17C was one of the most highly induced genes. Yet, this cytokine has no previous association with gonococcal disease nor any sexually transmitted infection and thus it was selected for further characterization in our model. We show that human Fallopian tubes express the IL-17C receptor (IL-17RE) on the epithelial surface and that treatment with purified IL-17C induces pro-inflammatory cytokine secretion in addition to sloughing of the epithelium and generalized tissue damage. These results demonstrate a previously unrecognized but critical role of IL-17C in the damaging inflammation induced by gonococci in a human explant model of PID. SignificanceWe performed untargeted transcriptional analysis of the human Fallopian tube response to Neisseria gonorrhoeae. Focusing on one upregulated transcript not previously identified in any context of gonococcal infection, we show that the human Fallopian tube secretes the inflammation amplifying cytokine IL-17C in response to N. gonorrhoeae challenge. IL-17C treatment of human Fallopian tubes stimulated pro-inflammatory cytokine secretion and was sufficient to elicit epithelial cell pathologies characteristic of pelvic inflammatory disease (sloughing, exfoliation). Thus, we identify IL-17C as a previously unrecognized component of the host response to gonococci that promotes inflammation and tissue damage within the human Fallopian tube.

microbiology↗

bioRxiv2022

Activity-based CRISPR Scanning Uncovers Allostery in DNA Methylation Maintenance Machinery

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite their fundamental importance, systematic identification of allosteric sites remains highly challenging. Here we perform CRISPR scanning on the essential maintenance methylation machinery--DNMT1 and its partner UHRF1--with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function mutations that increase DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our findings highlight the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, even beyond the direct drug target.

genomics↗

bioRxiv2022

Base editor scanning charts the DNMT3A activity landscape

DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation of this process is known to cause aberrant development and oncogenesis, yet how DNMT3A is regulated holistically by its three domains remains challenging to study. Here we integrate base editing with a DNA methylation reporter to perform in situ mutational scanning of DNMT3A in cells. We identify mutations throughout the protein that perturb function, including ones at an interdomain interface that block allosteric activation. Unexpectedly, we also find mutations in the PWWP domain, a histone reader, that modulate enzyme activity despite preserving histone recognition and protein stability. These effects arise from altered PWWP domain DNA affinity, which we show is a noncanonical function required for full activity in cells. Our findings highlight mechanisms of interdomain crosstalk and demonstrate a generalizable strategy to probe sequence-activity relationships of nonessential chromatin regulators.

biochemistry↗