Biology subjects

Fronick, C.

Publications and source records attributed to Fronick, C..

8 recordsLinked to original sources

bioRxiv2026

High-order enhancer hubs buffer allelic regulatory variation through kinetic compensation

Diploid genomes carry millions of heterozygous variants in cis-regulatory DNA, yet most genes produce similar RNA output from two parental alleles. How this balance is maintained is unclear. We developed Nanopore-HiChIP, a long-read method that maps high-order enhancer hubs on each haplotype. Over half of these enhancer hubs differ in chromatin architecture and transcription-factor occupancy between homologous chromosomes, but their target genes show substantially lower rates of allele-specific expression than genes lacking hub regulation. Single-cell kinetic modeling shows that burst frequency and burst size change in opposite directions, thereby preserving balanced transcriptional output. This hub-mediated kinetic buffering is enriched at haploinsufficient genes and coincides with smaller effects of expression quantitative trait loci. Enhancer hubs therefore absorb allelic regulatory variation through kinetic compensation, protecting dosage-sensitive transcription.

genomics↗

bioRxiv2025

Multi-site Assessment of Methods for Cell Preservation Upstream of Single Cell RNA Sequencing

Single cell RNA sequencing (scRNA-seq) is a revolutionary technique to identify cell types and their molecular phenotype in heterogeneous biological specimens. ScRNA-seq typically requires fresh, high quality single cell suspensions that are processed immediately to preserve their molecular profiles. This presents a challenge for samples with long preparation times and prevents collection at remote sites lacking the required instrumentation for sample processing. Recently, several commercial assays have been released that enable sample preservation at the time of collection either via fixation or cryopreservation, allowing for sample processing to occur months after the initial collection. The Association of Biomolecular Research Facilities (ABRF) DNA Sequencing (DSRG) and Genomics Bioinformatics (GBiRG) Research Groups have undertaken a cross-platform, multi-site study to assess the performance and reproducibility of three platforms: a) 10x Genomics FLEX, b) Parse Bioscience Evercode WT v2 and c) Honeycomb Bio HIVE. Total leukocytes were isolated from a single healthy individual using the EasySep RBC depletion reagent. Cells were then characterized by collecting a 21-color flow cytometry dataset for reference and the remaining material was used for scRNA-seq procedures where different sites then processed either the fixed or cryopreserved cells for each method. We evaluated performance of each method across traditional scRNA-seq quality control metrics and analysis applications, including gene/transcript detection sensitivity, cell type discovery and annotation, and differential expression. We demonstrate that data from the methods tested can be effectively integrated and produce concordant results with regard to cell type annotation and relative abundance, though we observe platform-specific differences in the expression of a subset of genes. Preservation-based methods also show better retention of fragile granulocyte populations compared with fresh samples processed using the 10x 3 workflow. The improvements to preservation methods are changing the way research is conducted and our thorough investigation into the performance of each method provides a valuable resource to help scientists determine the most appropriate single cell preservation workflow given their sample collection logistics and laboratory infrastructure constraints.

genomics↗

bioRxiv2025

Intermediately Methylated Regions in Normal Cells Are Epimutation Hotspots in Cancer

DNA methylation is altered in all cancers, but the mechanisms responsible for these changes are not well understood. Using data from 100 primary samples, we show that regions with intermediate methylation in normal hematopoietic cells are hotspots for stable, clonal epimutations in acute myeloid leukemia. Analysis of hematopoietic stem cell clones demonstrated that intermediate methylation at epimutation hotspots reflects random allele-specific methylation and expression at the single cell level, representing a previously unrecognized form of somatically acquired imprinting. We identified somatically imprinted regions in other tissues and show they are epimutation hotspots in other cancer types. This demonstrates that random allele-specific methylation is both a general property of normal cells, and a vulnerability that renders them susceptible to cooperating events and clonal selection during cancer.

genomics↗

bioRxiv2023

Endothelial cells are a key target of IFN-g during response to combined PD-1/CTLA-4 ICB treatment in a mouse model of bladder cancer

To explore mechanisms of response to combined PD-1/CTLA-4 immune checkpoint blockade (ICB) treatment in individual cell types, we generated scRNA-seq using a mouse model of invasive urothelial carcinoma with three conditions: untreated tumor, treated tumor, and tumor treated after CD4+ T cell depletion. After classifying tumor cells based on detection of somatic variants and assigning non-tumor cell types using SingleR, we performed differential expression analysis, overrepresentation analysis, and gene set enrichment analysis (GSEA) within each cell type. GSEA revealed that endothelial cells were enriched for upregulated IFN-g response genes when comparing treated cells to both untreated cells and cells treated after CD4+ T cell depletion. Functional analysis showed that knocking out IFNgR1 in endothelial cells inhibited treatment response. Together, these results indicated that IFN-g signaling in endothelial cells is a key mediator of ICB induced anti-tumor activity.

cancer biology↗

bioRxiv2021

Single Cell T Cell Receptor Repertoire Profiling for Dogs

BackgroundSpontaneous cancers in companion dogs are increasingly recognized as robust models of human disease. This recognition has led to translational clinical trials in companion dogs with osteosarcoma, lymphoma, melanoma, squamous cell carcinoma, and soft tissue sarcoma. The ability to precisely track tumor-specific immune responses in such clinical trials would benefit from reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). This technology defines clones of T cells reacting to immune interventions and can help identify the specific epitope of response. Single cell gene expression data give insights into the activity and polarization of the T cell. To date, scTCRseq has not been demonstrated for canine samples. MethodsSamples from two responding dogs in a trial of an autologous deglycosylated melanoma vaccine were selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. A single-cell suspension of cryopreserved peripheral blood mononuclear cells (PBMC) was prepared for 10X single cell sequencing. Full length 10X cDNA was amplified using a custom-designed nested PCR of the alpha/beta V(D)J region. A library made from this enriched product (scTCRseq) and a 10X gene expression (GEX) library (scRNAseq) were sequenced on the NovaSeq 6000. Results1,850-2,172 estimated V(D)J-expressing cells yielded 87-103.7 million reads with 73.8%-75.8% mapped to a V(D)J gene (beta/alpha chains ratio 1.5:1). 43 TRAJ, 29 TRAV, 12 TRBJ, and 22 TRBV gene segments were observed representing 72.9%, 51.8%, 100%, and 62.9% of all known V and J gene segments respectively. A large diversity of clonotypes was captured with 966-1,253 TRA/TRB clonotypes identified. Both dogs also exhibited a small number of highly abundant T cell clonotypes suggesting the presence of an anti-tumor T cell population. GEX enriched libraries successfully defined large clusters of CD8+ and CD4+ T cells that overlapped with V(D)J-expressing cells. DiscussionThe developed reagents successfully generated scTCRseq data, for the first time, which allowed the T cell repertoire to be surveyed in dogs responding to anti-tumor immunotherapy. These reagents will allow longitudinal tracking of anti-tumor T cell dynamics in canine cancer immunotherapy trials.

genomics↗

bioRxiv2020

A domestic cat whole exome sequencing resource for trait discovery

Over 94 million domestic cats are considered pets, who, as our companions, are also susceptible to cancers, common and rare diseases. Whole exome sequencing (WES) is a cost-effective strategy to study their putative disease-causing variants. Presented is ~35.8 Mb exome capture design based on the annotated Felis_catus_9.0 genome assembly, covering 201,683 regions of the cat genome. WES was conducted on 41 cats from various breeds with known and unknown diseases and traits, including 10 cats with prior whole genome sequence (WGS) data available, to test WES capture probe performance. A WES and WGS comparison was completed to understand variant discovery capability of each platform. At ~80x exome coverage, the percent of on-target base coverage >20x was 96.4% with an average of 10.4% off-target. For variant discovery, greater than 98% of WGS SNPs were also discovered by WES. Platform specific variants were mainly restricted to a small number of sex chromosome and olfactory receptor genes. Within the 41 cats with ~31 diseases and normal traits, 45 previously known disease or trait causal variants were observed, such as Persian progressive retinal degeneration and hydrocephalus. Novel candidate variants for polycystic kidney disease and atrichia in the Peterbald breed were also identified as well as a new cat patient with a known variant for cystinuria. These results show the discovery potential of deep exome sequencing to validate existing disease gene models and identify novel gene candidate alleles for many common and rare diseases in cats.

bioinformatics↗

bioRxiv2020

Harnessing Expressed Single Nucleotide Variation and Single Cell RNA Sequencing to Define Immune Cell Chimerism in the Rejecting Kidney Transplant

In solid organ transplantation, donor derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient and donor cells. To address this question, we performed paired whole exome sequencing of donor and recipient DNA and single cell RNA sequencing (scRNA-seq) of 5 human kidney transplant biopsy cores. Exome sequences were used to define single nucleotide variations (SNV) across all samples. By analyzing expressed SNVs in the scRNA-seq dataset we could define recipient vs. donor cell origin for all 81,139 cells. The leukocyte donor to recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages were characterized by inflammatory activation and donor macrophages by antigen presentation and complement signaling. Recipient origin T cells expressed cytotoxic and pro-inflammatory genes consistent with an effector cell phenotype whereas donor origin T cells are likely quiescent expressing oxidative phosphorylation genes relative to recipient T cells. Finally, both donor and recipient T cell clones were present within the rejecting kidney, suggesting lymphoid aggregation. Our results indicate that donor origin macrophages and T cells have distinct transcriptional profiles compared to their recipient counterparts and donor macrophages can persist for years post transplantation. This study demonstrates the power of this approach to accurately define leukocyte chimerism in a complex tissue such as the kidney transplant coupled with the ability to examine transcriptional profiles at single cell resolution.

genomics↗

bioRxiv2017

The prognostic effects of somatic mutations in ER-positive breast cancer

More than 50 genes are recurrently affected by somatic mutation in estrogen receptor positive (ER+) breast cancer but prognostic effects have not been definitively established. Primary tumor DNA was therefore subjected to targeted sequencing from 625 postmenopausal (UBC-TAM series) and 328 premenopausal (MA12 trial) hormone receptor-positive (HR+) patients. Independent validation of prognostic interactions was achieved using independent data from the METABRIC study. Associations between MAP3K1 and PIK3CA with luminal A status and TP53 mutations with Luminal B/non-luminal tumors were observed, validating the methodological approach. In UBC-TAM, NF1 frame-shift nonsense (FS/NS) mutation was validated as a poor outcome driver. For MA12, poor outcome associated with PIK3R1 mutation was similarly validated. DDR1 mutations were strongly associated with poor prognosis in UBC-TAM despite stringent false-discovery correction (q=0.0003). In conclusion, uncommon recurrent somatic mutations should be further explored to create a more complete explanation of the highly variable outcomes that typify ER+ breast cancer.

genomics↗