Biology subjects

Zahm, A. M.

Publications and source records attributed to Zahm, A. M..

7 recordsLinked to original sources

bioRxiv2026

keju: powerful and accurate inference in Massively Parallel Reporter Assays

Massively Parallel Reporter Assays (MPRAs) interrogate the regulatory function of thousands of designed genetic elements in parallel through linked DNA and RNA readouts using an engineered construct and attached minimal reporter. Given the complexity of MPRA experimental designs, several different sources of uncertainty complicate inference. We show that previous methods do not account for substantial differences in uncertainty levels between the DNA and RNA counts and between batches. Accordingly, we present keju, a hierarchical statistical model that estimates candidate transcription rate, differential activity between conditions, and effects from promoter composition for MPRA data. To maximize statistical power and improve false positive rate control, keju conditions on the DNA counts to model batch-specific and modality-specific uncertainty in the RNA counts. keju shows vastly improved sensitivity (59%) in simulations compared to previous methods (31% for MPRAnalyze and 9% for BCalm), and also has lower, more robust false positive rates, calling only 6.8% of unlabeled negative controls significant in real data (compared to 34% for MPRAnalyze and 12% for BCalm).

bioinformatics↗

bioRxiv2025

ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples

Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platforms performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of SARS-CoV-2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/647077v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a81075org.highwire.dtl.DTLVardef@d07f82org.highwire.dtl.DTLVardef@634beborg.highwire.dtl.DTLVardef@10d4b25_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Molecular basis of proton-sensing by G protein-coupled receptors

Three proton-sensing G protein-coupled receptors (GPCRs), GPR4, GPR65, and GPR68, respond to changes in extracellular pH to regulate diverse physiology and are implicated in a wide range of diseases. A central challenge in determining how protons activate these receptors is identifying the set of residues that bind protons. Here, we determine structures of each receptor to understand the spatial arrangement of putative proton sensing residues in the active state. With a newly developed deep mutational scanning approach, we determined the functional importance of every residue in proton activation for GPR68 by generating [~]9,500 mutants and measuring effects on signaling and surface expression. This unbiased screen revealed that, unlike other proton-sensitive cell surface channels and receptors, no single site is critical for proton recognition in GPR68. Instead, a network of titratable residues extend from the extracellular surface to the transmembrane region and converge on canonical class A GPCR activation motifs to activate proton-sensing GPCRs. More broadly, our approach integrating structure and unbiased functional interrogation defines a new framework for understanding the rich complexity of GPCR signaling. One-sentence summaryThe protonation networks governing activation of human pH-sensing GPCRs are uncovered by integrative cryo-EM and deep mutational scanning.

biochemistry↗

bioRxiv2023

Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters

Cellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery. This technology has found extensive use in biological research for applications including reporter gene assays, biomarker development, and programming synthetic circuits in living cells. However, a reliable and precise method for selecting minimally-sized synthetic promoters with desired background, amplitude, and stimulation response profiles has been elusive. In this study, we introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length. This comprehensive library allows for rapid identification of promoters with optimal transcriptional output parameters across multiple cell lines and stimuli. We showcase this librarys utility to identify promoters activated in unique cell types, and in response to metabolites, mitogens, cellular toxins, and agonism of both aminergic and non-aminergic GPCRs. We further show these promoters can be used in luciferase reporter assays, eliciting 50-100 fold dynamic ranges in response to stimuli. Our platform is effective, easily implemented, and provides a solution for selecting short-length promoters with precise performance for a multitude of applications.

synthetic biology↗

bioRxiv2019

In vivo screen identifies LXR agonism potentiates sorafenib killing of hepatocellular carcinoma

Existing drug therapies for hepatocellular carcinoma (HCC), including sorafenib, extend patient survival by only three months. We sought to identify novel druggable targets for use in combination with sorafenib to increase its efficacy. We implemented an in vivo genetic screening paradigm utilizing a library of 43 genes-of-interest expressed in the context of repopulation of the injured livers of Fumarylacetoacetate Hydrolase-deficient (Fah-/-) mice, which led to highly penetrant HCC. We then treated mice with vehicle or sorafenib to discover genetic determinants of sensitivity and resistance. Liver X Receptor alpha (LXR) emerged as a potential target. To examine LXR agonism in combination with sorafenib treatment, we added varying concentrations of sorafenib and LXR agonist drugs to HCC cell lines. We performed transcriptomic analysis to elucidate the mechanisms of HCC death. Fah-/- mice injected with the screening library developed HCC tumor clones containing Myc cDNA plus various other cDNAs. Treatment with sorafenib resulted in sorafenib-resistant HCCs that were significantly depleted in Nr1h3 cDNA, encoding LXR, suggesting that LXR activation is incompatible with tumor growth in the presence of sorafenib treatment in vivo. The combination of sorafenib and LXR agonism led to enhanced cell death as compared to monotherapy in multiple HCC cell lines, due to reduced expression of cell cycle regulators and increased expression of genes associated with apoptosis. Combination therapy also enhanced cell death in a sorafenib-resistant primary human HCC cell line. Our novel in vivo screen led to the discovery that LXR agonist drugs potentiate the efficacy of sorafenib in treating HCC.

cancer biology↗

bioRxiv2019

A high-content in vivo screen to identify microRNA epistasis in the repopulating mouse liver

Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing; yet the roles of individual miRNAs or their combinatorial effects in this process are largely unknown. In this study, we sought to identify miRNAs that regulate hepatocyte repopulation following toxic liver injury in a high-throughput manner using the Fah-/- mouse. We constructed plasmid pools encoding over 30,000 tough decoy (TuD) miRNA inhibitors designed to target hepatocyte miRNAs in a pairwise manner. Plasmid libraries were delivered to hepatocytes of Fah-/- mice at the time of liver injury via hydrodynamic tail vein injection and integrated transgene-containing transposons were quantified following repopulation via high-throughput sequencing. Changes in polysome-bound transcripts following miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Analysis of TuD abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly altered following repopulation. We classified a subset of miRNA-binding sites (MBSs) as having strong effect on liver repopulation, thus implicating the targeted hepatocyte miRNAs as regulators of this process. Furthermore, we generated a high-content map of pairwise interactions between 171 MBSs and identified both synergistic and redundant effects. Our study highlights the power of higher-order screens to uncover miRNA functions that would go undetected by individual miRNA perturbations, and provides a new paradigm for the study of epistasis of miRNA activities.

systems biology↗

bioRxiv2019

The dynamic chromatin architecture of the regenerating liver

Background & AimsThe adult liver is the main detoxification organ and is routinely exposed to environmental insults but retains the ability to restore its mass and function upon tissue damage. However, massive injury can lead to liver failure, and chronic injury causes fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, the transcriptional regulation of organ repair in the adult liver is incompletely understood.\n\nMethodsWe isolated nuclei from quiescent as well as repopulating hepatocytes in a mouse model of hereditary tyrosinemia, which recapitulates the injury and repopulation seen in toxic liver injury in humans. We then performed the assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) specifically in repopulating hepatocytes to identify differentially accessible chromatin regions and nucleosome positioning. Additionally, we employed motif analysis to predict differential transcription factor occupancy and validated the in silico results with chromatin immunoprecipitation followed by sequencing (ChIP-seq) for hepatocyte nuclear factor 4 (HNF4) and CCCTC-binding factor (CTCF).\n\nResultsChromatin accessibility in repopulating hepatocytes was increased in the regulatory regions of genes promoting proliferation and decreased in the regulatory regions of genes involved in metabolism. The epigenetic changes at promoters and liver enhancers correspond with regulation of gene expression, with enhancers of many liver function genes displaying a less accessible state during the regenerative process. Moreover, increased CTCF occupancy at promoters and decreased HNF4 binding at enhancers implicate these factors as key drivers of the transcriptomic changes in replicating hepatocytes that enable liver repopulation.\n\nConclusionsOur analysis of hepatocyte-specific epigenomic changes during liver repopulation identified CTCF and HNF4 as key regulators of hepatocyte proliferation and regulation of metabolic programs. Thus, liver repopulation in the setting of toxic injury makes use of both general transcription factors (CTCF) for promoter activation, and reduced binding by a hepatocyte-enriched factor (HNF4) to temporarily limit enhancer activity.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC=\"FIGDIR/small/664862v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (13K):\norg.highwire.dtl.DTLVardef@cefbc3org.highwire.dtl.DTLVardef@f2edc8org.highwire.dtl.DTLVardef@d90be9org.highwire.dtl.DTLVardef@19cf7a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗