Biology subjects

Choate, L. A.

Publications and source records attributed to Choate, L. A..

5 recordsLinked to original sources

bioRxiv2022

CRISPRa screen identifies a role for c-KIT signaling in tamoxifen resistance, potentially through upregulation of ABC transporters

Resistance to endocrine therapy is a common problem in patients with estrogen receptor alpha (ER) positive breast cancer. In this study, we took a non-biased genome-wide approach to identify novel mechanisms of endocrine resistance using a clustered regularly interspaced short palindromic repeats (CRISPR) activating (CRISPRa) screen. Results from the screen identified 109 candidate resistance-associated genes, with several of these genes, such as EGFR and SRC, having been previously associated with endocrine resistance. One candidate gene that has not been previously associated with endocrine resistance is the tyrosine kinase receptor, c-KIT. We further tested for associations between c-KIT and endocrine resistance and found that c-KIT overexpressing cells proliferate more rapidly in the presence of tamoxifen compared to control cell lines. To gain deeper insight into the potential role of c-KIT signaling in tamoxifen resistance, we next performed precision nuclear run-on and sequencing (PRO-seq) analysis of c-KIT overexpressing cells to identify downstream factors that may mediate the c-KIT response. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the overexpressed genes found that the only class of factors that was significantly induced by c-KIT was the ATP-binding cassette (ABC) transporters; specifically, ABCA1, ABCA4, and ABCG1. Interestingly, overexpression of two of these ABC transporters, ABCA1 and ABCG1, significantly correlated with worse prognosis in ER+ breast cancer patients following endocrine therapy. We then tested for potential therapeutic effects of c-KIT inhibition on endocrine resistance and found that the c-KIT inhibitor Gleevec appears to synergize with tamoxifen to suppress MCF-7-S cell growth. Together, our findings support the hypothesis that c-KIT signaling promotes endocrine resistance via the induction of ABC transporter activity. Additionally, our studies suggest that inhibition of c-KIT signaling may represent a novel strategy for preventing or overcoming endocrine resistance in ER+ patients.

cancer biology↗

bioRxiv2021

Genetic Dissection of the RNA Polymerase II Transcription Cycle

How DNA sequence affects the dynamics and position of RNA Polymerase II (Pol II) during transcription remains poorly understood. Here we used naturally occurring genetic variation in F1 hybrid mice to explore how DNA sequence differences affect the genome-wide distribution of Pol II. We measured the position and orientation of Pol II in eight organs collected from heterozygous F1 hybrid mice using ChRO-seq. Our data revealed a strong genetic basis for the precise coordinates of transcription initiation and promoter proximal pause, allowing us to redefine molecular models of core transcriptional processes. Our results implicate the strength of base pairing between A-T or G-C dinucleotides as key determinants to the position of Pol II initiation and pause. We report evidence that initiation site selection follows a stochastic process similar to brownian motion along the DNA template. We found widespread differences in the position of transcription termination, which impact the primary structure and stability of mature mRNA. Finally, we report evidence that allelic changes in transcription often affect mRNA and ncRNA expression across broad genomic domains. Collectively, we reveal how DNA sequences shape core transcriptional processes at single nucleotide resolution in mammals.

genomics↗

bioRxiv2020

Multiple stages of evolutionary change in anthrax toxin receptor expression in humans

The advent of animal husbandry and hunting increased human exposure to zoonotic pathogens. To understand how a zoonotic disease influenced human evolution, we studied changes in human expression of anthrax toxin receptor 2 (ANTXR2), which encodes a cell surface protein necessary for Bacillus anthracis virulence toxins to cause anthrax disease. In immune cells, ANTXR2 was 8-fold down-regulated in all available human samples compared to non-human primates, indicating regulatory changes early in the evolution of modern humans. We also observed multiple genetic signatures consistent with recent positive selection driving a European-specific decrease in ANTXR2 expression in several non-immune tissues affected by anthrax toxins. Our observations fit a model in which humans adapted to anthrax disease following early ecological changes associated with hunting and scavenging, as well as a second period of adaptation after the rise of modern agriculture.

genetics↗

bioRxiv2020

Accurate imputation of histone modifications using transcription

The role of histone modifications in transcription remains incompletely understood. Here we used experimental perturbations combined with sensitive machine learning tools that infer the distribution of histone marks using maps of nascent transcription. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3 consistent with a role for RNA in recruiting PRC2. A subset of DNase-I hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than a regulatory, role in transcription.

genomics↗

bioRxiv2018

Identification of regulatory elements from nascent transcription using dREG

Our genomes encode a wealth of transcription initiation regions (TIRs) that can be identified by their distinctive patterns of actively elongating RNA polymerase. We previously introduced dREG to identify TIRs using PRO-seq data. Here we introduce an efficient new implementation of dREG that uses PRO-seq data to identify both uni- and bidirectionally transcribed TIRs with 70% improvements in accuracy, 3-4-fold higher resolution, and >100-fold increases in computational efficiency. Using a novel strategy to identify TIRs based on their statistical confidence reveals extensive overlap with orthogonal assays, yet also reveals thousands of additional weakly-transcribed TIRs that were not identified by H3K27ac ChIP-seq or DNase-I-hypersensitivity. Novel TIRs discovered by dREG were often associated with RNA polymerase III initiation, bound by pioneer transcription factors, or located in broad domains marked by repressive chromatin modifications. We provide a web interface to dREG that can be used by the scientific community (http://dREG.DNASequence.org).

bioinformatics↗