Biology subjects

Day, C. R.

Publications and source records attributed to Day, C. R..

7 recordsLinked to original sources

bioRxiv2026

Protocol for measuring endocrine disruptive effects on transcriptional bursting using single-molecule imaging in human breast cancer cells

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol that integrates fixed-cell smFISH and live-cell single-molecule imaging to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines. This workflow enables measurement of burst size, burst initiation, and active allele frequency to determine how endocrine disruptor chemicals modulate transcriptional bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day, Yasar et al.1

cell biology↗

bioRxiv2026

Layered Single-Cell Heterogeneity in Hormone Receptor Signaling Across Mouse Organoids and Human ERα+ Cancer Cells

Hormone receptor signaling is often interpreted through receptor abundance as a proxy for hormone responsiveness, yet the determinants of single-cell variability in hormone response remain unclear. Using murine mammary organoids, we mapped estrogen (E2) and progesterone (P4) responses at single-cell resolution. Despite controlled 3D culture conditions, basal cell-derived organoids exhibit striking variability in hormone-induced transcriptional responses, with ER cells varying in the fraction of responsive genes engaged. This variability is not explained by receptor abundance alone. Instead, response magnitude correlates with expression of transcriptional co-regulators including Ncoa1, Ncor1, and Ncor2, suggesting that co-regulator balance contributes to variation in endocrine response magnitude. Organoids exhibit a mixed basal-luminal enhancer landscape and growth factor-dependent remodeling of ER and PR protein abundance. MCF7 cells show delayed activation kinetics and reach a lower response plateau. Together, these findings reveal that hormone response magnitude varies independently of receptor abundance in mammary organoids and follows distinct activation dynamics in human ER+ cancer cells.

cell biology↗

bioRxiv2025

WntA expression and wing transcriptomics illuminate the evolution of stripe patterns in skipper butterflies

Skippers (Hesperiidae) form a distinct lineage of butterflies where the developmental mechanisms of color patterning have seldom been studied. The wing patterns of skippers often consist of median stripes, and classic comparative morphology studies suggest these elements are homologous to the Central Symmetry System (CSS) found in nymphalid butterflies. Consistent with this hypothesis, we show here that expression of the signaling ligand gene WntA, a marker of the CSS in nymphalids, prefigures the position of the main wing patterns of the silver-spotted skipper Epargyreus clarus. Notably, WntA expression is associated with different color outputs in the forewing vs. hindwing, validating a theory of pattern homology from the mid-twentieth century. To further gain insights into the genes associated with this patterning process, we generated an annotated genome for E. clarus and performed an RNAseq study profiling gene expression along the proximo-distal (P-D) axis of early pupal wings. These data suggest that the transcription factor genes lobe, u-shaped, and odd-paired are expressed in restricted P-D sections of the wing similarly to WntA, indicating they may participate in the patterning of the CSS. In addition, inverted expression patterns of dachsous and four-jointed, as well as the expression of transcription factors with roles in specifying proximal (homothorax, tiptop/teashirt) and distal (vestigial, scalloped) section of ths Drosophila wing disk, suggest a deep conservation of wing P-D patterning process between Diptera and Lepidoptera. This work highlights the developmental homology between the CSS of Hesperiidae and Nymphalidae, and enriches our knowledge of patterning mechanisms in lepidopteran wings. Summary StatementThis study reveals how skipper butterfly wing stripes share deep developmental origins with other butterflies, offering fresh insight into wing pattern evolution.

developmental biology↗

bioRxiv2025

H3K27me3 chromatin heterogeneity reveals variable cell responses to estrogen and endocrine treatment

Gene expression heterogeneity generates subpopulations of tumor cells that can evade therapeutic pressure. This heterogeneity has been observed in both primary Estrogen Receptor alpha positive (ER+) breast tumors and cell lines. Therefore, understanding the mechanisms regulating expression heterogeneity is critical towards developing effective therapies. A key contributor to gene expression variability is the stochastic nature of transcription. Transcription occurs in a probabilistic, burst-like manner, in which gene activation occurs intermittently, producing RNA in pulses and interspersed with transcriptional off-periods. The estrogen-responsive gene TFF1 is expressed in the majority of ER breast tumors and exemplifies such heterogeneity, with transcriptional inactivity ranging from minutes to several days. Here, we identify the molecular mechanism underlying the wide range in TFF1 expression by analyzing cells sorted based on their TFF1 activity levels. We observed that TFF1 inactive (TFF1low) cells exhibit a repressive chromatin state marked by H3K27me3 at the TFF1 promoter and enhancer. Despite global similarity in ER binding, occupancy at the TFF1 regulatory elements was selectively reduced in TFF1low cells, resulting in fewer active alleles and diminished transcriptional bursting frequency. Conversely, TFF1high cells exhibited more active TFF1 alleles and hyperbursting. These cells also retained sensitivity to endocrine therapy, while TFF1low cells displayed reduced drug responsiveness. Genome-wide, differentially enriched H3K27me3 regions correlated with variable expression of estrogen-responsive genes, highlighting a broader regulatory mechanism that links chromatin state to expression variability. Together, our findings establish how repressive chromatin dynamics contribute to gene expression heterogeneity and endocrine resistance in ER breast cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/677184v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1194eedorg.highwire.dtl.DTLVardef@8cb67aorg.highwire.dtl.DTLVardef@202ad9org.highwire.dtl.DTLVardef@1dd222a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Single-nucleus transcriptomics of wing sexual dimorphism and scale cell specialization in sulphur butterflies

The evolution of sexual secondary characteristics necessitates regulatory factors that confer sexual identity to differentiating tissues and cells. In Colias eurytheme butterflies, males exhibit two specialized wing scale types -- UV-iridescent (UVI) and lanceolate scales -- absent in females and likely integral to male courtship behavior. This study investigates the regulatory mechanisms and single-nucleus transcriptomics underlying these two sexually dimorphic cell types during wing development. We show that Doublesex (Dsx) expression is itself dimorphic and required to repress the UVI cell state in females, while unexpectedly, UVI activation in males is independent from Dsx. In the melanic marginal band, Dsx is required in each sex to enforce the presence of lanceolate scales in males, and their absence in females. Single-nucleus RNAseq reveals that UV-iridescent and lanceolate scale cell precursors each show distinctive gene expression profiles at 40% of pupal development, with marker genes that include regulators of transcription, cell signaling, cytoskeletal patterning, and chitin secretion. Both male-specific cell types share a low expression of the Bric-a-brac (Bab) transcription factor, a key repressor of the UVI fate. Bab ChIP-seq profiling suggests that Bab binds the cis-regulatory regions of gene markers associated to UVI fate, including potential effector genes involved in the regulation of cytoskeletal processes and chitin secretion, and loci showing signatures of recent selective sweeps in an UVI-polymorphic population. These findings open new avenues for exploring wing patterning and scale development, shedding light on the mechanisms driving the specification of sex-specific cell states and the differentiation of specialized cell ultrastructures.

developmental biology↗

bioRxiv2023

ERα mediated gene state switching regulates the extent of the single-cell estrogen response

Gene regulation is complex, involving the coordination of hundreds of proteins that function to control genome accessibility, mediate enhancer-promoter interactions, and initiate transcription. At individual loci, transcriptional initiation is stochastic, resulting in short periods of nascent RNA synthesis known as transcriptional bursts. To understand how altered Estrogen Receptor function and cofactor recruitment regulates transcriptional bursting, we used single molecule imaging of estrogen responsive genes in Bisphenol A (BPA) treated cells. Using live cell imaging of the estrogen responsive TFF1 gene, we observe that cells treated with BPA exhibited burst initiation kinetics and burst sizes which were indistinguishable from cells induced with Estradiol (E2). However, we observed a 50% reduction in the number of active alleles in BPA treated cells. This effect is gene specific, as GREB1 was unperturbed. Although we observed no difference in chromatin accessibility, the TFF1 promoter exhibited an altered structure which coincided with reduced ER and cofactor binding. Lastly, deletion of the enhancer locus removed the BPA effect, indicating that enhancer function was perturbed. Our results demonstrate gene specific effects of altered ER recruitment and function which lead to a reduction of transcriptionally permissive states. Our work supports the model that the early estrogen response occurs from alleles in primed transcriptionally permissive states with additional inactive alleles contributing to the response over time.

cell biology↗

bioRxiv2023

Frizzled2 receives the WntA morphogen during butterfly wing pattern formation

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes that occur in a developing epithelium. While the secreted ligand WntA was shown to instruct the color pattern formation in butterflies, its modes of reception and signal transduction remain elusive. Butterfly genomes encode four homologues of the Frizzled-family of Wnt receptors. Here we show that CRISPR mosaic knock-outs of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss-of-function in multiple nymphalids. While WntA mosaic clones result in intermediate patterns of reduced size, consistently with a morphogen function, fz2 clones are cell-autonomous. Shifts in pupal expression in WntA crispants show that WntA and fz2 are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity (PCP) in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning, and shed light on the functional diversity of insect Frizzled receptors.

developmental biology↗