Biology subjects

Zamojski, M.

Publications and source records attributed to Zamojski, M..

8 recordsLinked to original sources

bioRxiv2026

Single-nucleus multiomic analysis reveals modulation of the gene regulatory circuit landscape in pituitary cell types during mouse estrous cycle.

Summary/AbstractThe estrous cycle transcriptional and chromatin dynamics in pituitary cell types have not been investigated. We report single-nucleus multiomics assays in 18 adult mouse pituitaries (102,069 post quality-control nuclei) across 6 cycle time points. Differential analysis revealed cycle stage-dependent epigenetic and transcriptional remodeling across the major pituitary cell populations. In gonadotropes and lactotropes, we identified temporal patterns of differential gene expression linked to various biological processes, notably neuronal and synaptic-related ontologies. Pseudotime trajectory analysis was consistent with a rapid transition of individual gonadotropes through different cellular states. In gonadotropes and lactotropes, we uncovered gene regulatory circuits whose activity varies between consecutive cycle time points. We experimentally validated a gonadotrope ETS2-driven circuit that differentially regulates Fshb gene expression between 2 and 9 am on estrus. Our data and analyses, available at https://rstudio-connect.hpc.mssm.edu/snpit_estrous_browser/, provide a window into gene regulatory mechanisms underlying estrous cycle stage transitions. HighlightsO_LIPituitary cells show chromatin and transcriptome plasticity across the estrous cycle C_LIO_LIWe identify stage-modulated gene regulatory circuits across pituitary cell types C_LIO_LIGonadotrope DEGs form distinct pathway-annotated temporal trajectory clusters C_LIO_LIWe validate a gonadotrope ETS2-driven circuit regulating the Fshb gene C_LI eTOC blurbZhang et al. conduct a single-nucleus multiomics analysis of mouse pituitaries in vivo across the estrous cycle. They reveal an epigenetic and transcriptomic plasticity in pituitary cell populations. In gonadotropes and lactotropes, they demonstrate that DEGs clustered by temporal trajectories are enriched for distinct biological processes and identify stage-modulated gene regulatory circuits. They experimentally validate a gonadotrope ETS2-driven circuit regulating Fshb expression. Their dynamic molecular atlas of the cycling pituitary captures key cis-regulatory mechanisms underlying estrous cycle stage transitions.

genomics↗

bioRxiv2025

Regulation of murine follicle-stimulating hormone β subunit transcription by newly identified enhancers

Activin-class ligands of the transforming growth factor {beta} family induce follicle-stimulating hormone (FSH) production by pituitary gonadotrope cells in mice via the actions of the transcription factors SMAD3, SMAD4, and FOXL2, which bind to cis-elements in the FSH{beta} subunit (Fshb) promoter. An enhancer region for murine Fshb transcription was identified in vitro. However, deletion of the region using CRISPR-Cas9 did not affect FSH synthesis or secretion in mice. Using single-nucleus ATAC-seq of whole murine pituitaries, we identified three additional open chromatin regions upstream of Fshb exclusively in gonadotropes. These regions, as well as the Fshb gene, were fully or partially closed in gonadotropes of FSH-deficient mice with genetically or pharmacologically inactivated activin type II receptors. The initially characterized enhancer region did not significantly alter basal or activin-stimulated murine Fshb promoter-reporter activity in homologous L{beta}T2 cells. In contrast, the other three open chromatin regions enhanced basal and activin A-stimulated Fshb promoter-reporter activity in L{beta}T2 cells, with the two most distal showing the greatest effects. These two regions were open, exhibited enrichment of the enhancer mark H3K27ac, and were bound by SMAD2/3 and FOXL2 in response to activin A in L{beta}T2 cells. The most distal enhancer exhibited strong FOXL2 and weak SMAD4 binding in gel shift assays. SMAD4, but not FOXL2, directly bound the other distal enhancer. Mutation of defined FOXL2 and SMAD4 cis-elements diminished enhancer activity in reporter assays in L{beta}T2 cells. Collectively, the data indicate that there may be as many as four activin-sensitive enhancers upstream of murine Fshb.

molecular biology↗

bioRxiv2025

Consensus Pituitary Atlas, a scalable resource for annotation, novel marker discovery and analyses in pituitary gland research

Previous single-cell profiling studies of the pituitary gland have yielded minimally reproducible insights largely due to their low statistical power and methodological inconsistencies. To address this problem, we generated a uniformly pre-processed Consensus Pituitary Atlas (CPA) using all existing mouse pituitary single-cell datasets (267 biological replicates, >1.1 million high-quality cells). The CPA revealed novel cell typing and lineage markers, including low-expression transcripts that previous analyses could not detect. The scale of the CPA enabled the development of machine learning models to automate and standardize cell type annotation and doublet identification for future studies. Leveraging the curated metadata, we identified sex-biased and age-dependent gene expression patterns at cell type resolution. To identify drivers of cell fates, first we determined consensus cell communication patterns. Secondly, we used RNA-sequencing and chromatin accessibility data to identify transcription factors associated with cell fates across modalities. The epitome platform acts as an interface with the CPA, allowing streamlined user-friendly analyses. HighlightsO_LIUniform processing of 267 mouse pituitary single-cell datasets (>1.1M cells) C_LIO_LIThe statistical power enabled cell type, sex- and age-specific marker discovery C_LIO_LIMachine learning models facilitate doublet detection and cell typing in new datasets C_LIO_LIepitome platform provides programming-free data access and visualizations C_LI

bioinformatics↗

bioRxiv2023

A Comparative Analysis of Imaging-Based Spatial Transcriptomics Platforms

Spatial transcriptomics is a rapidly evolving field, overwhelmed by a multitude of technologies. This study aims to offer a comparative analysis of datasets generated from leading in situ imaging platforms. We have generated spatial transcriptomics data from serial sections of prostate adenocarcinoma using the 10x Genomics Xenium and NanoString CosMx SMI platforms. Additionally, orthogonal single-nucleus RNA sequencing (snRNA-seq) was performed on the same FFPE tissue to establish a reference for the tumors transcriptional profiles. We assessed various technical aspects, such as reproducibility, sensitivity, dynamic range, cell segmentation, cell type annotation, and congruence with single-cell profiling. The practicality of assessing cellular organization and biomarker localization was evaluated. Although fewer genes are measured (CosMx: 960, Xenium: 377, with an overlap of 125), Xenium consistently demonstrates higher sensitivity, a broader dynamic range, and better alignment with single-cell reference profiles. Conversely, CosMxs out-of-the-box segmentation outperformed Xeniums, resulting in noticeable transcript misassignment in Xenium within certain tissue areas. However, the impact of this on the cells transcriptional profile was minimal. Together, this comprehensive comparison of two leading commercial platforms for spatial transcriptomics provides essential metrics for assessing their performance, offering invaluable insights for future research and technological advancements in this dynamic field.

genomics↗

bioRxiv2023

Integrated single-cell multiome analysis reveals muscle fiber-type gene regulatory circuitry modulated by endurance exercise

Endurance exercise is an important health modifier. We studied cell-type specific adaptations of human skeletal muscle to acute endurance exercise using single-nucleus (sn) multiome sequencing in human vastus lateralis samples collected before and 3.5 hours after 40 min exercise at 70% VO2max in four subjects, as well as in matched time of day samples from two supine resting circadian controls. High quality same-cell RNA-seq and ATAC-seq data were obtained from 37,154 nuclei comprising 14 cell types. Among muscle fiber types, both shared and fiber-type specific regulatory programs were identified. Single-cell circuit analysis identified distinct adaptations in fast, slow and intermediate fibers as well as LUM-expressing FAP cells, involving a total of 328 transcription factors (TFs) acting at altered accessibility sites regulating 2,025 genes. These data and circuit mapping provide single-cell insight into the processes underlying tissue and metabolic remodeling responses to exercise.

genomics↗

bioRxiv2023

Peak-agnostic high-resolution cis-regulatory circuitry mapping using single cell multiome data

Single same cell RNAseq/ATACseq multiome data provide unparalleled potential to develop high resolution maps of the cell-type specific transcriptional regulatory circuitry underlying gene expression. We present CREMA, a framework that recovers the full cis-regulatory circuitry by modeling gene expression and chromatin activity in individual cells without peak-calling or cell type labeling constraints. We demonstrate that CREMA overcomes the limitations of existing methods that fail to identify about half of functional regulatory elements which are outside the called chromatin "peaks". These circuit sites outside called peaks are shown to be important cell type specific functional regulatory loci, sufficient to distinguish individual cell types. Analysis of mouse pituitary data identifies a Gata2-circuit for the gonadotrope-enriched disease-associated Pcsk1 gene, which is experimentally validated by reduced gonadotrope expression in a gonadotrope conditional Gata2-knockout model. We present a web accessible human immune cell regulatory circuit resource, and provide CREMA as an R package.

bioinformatics↗

bioRxiv2021

Single nucleus pituitary transcriptomic and epigenetic landscape reveals human stem cell heterogeneity with diverse regulatory mechanisms

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus (sn) RNAseq and ATACseq resource from pediatric, adult, and aged pituitaries (snpituitaryatlas.princeton.edu) and characterize cell type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors (TFs) that are significantly associated with gene expression in PSCs compared to other cell types and within PSCs. Modeling the heterogeneous expression of two markers for committing cell lineages among PSCs shows significant correlation with regulatory domain accessibility for GATA3, but with TF expression for POMC. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes.

cell biology↗

bioRxiv2020

Single nucleus multi-omics regulatory atlas of the murine pituitary

The pituitary regulates growth, reproduction and other endocrine systems. To investigate transcriptional network epigenetic mechanisms, we generated paired single nucleus (sn) transcriptome and chromatin accessibility profiles in single mouse pituitaries and genome-wide sn methylation datasets. Our analysis provided insight into cell type epigenetics, regulatory circuit and gene control mechanisms. Latent variable pathway analysis detected corresponding transcriptome and chromatin accessibility programs showing both inter-sexual and inter-individual variation. Multi-omics analysis of gene regulatory networks identified cell type-specific regulons whose composition and function were shaped by the promoter accessibility state of target genes. Co-accessibility analysis comprehensively identified putative cis-regulatory regions, including a domain 17kb upstream of Fshb that overlapped the fertility-linked rs11031006 human polymorphism. In vitro CRISPR-deletion at this locus increased Fshb levels, supporting this domains inferred regulatory role. The sn pituitary multi-omics atlas (snpituitaryatlas.princeton.edu) is a public resource for elucidating cell type-specific gene regulatory mechanisms and principles of transcription circuit control.

cell biology↗