Biology subjects

van Amerongen, R.

Publications and source records attributed to van Amerongen, R..

11 recordsLinked to original sources

bioRxiv2025

Mechanistic dissection of GRHL2 and PR transcriptional co-regulation in breast cells

Gene expression is controlled by complex transcriptional networks in which transcription factors and their cognate enhancer elements integrate developmental and environmental cues. The progesterone receptor (PR), a hormone-activated transcription factor, is essential for breast development and physiology, yet how it engages with the chromatin and lineage-specific cofactors remains unclear. Using an unbiased approach, we identify the epithelial transcription factor grainyhead-like 2 (GRHL2) as a key co-regulator of PR activity in hormone responsive breast cancer cells. We show that GRHL2 interacts with PR in a progesterone-independent manner. Upon progesterone stimulation, GRHL2 and PR are both recruited to distal enhancer elements of target genes. Furthermore, GRHL2- and PR-bound elements connect spatially through chromatin looping to regulate shared targets. These findings uncover a previously unrecognized mechanism by which GRHL2 and PR coordinate gene regulation through both chromatin binding and 3D genome architecture modification, positioning GRHL2 as a crucial modulator of steroid hormone receptor function.

genomics↗

bioRxiv2025

GRHL and PGR control WNT4 expression in the mammary gland via 3D looping of conserved and species-specific enhancers

WNT4 is critical for epithelial side branching in the mammary gland. In both mice and humans, its expression is tightly regulated and restricted to hormone-responsive, mature luminal cells. Although generally assumed to act downstream of progesterone, the molecular mechanisms that control lineage-specific induction of WNT4 gene expression remain unknown. Here we functionally dissect the cis-acting enhancer network and spatiotemporal transcriptional mechanisms that regulate WNT4 expression in the mouse mammary gland and the human breast. We identify Grainyhead-like (GRHL) proteins as luminal-specific pioneer factors that bind to a conserved distal chromatin hub. The progesterone receptor (PGR) binds to different cis-acting elements that contact the hub via 3D chromatin looping to induce WNT4 expression. More generally, this model explains how the interplay between endocrine signaling and dynamic molecular interactions at the physical chromatin level can be translated into robust gene expression patterns on the tissue scale. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/648333v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@ddca48org.highwire.dtl.DTLVardef@10b3d69org.highwire.dtl.DTLVardef@ecea45org.highwire.dtl.DTLVardef@86d00_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Transcriptomic analysis of pubertal and adult virgin mouse mammary epithelial and stromal cell populations

Conflicting data exist as to how mammary epithelial cell proliferation changes during the reproductive cycle. To study the effect of endogenous hormone fluctuations on gene expression in the mouse mammary gland, we performed bulk RNAseq analyses of epithelial and stromal cell populations that were isolated either during puberty or at different stages of the adult virgin estrous cycle. Our data confirm prior findings that proliferative changes do not occur in every mouse in every cycle. We also show that during the estrous cycle the main gene expression changes occur in adipocytes and other stromal cells. Finally, we present a comprehensive overview of the Wnt gene expression landscape in different mammary gland cell types in pubertal and adult mice. This work contributes to understanding the effects of physiological hormone fluctuations and locally produced signaling molecules on gene expression changes in the mammary gland during the reproductive cycle and should be a useful resource for future studies investigating gene expression patterns in different cell types across different developmental timepoints.

developmental biology↗

bioRxiv2023

A molecular toolbox to study progesterone receptor signaling

Progesterone receptor (PR) signaling is required for mammary gland development and homeostasis. A major bottleneck in studying PR signaling is the lack of sensitive assays to measure and visualize PR pathway activity both quantitatively and spatially. Here, we develop new tools to study PR signaling in human breast epithelial cells. First, we generate optimized Progesterone Responsive Element (PRE)-luciferase constructs and demonstrate that these new reporters are a powerful tool to quantify PR signaling activity across a wide range of progesterone concentrations in two luminal breast cancer cell lines, MCF7 and T47D. We also describe a fluorescent lentiviral PRE-GFP reporter as a novel tool to visualize PR signaling at the single-cell level. Our reporter constructs are sensitive to physiological levels of progesterone. Second, we show that low background signaling, and high levels of PR expression are a prerequisite for robustly measuring PR signaling. Increasing PR expression by transient transfection, stable overexpression in MCF7 or clonal selection in T47D, drastically improves both the dynamic range of luciferase reporter assays, and the induction of endogenous PR target genes as measured by qRT-PCR. We find that the PR signaling response differs per cell line, target gene and hormone concentration used. Taken together, our tools allow a more rationally designed approach for measuring PR signaling in breast epithelial cells.

cell biology↗

bioRxiv2022

Convergent deployment of ancestral programs during the evolution of mammalian flight membranes

Lateral flight membranes, or patagia, have evolved repeatedly in diverse mammalian lineages. While little is known about patagium development, its recurrent evolution may suggest a shared molecular basis. By combining transcriptomics, developmental experiments, and mouse transgenics, we demonstrate that lateral WNT5A expression in the marsupial sugar glider (Petaurus breviceps) promotes the differentiation of its patagium primordium. We further show that this function of WNT5A reprises ancestral roles in skin morphogenesis predating mammalian flight and has been convergently employed during patagium evolution in eutherian bats. Moreover, we find that many genes involved in limb development have been re-deployed during patagium outgrowth in both the sugar glider and bat. Taken together, our findings reveal that deeply conserved molecular toolkits underpin the evolutionary transition to flight in mammals.

developmental biology↗

bioRxiv2021

Hyperactive WNT/CTNNB1 signaling induces a competing cell proliferation and epidermal differentiation response in the mouse mammary epithelium

In the past forty years, the WNT/CTNNB1 signaling pathway has emerged as a key player in mammary gland development and homeostasis. While also evidently involved in breast cancer, much unclarity continues to surround its precise role in mammary tumor formation and progression. This is largely due to the fact that the specific and direct effects of hyperactive WNT/CTNNB1 signaling on the mammary epithelium remain unknown. Here we use a primary mouse mammary organoid culture system to close this fundamental knowledge gap. We show that hyperactive WNT/CTNNB1 signaling induces competing cell proliferation and differentiation responses. While proliferation is dominant at lower levels of WNT/CTNNB1 signaling activity, higher levels cause reprogramming towards an epidermal cell fate. We show that this involves de novo activation of the epidermal differentiation cluster (EDC) locus and we identify master regulatory transcription factors that likely control the process. This is the first time that the molecular and cellular dose-response effects of WNT/CTNNB1 signaling in the mammary epithelium have been dissected in such detail. Our analyses reveal that the mammary epithelium is exquisitely sensitive to small changes in WNT/CTNNB1 signaling and offer a mechanistic explanation for the squamous differentiation that is observed in some WNT/CTNNB1 driven tumors.

cancer biology↗

bioRxiv2020

How to use online tools to generate new hypotheses for mammary gland biology research: a case study for Wnt7b

An increasing number of -omics datasets, generated by labs all across the world, are becoming available. They contain a wealth of data that are largely unexplored. Not every scientist, however, will have access to the required resources and expertise to analyze such data from scratch. Luckily, a growing number of investigators is dedicating their time and effort to the development of user friendly, online applications that allow researchers to use and investigate these datasets. Here, we will illustrate the usefulness of such an approach. Using regulation of Wnt7b as an example, we will highlight a selection of accessible tools and resources that are available to researchers in the area of mammary gland biology. We show how they can be used for in silico analyses of gene regulatory mechanisms, resulting in new hypotheses and providing leads for experimental follow up. We also call out to the mammary gland community to join forces in a coordinated effort to generate and share additional tissue-specific -omics datasets and thereby expand the in silico toolbox.

developmental biology↗

bioRxiv2020

Quantitative live-cell imaging yields novel insight into endogenous WNT/CTNNB1 signaling dynamics

WNT/CTNNB1 signaling regulates tissue development and homeostasis in all multicellular animals. Multiple aspects of the underlying molecular mechanism remain poorly understood and critical information on endogenous WNT/CTNNB1 signaling dynamics is missing. Here we combine CRISPR/Cas9-mediated genome editing and quantitative live-cell microscopy to measure diffusion characteristics of fluorescently tagged, endogenous CTNNB1 in human cells with high spatiotemporal resolution under both physiological and oncogenic conditions. State-of-the-art functional imaging reveals that a substantial fraction of CTNNB1 resides in slow-diffusing complexes in the cytoplasm, irrespective of the activation status of the pathway. The identity of this cytoplasmic CTNNB1 complex changes according to the phosphorylation status of CTNNB1 as it undergoes a major reduction in size when WNT/CTNNB1 is (hyper)activated. We also measure the concentration of complexed and free CTNNB1 in both the cytoplasm and the nucleus before and after WNT stimulation, and use these parameters to build a minimal computational model of WNT/CTNNB1 signaling. Using this integrated experimental and computational approach, our work reveals that WNT pathway activation regulates the dynamic distribution of CTNNB1 across different functional pools by modulating three regulatory nodes: the cytoplasmic destruction complex, nucleocytoplasmic shuttling and nuclear retention.

cell biology↗

bioRxiv2020

MEIS-WNT5A axis regulates development of 4th ventricle choroid plexus

The choroid plexus (ChP) produces cerebrospinal fluid and forms a critical barrier between the brain and the circulation. While the ChP forms in each brain ventricle, it adopts a different shape in each one and remarkably little is known about the mechanisms underlying its development. Here, we show that epithelial WNT5A is critical for determining fourth ventricle (4V) ChP morphogenesis and size. Systemic Wnt5a knockout, or forced WNT5A overexpression beginning at E10.5, profoundly reduced the size and development of ChP in all ventricles. However, conditional deletion of Wnt5a expression in Foxj1-expressing epithelial cells affected only the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical Wnt signaling via Ror1/Ror2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS1 and MEIS2 as upstream regulators of Wnt5a in the 4V ChP epithelium.

developmental biology↗

bioRxiv2020

A novel Axin2 knock-in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/{beta}-catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here we report the generation and characterization of a new knock-in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi-cistronic targeting cassette at the 3 end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock-in allele expresses a bright fluorescent reporter (3xNLS-SGFP2) and a doxycycline-inducible driver for lineage tracing (rtTA3). We show that the Axin2P2A-rtTA3-T2A-3xNLS-SGFP2 strain labels WNT/CTNNB1 cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/024182v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@315f28org.highwire.dtl.DTLVardef@15e5c04org.highwire.dtl.DTLVardef@1eb40a7org.highwire.dtl.DTLVardef@1e1ad8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

TMEM98 is a negative regulator of FRAT mediated Wnt/β-catenin signalling

Wnt/{beta}-catenin signalling is crucial for maintaining the balance between cell proliferation and differentiation, both during tissue morphogenesis and during tissue maintenance in postnatal life. Whereas the signalling activities of the core Wnt/{beta}-catenin pathway components are understood in great detail, far less is known about the precise role and regulation of the many different modulators of Wnt/{beta}-catenin signalling that have been identified to date.\n\nHere we describe TMEM98, a putative transmembrane protein of unknown function, as an interaction partner and regulator of the GSK3-binding protein FRAT2. We show that TMEM98 reduces FRAT2 protein levels and, accordingly, inhibits the FRAT2-mediated induction of {beta}-catenin/TCF signalling. We also characterize the intracellular trafficking of TMEM98 in more detail and show that it is recycled between the plasma membrane and the Golgi. Together, our findings not only reveal a new layer of regulation for Wnt/{beta}-catenin signalling, but also a new biological activity for TMEM98.

cell biology↗