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Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mammary gland is an outstanding system to study the regulatory mechanisms governing adult epithelial stem cell activity. Stem cells in the basal layer of the mammary gland fuel the morphogenesis and regeneration of a complex epithelial network during development and upon transplantation. The self-renewal of basal stem/progenitor cells is subjected to regulation by both cell-intrinsic and extrinsic mechanisms. Nfatc1 is a transcription factor that regulates breast tumorigenesis and metastasis, but its role in mammary epithelial development and stem cell function has not been investigated. Here we show that Nfatc1 is expressed in a small subset of mammary basal epithelial cells and its epithelial-specific deletion results in mild defects in side branching and basal-luminal cell balance. Moreover, Nfatc1-deficient basal cells exhibit reduced colony forming ability in vitro and somewhat compromised regenerative potential upon transplantation. Thus, our study provides evidence for a detectable yet non-essential role of Nfatc1 in mammary epithelial morphogenesis and basal stem/progenitor cell self-renewal., (© 2021. The Author(s).)

Stem cells that reside in the bulge of adult mouse hair follicles are a leading model of tissue stem cell research. Ex vivo culturing, molecular and cell biological characterizations, as well as genetic manipulation of fluorescence-activated cell sorting-isolated bulge stem cells offer a useful experimental pipeline to complement in vivo studies. Here we describe detailed methods for culturing, immunostaining, live cell imaging, and adenoviral infection of bulge stem cells for downstream applications such as in vitro clonal and in vivo patch assays.

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFβ and Wnt/β-catenin. Particularly, it activates Wnt/β-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

Understanding the epigenetic mechanisms that control the activation of adult stem cells holds the promise of tissue and organ regeneration. Hair follicle stem cells have emerged as a prime model to study stem cell activation. Wnt/β-catenin signaling controls multiple aspects of skin epithelial regeneration, with its excessive activity promoting the hyperactivation of hair follicle stem/progenitor cells and tumorigenesis. The contribution of chromatin factors in regulating Wnt/β-catenin pathway function in these processes is unknown. Here, we show that chromatin effector Pygopus homolog 2 (Pygo2) produced by the epithelial cells facilitates depilation-induced hair regeneration, as well as β-catenin-induced activation of hair follicle stem/early progenitor cells and trichofolliculoma-like skin hyperplasia. Pygo2 maximizes the expression of Wnt/β-catenin targets, but is dispensable for β-catenin-mediated expansion of LIM/homeobox protein Lhx2(+) cells, in the stem/early progenitor cell compartment of the hair follicle. Moreover, β-catenin and Pygo2 converge to induce the accumulation and acetylation of tumor suppressor protein p53 upon the cell cycle entry of hair follicle early progenitor cells and in cultured keratinocytes. These findings identify Pygo2 as an important regulator of Wnt/β-catenin function in skin epithelia and p53 activation as a prominent downstream event of β-catenin/Pygo2 action in stem cell activation.

Background: Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells., Results: We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis., Conclusion: Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

Epigenetic mechanisms regulating lineage differentiation of mammary stem cells (MaSCs) remain poorly understood. Pygopus 2 (Pygo2) is a histone methylation reader and a context-dependent Wnt/β-catenin coactivator. Here we provide evidence for Pygo2's function in suppressing luminal/alveolar differentiation of MaSC-enriched basal cells. We show that Pygo2-deficient MaSC/basal cells exhibit partial molecular resemblance to luminal cells, such as elevated Notch signaling and reduced mammary repopulating capability upon transplantation. Inhibition of Notch signaling suppresses basal-level and Pygo2-deficiency-induced luminal/alveolar differentiation of MaSC/basal cells, whereas activation of Wnt/β-catenin signaling suppresses luminal/alveolar differentiation and Notch3 expression in a Pygo2-dependent manner. We show that Notch3 is a direct target of Pygo2 and that Pygo2 is required for β-catenin binding and maintenance of a poised/repressed chromatin state at the Notch3 locus in MaSC/basal cells. Together, our data support a model where Pygo2-mediated chromatin regulation connects Wnt signaling and Notch signaling to restrict the luminal/alveolar differentiation competence of MaSC/basal cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Transcriptional regulation is fundamentally important for the progression of tissue stem cells through different stages of development and differentiation. Mammalian skin epidermis is an excellent model system to study such regulatory mechanisms due to its easy accessibility, stereotypic spatial arrangement, and availability of well-established cell type/lineage differentiation markers. Moreover, epidermis is one of the few mammalian tissues the stem cells of which can be maintained and propagated in culture to generate mature cell types and a functional tissue (reviewed in [1]), offering in vitro and ex vivo platforms to probe deep into the underlying cell and molecular mechanisms of biological functions.

Epithelial stem cells, such as those present in mammalian skin, intestine, or mammary gland, are tissue stem cells capable of both long-term self-renewal and multi-lineage differentiation. Here we review studies implicating epigenetic control mechanisms in mammalian epithelial stem cell development and homeostasis. We also provide an update of recent progresses in the involvement of canonical Wnt signaling and note an interesting link between the Wnt pathway and chromatin regulation in epithelial stem cells. We anticipate that epigenetic and epigenomic studies of these cells will increase exponentially in the near future., ((c) 2010 Wiley-Liss, Inc.)

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain-containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/beta-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues beta-catenin-induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/beta-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.

Transcriptional control plays a key role in regulating epidermal proliferation and differentiation. Although ample information has been obtained on how epidermal homeostasis is controlled in adult skin, less is known about the control of proliferation/differentiation of epidermal stem/progenitor cells in the developing embryo. Ovol1, encoding a zinc finger protein homologous to Drosophila melanogaster Ovo, is expressed in embryonic epidermal progenitor cells that are transiting from proliferation to terminal differentiation. In this study, we demonstrate a function for Ovol1 in interfollicular epidermal development. In its absence, developing epidermis fails to properly restrict the proliferative potential of progenitor cells, and cultured keratinocytes fail to efficiently undergo growth arrest in response to extrinsic growth-inhibitory signals. We present molecular evidence that c-myc expression is up-regulated in Ovol1-deficient suprabasal cells and that Ovol1 represses c-myc transcription by directly binding to its promoter. Collectively, our findings indicate that Ovol1 is required for proliferation exit of committed epidermal progenitor cells and identify c-myc as an Ovol1 target.

Stem-cell containing mammary basal epithelial cells exist in a quasi-mesenchymal transcriptional state characterized by simultaneous expression of typical epithelial genes and typical mesenchymal genes. Whether robust maintenance of such a transcriptional state is required for adult basal stem cells to fuel self-renewal and regeneration remains unclear. In this work, we utilized SMA-CreER to direct efficient basal cell-specific deletion of Ovol2, which encodes a transcription factor that inhibits epithelial-to-mesenchymal transition (EMT), in adult mammary gland. We identified a basal cell-intrinsic role of Ovol2 in promoting epithelial, and suppressing mesenchymal, molecular traits. Interestingly, Ovol2-deficient basal cells display minimal perturbations in their ability to support tissue homeostasis, colony formation, and transplant outgrowth. These findings underscore the ability of adult mammary basal cells to tolerate molecular perturbations associated with altered epithelia-mesenchymal plasticity without drastically compromising their self-renewal potential.

The mammary epithelial cell (MEC) system is a bilayered ductal epithelium of luminal and basal cells, maintained by a lineage of stem and progenitor populations. Here, we used integrated single-cell transcriptomics and chromatin accessibility analysis to reconstruct the cell types of the mouse MEC system and their underlying gene regulatory features in an unbiased manner. We define differentiation states within the secretory type of luminal cells, which forms a continuous spectrum of general luminal progenitor and lactation-committed progenitor cells. By integrating single-cell transcriptomics and chromatin accessibility landscapes, we identify cis- and trans-regulatory elements that are differentially activated in the specific epithelial cell types and our newly defined luminal differentiation states. Our work provides a resource to reveal cis/trans-regulatory elements associated with MEC identity and differentiation that will serve as a reference to determine how the chromatin accessibility landscape changes during breast cancer.

Directional migration is inherently important for epithelial tissue regeneration and repair, but how it is precisely controlled and coordinated with cell proliferation is unclear. Here, we report that Ovol2, a transcriptional repressor that inhibits epithelial-to-mesenchymal transition (EMT), plays a crucial role in adult skin epithelial regeneration and repair. Ovol2-deficient mice show compromised wound healing characterized by aberrant epidermal cell migration and proliferation, as well as delayed anagen progression characterized by defects in hair follicle matrix cell proliferation and subsequent differentiation. Epidermal keratinocytes and bulge hair follicle stem cells (Bu-HFSCs) lacking Ovol2 fail to expand in culture and display molecular alterations consistent with enhanced EMT and reduced proliferation. Live imaging of wound explants and Bu-HFSCs reveals increased migration speed but reduced directionality, and post-mitotic cell cycle arrest. Remarkably, simultaneous deletion of Zeb1 encoding an EMT-promoting factor restores directional migration to Ovol2-deficient Bu-HFSCs. Taken together, our findings highlight the important function of an Ovol2-Zeb1 EMT-regulatory circuit in controlling the directional migration of epithelial stem and progenitor cells to facilitate adult skin epithelial regeneration and repair.

The mammalian skin epidermis is a stratified epithelium composed of multiple layers of epithelial cells that exist in appropriate sizes and proportions, and with distinct boundaries separating each other. How the epidermis develops from a single layer of committed precursor cells to form a complex multilayered structure of multiple cell types remains elusive. Here, we construct stochastic, three-dimensional, and multiscale models consisting of a lineage of multiple cell types to study the control of epidermal development. Symmetric and asymmetric cell divisions, stochastic cell fate transitions within the lineage, extracellular morphogens, cell-to-cell adhesion forces, and cell signaling are included in model. A GPU algorithm was developed and implemented to accelerate the simulations. These simulations show that a balance between cell proliferation and differentiation during lineage progression is crucial for the development and maintenance of the epidermal tissue. We also find that selective intercellular adhesion is critical to sharpening the boundary between layers and to the formation of a highly ordered structure. The long-range action of a morphogen provides additional feedback regulations, enhancing the robustness of overall layer formation. Our model is built upon previous experimental findings revealing the role of Ovol transcription factors in regulating epidermal development. Direct comparisons of experimental and simulation perturbations show remarkable consistency. Taken together, our results highlight the major determinants of a well-stratified epidermis: balanced proliferation and differentiation, and a combination of both short- (symmetric/asymmetric division and selective cell adhesion) and long-range (morphogen) regulations. These underlying principles have broad implications for other developmental or regenerative processes leading to the formation of multilayered tissue structures, as well as for pathological processes such as epidermal wound healing.

Mammary gland branching morphogenesis and ductal homeostasis relies on mammary stem cell function for the maintenance of basal and luminal cell compartments. The mechanisms of transcriptional regulation of the basal cell compartment are currently unknown. We explored these mechanisms in the basal cell compartment and identified the Co-factor of LIM domains (CLIM/LDB/NLI) as a transcriptional regulator that maintains these cells. Clims act within the basal cell compartment to promote branching morphogenesis by maintaining the number and proliferative potential of basal mammary epithelial stem cells. Clim2, in a complex with LMO4, supports mammary stem cells by directly targeting the Fgfr2 promoter in basal cells to increase its expression. Strikingly, Clims also coordinate basal-specific transcriptional programs to preserve luminal cell identity. These basal-derived cues inhibit epidermis-like differentiation of the luminal cell compartment and enhance the expression of luminal cell-specific oncogenes ErbB2 and ErbB3. Consistently, basal-expressed Clims promote the initiation and progression of breast cancer in the MMTV-PyMT tumor model, and the Clim-regulated branching morphogenesis gene network is a prognostic indicator of poor breast cancer outcome in humans.

Breast cancer is a heterogenous disease that can be classified into multiple subtypes including the most aggressive basal-like and triple-negative subtypes. Understanding the heterogeneity within the normal mammary basal epithelial cells holds the key to inform us about basal-like cancer cell differentiation dynamics as well as potential cells of origin. Although it is known that the mammary basal compartment contains small pools of stem cells that fuel normal tissue morphogenesis and regeneration, a comprehensive yet focused analysis of the transcriptional makeup of the basal cells is lacking. We used single-cell RNA-sequencing and multiplexed RNA in-situ hybridization to characterize mammary basal cell heterogeneity. We used bioinformatic and computational pipelines to characterize the molecular features as well as predict differentiation dynamics and cell–cell communications of the newly identified basal cell states. We used genetic cell labeling to map the in vivo fates of cells in one of these states. We identified four major distinct transcriptional states within the mammary basal cells that exhibit gene expression signatures suggestive of different functional activity and metabolic preference. Our in vivo labeling and ex vivo organoid culture data suggest that one of these states, marked by Egr2 expression, represents a dynamic transcriptional state that all basal cells transit through during pubertal mammary morphogenesis. Our study provides a systematic approach to understanding the molecular heterogeneity of mammary basal cells and identifies previously unknown dynamics of basal cell transcriptional states. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]