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Skin epidermis constitutes the outer permeability barrier that protects the body from dehydration, heat loss, and myriad external assaults. Mechanisms that maintain barrier integrity in constantly challenged adult skin and how epidermal dysregulation shapes the local immune microenvironment and whole-body metabolism remain poorly understood. Here, we demonstrate that inducible and simultaneous ablation of transcription factor-encoding Ovol1 and Ovol2 in adult epidermis results in barrier dysregulation through impacting epithelial-mesenchymal plasticity and inflammatory gene expression. We find that aberrant skin immune activation then ensues, featuring Langerhans cell mobilization and T cell responses, and leading to elevated levels of secreted inflammatory factors in circulation. Finally, we identify failure to gain body weight and accumulate body fat as long-term consequences of epidermal-specific Ovol1/2 loss and show that these global metabolic changes along with the skin barrier/immune defects are partially rescued by immunosuppressant dexamethasone. Collectively, our study reveals key regulators of adult barrier maintenance and suggests a causal connection between epidermal dysregulation and whole-body metabolism that is in part mediated through aberrant immune activation.

The noninflamed microenvironment in prostate cancer represents a barrier to immunotherapy. Genetic alterations underlying cancer cell-intrinsic oncogenic signaling are increasingly appreciated for their role in shaping the immune landscape. Recently, we identified Pygopus 2 (PYGO2) as the driver oncogene for the amplicon at 1q21.3 in prostate cancer. Here, using transgenic mouse models of metastatic prostate adenocarcinoma, we found that Pygo2 deletion decelerated tumor progression, diminished metastases, and extended survival. Pygo2 loss augmented the activation and infiltration of cytotoxic T lymphocytes (CTLs) and sensitized tumor cells to T cell killing. Mechanistically, Pygo2 orchestrated a p53/Sp1/Kit/Ido1 signaling network to foster a microenvironment hostile to CTLs. Genetic or pharmacological inhibition of Pygo2 enhanced the antitumor efficacy of immunotherapies using immune checkpoint blockade (ICB), adoptive cell transfer, or agents inhibiting myeloid-derived suppressor cells. In human prostate cancer samples, Pygo2 expression was inversely correlated with the infiltration of CD8+ T cells. Analysis of the ICB clinical data showed association between high PYGO2 level and worse outcome. Together, our results highlight a potential path to improve immunotherapy using Pygo2-targeted therapy for advanced prostate cancer.

Delayed and often impaired wound healing in the elderly presents major medical and socioeconomic challenges. A comprehensive understanding of the cellular/molecular changes that shape complex cell-cell communications in aged skin wounds is lacking. Here, we use single-cell RNA sequencing to define the epithelial, fibroblast, immune cell types, and encompassing heterogeneities in young and aged skin during homeostasis and identify major changes in cell compositions, kinetics, and molecular profiles during wound healing. Our comparative study uncovers a more pronounced inflammatory phenotype in aged skin wounds, featuring neutrophil persistence and higher abundance of an inflammatory/glycolytic Arg1Hi macrophage subset that is more likely to signal to fibroblasts via interleukin (IL)-1 than in young counterparts. We predict systems-level differences in the number, strength, route, and signaling mediators of putative cell-cell communications in young and aged skin wounds. Our study exposes numerous cellular/molecular targets for functional interrogation and provides a hypothesis-generating resource for future wound healing studies.

Skin epidermis constitutes the exterior barrier that protects the body from dehydration and environmental assaults. Barrier defects underlie common inflammatory skin diseases, but the molecular mechanisms that maintain barrier integrity and regulate epidermal-immune cell cross-talk in inflamed skin are not fully understood. In this study, we show that skin epithelia-specific deletion of Ovol1, which encodes a skin disease‒linked transcriptional repressor, impairs the epidermal barrier and aggravates psoriasis-like skin inflammation in mice in part by enhancing neutrophil accumulation and abscess formation. Through molecular studies, we identify IL-33, a cytokine with known pro-inflammatory and anti-inflammatory activities, and Cxcl1, a neutrophil-attracting chemokine, as potential weak and strong direct targets of Ovol1, respectively. Furthermore, we provide functional evidence that elevated Il33 expression reduces disease severity in imiquimod-treated Ovol1-deficient mice, whereas persistent accumulation and epidermal migration of neutrophils exacerbate it. Collectively, our study uncovers the importance of an epidermally expressed transcription factor that regulates both the integrity of the epidermal barrier and the behavior of neutrophils in psoriasis-like inflammation.

The Mammary gland undergoes complicated epithelial remodeling to form lobuloalveoli during pregnancy, in which basal epithelial cells remarkably increase to form a basket-like architecture. However, it remains largely unknown how dormant mammary basal stem/progenitor cells involve in lobuloalveolar development. Here, we show that Nfatc1 expression marks a rare population of mammary epithelial cells with the majority being basal epithelial cells. Nfatc1 reporter-marked basal epithelial cells are relatively dormant mammary stem/progenitor cells. Although Nfatc1 reporter-marked basal epithelial cells have limited contribution to the homeostasis of mammary epithelium, they divide rapidly during pregnancy and contribute to lobuloalveolar development. Furthermore, Nfatc1 reporter-marked basal epithelial cells are preferentially used for multiple pregnancies. Using single-cell RNA-seq analysis, we identify multiple functionally distinct clusters within the Nfatc1 reporter-marked cell-derived progeny cells during pregnancy. Taken together, our findings underscore Nfatc1 reporter-marked basal cells as dormant stem/progenitor cells that contribute to mammary lobuloalveolar development during pregnancy.

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.

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.

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.

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.

Psoriasis is a common inflammatory skin disease characterized by aberrant inflammation and epidermal hyperplasia. Molecular mechanisms that regulate psoriasis-like skin inflammation remain to be fully understood. Here, we show that the expression of Ovol1 (encoding ovo-like 1 transcription factor) is upregulated in psoriatic skin, and its deletion results in aggravated psoriasis-like skin symptoms following stimulation with imiquimod. Using bulk and single-cell RNA sequencing, we identify molecular changes in the epidermal, fibroblast, and immune cells of Ovol1-deficient skin that reflect an altered course of epidermal differentiation and enhanced inflammatory responses. Furthermore, we provide evidence for excessive full-length IL-1α signaling in the microenvironment of imiquimod-treated Ovol1-deficient skin that functionally contributes to immune cell infiltration and epidermal hyperplasia. Collectively, our study uncovers a protective role for OVOL1 in curtailing psoriasis-like inflammation and the associated skin pathology.

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

Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.

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.

It is known that LGR4 plays an important role in hair follicle (HF) development, but the impact of LGR4 on the hair cycle is still unclear. In this study, we have found that K14-Cre-mediated skin epithelia-specific deletion of Lgr4 results in delayed anagen entry during the physiological hair cycle and compromised HF regeneration upon transplantation. We show that, although Lgr4 deletion does not appear to affect the number of quiescent HF stem cells, it leads to reduced numbers of LGR5+ and actively proliferating stem cells in the HFs. Moreover, LGR4-deficient HFs show molecular changes consistent with decreased mTOR and Wnt signaling but upregulated BMP signaling. Importantly, the reactivation of the protein kinase B pathway by injecting the protein kinase B activator SC79 in Lgr4-/- mice can effectively reverse the hair cycle delay. Together, these data suggest that LGR4 promotes the normal hair cycle by activating HF stem cells and by influencing the activities of multiple signaling pathways that are known to regulate HF stem cells. Our study also implicates LGR4 as a potential target for treating hair disorder in the future.

Our knowledge of transcriptional heterogeneities in epithelial stem and progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem and progenitor potential, but a comprehensive dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell state in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses predict a quasi-linear differentiation hierarchy where basal cells progress from Col17a1Hi/Trp63Hi state to early-response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal transcriptional states. Our study provides a systematic view of epidermal cellular dynamics, supporting a revised "hierarchical-lineage" model of homeostasis.

The transition of epithelial cells into a mesenchymal state (epithelial-to-mesenchymal transition or EMT) is a highly dynamic process implicated in various biological processes. During EMT, cells do not necessarily exist in 'pure' epithelial or mesenchymal states. There are cells with mixed (or hybrid) features of the two, which are termed as the intermediate cell states (ICSs). While the exact functions of ICS remain elusive, together with EMT it appears to play important roles in embryogenesis, tissue development, and pathological processes such as cancer metastasis. Recent single cell experiments and advanced mathematical modeling have improved our capability in identifying ICS and provided a better understanding of ICS in development and disease. Here, we review the recent findings related to the ICS in/or EMT and highlight the challenges in the identification and functional characterization of ICS.

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.

Cutaneous wound healing occurs in distinct yet overlapping steps with the end goal of reforming a stratified epithelium to restore epidermal barrier function. A key component of this process is re-epithelialization, which involves the proliferation and migration of epidermal keratinocytes surrounding the wound. This spatiotemporally controlled process resembles aspects of the epithelial-to-mesenchymal transition (EMT) process and is thus proposed to involve a partial EMT. Here, we review current literature on the cellular and molecular changes that occur during, and the known or potential regulatory factors of cutaneous wound re-epithelialization and EMT to highlight their similarities and differences. We also discuss possible future directions toward a better understanding of the underlying regulatory mechanisms with implications for developing new therapeutics to improve wound repair in humans. Developmental Dynamics 247:473-480, 2018. © 2017 Wiley Periodicals, Inc.

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.

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.

Lgr4 is a member of the leucine-rich, G protein-coupled receptor family of proteins, and has recently been shown to augment Wnt/β-catenin signaling via binding to Wnt agonists R-spondins. It plays an important role in skin development, but its involvement in skin tumorigenesis is unclear. Here, we report that mice deficient for Lgr4 are resistant to 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced keratinocyte proliferation and papilloma formation. We show that TPA treatment activates MEK1, ERK1/2 and downstream effector AP-1 in wild-type (WT) epidermal cells and mice, but not in cells or mice where Lgr4 is depleted. Wnt/β-catenin signaling is also dramatically activated by TPA treatment, and this activation is abolished when Lgr4 is deleted. We provide evidences that blocking both MEK1/ERK1/2 and Wnt/β-catenin pathways prevents TPA-induced increase in the expression of Ccnd1 (cyclin D1), a known Wnt/β-catenin target gene, and that the activation of MEK1/ERK1/2 pathway lies upstream of Wnt/β-catenin signal pathway. Collectively, our findings identify Lgr4 as a critical positive factor for skin tumorigenesis by mediating the activation of MEK1/ERK1/2 and Wnt/β-catenin pathways.

Reversible epithelial-to-mesenchymal transition (EMT) is central to tissue development, epithelial stemness, and cancer metastasis. While many regulatory elements have been identified to induce EMT, the complex process underlying such cellular plasticity remains poorly understood. Utilizing a systems biology approach integrating modeling and experiments, we found multiple intermediate states contributing to EMT and that the robustness of the transitions is modulated by transcriptional factor Ovol2. In particular, we obtained evidence for a mutual inhibition relationship between Ovol2 and EMT inducer Zeb1, and observed that adding this regulation generates a novel four-state system consisting of two distinct intermediate phenotypes that differ in differentiation propensities and are favored in different environmental conditions. We identified epithelial cells that naturally exist in an intermediate state with bidirectional differentiation potential, and found the balance between EMT-promoting and -inhibiting factors to be critical in achieving and selecting between intermediate states. Our analysis suggests a new design principle in controlling cellular plasticity through multiple intermediate cell fates and underscores the critical involvement of Ovol2 and its associated molecular regulations.

Pygopus 2 (Pygo2/PYGO2) is an evolutionarily conserved coactivator and chromatin effector in the Wnt/β-catenin signaling pathway that regulates cell growth and differentiation in various normal and malignant tissues. Although PYGO2 is highly overexpressed in a number of human cancers, the molecular mechanism underlying its deregulation is largely unknown. Here we report that Pygo2 protein is degraded through the ubiquitin/proteasome pathway and is posttranslationally stabilized through phosphorylation by activated phosphatidylinositol 3-kinase/Akt signaling. Specifically, Pygo2 is stabilized upon inhibition of the proteasome, and its intracellular level is regulated by Cullin 4 (Cul4) and DNA damage-binding protein 1 (DDB1), components of the Cul4-DDB1 E3 ubiquitin ligase complex. Furthermore, Pygo2 is phosphorylated at multiple residues, and Akt-mediated phosphorylation at serine 48 leads to its decreased ubiquitylation and increased stability. Finally, we provide evidence that Akt and its upstream growth factors act in parallel with Wnt to stabilize Pygo2. Taken together, our findings highlight chromatin regulator Pygo2 as a common node downstream of oncogenic Wnt and Akt signaling pathways and underscore posttranslational modification, particularly phosphorylation and ubiquitylation, as a significant mode of regulation of Pygo2 protein expression.

Skin is a complex organ tasked with, among other functions, protecting the body from the outside world. Its outermost protective layer, the epidermis, is comprised of multiple cell layers that are derived from a single-layered ectoderm during development. Using a new stochastic, multi-scale computational modelling framework, the anisotropic subcellular element method, we investigate the role of cell morphology and biophysical cell-cell interactions in the formation of this layered structure. This three-dimensional framework describes interactions between collections of hundreds to thousands of cells and (i) accounts for intracellular structure and morphology, (ii) easily incorporates complex cell-cell interactions and (iii) can be efficiently implemented on parallel architectures. We use this approach to construct a model of the developing epidermis that accounts for the internal polarity of ectodermal cells and their columnar morphology. Using this model, we show that cell detachment, which has been previously suggested to have a role in this process, leads to unpredictable, randomized stratification and that this cannot be abrogated by adjustment of cell-cell adhesion interaction strength. Polarized distribution of cell adhesion proteins, motivated by epithelial polarization, can however eliminate this detachment, and in conjunction with asymmetric cell division lead to robust and predictable development.

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.

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.

During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such a mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here, we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/Ovol2-deficient epidermal cells fail to undertake α-catenin-driven actin cytoskeletal reorganization and adhesive maturation and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations and defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-α-catenin sequential repression and highlight Ovol1 and Ovol2 as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity.

Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition [EMT]). However, how epithelial plasticity is kept in check in epithelial cells during tissue development and regeneration remains to be fully understood. Here we show that restricting the EMT of mammary epithelial cells by transcription factor Ovol2 is required for proper morphogenesis and regeneration. Deletion of Ovol2 blocks mammary ductal morphogenesis, depletes stem and progenitor cell reservoirs, and leads epithelial cells to undergo EMT in vivo to become nonepithelial cell types. Ovol2 directly represses myriad EMT inducers, and its absence switches response to TGF-β from growth arrest to EMT. Furthermore, forced expression of the repressor isoform of Ovol2 is able to reprogram metastatic breast cancer cells from a mesenchymal to an epithelial state. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity in development and cancer.

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.

Abstract Background Simulation of sophisticated biological models requires considerable computational power. These models typically integrate together numerous biological phenomena such as spatially-explicit heterogeneous cells, cell-cell interactions, cell-environment interactions and intracellular gene networks. The recent advent of programming for graphical processing units (GPU) opens up the possibility of developing more integrative, detailed and predictive biological models while at the same time decreasing the computational cost to simulate those models. Results We construct a 3D model of epidermal development and provide a set of GPU algorithms that executes significantly faster than sequential central processing unit (CPU) code. We provide a parallel implementation of the subcellular element method for individual cells residing in a lattice-free spatial environment. Each cell in our epidermal model includes an internal gene network, which integrates cellular interaction of Notch signaling together with environmental interaction of basement membrane adhesion, to specify cellular state and behaviors such as growth and division. We take a pedagogical approach to describing how modeling methods are efficiently implemented on the GPU including memory layout of data structures and functional decomposition. We discuss various programmatic issues and provide a set of design guidelines for GPU programming that are instructive to avoid common pitfalls as well as to extract performance from the GPU architecture. Conclusions We demonstrate that GPU algorithms represent a significant technological advance for the simulation of complex biological models. We further demonstrate with our epidermal model that the integration of multiple complex modeling methods for heterogeneous multicellular biological processes is both feasible and computationally tractable using this new technology. We hope that the provided algorithms and source code will be a starting point for modelers to develop their own GPU implementations, and encourage others to implement their modeling methods on the GPU and to make that code available to the wider community.

Cytokeratins are intermediate filament proteins found in most epithelial cells including the mammary epithelium. Specific cytokeratin expression has been found to mark different epithelial cell lineages and also to associate with putative mammary stem/progenitor cells. However, a comparative analysis of the expression of cytokaratins during embryonic and postnatal mammary development is currently lacking. Moreover, it is not clear whether the different classes of putative mammary stem/progenitor cells exist during embryonic development. Here, we use double/triple-label immunofluorescence and immunohistochemistry to systematically compare the expression of cytokeratin 5 (K5), cytokeratin 6 (K6), cytokeratin 8 (K8), cytokeratin 14 (K14) and cytokeratin 19 (K19) in embryonic and early postnatal mouse mammary glands. We show that K6+ and K8+/K14+ putative mammary progenitor cells arise during embryogenesis with distinct temporal and spatial distributions. Moreover, we describe a transient disconnection of the expression of K5 and K14, two cytokeratins that are often co-expressed, during the first postnatal weeks of mammary development. Finally, we report that cytokeratin expression in cultured primary mammary epithelial cells mimics that during the early stages of postnatal mammary development. These studies demonstrate an embryonic origin of putative mammary stem/progenitor cells. Moreover, they provide additional insights into the use of specific cytokeratins as markers of mammary epithelial differentiation, or the use of their promoters to direct gene overexpression or ablation in genetic studies of mouse mammary development.

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.

Ovol1 belongs to a family of evolutionarily conserved zinc finger proteins that act downstream of key developmental signaling pathways such as Wnt and TGF-β/BMP. It plays important roles in epithelial and germ cell development, particularly by repressing c-Myc and Id2 genes and modulating the balance between proliferation and differentiation of progenitor cells. In this study, we show that Ovol1 negatively regulates its own expression by binding to and repressing the activity of its promoter. We further demonstrate that Ovol1 uses both passive and active repression mechanisms to auto-repress: (1) it antagonizes transcriptional activation of c-Myb, a known positive regulator of proliferation, by competing for DNA binding; (2) it recruits histone deacetylase activity to the promoter via an N-terminal SNAG repressor domain. At Ovol1 cognate sites in the endogenous Ovol1 promoter, c-Myb binding correlates with increased histone acetylation, whereas the expression of Ovol1 correlates with a displacement of c-Myb from the DNA and decreased histone acetylation. Collectively, our data suggest that Ovol1 restricts its own expression by counteracting c-Myb activation and histone acetylation of the Ovol1 promoter.

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.