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Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell behavior in disease.

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type-specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells' roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription-polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell-specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.

Limb-bud and heart (LBH) is a key transcriptional regulator in vertebrates with pivotal roles in embryonic development and human disease. Herein, using a diverse array of biophysical techniques, we report the first structural characterization of LBH pertinent to its biological function. Our data reveal that LBH is structurally disordered with no discernable secondary or tertiary structure and exudes rod-like properties in solution. Consistent with these observations, we also demonstrate that LBH is conformationally flexible and thus may be capable of adapting distinct conformations under specific physiological contexts. We propose that LBH is a member of the intrinsically disordered protein (IDP) family, and that conformational plasticity may play a significant role in modulating LBH-dependent transcriptional processes.

Epigenetic regulation of differentiation-related genes is poorly understood. We previously reported that transcription factors GATA6 and Sp1 interact with and activate the rat proximal 358-bp promoter/enhancer (p358P/E) of lung alveolar epithelial type I (AT1) cell-specific gene aquaporin-5 (Aqp5). In this study, we found that histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) increased AQP5 expression and Sp1-mediated transcription of p358P/E. HDAC3 overexpression inhibited Sp1-mediated Aqp5 activation, while HDAC3 knockdown augmented AQP5 protein expression. Knockdown of GATA6 or transcriptional co-activator/histone acetyltransferase p300 decreased AQP5 expression, while p300 overexpression enhanced p358P/E activation by GATA6 and Sp1. GATA6 overexpression, SAHA treatment or HDAC3 knockdown increased histone H3 (H3) but not histone H4 (H4) acetylation within the homologous p358P/E region of mouse Aqp5. HDAC3 binds to Sp1 and HDAC3 knockdown increased interaction of GATA6/Sp1, GATA6/p300 and Sp1/p300. These results indicate that GATA6 and HDAC3 control Aqp5 transcription via modulation of H3 acetylation/deacetylation, respectively, through competition for binding to Sp1, and suggest that p300 modulates acetylation and/or interacts with GATA6/Sp1 to regulate Aqp5 transcription. Cooperative interactions among transcription factors and histone modifications regulate Aqp5 expression during alveolar epithelial cell transdifferentiation, suggesting that HDAC inhibitors may enhance repair by promoting acquisition of AT1 cell phenotype.

The identification of multipotent mammary stem cells (MaSCs) has provided an explanation for the unique regenerative capacity of the mammary gland throughout adult life. However, it remains unclear what genes maintain MaSCs and control their specification into the two epithelial lineages: luminal and basal. LBH is a novel transcription co-factor in the WNT pathway with hitherto unknown physiological function. LBH is expressed during mammary gland development and aberrantly overexpressed in aggressive ‘basal’ subtype breast cancers. Here, we have explored the in vivo role of LBH in mammopoiesis. We show that in postnatal mammary epithelia, LBH is predominantly expressed in the Lin−CD29highCD24+ basal MaSC population. Upon conditional inactivation of LBH, mice exhibit pronounced delays in mammary tissue expansion during puberty and pregnancy, accompanied by increased luminal differentiation at the expense of basal lineage specification. These defects could be traced to a severe reduction in the frequency and self-renewal/differentiation potential of basal MaSCs. Mechanistically, LBH induces expression of key epithelial stem cell transcription factor ΔNp63 to promote a basal MaSC state and repress luminal differentiation genes, mainly that encoding estrogen receptor α (Esr1/ERα). Collectively, these studies identify LBH as an essential regulator of basal MaSC expansion/maintenance, raising important implications for its potential role in breast cancer pathogenesis.

Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation., Author Summary Understanding the role of epigenetic control of gene expression is critical to the full description of biological processes, such as development and regeneration. Herein we utilize the differentiation of cells from the distal lung to gain insight into the correlation between the epigenetic landscape, molecular signaling events, and eventual changes in transcription and phenotype. We found that by integrating epigenetic profiling with whole genome transcriptomic data we were able to determine which molecular signaling events were activated and repressed during adult alveolar epithelial cell differentiation, and we identified epigenetic changes that contributed to these changes. Furthermore, we validated the role of one of these predicted but not previously identified pathways, retinoid X receptor signaling, in this process.

The T box transcription factor TBX2, a master regulator of organogenesis, is aberrantly amplified in aggressive human epithelial cancers. While it has been shown that overexpression of TBX2 can bypass senescence, a failsafe mechanism against cancer, its potential role in tumor invasion has remained obscure. Here we demonstrate that TBX2 is a strong cell-autonomous inducer of the epithelial-mesenchymal transition (EMT), a latent morphogenetic program that is key to tumor progression from noninvasive to invasive malignant states. Ectopic expression of TBX2 in normal HC11 and MCF10A mammary epithelial cells was sufficient to induce morphological, molecular, and behavioral changes characteristic of EMT. These changes included loss of epithelial adhesion and polarity gene (E-cadherin, ß-catenin, ZO1) expression, and abnormal gain of mesenchymal markers (N-cadherin, Vimentin), as well as increased cell motility and invasion. Conversely, abrogation of endogenous TBX2 overexpression in the malignant human breast carcinoma cell lines MDA-MB-435 and MDA-MB-157 led to a restitution of epithelial characteristics with reciprocal loss of mesenchymal markers. Importantly, TBX2 inhibition abolished tumor cell invasion and the capacity to form lung metastases in a Xenograft mouse model. Meta-analysis of gene expression in over one thousand primary human breast tumors further showed that high TBX2 expression was significantly associated with reduced metastasis-free survival in patients, and with tumor subtypes enriched in EMT gene signatures, consistent with a role of TBX2 in oncogenic EMT. ChIP analysis and cell-based reporter assays further revealed that TBX2 directly represses transcription of E-cadherin, a tumor suppressor gene, whose loss is crucial for malignant tumor progression. Collectively, our results uncover an unanticipated link between TBX2 deregulation in cancer and the acquisition of EMT and invasive features of epithelial tumor cells.

Increasing evidence suggests that embryonic development and tumorigenesis share some of the same molecular mechanisms. In particular, aberrant reactivation of latent developmental signaling pathways and transcription factors in tumor cells has been associated with and shown to play causal roles in advanced-stage, invasive cancers (5, 6, 80). Limb-bud and heart (LBH) is a highly conserved, novel tissue-specific transcription cofactor in vertebrates with important roles in embryonic development (7, 8, 19). We have previously identified Lbh as a novel mouse gene with unique spatiotemporal expression during early embryogenesis that reflects pattern formation in the developing limb buds and heart (8). Lbh encodes a small acidic protein (molecular mass, 12.3 kDa) that contains a conserved putative nuclear localization signal and a glutamate-rich putative transcriptional activation domain but lacks a DNA binding domain (DBD) (8). In mammalian reporter assays, LBH has both transcriptional activator and corepressor functions (7, 8). Recent biophysical analysis has revealed a high degree of structural disorder in LBH, suggesting that conformational plasticity may play a significant role in modulating LBH-dependent transcriptional processes (2). Aberrant gain of LBH function is associated with partial trisomy 2p syndrome (7), a rare human autosomal disorder that is characterized by multiple congenital anomalies, including cardiovascular, skeletal, and postaxial limb defects (46). Partial trisomy 2p syndrome patients harbor a triplication of chromosomal region 2p23, where LBH maps, indicating that increased LBH gene dosage is pathological in humans (7). Transgenic misexpression of Lbh during embryonic heart development in mice phenocopies congenital heart disease observed in these patients (7) and indicates that Lbh functions to attenuate cardiac chamber differentiation through corepression of key cardiac transcription factors NKX2.5 and TBX5 (7). Interestingly, gain of function of LBH during mouse heart development also causes various growth defects, such as ventricular hyperplasia and increased cardiac valve formation, as well as the abnormally sustained self-renewal of cardiomyocytes after birth, suggesting that LBH has promitogenic activity (7). Consistent with these findings, retroviral overexpression of Lbh in chicken embryonic limbs leads to increased cell proliferation of immature chondrocytes and markedly delays bone differentiation (19). However, the regulatory pathways acting upstream of LBH and its role in adult development have remained obscure. Wnt signaling plays a fundamental role in embryonic development by regulating pattern formation, cell proliferation, differentiation, and migration (44). Wnt ligands are secreted lipid-modified glycoproteins that act as morphogens and elicit different cell behaviors depending on whether receptor interaction activates a canonical β-catenin-dependent transduction pathway or other β-catenin-independent noncanonical pathways (39, 73). In adults, canonical Wnt signaling promotes the self-renewal and maintenance of stem cells required for normal tissue homeostasis (52), a function that becomes oncogenic when this pathway is deregulated (18). Activation of canonical Wnt signaling leads to the stabilization of cytoplasmic β-catenin and its subsequent translocation into the nucleus, where it forms a heteromeric complex with DNA-binding proteins of the T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family to activate target gene transcription (18). In the absence of Wnt, TCF/LEF factors are bound to target gene promoters but act as transcriptional repressors by forming a complex with Groucho/Grg/TLE corepressors (18). The amplitude of canonical Wnt signaling is autoregulated via multiple positive and negative feedback mechanisms that include TCF/LEF factors themselves and the secreted antagonist Dickkopf 1 (DKK1), respectively (13, 18, 26, 51). In addition, Wnt ligands can activate multiple noncanonical signaling pathways, including the planar cell polarity (PCP) and Wnt/Ca2+ pathways and, in the case of Wnt7a in limb development, a pathway involving the homeodomain transcription factor Lmx1b (37, 62). Genetic studies of mice first indicated that canonical Wnt signaling is oncogenic and implicated in breast cancer. Aberrant activation of Wnt1 through proviral integration of the mouse mammary tumor virus (MMTV) causes aggressive mammary tumors in mice (53, 71). Similarly, transgenic overexpression of stabilized β-catenin in mouse mammary glands results in formation of invasive mammary adenocarcinomas (33, 69). Abnormal activation of the canonical Wnt pathway is also associated with human breast cancer (41) and is most common in a highly aggressive subtype of breast cancers known as basal carcinomas (22, 28, 38, 64). This tumor subtype accounts for 15 to 20% of breast cancers and is characterized by early onset and a highly invasive, poorly differentiated (keratin 5/6-positive) tumor phenotype. Basal-like breast cancers have a poor clinical outcome and represent a challenge for therapeutic intervention due to their lack of expression of the therapeutic targets estrogen receptor (ER), progesterone receptor (PR), and the ERBB2 oncogene (57, 65, 66), thus emphasizing the need for identification of new tumor-specific markers. The spatiotemporal expression pattern of Lbh during mouse embryogenesis (8) led us to hypothesize that Lbh may be controlled by morphogenic signaling pathways that orchestrate cell specification and pattern formation. Using a combination of molecular, mammalian tissue culture, mouse genetics, and in silico analyses, we set out to identify the molecular pathways operating upstream of LBH. In doing so, we discovered that LBH expression in epithelial development is tightly controlled by an antagonistic relationship between canonical Wnt/β-catenin and noncanonical Wnt7a signaling. Whereas LBH transcription is induced by Wnt/β-catenin signaling via four conserved TCF/LEF binding sites in the LBH gene locus, this induction is efficiently blocked by Wnt7a. Given the parallels between Wnt signaling in development and cancer, we hypothesized that LBH, as a canonical Wnt target gene, might be deregulated in breast cancer. Indeed, we found that LBH is aberrantly overexpressed in mammary tumors of MMTV-Wnt1 transgenic mice as well as in highly aggressive basal subtype human breast cancers. Overexpression of Lbh in HC11 mammary epithelial cells (MECs) further demonstrates that LBH suppresses terminal cell differentiation, an effect that could contribute to Wnt-induced tumorigenesis. Collectively, our data suggest that LBH is a direct Wnt target gene that is reactivated in a particularly lethal form of human breast cancer.