Delivery of siRNA therapeutics using cowpea chlorotic mottle virus-like particles.

While highly promising in medicine, gene therapy requires delivery agents to protect and target nucleic acid therapeutics. We developed a plant viral siRNA delivery platform making use of self-assembling cowpea chlorotic mottle virus (CCMV). CCMV was loaded with siRNAs targeting GFP or FOXA1; to further enhance cell uptake and intracellular trafficking, resulting in more efficient gene knockdown, we appended CCMV with a cell penetrating peptide (CPP), specifically M-lycotoxin peptide L17E.

Foxa1 and Foxa2 in thymic epithelial cells (TEC) regulate medullary TEC and regulatory T-cell maturation.

The Foxa1 and Foxa2 transcription factors are essential for mouse development. Here we show that they are expressed in thymic epithelial cells (TEC) where they regulate TEC development and function, with important consequences for T-cell development. TEC are essential for T-cell differentiation, lineage decisions and repertoire selection. Conditional deletion of Foxa1 and Foxa2 from murine TEC led to a smaller thymus with a greater proportion of TEC and a greater ratio of medullary to cortical TEC. Cell-surface MHCI expression was increased on cortical TEC in the conditional Foxa1Foxa2 knockout thymus, and MHCII expression was reduced on both cortical and medullary TEC populations. These changes in TEC differentiation and MHC expression led to a significant reduction in thymocyte numbers, reduced positive selection of CD4+CD8+ cells to the CD4 lineage, and increased CD8 cell differentiation. Conditional deletion of Foxa1 and Foxa2 from TEC also caused an increase in the medullary TEC population, and increased expression of Aire, but lower cell surface MHCII expression on Aire-expressing mTEC, and increased production of regulatory T-cells. Thus, Foxa1 and Foxa2 in TEC promote positive selection of CD4SP T-cells and modulate regulatory T-cell production and activity, of importance to autoimmunity.

FOXA1 deletion in luminal epithelium causes prostatic hyperplasia and alteration of differentiated phenotype.

The forkhead box (Fox) superfamily of transcription factors has essential roles in organogenesis and tissue differentiation. Foxa1 and Foxa2 are expressed during prostate budding and ductal morphogenesis, whereas Foxa1 expression is retained in adult prostate epithelium. Previous characterization of prostatic tissue rescued from embryonic Foxa1 knockout mice revealed Foxa1 to be essential for ductal morphogenesis and epithelial maturation. However, it is unknown whether Foxa1 is required to maintain the differentiated status in adult prostate epithelium. Here, we employed the PBCre4 transgenic system and determined the impact of prostate-specific Foxa1 deletion in adult murine epithelium. PBCre4/Foxa1(loxp/loxp) mouse prostates showed progressive florid hyperplasia with extensive cribriform patterning, with the anterior prostate being most affected. Immunohistochemistry studies show mosaic Foxa1 KO consistent with PBCre4 activity, with Foxa1 KO epithelial cells specifically exhibiting altered cell morphology, increased proliferation, and elevated expression of basal cell markers. Castration studies showed that, while PBCre4/Foxa1(loxp/loxp) prostates did not exhibit altered sensitivity in response to hormone ablation compared with control prostates, the number of Foxa1-positive cells in mosaic Foxa1 KO prostates was significantly reduced compared with Foxa1-negative cells following castration. Unexpectedly, gene expression profile analyses revealed that Foxa1 deletion caused abnormal expression of seminal vesicle-associated genes in KO prostates. In summary, these results indicate Foxa1 expression is required for the maintenance of prostatic cellular differentiation.

Foxa1 and foxa2 are required for the maintenance of dopaminergic properties in ventral midbrain neurons at late embryonic stages.

The maintained expression of transcription factors throughout the development of mesodiencephalic dopaminergic (mDA) neurons suggests multiple roles at various stages in development. Two members of the forkhead/winged helix transcription factor family, Foxa1 and Foxa2, have been recently shown to have an important influence in the early development of mDA neurons. Here we present data demonstrating that these genes are also involved in the later maintenance of the mDA system. We conditionally removed both genes in postmitotic mDA neurons using the dopamine transporter-cre mouse. Deletion of both Foxa1 and Foxa2 resulted in a significant reduction in the number of tyrosine hydroxylase (TH)-positive mDA neurons. The decrease was predominantly observed in the substantia nigra region of the mDA system, which led to a loss of TH+ fibers innervating the striatum. Further analysis demonstrated that the reduction in the number of TH+ cells in the mutant mice was not due to apoptosis or cell-fate change. Using reporter mouse lines, we found that the mDA neurons were still present in the ventral midbrain, but that they had lost much of their dopaminergic phenotype. The majority of these neurons remained in the ventral mesencephalon until at least 18 months of age. Chromatin immunoprecipitation suggested that the loss of the mDA phenotype is due to a reduction in the binding of the nuclear orphan receptor, Nurr-1 to the promoter region of TH. These results extend previous findings and demonstrate a later role for Foxa genes in regulating the maintenance of dopaminergic phenotype in mDA neurons.

Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA.

Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs, but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA) based on global nuclear run-on sequencing (GRO-seq) analysis, with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer-promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.

Loss of ERα and FOXA1 expression in a progression model of luminal type breast cancer: insights from PyMT transgenic mouse model.

The classification of breast cancer into multiple molecular subtypes has necessitated the need for biomarkers that can assess tumor progression and the effects of chemopreventive agents on specific breast cancer subtypes. The goal of this study was to identify biomarkers whose expression are altered along with estrogen receptor α (ERα) in the polyoma middle-T antigen (PyMT) transgenic model of breast cancer and to investigate the chemopreventive activity of phenethyl isothiocyanate (PEITC). The diet of PyMT female mice was fortified with PEITC (8 mmol/kg) and the mammary streak and/or gross tumors and metastases in lungs were subjected to immunohistochemical analyses for ERα, FOXA1, and GATA-3. FOXA1 is associated with luminal type A cancers, while GATA-3 is a marker of luminal progenitor cell differentiation. In both control and PEITC-treated groups, there was a progressive loss of ERα and FOXA1 but persistence of GATA-3 expression indicating that the tumors retain luminal phenotype. Overall, the PyMT induced tumors exhibited the entire gamut of phenotypes from ERα+/FOXA1+/GATA-3+ tumors in the early stage to ERα±/FOXA1-/GATA-3+ in the late stage. Thus, PyMT model serves as an excellent model for studying progression of luminal subtype tumors. PEITC treated animals had multiple small tumors, indicating delay in tumor progression. Although these tumors were histologically similar to those in controls, there was a lower expression of these biomarkers in normal luminal cells indicating delay in tumor initiation. In in vitro studies, PEITC depleted AldeFluor-positive putative stem/progenitor cells, which may partly be responsible for the delay in tumor initiation.

Foxa1 and Foxa2 control the differentiation of goblet and enteroendocrine L- and D-cells in mice.

BACKGROUND & AIMS: The winged helix transcription factors Foxa1 and Foxa2 are expressed in all epithelia of the gastrointestinal tract from its embryonic origin into adulthood. In vitro studies have shown that Foxa1/a2 can transactivate the promoters of Mucin 2 (Muc2), which is expressed in goblet cells, and of preproglucagon, which is expressed in enteroendocrine cells. These findings suggest Foxa1/a2 as critical factors in the differentiation of gut epithelial cells. METHODS: Mice with intestine-specific simultaneous deletion of Foxa1 and Foxa2 were derived using the Cre-loxP system and analyzed using histologic and molecular means. RESULTS: Both Foxa1 and Foxa2 were deleted successfully in the epithelia of the small intestine and colon using Villin-Cre mice. Immunohistochemical staining showed that Foxa1/a2 mutants lack glucagon-like peptide-1- and peptide-2-expressing cells (L-cells), and have reduced numbers of somatostatin (D-cells) and peptide YY-expressing cells (L-cells). Preproglucagon, somatostatin, and peptide YY messenger RNA (mRNA) levels also were reduced significantly in Foxa1/a2 mutants. Thus, Foxa1 and Foxa2 are essential regulators of these enteroendocrine lineages in vivo. The mRNA levels of transcription factors Islet-1 and Pax6 were reduced significantly in the small intestine, showing that Foxa1 and Foxa2 impact on a transcription factor network in the enteroendocrine lineage. In addition, deletion of Foxa1/a2 caused a reduction in goblet cell number with altered expression of the secretory mucins Muc2, Mucin5b, Mucin5ac, and Mucin 6. CONCLUSIONS: The winged helix factors Foxa1 and Foxa2 are essential members of the transcription factor network that govern secretory cell differentiation in the mammalian gastrointestinal tract.

Foxa1 and Foxa2 regulate bile duct development in mice.

The forkhead box proteins A1 and A2 (Foxa1 and Foxa2) are transcription factors with critical roles in establishing the developmental competence of the foregut endoderm and in initiating liver specification. Using conditional gene ablation during a later phase of liver development, we show here that deletion of both Foxa1 and Foxa2 (Foxa1/2) in the embryonic liver caused hyperplasia of the biliary tree. Abnormal bile duct formation in Foxa1/2-deficient liver was due, at least in part, to activation of IL-6 expression, a proliferative signal for cholangiocytes. The glucocorticoid receptor is a negative regulator of IL-6 transcription; in the absence of Foxa1/2, the glucocorticoid receptor failed to bind to the IL-6 promoter, causing enhanced IL-6 expression. Thus, after liver specification, Foxa1/2 are required for normal bile duct development through prevention of excess cholangiocyte proliferation. Our data suggest that Foxa1/2 function as terminators of bile duct expansion in the adult liver through inhibition of IL-6 expression.

Foxa1-deficient mice exhibit impaired insulin secretion due to uncoupled oxidative phosphorylation.

Foxa1 (formerly hepatic nuclear factor 3alpha) belongs to the family of Foxa genes that are expressed in early development and takes part in the differentiation of endoderm-derived organs and the regulation of glucose homeostasis. Foxa1-/- pups are growth retarded and hypoglycemic but glucose intolerant in response to an intraperitoneal glucose challenge. However, the mechanism of glucose intolerance in this model has not been investigated. Here, we show that Foxa1-/- islets exhibit decreased glucose-stimulated insulin release in islet perifusion experiments and have significantly reduced pancreatic insulin and glucagon content. Moreover, Foxa1-/- beta-cells exhibit attenuated calcium influx in response to glucose and glyburide, suggesting an insulin secretion defect either at the level or upstream of the ATP-sensitive K+ channel. Intracellular ATP levels after incubation with 10 mmol/l glucose were about 2.5 times lower in Foxa1-/- islets compared with controls. This diminished ATP synthesis could be explained by increased expression of the mitochondrial uncoupling protein uncoupling protein 2 (UCP2) in Foxa1-deficient islets, resulting in partially uncoupled mitochondria. Chromatin immunoprecipitation assays indicate that UCP2 is a direct transcriptional target of Foxa1 in vivo. Thus, we have identified a novel function for Foxa1 in the regulation of oxidative phosphorylation in pancreatic beta-cells.

Stage-specific regulation of respiratory epithelial cell differentiation by Foxa1.

Foxa1 is a member of the winged helix family of transcription factors that is expressed in epithelial cells of the conducting airways and in alveolar type II cells of the lung. To determine the role of Foxa1 during lung morphogenesis, histology and gene expression were assessed in lungs from Foxa1-/- gene-targeted mice from embryonic day (E) 16.5 to postnatal day (PN) 13. Deletion of Foxa1 perturbed maturation of the respiratory epithelium at precise times during lung morphogenesis. While dilatation of peripheral lung saccules was delayed in Foxa1-/- mice at E16.5, sacculation was unperturbed later in development (E17.5-E18.5). At PN5, alveolarization was markedly delayed in Foxa1-/- mice; however, by PN13 lung histology was comparable to wild-type controls. Clara cell secretory protein (CCSP), prosurfactant protein (SP)-C, and SP-B protein content and immunostaining were decreased in Foxa1-/- mice between E16.5 and E18.5 but normalized after birth. Timing and sites of expression of thyroid transcription factor-1, Foxj1, and beta-tubulin were unaltered in lungs of Foxa1-/- mice. In vitro, Foxa1 regulated the activity of CCSP and SP-A, SP-B, SP-C, and SP-D promoters as assessed by luciferase reporter assays in HeLa, H441, and MLE15 cells. Although Foxa1 regulates respiratory epithelial differentiation and structural maturation of the lung at precise developmental periods, the delay in maturation is subsequently compensated at times to enable respiratory function and restore normal lung structure after birth.