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Epicardium-derived cells (EPDCs) contribute cardiovascular cell types during development and in adulthood respond to Thymosin β4 (Tβ4) and myocardial infarction (MI) by reactivating a fetal gene programme to promote neovascularization and cardiomyogenesis. The mechanism for epicardial gene (re-)activation remains elusive. Here we reveal that BRG1, the essential ATPase subunit of the SWI/SNF chromatin-remodelling complex, is required for expression of Wilms' tumour 1 (Wt1), fetal EPDC activation and subsequent differentiation into coronary smooth muscle, and restores Wt1 activity upon MI. BRG1 physically interacts with Tβ4 and is recruited by CCAAT/enhancer-binding protein β (C/EBPβ) to discrete regulatory elements in the Wt1 locus. BRG1-Tβ4 co-operative binding promotes optimal transcription of Wt1 as the master regulator of embryonic EPDCs. Moreover, chromatin immunoprecipitation-sequencing reveals BRG1 binding at further key loci suggesting SWI/SNF activity across the fetal epicardial gene programme. These findings reveal essential functions for chromatin-remodelling in the activation of EPDCs during cardiovascular development and repair.

The transcriptional coregulators PGC-1α and PGC-1β modulate the expression of numerous partially overlapping genes involved in mitochondrial biogenesis and energetic metabolism. The physiological role of PGC-1β is poorly understood in skeletal muscle, a tissue of high mitochondrial content to produce ATP levels required for sustained contractions. Here we determine the physiological role of PGC-1β in skeletal muscle using mice, in which PGC-1β is selectively ablated in skeletal myofibres at adulthood (PGC-1β((i)skm-/-) mice). We show that myofibre myosin heavy chain composition and mitochondrial number, muscle strength and glucose homeostasis are unaffected in PGC-1β((i)skm-/-) mice. However, decreased expression of genes controlling mitochondrial protein import, translational machinery and energy metabolism in PGC-1β((i)skm-/-) muscles leads to mitochondrial structural and functional abnormalities, impaired muscle oxidative capacity and reduced exercise performance. Moreover, enhanced free-radical leak and reduced expression of the mitochondrial anti-oxidant enzyme Sod2 increase muscle oxidative stress. PGC-1β is therefore instrumental for skeletal muscles to cope with high energetic demands.

Tumourigenesis within the intestine is potently driven by deregulation of the Wnt pathway, a process epigenetically regulated by the chromatin remodelling factor Brg1. We aimed to investigate this interdependency in an in vivo setting and assess the viability of Brg1 as a potential therapeutic target. Using a range of transgenic approaches, we deleted Brg1 in the context of Wnt-activated murine small intestinal epithelium. Pan-epithelial loss of Brg1 using VillinCreERT2 and AhCreERT transgenes attenuated expression of Wnt target genes, including a subset of stem cell-specific genes and suppressed Wnt-driven tumourigenesis improving animal survival. A similar increase in survival was observed when Wnt activation and Brg1 loss were restricted to the Lgr5 expressing intestinal stem cell population. We propose a mechanism whereby Brg1 function is required for aberrant Wnt signalling and ultimately for the maintenance of the tumour initiating cell compartment, such that loss of Brg1 in an Apc-deficient context suppresses adenoma formation. Our results highlight potential therapeutic value of targeting Brg1 and serve as a proof of concept that targeting the cells of origin of cancer may be of therapeutic relevance.

Numerous transcriptional regulators of neurogenesis have been identified in the developing and adult brain, but how neurogenic fate is programmed at the epigenetic level remains poorly defined. Here, we report that the transcription factor Pax6 directly interacts with the Brg1-containing BAF complex in adult neural progenitors. Deletion of either Brg1 or Pax6 in the subependymal zone (SEZ) causes the progeny of adult neural stem cells to convert to the ependymal lineage within the SEZ while migrating neuroblasts convert to different glial lineages en route to or in the olfactory bulb (OB). Genome-wide analyses reveal that the majority of genes downregulated in the Brg1 null SEZ and OB contain Pax6 binding sites and are also downregulated in Pax6 null SEZ and OB. Downstream of the Pax6-BAF complex, we find that Sox11, Nfib, and Pou3f4 form a transcriptional cross-regulatory network that drives neurogenesis and can convert postnatal glia into neurons. Taken together, elements of our work identify a tripartite effector network activated by Pax6-BAF that programs neuronal fate., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Schwann cells produce myelin sheaths and thereby permit rapid saltatory conductance in the vertebrate peripheral nervous system. Their stepwise differentiation from neural crest cells is driven by a defined set of transcription factors. How this is linked to chromatin changes is not well understood. Here we show that the glial transcription factor Sox10 functions in Schwann cells by recruiting Brg1-containing chromatin-remodeling complexes via Baf60a to regulatory regions of Oct6 and Krox20 genes. It thereby stimulates expression of these transcriptional regulators that then cooperate with Sox10 to convert immature into myelinating Schwann cells. The functional interaction between Sox10 and Brg1 is evident from gain- and loss-of-function studies, similar neuropathies in the corresponding mouse mutants, and an aggravated neuropathy in compound mutants. Our results demonstrate that the transcription factor-mediated recruitment of the chromatin-remodeling machinery to specific genomic loci is an essential driving force for Schwann cell differentiation and myelination., (Copyright © 2012 Elsevier Inc. All rights reserved.)

SWI/SNF ATP-dependent chromatin-remodeling complexes containing either Brahma-related gene 1 (Brg1) or Brahma (Brm) play important roles in mammalian development. In this study we examined the roles of Brg1 and Brm in smooth muscle development, in vivo, through generation and analysis of mice harboring a smooth muscle-specific knockout of Brg1 on wild-type and Brm null backgrounds. Knockout of Brg1 from smooth muscle in Brg1(flox/flox) mice expressing Cre recombinase under the control of the smooth muscle myosin heavy-chain promoter resulted in cardiopulmonary defects, including patent ductus arteriosus, in 30 to 40% of the mice. Surviving knockout mice exhibited decreased expression of smooth muscle-specific contractile proteins in the gastrointestinal tract, impaired contractility, shortened intestines, disorganized smooth muscle cells, and an increase in apoptosis of intestinal smooth muscle cells. Although Brm knockout mice had normal intestinal structure and function, knockout of Brg1 on a Brm null background exacerbated the effects of knockout of Brg1 alone, resulting in an increase in neonatal lethality. These data show that Brg1 and Brm play critical roles in regulating development of smooth muscle and that Brg1 has specific functions within vascular and gastrointestinal smooth muscle that cannot be performed by Brm.

Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2-5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.

Alterations in chromatin play an important role in oncogenic transformation, although the underlying mechanisms are often poorly understood. The SWI/SNF complex contributes to epigenetic regulation by using the energy of ATP hydrolysis to remodel chromatin and thus regulate transcription of target genes. SNF5, a core subunit of the SWI/SNF complex, is a potent tumor suppressor that is specifically inactivated in several types of human cancer. However, the mechanism by which SNF5 mutation leads to cancer and the role of SNF5 within the SWI/SNF complex remain largely unknown. It has been hypothesized that oncogenesis in the absence of SNF5 occurs due to a loss of function of the SWI/SNF complex. Here, we show, however, distinct effects for inactivation of Snf5 and the ATPase subunit Brg1 in primary cells. Further, using both human cell lines and mouse models, we show that cancer formation in the absence of SNF5 does not result from SWI/SNF inactivation but rather that oncogenesis is dependent on continued presence of BRG1. Collectively, our results show that cancer formation in the absence of SNF5 is dependent on the activity of the residual BRG1-containing SWI/SNF complex. These findings suggest that, much like the concept of oncogene addiction, targeted inhibition of SWI/SNF ATPase activity may be an effective therapeutic approach for aggressive SNF5-deficient human tumors.

Purpose: Hepatoblastoma represents the most frequent malignant liver tumor in childhood. The phosphatidylinositol-3'-kinase (PI3K)/AKT pathway is crucial in downstream signaling of multiple receptor tyrosine kinases of pathogenic importance in hepatoblastoma. Increased PI3K/AKT signaling pathway activity and activating mutations of PIK3CA, encoding a PI3K catalytic subunit, have been reported in different childhood tumors. The current study was done to analyze the role of PI3K/AKT signaling in hepatoblastoma., Experimental Design: Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT protein, its targets p-(Ser9)-GSK-3beta and p-(Ser2448)-mTOR, as well as the cell cycle regulators Cyclin D1, p27(KIP1), and p21(CIP1) were done and the PIK3CA gene was screened for mutations. In vitro, two hepatoblastoma cell lines treated with the PI3K inhibitor LY294002 were analyzed for AKT and GSK-3beta phosphorylation, cell proliferation, and apoptosis. Additionally, simultaneous treatments of hepatoblastoma with LY294002 and cytotoxic drugs were carried out., Results: Most tumors strongly expressed p-AKT, p-GSK-3beta, and p-mTOR; subgroups showed significant Cyclin D1, p27(KIP1), and p21(CIP1) expression. One hepatoblastoma carried an E545A mutation in the PIK3CA gene. In vitro, PI3K inhibition diminished hepatoblastoma cell growth being accompanied by reduced AKT and GSK-3beta phosphorylation. Flow cytometry and 4', 6-diamidino-2-phenylindole stainings showed that PI3K pathway inhibition leads to a substantial increase in apoptosis and a decrease in cellular proliferation linked to reduced Cyclin D1 and increased p27(KIP1) levels. Simultaneous treatment of hepatoblastoma cell lines with LY294002 and cytotoxic drugs resulted in positive interactions., Conclusions: Our findings imply that PI3K signaling plays an essential role in growth control of hepatoblastoma and might be successfully targeted in multimodal therapeutic strategies.

Switch (SWI)/sucrose nonfermentable (SNF) is an evolutionarily conserved complex with ATPase function, capable of regulating nucleosome position to alter transcriptional programs within the cell. It is known that the SWI/SNF complex is responsible for regulation of many genes involved in cell cycle control and proliferation, and it has recently been implicated in cancer development. The ATPase action of SWI/SNF is conferred through either the brahma-related gene 1 (Brg1) or brahma (Brm) subunit of the complex, and it is of central importance to the modification of nucleosome position. In this study, the role of the Brg1 and Brm subunits were examined as they relate to chromatin structure and organization. Deletion of the Brg1 ATPase results in dissolution of pericentromeric heterochromatin domains and a redistribution of histone modifications associated with these structures. This effect was highly specific to Brg1 and is not reproduced by the loss of Brm or SNF5/BAF47/INI1. Brg1 deficiency is associated with the appearance of micronuclei and aberrant mitoses that are a by-product of dissociated chromatin structure. Thus, Brg1 plays a critical role in maintaining chromatin structural integrity.

Brahma-related gene 1 (BRG1) is a catalytic subunit of the switch in mating type/sucrose nonfermentation complex and plays an important role in cancer development. Mouse homozygous knockout experiments testing the role of BRG1 in tumorigenesis have been hampered because BRG1 inactivation is embryonic lethal. To bypass this constraint, we developed a lung-specific conditional knockout of BRG1 and examined the effect of BRG1 inactivation in an ethyl carbamate lung carcinogenesis mouse model. We found that the heterozygous loss of BRG1 resulted in increases in both the number and size of tumors when compared with controls. In contrast, when both BRG1 alleles were inactivated, neither the number nor the size of tumors increased compared with controls. In mouse lung tissue where BRG1 was homozygously inactivated, immunostaining for apoptotic markers showed significant increase in Apo-BrdUrd and cleaved caspase-3. These data indicate that a loss of cell viability underlies why biallelic inactivation of BRG1 does not increase tumorigenesis. We also examined mice when exposed to the carcinogen ethyl carbamate and then subjected to BRG1 inactivation. In these cells, loss of BRG1 after carcinogen exposure potentiated tumor development. A subset of tumors retained BRG1 expression, whereas others showed either partial or complete loss of BRG1 expression. Tumors completely devoid of BRG1 expression were significantly larger and expressed higher levels of two markers of proliferation, proliferating cell nuclear antigen and Ki67. Although biallelic inactivation of BRG1 could not initiate tumor development in untransformed cells, our results indicate that transformation and tumor progression are greatly affected by loss of BRG1.

Hepatocellular carcinoma (HCC) is a major cause of death worldwide. Here, we provide evidence that the ligand-dependent nuclear receptor co-regulator Trim24 (also known as Tif1alpha) functions in mice as a liver-specific tumor suppressor. In Trim24-null mice, hepatocytes fail to execute proper cell cycle withdrawal during the neonatal-to-adult transition and continue to cycle in adult livers, becoming prone to a continuum of cellular alterations that progress toward metastatic HCC. Using pharmacological approaches, we show that inhibition of retinoic acid signaling markedly reduces hepatocyte proliferation in Trim24-/- mice. We further show that deletion of a single retinoic acid receptor alpha (Rara) allele in a Trim24-null background suppresses HCC development and restores wild-type expression of retinoic acid-responsive genes in the liver, thus demonstrating that in this genetic background Rara expresses an oncogenic activity correlating with a dysregulation of the retinoic acid signaling pathway. Our results not only provide genetic evidence that Trim24 and Rara co-regulate hepatocarcinogenesis in an antagonistic manner but also suggest that aberrant activation of Rara is deleterious to liver homeostasis.

Liver receptor homolog-1 (LRH-1) is a nuclear receptor involved in intestinal lipid homeostasis and cell proliferation. Here we show that haploinsufficiency of LRH-1 predisposes mice to the development of intestinal inflammation. Besides the increased inflammatory response, LRH-1 heterozygous mice exposed to 2,4,6-trinitrobenzene sulfonic acid show lower local corticosterone production as a result of an impaired intestinal expression of the enzymes CYP11A1 and CYP11B1, which control the local synthesis of corticosterone in the intestine. Local glucocorticoid production is strictly enterocyte-dependent because it is robustly reduced in epithelium-specific LRH-1-deficient mice. Consistent with these findings, colon biopsies of patients with Crohn's disease and ulcerative colitis show reduced expression of LRH-1 and genes involved in the production of glucocorticoids. Hence, LRH-1 regulates intestinal immunity in response to immunological stress by triggering local glucocorticoid production. These findings underscore the importance of LRH-1 in the control of intestinal inflammation and the pathogenesis of inflammatory bowel disease.

Mice in which peroxisome proliferator-activated receptor beta (PPARbeta) is selectively ablated in skeletal muscle myocytes were generated to elucidate the role played by PPARbeta signaling in these myocytes. These somatic mutant mice exhibited a muscle fiber-type switching toward lower oxidative capacity that preceded the development of obesity and diabetes, thus demonstrating that PPARbeta is instrumental in myocytes to the maintenance of oxidative fibers and that fiber-type switching is likely to be the cause and not the consequence of these metabolic disorders. We also show that PPARbeta stimulates in myocytes the expression of PGC1alpha, a coactivator of various transcription factors, known to play an important role in slow muscle fiber formation. Moreover, as the PGC1alpha promoter contains a PPAR response element, the effect of PPARbeta on the formation and/or maintenance of slow muscle fibers can be ascribed, at least in part, to a stimulation of PGC1alpha expression at the transcriptional level.

Previously we identified a transcription factor, LPS-Induced TNF-alpha Factor (LITAF), mediating inflammatory cytokine expression in LPS-induced processes. To characterize the role of LITAF in vivo, we generated a macrophage-specific LITAF-deficient mouse (macLITAF(-/-)). Our data demonstrate that in macrophages (i) several cytokines (such as TNF-alpha, IL-6, sTNF-RII, and CXCL16) are induced at lower levels in macLITAF(-/-) compared with LITAF(+/+) control macrophages; (ii) macLITAF(-/-) mice are more resistant to LPS-induced lethality. To further identify LITAF signaling pathways, we tested mouse TLR-2(-/-), -4(-/-), and -9(-/-) and WT peritoneal macrophages exposed to LPS. Using these cells, we now show that LITAF expression can be induced after challenge either with LPS from Porphyromonas gingivalis via agonism at TLR-2, or with LPS from Escherichia coli via agonism at TLR-4, both requiring functional MyD88. We also show that, in response to LPS, the MyD88-dependent LITAF pathway differs from the NF-kappaB pathway. Furthermore, using a kinase array, p38alpha was found to mediate LITAF phosphorylation and the inhibition of p38alpha with a p38-specific inhibitor (SB203580) blocked LITAF nuclear translocation and reduced LPS-induced TNF-alpha protein levels. Finally, macLITAF(-/-) macrophages rescued by LITAF cDNA transfection restored levels of TNF-alpha similar to those observed in WT cells. We conclude that LITAF is an important mediator of the LPS-induced inflammatory response that can be distinguished from NF-kappaB pathway and that p38alpha is the specific kinase involved in the pathway linking LPS/MyD88/LITAF to TNF.

Epigenetic alterations in cell-type-specific gene expression control the transition of neural stem cells (NSCs) from predominantly neurogenic to predominantly gliogenic phases of differentiation, but how this switch occurs is unclear. Here, we show that brahma-related gene 1 (Brg1), an ATP-dependent chromatin remodeling factor, is required for the repression of neuronal commitment and the maintenance of NSCs in a state that permits them to respond to gliogenic signals. Loss of Brg1 in NSCs in conditional brg1 mutant mice results in precocious neuronal differentiation, such that cells in the ventricular zone differentiate into post-mitotic neurons before the onset of gliogenesis. As a result, there is a dramatic failure of astrocyte and oligodendrocyte differentiation in these animals. The ablation of brg1 in gliogenic progenitors in vitro also prevents growth-factor-induced astrocyte differentiation. Furthermore, proteins implicated in the maintenance of stem cells, including Sox1, Pax6 and Musashi-1, are dramatically reduced in the ventricular zones of brg1 mutant mice. We conclude that Brg1 is required to repress neuronal differentiation in NSCs as a means of permitting glial cell differentiation in response to gliogenic signals, suggesting that Brg1 regulates the switch from neurogenesis to gliogenesis.

Animal SWI2/SNF2 protein complexes containing either the brahma (BRM) or brahma-related gene 1 (BRG1) ATPase are involved in nucleosome remodelling and may control the accessibility of sequence-specific transcription factors to DNA. In vitro studies have indicated that BRM and BRG1 could regulate the expression of distinct sets of genes. However, as mice lacking BRM are viable and fertile, BRG1 might efficiently compensate for BRM loss. By contrast, as Brg1-null fibroblasts are viable but Brg1-null embryos die during the peri-implantation stage, BRG1 might exert cell-specific functions. To further investigate the in vivo role of BRG1, we selectively ablated Brg1 in keratinocytes of the forming mouse epidermis. We show that BRG1 is selectively required for epithelial-mesenchymal interactions in limb patterning, and during keratinocyte terminal differentiation, in which BRM can partially substitute for BRG1. By contrast, neither BRM nor BRG1 are essential for the proliferation and early differentiation of keratinocytes, which may require other ATP-dependent nucleosome-remodelling complexes. Finally, we demonstrate that cell-specific targeted somatic mutations can be created at various times during the development of mouse embryos cell-specifically expressing the tamoxifen-activatable Cre-ER(T2) recombinase.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

The peroxisome proliferator-activated receptor gamma (PPARgamma) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARgamma functions in mature adipocytes is precluded by lethality of PPARgamma(-/-) fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARgamma in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER(T2) recombination system. We show that mature PPARgamma-null white and brown adipocytes die within a few days and are replaced by newly formed PPARgamma-positive adipocytes, demonstrating that PPARgamma is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARgamma antagonists could be used to acutely reduce obesity.

Vitamin A (retinol) actions in eye development are mediated by retinoic acid receptors (RARs and RXRs). Using the Cre/loxP system, we have selectively ablated RXR alpha in the retinal pigment epithelium (RPE), a cell monolayer critically involved in visual retinoid renewal and phagocytosis of photoreceptor outer segments. In the mutant (RXR alpha (rpe-/-)) mice, RPE cells are morphologically and functionally abnormal and display decreased expression of proteins involved in the visual retinoid cycle, namely RPE65, CRALBP, and RGR. RXR alpha (rpe-/-) mice also show alterations of photoreceptor cells including: 1) decrease in their number; 2) outer segment shortening and disorganization, and 3) reduced light responses in electroretinograms. These results indicate that RXR alpha is required for normal maturation of the RPE, which is known to play essential roles in photoreceptor cell function and survival, and point to a possible involvement of RXR alpha signaling pathways in the RPE in human retinal diseases.

T cells develop through distinct stages directed by a series of signals. We explored the roles of SWI/SNF-like BAF chromatin remodeling complexes in this process by progressive deletion of the ATPase subunit, Brg, through successive stages of early T cell development. Brg-deficient cells were blocked at each of the developmental transitions examined. Bcl-xL overexpression suppressed cell death without relieving the developmental blockades, leading to the accumulation of Brg-deleted cells that were unexpectedly cell cycle arrested. These defects resulted partly from the disruptions of pre-TCR and potentially Wnt signaling pathways controlling the expression of genes such as c-Kit and c-Myc critical for continued development. Our studies indicate that BAF complexes dynamically remodel chromatin to propel sequential developmental transitions in response to external signals.

To investigate the roles of retinoic acid (RA) receptors (RARs) in the physiology of epidermis that does not express RAR beta, conditional spatio-temporally controlled somatic mutagenesis was used to selectively ablate RAR alpha in keratinocytes of RAR gamma-null mice. Keratinocyte proliferation was maintained in adult mouse epidermis lacking both RAR alpha and RAR gamma, as well as in RAR beta-null mice. All RAR-mediated signalling pathways are therefore dispensable in epidermis for homeostatic keratinocyte renewal. However, topical treatment of mouse skin with selective retinoids indicated that RXR/RAR gamma heterodimers, in which RXR transcriptional activity was subordinated to that of its RAR gamma partner, were required for retinoid-induced epidermal hyperplasia, whereas RXR homodimers and RXR/RAR alpha heterodimers were not involved. RA-induced keratinocyte proliferation was studied in mutant mice in which RXR alpha, RXR alpha and RAR alpha, RAR gamma, or RXR alpha and RAR gamma genes were specifically disrupted in either basal or suprabasal keratinocytes. We demonstrate that the topical retinoid signal is transduced by RXR alpha/RAR gamma heterodimers in suprabasal keratinocytes, which, in turn, stimulate proliferation of basal keratinocytes via a paracrine signal that may be heparin-binding EGF-like growth factor.

The gene for transcriptional intermediary factor 1beta (TIF1beta) encodes a transcriptional co-repressor known to play essential roles in chromatin remodeling as well as in early embryonic development. During spermatogenesis, TIF1beta is preferentially associated with heterochromatin structures of Sertoli cells and round spermatids, as well as with meiotic chromosomes. Its expression is tightly regulated within spermatocyte and spermatid populations, and it is undetectable in spermatogonia. Spatiotemporally controlled ablation of TIF1beta by using a germ cell lineage-specific CreER(T)/loxP system leads to testicular degeneration. This degeneration is not due to impairment of chromatin remodeling processes during meiosis and spermiogenesis, as TIF1beta-deficient spermatocytes are able to complete their differentiation into spermatozoa. It rather occurs as a consequence of shedding of immature germ cells (spermatocytes and spermatids), and disappearance of stem spermatogonia. These results indicate that TIF1beta has important functions in the homeostasis of the seminiferous epithelium, and probably plays a crucial role in the network of paracrine interactions between germ cell subpopulations and/or Sertoli cells.

X-linked adrenal hypoplasia congenita (AHC) is caused by mutations in the NR0B1 gene. This gene encodes an orphan member of the nuclear receptor superfamily, DAX1. Ongoing efforts in our laboratory have identified nine novel NR0B1 mutations in X-linked AHC patients (Y81X, 343delG, 457delT, 629delG, L295P, 926-927delTG, 1130delA, 1141-1155del15, and E428X). Two additional families segregate previously identified NR0B1 mutations (501delA and R425T). Sequence analysis of the mitochondrial D-loop indicates that the 501delA family is unrelated through matrilineal descent to our previously analyzed 501delA family., (Copyright 2001 Wiley-Liss, Inc.)

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. We show that keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation.

Retinoid X receptors (RXRs) are involved in a number of signaling pathways as heterodimeric partners of numerous nuclear receptors. Hepatocytes express high levels of the RXRalpha isotype, as well as several of its putative heterodimeric partners. Germ-line disruption (knockout) of RXRalpha has been shown to be lethal in utero, thus precluding analysis of its function at later life stages. Hepatocyte-specific disruption of RXRalpha during liver organogenesis has recently revealed that the presence of hepatocytes is not mandatory for the mouse, at least under normal mouse facility conditions, even though a number of metabolic events are impaired [Wan, Y.-J., et al. (2000) Mol. Cell. Biol. 20, 4436-4444]. However, it is unknown whether RXRalpha plays a role in the control of hepatocyte proliferation and lifespan. Here, we report a detailed analysis of the liver of mice in which RXRalpha was selectively ablated in adult hepatocytes by using the tamoxifen-inducible chimeric Cre recombinase system. Our results show that the lifespan of adult hepatocytes lacking RXRalpha is shorter than that of their wild-type counterparts, whereas proliferative hepatocytes of regenerating liver exhibit an even shorter lifespan. These lifespan shortenings are accompanied by increased polyploidy and multinuclearity. We conclude that RXRalpha plays important cell-autonomous function(s) in the mechanism(s) involved in the lifespan of hepatocytes and liver regeneration.

RXR-alpha is the most abundant of the three retinoid X receptors (RXRs) in the epidermis. In this study, we have used Cre-mediated recombination to selectively disrupt the mouse gene for RXR-alpha in epidermal and hair follicle keratinocytes. We show that RXR-alpha is apparently dispensable for prenatal epidermal development, while it is involved in postnatal skin maturation. After the first hair pelage, mutant mice develop a progressive alopecia, histologically characterised by the destruction of hair follicle architecture and the formation of utriculi and dermal cysts in adult mice. Our results demonstrate that RXR-alpha plays a key role in anagen initiation during the hair follicle cycle. In addition, RXR-alpha ablation results in epidermal interfollicular hyperplasia with keratinocyte hyperproliferation and aberrant terminal differentiation, accompanied by an inflammatory reaction of the skin. Our data not only provide genetic evidence that RXR-alpha/VDR heterodimers play a major role in controlling hair cycling, but also suggest that additional signalling pathways mediated by RXR-alpha heterodimerised with other nuclear receptors are involved in postnatal hair follicle growth, and homeostasis of proliferation/differentiation of epidermal keratinocytes and of the skin's immune system.

Retinoid X receptor alpha (RXRalpha) is involved in multiple signaling pathways, as a heterodimeric partner of several nuclear receptors. To investigate its function in energy homeostasis, we have selectively ablated the RXRalpha gene in adipocytes of 4-week-old transgenic mice by using the tamoxifen-inducible Cre-ERT2 recombination system. Mice lacking RXRalpha in adipocytes were resistant to dietary and chemically induced obesity and impaired in fasting-induced lipolysis. Our results also indicate that RXRalpha is involved in adipocyte differentiation. Thus, our data demonstrate the feasibility of adipocyte-selective temporally controlled gene engineering and reveal a central role of RXRalpha in adipogenesis, probably as a heterodimeric partner for peroxisome proliferator-activated receptor gamma.

Mammalian SWI/SNF complexes utilize either brahma (Brm) or brahma-related gene 1 (Brg1) catalytic subunits to remodel nucleosomes in an ATP-dependent manner. Brm was previously shown to be dispensable, suggesting that Brm and Brg1 are functionally redundant. To test this hypothesis, we have generated a Brg1 null mutation by gene targeting, and, surprisingly, homozygotes die during the periimplantation stage. Furthermore, blastocyst outgrowth studies indicate that neither the inner cell mass nor trophectoderm survives. However, experiments with other cell types demonstrate that Brg1 is not a general cell survival factor. In addition, Brg1 heterozygotes are predisposed to exencephaly and tumors. These results provide evidence that biochemically similar chromatin-remodeling complexes have dramatically different functions during mammalian development.

Nuclear receptors for retinoids (RARs) and vitamin D (VDR), and for some other ligands (TRs, PPARs and LXRs), maybe critical in the development and homeostasis of mammalian epidermis. It is believed that these receptors form heterodimers with retinoid X receptors (RXRs) to act as transcriptional regulators. However, most genetic approaches aimed at establishing their physiological functions in the skin have been inconclusive owing either to pleiotropic effects and redundancies between receptor isotypes in gene knockouts, or to equivocal interpretation of dominant-negative mutant studies in transgenic mice. Moreover, knockout of RXRalpha, the main skin RXR isotype, is lethal in utero before skin formation. Here we have resolved these problems by developing an efficient technique to create spatiotemporally controlled somatic mutations in the mouse. We used tamoxifen-inducible Cre-ER(T) recombinases to ablate RXRalpha selectively in adult mouse keratinocytes. We show that RXRalpha has key roles in hair cycling, probably through RXR/VDR heterodimers, and in epidermal keratinocyte proliferation and differentiation.

Fasting is associated with significant changes in nutrient metabolism, many of which are governed by transcription factors that regulate the expression of rate-limiting enzymes. One factor that plays an important role in the metabolic response to fasting is the peroxisome proliferator-activated receptor alpha (PPARalpha). To gain more insight into the role of PPARalpha during fasting, and into the regulation of metabolism during fasting in general, a search for unknown PPARalpha target genes was performed. Using subtractive hybridization (SABRE) comparing liver mRNA from wild-type and PPARalpha null mice, we isolated a novel PPARalpha target gene, encoding the secreted protein FIAF (for fasting induced adipose factor), that belongs to the family of fibrinogen/angiopoietin-like proteins. FIAF is predominantly expressed in adipose tissue and is strongly up-regulated by fasting in white adipose tissue and liver. Moreover, FIAF mRNA is decreased in white adipose tissue of PPARgamma +/- mice. FIAF protein can be detected in various tissues and in blood plasma, suggesting that FIAF has an endocrine function. Its plasma abundance is increased by fasting and decreased by chronic high fat feeding. The data suggest that FIAF represents a novel endocrine signal involved in the regulation of metabolism, especially under fasting conditions.

The two alleles of the 30 kDa TATA-binding protein associated factor (TAF(II)30) gene, have been targeted by homologous recombination in murine F9 embryonal carcinoma cells and subsequently disrupted using a Cre recombinase-loxP strategy. The TAF(II)30-null cells are not viable, but are rescued by the expression of human TAF(II)30. Cells lacking TAF(II)30 are blocked in G(1)/G(0) phase of the cell cycle and undergo apoptosis. In agreement with the G(1) arrest phenotype, the expression of cyclin E is impaired and the retinoblastoma protein is hypophosphorylated in the TAF(II)30-null cells. Interestingly, retinoic acid (RA) treatment prevented TAF(II)30-null cell death and induced primitive endodermal differentiation. In contrast, the RA- and cAMP-induced parietal endodermal differentiation was impaired in the TAF(II)30-null cells. Thus, TAF(II)30 is not indispensable for class II gene transcription in general, but seems to be required for the expression of a subset of genes.

We have cloned a mouse cDNA homolog of P450RAI, a cytochrome P450 belonging to a new family (CYP26), which has previously been isolated from zebrafish and human cDNAs and found to encode a retinoic acid-inducible retinoic acid hydroxylase activity. The cross-species conservation of the amino acid sequence is high, particularly between the mouse and the human enzymes, in which it is over 90%. Like its human and zibrafish counterparts, the mouse P450RAI cDNA catalyzes metabolism of retinoic acid into 4-OH-retinoic acid, 4-oxo-retinoic acid, 18-OH-retinoic acid, and unidentified water-soluble metabolites when transfected into COS-1 cells. Retinoic acid-inducible retinoic acid metabolism has previously been observed in F9 murine embryonal carcinoma cells and some derivatives lacking retinoid receptors. We were interested in determining whether P450RAI could be responsible for retinoic acid metabolism in F9 cells and in studying the effect of retinoid receptor ablation on P450RAI expression. In wild-type F9 cells and derivatives lacking RAR gamma, RAR alpha, and/or RXR alpha, we observed a direct relationship between the level of retinoic acid metabolic activity and retinoic acid-induced P450RAI mRNA. These experiments, as well as others using synthetic receptor subtype-specific retinoids, suggest that the RAR gamma and RXR alpha receptors mediate the effects of retinoic acid on the expression of the P450RAI gene.

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)alpha and retinoic acid receptor (RAR)alpha genes or the RXRalpha and RARgamma genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRalpha-/-, RARalpha-/-, and RARgamma-/- cells. RXRalpha-/-/ RARalpha-/- cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRalpha-/- cells. In contrast, RXRalpha-/-/RARgamma-/- cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRalpha and RARgamma represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype- and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.

The yeast and animal SNF-SWI and related multiprotein complexes are thought to play an important role in processes, such as transcription factor binding to regulatory elements, which require nucleosome remodeling in order to relieve the repressing effect of packaging DNA in chromatin. There are two mammalian homologs of the yeast SNF2-SWI2 subunit protein, SNF2alpha-brm and SNF2beta-BRG1, and overexpression of either one of them has been shown to enhance transcriptional activation by glucocorticoid, estrogen, and retinoic acid (RA) receptors in transiently transfected cells. We have investigated here the function of SNF2beta-BRG1 in the RA receptor-retinoid X receptor-mediated transduction of the retinoid signal in F9 embryonal carcinoma (EC) cells which differentiate into endodermal-like cells upon RA treatment. The two SNF2beta-BRG1 alleles have been targeted by homologous recombination and subsequently disrupted by using a conditional Cre recombinase. We show that F9 EC cells inactivated on both SNF2beta alleles are not viable and that heterozygous mutant cells are affected in proliferation but not in RA-induced differentiation. Thus, in F9 EC cells, SNF2beta-BRG1 appears to play an essential role in basal processes involved in cell proliferation, in addition to its putative role in the activation of transcription mediated by nuclear receptors.

The F9 murine embryonal carcinoma cell line represents a well-established system for the study of retinoid signaling in vivo. We have investigated the functional specificity of different retinoid X receptor (RXR)-retinoic acid (RA) receptor (RAR) isotype pairs for the control of expression of endogenous RA-responsive genes, by using wild-type (WT), RXR alpha(-/-), RAR alpha(-/-), RAR gamma(-/-), RXR alpha(-/-)-RAR alpha(-/-), and RXR alpha(-/-)-RAR gamma(-/-) F9 cells, as well as panRXR and RAR isotype (alpha, beta, and gamma)-selective retinoids. We show that in these cells the control of expression of different sets of RA-responsive genes is preferentially mediated by distinct RXR-RAR isotype combinations. Our data support the conclusion that RXR-RAR heterodimers are the functional units transducing the retinoid signal and indicate in addition that these heterodimers exert both specific and redundant functions on the expression of particular sets of RA-responsive genes. We also show that the presence of a given receptor isotype can hinder the activity of another isotype and therefore that functional redundancy between retinoid receptor isotypes can be artifactually generated by gene knockouts.

The F9 murine embryonal carcinoma (EC) cell line, a well established model system for the study of retinoic acid (RA)-induced differentiation, differentiates into cells resembling three types of extra-embryonic endoderm (primitive, parietal and visceral), depending on the culture conditions and RA concentration used. A number of previously identified genes are differentially expressed during this process and serve as markers for the different endodermal cell types. Differentiation is also accompanied by a decreased rate of proliferation and an apoptotic response. Using homologous recombination, we have disrupted both alleles of the retinoid X receptor (RXR) alpha gene in F9 cells to investigate its role in mediating these responses. The loss of RXRalpha expression impaired the morphological differentiation of F9 EC cells into primitive and parietal endoderm, but has little effect on visceral endodermal differentiation. Concomitantly the inducibility of most primitive and parietal endoderm differentiation-specific genes was impaired, while several genes upregulated during visceral endodermal differentiation were induced normally. We also demonstrate that RXRalpha is required for both the anti-proliferative and apoptotic responses in RA-treated F9 cells. Additionally, we provide further evidence that retinoic acid receptor (RAR)-RXR heterodimers are the functional units transducing the effects of retinoids in F9 cells.

We have previously demonstrated that the human oestrogen receptor (hER) contains two transcriptional activation functions located in the N-terminal region (TAF-1) and in the hormone binding domain (TAF-2), which can act both independently and synergistically in a promoter- and cell-specific manner in animal cells. We have also demonstrated that hER can activate transcription from chimaeric oestrogen-responsive GAL1 promoters in yeast, and shown that transcriptional activation was due to TAF-1, whereas TAF-2 showed little, if any, transcriptional activity on these promoters. By using a more complex promoter derived from the URA3 gene, we now show that TAF-2 is also active in yeast, and that the activities of TAF-1 and TAF-2 are promoter-context-specific in yeast. We also confirm that the agonistic activity of 4-hydroxytamoxifen (OHT) can be ascribed to the activity of TAF-1.

Using a transient co-transfection system we have demonstrated that response elements for estrogen (ER), thyroid hormone (TR) and retinoic acid receptors (RAR) are closely related. Thyroid hormone-induced activation of transcription was observed in CV1 cells and not in HeLa cells, suggesting the existence of cell-specific transcription factors necessary for the response. By contrast to its cellular counterpart (c-erbA/cTR alpha) the oncogene protein gag v-erbA is unable to activate gene transcription from different response elements derived from the rat growth hormone (rGH) gene promoter. A chimeric construct consisting of the ER in which the DNA binding domain has been replaced by that of cTR alpha was able to stimulate the reporter gene. In contrast, a construct in which ER DNA binding domain has been replaced by that of gag v-erbA did not activate gene transcription. These results lead us to the conclusion that the mutated DNA binding domain of v-erbA is in part responsible for the lack of transcriptional activation and in repression of gene expression. This is due in large part to the Gly73----Ser mutation which corresponds to the position of one of the three discriminating amino acids that are thought to interact with a specific base of the response element.