Winged Helix Transcription Factor Research Articles

FOXP1 inhibits cell growth and attenuates tumorigenicity of neuroblastoma.

BackgroundSegmental genomic copy number alterations, such as loss of 11q or 3p and gain of 17q, are well established markers of poor outcome in neuroblastoma, and have been suggested to comprise tumor suppressor genes or oncogenes, respectively. The gene forkhead box P1 (FOXP1) maps to chromosome 3p14.1, a tumor suppressor locus deleted in many human cancers including neuroblastoma. FoxP1 belongs to a family of winged-helix transcription factors that are involved in processes of cellular proliferation, differentiation and neoplastic transformation.MethodsMicroarray expression profiles of 476 neuroblastoma specimens were generated and genes differentially expressed between favorable and unfavorable neuroblastoma were identified. FOXP1 expression was correlated to clinical markers and patient outcome. To determine whether hypermethylation is involved in silencing of FOXP1, methylation analysis of the 5′ region of FOXP1 in 47 neuroblastomas was performed. Furthermore, FOXP1 was re-expressed in three neuroblastoma cell lines to study the effect of FOXP1 on growth characteristics of neuroblastoma cells.ResultsLow expression of FOXP1 is associated with markers of unfavorable prognosis like stage 4, age >18 months and MYCN amplification and unfavorable gene expression-based classification (P < 0.001 each). Moreover, FOXP1 expression predicts patient outcome accurately and independently from well-established prognostic markers. Array-based CGH analysis of 159 neuroblastomas revealed that heterozygous loss of the FOXP1 locus was a rare event (n = 4), but if present, was associated with low FOXP1 expression. By contrast, DNA methylation analysis in 47 neuroblastomas indicated that hypermethylation is not regularly involved in FOXP1 gene silencing. Re-expression of FoxP1 significantly impaired cell proliferation, viability and colony formation in soft agar. Furthermore, induction of FOXP1 expression led to cell cycle arrest and apoptotic cell death of neuroblastoma cells.ConclusionsOur results suggest that down-regulation of FOXP1 expression is a common event in high-risk neuroblastoma pathogenesis and may contribute to tumor progression and unfavorable patient outcome.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-840) contains supplementary material, which is available to authorized users.

Changes in Nucleosome Occupancy Associated with Metabolic Alterations in Aged Mammalian Liver

Aging is accompanied by physiological impairments, which, in insulin-responsive tissues, including the liver, predispose individuals to metabolic disease. However, the molecular mechanisms underlying these changes remain largely unknown. Here, we analyze genome-wide profiles of RNA and chromatin organization in the liver of young (3 months) and old (21 months) mice. Transcriptional changes suggest that derepression of the nuclear receptors PPARα, PPARγ, and LXRα in aged mouse liver leads to activation of targets regulating lipid synthesis and storage, whereas age-dependent changes in nucleosome occupancy are associated with binding sites for both known regulators (forkhead factors and nuclear receptors) and candidates associated with nuclear lamina (Hdac3 and Srf) implicated to govern metabolic function of aging liver. Winged-helix transcription factor Foxa2 and nuclear receptor corepressor Hdac3 exhibit a reciprocal binding pattern at PPARα targets contributing to gene expression changes that lead to steatosis in aged liver.

Systematic discovery of novel ciliary genes through functional genomics in the zebrafish

Cilia are microtubule-based hair-like organelles that play many important roles in development and physiology, and are implicated in a rapidly expanding spectrum of human diseases, collectively termed ciliopathies. Primary ciliary dyskinesia (PCD), one of the most prevalent of ciliopathies, arises from abnormalities in the differentiation or motility of the motile cilia. Despite their biomedical importance, a methodical functional screen for ciliary genes has not been carried out in any vertebrate at the organismal level. We sought to systematically discover novel motile cilia genes by identifying the genes induced by Foxj1, a winged-helix transcription factor that has an evolutionarily conserved role as the master regulator of motile cilia biogenesis. Unexpectedly, we find that the majority of the Foxj1-induced genes have not been associated with cilia before. To characterize these novel putative ciliary genes, we subjected 50 randomly selected candidates to a systematic functional phenotypic screen in zebrafish embryos. Remarkably, we find that over 60% are required for ciliary differentiation or function, whereas 30% of the proteins encoded by these genes localize to motile cilia. We also show that these genes regulate the proper differentiation and beating of motile cilia. This collection of Foxj1-induced genes will be invaluable for furthering our understanding of ciliary biology, and in the identification of new mutations underlying ciliary disorders in humans.

Systematic discovery of novel ciliary genes through functional genomics in the zebrafish.

Cilia are microtubule-based hair-like organelles that play many important roles in development and physiology, and are implicated in a rapidly expanding spectrum of human diseases, collectively termed ciliopathies. Primary ciliary dyskinesia (PCD), one of the most prevalent of ciliopathies, arises from abnormalities in the differentiation or motility of the motile cilia. Despite their biomedical importance, a methodical functional screen for ciliary genes has not been carried out in any vertebrate at the organismal level. We sought to systematically discover novel motile cilia genes by identifying the genes induced by Foxj1, a winged-helix transcription factor that has an evolutionarily conserved role as the master regulator of motile cilia biogenesis. Unexpectedly, we find that the majority of the Foxj1-induced genes have not been associated with cilia before. To characterize these novel putative ciliary genes, we subjected 50 randomly selected candidates to a systematic functional phenotypic screen in zebrafish embryos. Remarkably, we find that over 60% are required for ciliary differentiation or function, whereas 30% of the proteins encoded by these genes localize to motile cilia. We also show that these genes regulate the proper differentiation and beating of motile cilia. This collection of Foxj1-induced genes will be invaluable for furthering our understanding of ciliary biology, and in the identification of new mutations underlying ciliary disorders in humans.

Nitroso-redox balance and mitochondrial homeostasis are regulated by STOX1, a pre-eclampsia-associated gene.

Storkhead box 1 (STOX1) is a winged-helix transcription factor that is implicated in the genetic forms of a high-prevalence human gestational disease, pre-eclampsia. STOX1 overexpression confers pre-eclampsia-like transcriptomic features to trophoblastic cell lines and pre-eclampsia symptoms to pregnant mice. The aim of this work was to evaluate the impact of STOX1 on free radical equilibrium and mitochondrial function, both in vitro and in vivo. Transcriptome analysis of STOX1-transgenic versus nontransgenic placentas at 16.5 days of gestation revealed alterations of mitochondria-related pathways. Placentas overexpressing STOX1 displayed altered mitochondrial mass and were severely biased toward protein nitration, indicating nitroso-redox imbalance in vivo. Trophoblast cells overexpressing STOX1 displayed an increased mitochondrial activity at 20% O2 and in hypoxia, despite reduction of the mitochondrial mass in the former. STOX1 overexpression is, therefore, associated with hyperactive mitochondria, resulting in increased free radical production. Moreover, nitric oxide (NO) production pathways were activated, resulting in peroxynitrite formation. At low oxygen pressure, STOX1 overexpression switched the free radical balance from reactive oxygen species (ROS) to reactive nitrogen species (RNS) in the placenta as well as in a trophoblast cell line. In pre-eclamptic placentas, NO interacts with ROS and generates peroxynitrite and nitrated proteins as end products. This process will deprive the maternal organism of NO, a crucial vasodilator molecule. Our data posit STOX1 as a genetic switch in the ROS/RNS balance and suggest an explanation for elevated blood pressure in pre-eclampsia.

Activation of MAPK and FoxO by manganese (Mn) in rat neonatal primary astrocyte cultures.

Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinson's disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl2 led to increased levels of FoxO (dephosphorylated and phosphorylated) compared with control cells (P<0.01). p-FoxO disappeared from the cytosol upon Mn exposure. Pre-treatment of cultured cells with (R)-(−)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events compensating for Mn-induced neurotoxicity and may serve as valuable targets for neuroprotection in the setting of Mn-induced neurodegeneration.

FoxD3 regulates cranial neural crest EMT via downregulation of tetraspanin18 independent of its functions during neural crest formation

The scaffolding protein tetraspanin18 (Tspan18) maintains epithelial cadherin-6B (Cad6B) to antagonize chick cranial neural crest epithelial-to-mesenchymal transition (EMT). For migration to take place, Tspan18 must be downregulated. Here, we characterize the role of the winged-helix transcription factor FoxD3 in the control of Tspan18 expression. Although we previously found that Tspan18 mRNA persists several hours past the stage it would normally be downregulated in FoxD3-deficient neural folds, we now show that Tspan18 expression eventually declines. This indicates that while FoxD3 is crucial for initial downregulation of Tspan18, other factors subsequently impact Tspan18 expression. Remarkably, the classical EMT transcription factor Snail2 is not one of these factors. As in other vertebrates, FoxD3 is required for chick cranial neural crest specification and migration, however, FoxD3 has surprisingly little impact on chick cranial neural crest cell survival. Strikingly, Tspan18 knockdown rescues FoxD3-dependent neural crest migration defects, although neural crest specification is still deficient. This indicates that FoxD3 promotes cranial neural crest EMT by eliciting Tspan18 downregulation separable from its Tspan18-independent activity during neural crest specification and survival.

Increased CD4+CD25+ regulatory T cells correlate with poor short-term outcomes in hepatitis B virus-related acute-on-chronic liver failure patients

The roles of CD4(+)CD25(+) regulatory T cells (Treg) in chronicity of hepatitis B virus (HBV) infection have been confirmed. We aimed to explore alteration of Treg in patients with HBV-related acute-on-chronic liver failure (ACLF). Thirty-two HBV-related ACLF patients, 44 chronic hepatitis B patients, and 41 healthy controls were recruited. We detected frequencies of peripheral Treg and intrahepatic forkhead winged helix transcription factor (Foxp3)(+) cells. Inhibitory activity of Treg was assessed by functional suppression assays. Serum interferon-γ and interleukin-10 were also determined. Peripheral Treg and intrahepatic Foxp3(+) cells were more markedly increased in ACLF than chronic hepatitis B and controls (all p<0.001), and the Foxp3(+) cells located predominantly in the portal areas. The Treg frequency was positively correlated with HBV DNA load, international normalized ratio, model of end stage liver disease score, and serum interleukin-10 level in ACLF patients. Functional assays invitro demonstrated that ACLF patients exhibited higher suppressive effects of Treg on proliferations of autologous CD4(+)CD25(-) T cells than controls. On logistic regression, prolonged international normalized ratio and higher peripheral Treg frequency predicted 30-day survival of ACLF. The patients with HBV-related ACLF exhibit increased amounts of Treg, of which redistribution from periphery to liver seems to modulate liver inflammation. Higher Treg amounts are associated with more severe liver disease in ACLF, and its level in combination with international normalized ratio may assist prediction of short-term outcomes of HBV-related ACLF.

Studying forkhead box protein A1–DNA interaction and ligand inhibition using gold nanoparticles, electrophoretic mobility shift assay, and fluorescence anisotropy

Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. Conventional analytical methods—electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA)—as well as a gold nanoparticles (AuNPs)-based assay were used to study DNA binding properties of FoxA1 and ligand interruption of FoxA1–DNA binding. In the AuNPs assay, the distinct ability of protein–DNA complex to protect AuNPs against salt-induced aggregation was exploited to screen sequence selectivity and determine the binding affinity constant based on AuNPs color change and absorbance spectrum shift. Both conventional EMSA and FA and the AuNPs assay suggested that FoxA1 binds to DNA in a core sequence-dependent manner and the flanking sequence also played a role to influence the affinity. The EMSA and AuNPs were found to be more sensitive than FA in differentiation of sequence-dependent affinity. With the addition of a spin filtration step, AuNPs assay has been extended for studying small molecular ligand inhibition of FoxA1–DNA interactions enabling drug screening. The results correlate very well with those obtained using FA.

Unraveling the Role of FOXQ1 in Colorectal Cancer Metastasis

Malignant cell transformation, invasion, and metastasis are dependent on the coordinated rewiring of gene expression. A major component in the scaffold of these reprogramming events is one in which epithelial cells lose intercellular connections and polarity to adopt a more motile mesenchymal phenotype, which is largely supported by a robust transcriptional machinery consisting mostly of developmental transcription factors. This study demonstrates that the winged helix transcription factor, FOXQ1, contributes to this rewiring process, in part by directly modulating the transcription of TWIST1, itself a key mediator of metastasis that transcriptionally regulates the expression of important molecules involved in epithelial-to-mesenchymal transition. Forced expression and RNA-mediated silencing of FOXQ1 led to enhanced and suppressed mRNA and protein levels of TWIST1, respectively. Mechanistically, FOXQ1 enhanced the reporter activity of TWIST1 and directly interacted with its promoter. Furthermore, enhanced expression of FOXQ1 resulted in increased migration and invasion in colorectal cancer cell lines, whereas knockdown studies showed the opposite effect. Moreover, using the in vivo chicken chorioallantoic membrane metastasis assay model, FOXQ1 significantly enhanced distant metastasis with minimal effects on tumor growth. These findings reveal FOXQ1 as a modulator of TWIST1-mediated metastatic phenotypes and support its potential as a biomarker of metastasis.

The Winged Helix Transcription Factor Foxa3 Regulates Adipocyte Differentiation and Depot-Selective Fat Tissue Expansion

Conversion of mesenchymal stem cells into terminally differentiated adipocytes progresses sequentially through regulated transcriptional steps. While it is clear that the late phases of adipocyte maturation are governed by the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), less is known about the transcriptional control of the initial stages of differentiation. To identify early regulators, we performed a small interfering RNA (siRNA) screen of Forkhead-box genes in adipocytes and show here for the first time that the winged helix factor Foxa3 promotes adipocyte differentiation by cooperating with C/EBPβ and -δ to transcriptionally induce PPARγ expression. Furthermore, we demonstrate that mice with genetic ablation of Foxa3 have a selective decrease in epididymal fat depot and a cell-autonomous defect to induce PPARγ specifically in their visceral adipocytes. In obese subjects, FOXA3 is differentially expressed in visceral and subcutaneous adipose depots. Overall, our study implicates Foxa3 in the regulation of adipocyte differentiation and depot-selective adipose tissue expansion.

Abstract A34: Nucleosome dynamics of cell differentiation

Abstract Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions. Currently, we determine whether chromatin remodeling complexes are involved in nucleosome occupation. Citation Format: Zhaoyu Li. Nucleosome dynamics of cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A34.

Abstract 2703: A direct regulation of Twist1 by FOXQ1 promotes colorectal cancer metastasis.

Abstract Abstract Malignant cell transformation, invasion and metastasis are dependent on the coordinated rewiring of gene expression. A major component in the scaffold of these reprogramming events is one in which epithelial cells lose intercellular connections and polarity and adopt a motile mesenchymal phenotype, one that is largely supported by a robust transcriptional machinery consisting mostly of developmental transcription factors. We investigated the winged helix transcription factor, FOXQ1, identified from an oligonucleotide microarray expression analysis, as being concomitantly deregulated in both epithelial and stromal compartments. We found that FOXQ1 contributes to epithelial to mesenchymal transition (EMT), in part by directly affecting the transcription of Twist1, itself a key mediator of metastasis that transcriptionally regulates the expression of important molecules involved in EMT. Reporter gene assays and chromatin immunoprecipitation experiments showed that FOXQ1 regulated and directly interacted with the Twist1 promoter, leading to a suppression of E-cadherin and increased expression of mesenchymal markers. Forced expression and RNAi mediated silencing of FOXQ1 led to increased and decreased levels of Twist1 mRNA and protein levels, respectively, with concomitant effects on migration, invasion and in vivo metastasis of colorectal cancer cells. Moreover, FOXQ1 mRNA and protein expression correlated in a series of resected patient samples. Conclusively our results show that FOXQ1 is a novel mediator of colorectal cancer metastasis and joins the network of transcription factors that orchestrate EMT. Citation Format: Mohammed Abba, Nitin Patil, Kabeer Rasheed, Laura D. Nelson, Giridhar Mudduluru, Jörg H. Leupold, Heike Allgayer. A direct regulation of Twist1 by FOXQ1 promotes colorectal cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2013-2703

Tetraspanin18 is a FoxD3-responsive antagonist of cranial neural crest epithelial-to-mesenchymal transition that maintains cadherin-6B protein.

During epithelial-to-mesenchymal transition (EMT), tightly associated, polarized epithelial cells become individual mesenchymal cells capable of migrating. Here, we investigate the role of the transmembrane protein tetraspanin18 (Tspan18) in chick cranial neural crest EMT. Tspan18 mRNA is expressed in premigratory cranial neural crest cells, but is absent from actively migrating neural crest cells. Tspan18 knockdown leads to a concomitant loss of cadherin-6B (Cad6B) protein, whereas Cad6B protein persists when Tspan18 expression is extended. The temporal profile of Cad6B mRNA downregulation is unaffected in these embryos, which indicates that Tspan18 maintains Cad6B protein levels and reveals that Cad6B is regulated by post-translational mechanisms. Although downregulation of Tspan18 is necessary, it is not sufficient for neural crest migration: the timing of neural crest emigration, basal lamina breakdown and Cad7 upregulation proceed normally in Tspan18-deficient cells. This emphasizes the need for coordinated transcriptional and post-translational regulation of Cad6B during EMT and illustrates that Tspan18-antagonized remodeling of cell-cell adhesions is only one step in preparation for cranial neural crest migration. Unlike Cad6B, which is transcriptionally repressed by Snail2, Tspan18 expression is downstream of the winged-helix transcription factor FoxD3, providing a new transcriptional input into cranial neural crest EMT. Together, our data reveal post-translational regulation of Cad6B protein levels by Tspan18 that must be relieved by a FoxD3-dependent mechanism in order for cranial neural crest cells to migrate. These results offer new insight into the molecular mechanisms of cranial neural crest EMT and expand our understanding of tetraspanin function relevant to metastasis.

Foxa2 and H2A.Z Mediate Nucleosome Depletion during Embryonic Stem Cell Differentiation

Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiationisdependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions.

Evolutionarily Ancient Association of the FoxJ1 Transcription Factor with the Motile Ciliogenic Program

It is generally believed that the last eukaryotic common ancestor (LECA) was a unicellular organism with motile cilia. In the vertebrates, the winged-helix transcription factor FoxJ1 functions as the master regulator of motile cilia biogenesis. Despite the antiquity of cilia, their highly conserved structure, and their mechanism of motility, the evolution of the transcriptional program controlling ciliogenesis has remained incompletely understood. In particular, it is presently not known how the generation of motile cilia is programmed outside of the vertebrates, and whether and to what extent the FoxJ1-dependent regulation is conserved. We have performed a survey of numerous eukaryotic genomes and discovered that genes homologous to foxJ1 are restricted only to organisms belonging to the unikont lineage. Using a mis-expression assay, we then obtained evidence of a conserved ability of FoxJ1 proteins from a number of diverse phyletic groups to activate the expression of a host of motile ciliary genes in zebrafish embryos. Conversely, we found that inactivation of a foxJ1 gene in Schmidtea mediterranea, a platyhelminth (flatworm) that utilizes motile cilia for locomotion, led to a profound disruption in the differentiation of motile cilia. Together, all of these findings provide the first evolutionary perspective into the transcriptional control of motile ciliogenesis and allow us to propose a conserved FoxJ1-regulated mechanism for motile cilia biogenesis back to the origin of the metazoans.

Identification and characterization of the vertebrate motile ciliome

Our previous work with the zebrafish embryo had uncovered a master regulatory role for the winged-helix transcription factor FoxJ1 in motile ciliogenesis. In humans, dysfunction of motile cilia leads to Primary Ciliary Dyskinesia (PCD). Mutations in only a few genes have thus far been associated with PCD. To identify new genes that are required for motile cilia formation and function, we performed a genome-wide screen to identify the targets of FoxJ1. Approximately 650 genes with mammalian orthologs were found to be up-regulated in response to FoxJ1 over-exopression in zebrafish embryos. Remarkably, all of the genes that have yet been linked with PCD figure on our list of FoxJ1 targets, implying that many of the new genes that we have identified could be mutated in the uncharacterized cases of PCD. To determine how the novel FoxJ1 targets affect cilia, we have carried out systematic gene knock-down using morpholinos, and assayed for perturbations in ciliary architecture and the developmental and physiological read-outs of defective ciliary activity. In addition, using GFP-tagged versions of the proteins, we have performed localization studies with respect to the ciliary apparatus, and employed the yeast 2 hybrid assay to identify interacting partners. Finally, with chromatin immunoprecipitation (ChIP) we have surveyed for FoxJ1 binding sites throughout the zebrafish genome. Our findings indicate that the screen has successfully identified a very large number of novel genes required for ciliary development and function. We believe that these novel ciliary genes will help us to better understand the causes of human ciliopathies. http://www.imcb.a-star.edu.sg/php/sudipto.php

RPA Assists HSF1 Access to Nucleosomal DNA by Recruiting Histone Chaperone FACT

Transcription factor access to regulatory elements is prevented by the nucleosome. Heat shock factor 1 (HSF1) is a winged helix transcription factor that plays roles in control and stressed conditions by gaining access to target elements, but mechanisms of HSF1 access are not well known in mammalian cells. Here, we show the physical interaction between the wing motif of human HSF1 and replication protein A (RPA), which is involved in DNA metabolism. Depletion of RPA1 abolishes HSF1 access to the promoter of HSP70 in unstressed condition and delays its rapid activation in response to heat shock. The HSF1-RPA complex leads to preloading of RNA polymerase II and opens the chromatin structure by recruiting a histone chaperone, FACT. Furthermore, this interaction is required for melanoma cell proliferation. These results provide a mechanism of constitutive HSF1 access to nucleosomal DNA, which is important for both basal and inducible gene expression.

Why men are at higher risk for hepatocellular carcinoma?

Despite recent breakthroughs in the management of hepatocellular carcinoma (HCC), it is still the fastest-rising cause of cancer-related death in the United States [1]. Unfortunately, most patients are diagnosed at intermediate-advanced stages, when curative therapies are not a primary option. HCC natural history is fairly well known, since most tumors arise from a traceable underlying liver disease (i.e., cirrhosis), allowing for early diagnosis in the setting of surveillance programs. In patients with cirrhosis, there is a striking gender imbalance in HCC incidence, with a solid predominance for males regardless of geographic area, etiologic factor, and ethnicity. Male to female ratio averages between 2:1 and >4:1 across studies [2]. These discrepancies are not merely explained by gender-specific differences in exposure to cirrhosis risk factors (e.g., viral hepatitis or alcohol abuse), since experimental models of chemically-induced HCC also demonstrate a clear male predominance, although molecular mechanisms behind gender bias remain unknown. There is also preliminary evidence suggesting that specific genomic profiles, once HCC has developed, are predominant according to patient’s gender, reflected by an enrichment in certain molecular subclasses [3,4]. In a recent study by Li et al. [5], the authors report a central role for the winged helix transcription factors Foxa1/Foxa2 in controlling estrogen and androgen signaling via the recruitment of estrogen receptor alpha (ERα) and androgen receptor (AR) to hepatocyte target genes. They found that the effects of sex hormones in the liver are Foxa1/Foxa2-dependent and provide a novel molecular mechanism responsible for gender dimorphism in HCC. Sexually dimorphic HCC was completely reversed in Foxa1- and Foxa2-deficient mice after chemically-induced (diethylnitrosamine) HCC. The authors also included human samples in their analysis, and despite the fact that no mutations in any of the FOXA genes have been reported so far in HCC, the authors provide evidence that certain single nucleotide polymorphisms at FOXA2 binding sites reduce its affinity with ERα, and this correlates with increased incidence of HCC development in women. A similar gender bias occurrence was observed in a recent forward genetic screen using the Sleeping Beauty transposon insertional mutagenesis system [6]. Overall, the study by Li et al. [5] reveals a novel interplay between Foxa and hormone receptors binding sites that justifies gender disparities in HCC. Molecular data indicate that androgens contribute to the HCC development by acting as tumor promoters via induction of DNA damage and oxidative stress [7]. Conversely, estrogens have been suggested to act as general suppressors of HCC through reduction in the proinflammatory effects of MyD88-mediated secretion of IL6 [8]. In addition, estrogens seem to exert different tumorigenic properties depending on the tissue type, since contrary to the epidemiological data in cirrhotic women, chronic exposure to estrogens favors carcinogenesis in breast and uterus. Paradoxically, both androgens (i.e., anabolic steroids) and estrogens (e.g., oral contraceptives) are able to induce benign liver tumors (i.e., adenomas) and ultimately HCC in individuals without underlying liver disease (Fig. 1). Overall, the effects of estrogens in terms of cancer development are largely tissue and environment-dependent, protective in experimental models of cirrhosis but inducing oncogenesis in the normal liver, and promoting tumor growth in full-blown HCC. Strikingly, the study by Li et al. [5] found that tumor load in female Foxa-deficient mice far exceeded that of control males, suggesting that estrogens could have a pro-tumorigenic effect in the absence of Foxa1/Foxa2. Whether Foxa is the link that explains these opposing effects of estrogens requires further investigation. Background inflammation in cirrhosis could play an additional confounding role, emphasized by the crosstalk between IL6-STAT signaling and estrogens in experimental models of HCC [8]. Fig. 1 Summary of the role of sex hormones in HCC From a therapeutic standpoint, several studies have tried to evaluate the role of anti-estrogen therapy (e.g., tamoxifen) in patients with overt HCC. Evidence of expression of ER in some tumors and experimental data of estrogen-dependent HCC growth provided the scientific rational for this approach. Most of the studies were small trials, conducted in the 1990s and including a heterogeneous patient population. A meta-analysis including close to 700 patients revealed no impact of tamoxifen in 1-year survival in patients with HCC [9]. Data on anti-androgens or a possible role of hormone therapy in the chemo-preventive scenario are even less informative. In summary, we are at the beginning of unraveling the complex molecular mechanisms behind gender disparities in HCC. Among them, estrogens seem to play a role in IL6 production and modulation of gene expression through FOXA transcription factors, what prevents HCC development in experimental models of HCC. However, more data are still needed to define the implication of sexual hormones in the molecular pathogenesis of HCC. This could provide the proof-of-principle to rescue hormone-based drugs for further evaluation as systemic therapies of HCC in certain clinical contexts.

Foxa2 mediates critical functions of prechordal plate in patterning and morphogenesis and is cell autonomously required for early ventral endoderm morphogenesis

Summary Axial mesendoderm is comprised of prechordal plate and notochord. Lack of a suitable Cre driver has hampered the ability to genetically dissect the requirement for each of these components, or genes expressed within them, to anterior patterning. Here, we have utilized Isl1-Cre to investigate roles of the winged helix transcription factor Foxa2 specifically in prechordal plate and ventral endoderm. Foxa2loxP/loxP; Isl1-Cre mutants died at 13.5 dpc, exhibiting aberrations in anterior neural tube and forebrain patterning, and in ventral foregut morphogenesis and cardiac fusion. Molecular analysis of Foxa2loxP/loxP; Isl1-Cre mutants indicated that Foxa2 is required in Isl1 lineages for expression of notochord and dorsal foregut endoderm markers, Shh. Brachyury, and Hlxb9. Our results support a requirement for Foxa2 in prechordal plate for notochord morphogenesis, axial patterning, and patterning of dorsal foregut endoderm. Loss of Foxa2 in ventral endoderm resulted in reduced expression of Sox17, Gata4, and ZO proteins, accounting at least in part for observed lack of foregut fusion, cardia bifida, and increased apoptosis of ventral endoderm.