Regulatory Factor X Research Articles

Pan-cancer investigation of RFX family and associated genes identifies RFX8 as a therapeutic target in leukemia

BackgroundSeveral transcription factors and co-factors are encoded by the RFX (Regulatory Factor X) family (RFX1-8) and associated genes (RFXAP and RFXANK). Increasing evidence suggests that the RFX family and associated genes are involved in the development and progression of cancer. However, no prior research has focused on a multi-omic analysis of these genes to evaluate their role in tumor progression. MethodsUsing combined TCGA and GTEx pan-cancer data, we investigated the expression patterns and survival profiles of these ten genes. We then focused on RFX8 to analyze its clinicopathological and therapeutic features. Finally, we conducted experimental validation of RFX8 function in acute myeloid leukemia (AML). ResultsRFX5 and RFXANK showed higher expression levels, while RFX6 showed lower expression levels in most types of cancer, with RFX8 being the most upregulated in LAML. RFX2 and RFXAP demonstrated prognostic significance in eight types of cancer, and RFX8 showed significance in six types of cancer. The expression of these ten genes exhibited specific characteristics in immune subtypes, tumor microenvironment, and stemness. The expression of RFX8 was correlated with various tumor stages, microsatellite instability (MSI), tumor mutation burden (TMB), immune cell infiltration, and immune-checkpoint expression. Additionally, RFX8 was found to regulate tumorigenesis and sensitivity to chelerythrine in AML. ConclusionsOur work delineated the landscape of the RFX family and associated genes in the pan-cancer context and the specific role of RFX8 in AML. These findings might offer cues for further investigations of these genes in cancer biology.

RFX transcription factor in the human-associated yeast Candida albicans regulates adhesion to oral epithelium.

Adhesion to mucosal surfaces is a critical step in many bacterial and fungal infections. Here, using a mouse model of oral infection by the human fungal pathobiont Candida albicans, we report the identification of a novel regulator of C. albicans adhesion to the oral mucosa. The regulator is a member of the regulatory factor X (RFX) family of transcription factors, which control cellular processes ranging from genome integrity in model yeasts to tissue differentiation in vertebrates. Mice infected with the C. albicans rfx1 deletion mutant displayed increased fungal burden in tongues compared to animals infected with the reference strain. High-resolution imaging revealed RFX1 transcripts being expressed by C. albicans cells during infection. Concomitant with the increase in fungal burden, the rfx1 mutant elicited an enhanced innate immune response. Transcriptome analyses uncovered HWP1, a gene encoding an adhesion protein that mediates covalent attachment to buccal cells, as a major RFX1-regulated locus. Consistent with this result, we establish that C. albicans adhesion to oral cells is modulated by RFX1 in an HWP1-dependent manner. Our findings expand the repertoire of biological processes controlled by the RFX family and illustrate a mechanism whereby C. albicans can adjust adhesion to the oral epithelium.

The C. elegans regulatory factor X (RFX) DAF-19M module: A shift from general ciliogenesis to cell-specific ciliary and behavioral specialization.

Cilia are important for the interaction with environments and the proper function of tissues. While the basic structure of cilia is well conserved, ciliated cells have various functions. To understand the distinctive identities of ciliated cells, the identification of cell-specific proteins and its regulation is essential. Here, we report the mechanism that confers a specific identity on IL2 neurons in Caenorhabditis elegans, neurons important for the dauer larva-specific nictation behavior. We show that DAF-19M, an isoform of the sole C. elegans RFX transcription factor DAF-19, heads a regulatory subroutine, regulating target genes through an X-box motif variant under the control of terminal selector proteins UNC-86 and CFI-1 in IL2 neurons. Considering the conservation of DAF-19M module in IL2 neurons for nictation and in male-specific neurons for mating behavior, we propose the existence of an evolutionarily adaptable, hard-wired genetic module for distinct behaviors that share the feature "recognizing the environment."

The First Report of a Missense Variant in RFX2 Causing Non-Syndromic Tooth Agenesis in a Consanguineous Pakistani Family.

Background: The syndromic and non-syndromic congenital missing teeth phenotype is termed tooth agenesis. Since tooth agenesis is a heterogeneous disorder hence, the patients show diverse absent teeth phenotypes. Thus identifying novel genes involved in the morphogenesis of ectodermal appendages, including teeth, paves the way for establishing signaling pathways. Methods and Results: We have recruited an autosomal recessive non-syndromic tooth agenesis family with two affected members. The exome sequencing technology identified a novel missense sequence variant c.1421T > C; p.(Ile474Thr) in a regulatory factor X (RFX) family member (RFX2, OMIM: 142,765). During the data analysis eight rare variants on various chromosomal locations were identified, but the co-segregation analysis using Sanger sequencing confirmed the segregation of only two variants RFX2: c.1421T > C; p.(Ile474Thr), DOHH: c.109C > G; p.(Pro37Ala) lying in a common 7.1 MB region of homozygosity on chromosome 19p13.3. Furthermore, the online protein prediction algorithms and protein modeling analysis verified the RFX2 variant as a damaging genetic alteration and ACMG pathogenicity criteria classified it as likely pathogenic. On the other hand, the DOHH variant showed benign outcomes. Conclusion: RFX2 regulates the Hedgehog and fibroblast growth factor signaling pathways, which are involved in the epithelial and mesenchymal interactions during tooth development. Prior animal model studies have confirmed the expression of rfx2 at a developmental stage governing mouth formation. Moreover, its regulatory role and close association with ciliary and non-ciliary genes causing various dental malformations makes it a potential candidate gene for tooth agenesis phenotype. Further studies will contribute to exploring the direct role of RFX2 in human tooth development.

The Joubert syndrome protein CEP41 is excluded from the distal segment of cilia in C. elegans.

Rare diseases are a fundamental issue in today’s world, affecting more than 300 million individuals worldwide. According to data from Orphanet and OMIM, about 50-60 new conditions are added to the list of over 6,000 clinically distinct diseases each year, rendering disease diagnosis and treatment even more challenging. Ciliopathies comprise a heterogeneous category of rare diseases made up of over 35 distinct diseases, including Joubert syndrome (JBTS; OMIM 213300), that are caused by functional and structural defects in cilia. JBTS is an autosomal recessive condition characterized by a range of symptoms, including cerebellar vermis hypoplasia and poor muscle tone. There are now a total of 38 genes that cause JBTS, almost all of which encode protein products that are found in cilia and cilia-associated compartments, such as the basal body and transition zone. CEP41 is a JBTS-associated protein that is found in cilia and the basal body of mammals, but its localization in other ciliary organisms remains elusive. C. elegans is an excellent model organism for studying the molecular mechanisms of rare diseases like JBTS. We, therefore, decided to use C. elegans to identify the localization of CEP41. Our microscopy analysis revealed that CEPH-41(CEntrosomal Protein Homolog 41) not only localizes to cilia but is excluded from the distal segment of the amphid and phasmid cilia in C. elegans. Furthermore, we discovered a putative X-box motif located in the promoter of ceph-41 and the expression of ceph-41 is regulated by DAF-19, a sole Regulatory Factor X (RFX) transcription factor.

NLRC5 promotes transcription of BTN3A1-3 genes and Vγ9Vδ2 Tcell-mediated killing.

SummaryBTN3A molecules—BTN3A1 in particular—emerged as important mediators of Vγ9Vδ2 T cell activation by phosphoantigens. These metabolites can originate from infections, e.g. with Mycobacterium tuberculosis, or by alterations in cellular metabolism. Despite the growing interest in the BTN3A genes and their high expression in immune cells and various cancers, little is known about their transcriptional regulation. Here we show that these genes are induced by NLRC5, a regulator of MHC class I gene transcription, through an atypical regulatory motif found in their promoters. Accordingly, a robust correlation between NLRC5 and BTN3A gene expression was found in healthy, in M. tuberculosis-infected donors' blood cells, and in primary tumors. Moreover, forcing NLRC5 expression promoted Vγ9Vδ2 T-cell-mediated killing of tumor cells in a BTN3A-dependent manner. Altogether, these findings indicate that NLRC5 regulates the expression of BTN3A genes and hence open opportunities to modulate antimicrobial and anticancer immunity.

Neuronal NLRC5 regulates MHC class I expression in Neuro-2a cells and also during hippocampal development.

Major histocompatibility Complex class I (MHC I) molecules are ubiquitously expressed, being found in most nucleated cells, where they are central mediators of both the adaptive and innate immune responses. Recent studies have shown that MHC I are also expressed in the developing brain where they participate in synapse elimination and plasticity. Up-regulation of MHC I within the developing brain has been reported, however, the mechanism(s) regulating this developmental up-regulation of neuronal MHC I remains unknown. Here, we show NLR family CARD domain containing 5 (NLRC5), a newly identified member of the NLR family, is widely expressed in hippocampal neurons, and the expression pattern of NLRC5 coincides with increased MHC I mRNA in the developing hippocampus. Using a luciferase assay in Neuro-2a cells we demonstrate that NLRC5 can induce the activation of MHC I and this induction requires the W/S-X-Y motif. Further studies show that transcription factors regulatory factor X (RFX) and CREB1, which bind to X1 and X2 box, are crucial for NLRC5-mediated induction. Moreover immunoprecipitation experiments reveal that NLRC5 interacts with RFX subunits RFX5 and RFXANK. Knockout of Nlrc5 dramatically impairs basal expression of MHC I in mouse hippocampus. Taken together, our findings identify NLRC5 as a key regulator of MHC I up-regulation in the developing hippocampus and suggest an important role for NLRC5 in neurons. Cover Image for this issue: doi: 10.1111/jnc.14729.

The transcription factor TpRfx1 is an essential regulator of amylase and cellulase gene expression in Talaromyces pinophilus

BackgroundPerfect and low cost of fungal amylolytic and cellulolytic enzymes are prerequisite for the industrialization of plant biomass biorefinergy to biofuels. Genetic engineering of fungal strains based on regulatory network of transcriptional factors (TFs) and their targets is an efficient strategy to achieve the above described aim. Talaromyces pinophilus produces integrative amylolytic and cellulolytic enzymes; however, the regulatory mechanism associated with the expression of amylase and cellulase genes in T. pinophilus remains unclear. In this study, we screened for and identified novel TFs regulating amylase and/or cellulase gene expression in T. pinophilus 1-95 through comparative transcriptomic and genetic analyses.ResultsComparative analysis of the transcriptomes from T. pinophilus 1-95 grown on media in the presence and absence of glucose or soluble starch as the sole carbon source screened 33 candidate TF-encoding genes that regulate amylase gene expression. Thirty of the 33 genes were successfully knocked out in the parental strain T. pinophilus ∆TpKu70, with seven of the deletion mutants firstly displaying significant changes in amylase production as compared with the parental strain. Among these, ∆TpRfx1 (TpRfx1: Talaromyces pinophilus Rfx1) showed the most significant decrease (81.5%) in amylase production, as well as a 57.7% reduction in filter paper cellulase production. Real-time quantitative reverse transcription PCR showed that TpRfx1 dynamically regulated the expression of major amylase and cellulase genes during cell growth, and in vitro electrophoretic mobility shift assay revealed that TpRfx1 bound the promoter regions of genes encoding α-amylase (TP04014/Amy13A), glucoamylase (TP09267/Amy15A), cellobiohydrolase (TP09412/cbh1), β-glucosidase (TP05820/bgl1), and endo-β-1,4-glucanase (TP08514/eg1). TpRfx1 protein containing a regulatory factor X (RFX) DNA-binding domain belongs to RFX family.ConclusionWe identified a novel RFX protein TpRFX1 that directly regulates the expression of amylase and cellulase genes in T. pinophilus, which provides new insights into the regulatory mechanism of fungal amylase and cellulase gene expression.

Genetic regulatory signatures underlying islet gene expression and type 2 diabetes

Genome-wide association studies (GWAS) have identified >100 independent SNPs that modulate the risk of type 2 diabetes (T2D) and related traits. However, the pathogenic mechanisms of most of these SNPs remain elusive. Here, we examined genomic, epigenomic, and transcriptomic profiles in human pancreatic islets to understand the links between genetic variation, chromatin landscape, and gene expression in the context of T2D. We first integrated genome and transcriptome variation across 112 islet samples to produce dense cis-expression quantitative trait loci (cis-eQTL) maps. Additional integration with chromatin-state maps for islets and other diverse tissue types revealed that cis-eQTLs for islet-specific genes are specifically and significantly enriched in islet stretch enhancers. High-resolution chromatin accessibility profiling using assay for transposase-accessible chromatin sequencing (ATAC-seq) in two islet samples enabled us to identify specific transcription factor (TF) footprints embedded in active regulatory elements, which are highly enriched for islet cis-eQTL. Aggregate allelic bias signatures in TF footprints enabled us de novo to reconstruct TF binding affinities genetically, which support the high-quality nature of the TF footprint predictions. Interestingly, we found that T2D GWAS loci were strikingly and specifically enriched in islet Regulatory Factor X (RFX) footprints. Remarkably, within and across independent loci, T2D risk alleles that overlap with RFX footprints uniformly disrupt the RFX motifs at high-information content positions. Together, these results suggest that common regulatory variations have shaped islet TF footprints and the transcriptome and that a confluent RFX regulatory grammar plays a significant role in the genetic component of T2D predisposition.

Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs.

DYX1C1, DCDC2, and KIAA0319 are three of the most replicated dyslexia candidate genes (DCGs). Recently, these DCGs were implicated in functions at the cilium. Here, we investigate the regulation of these DCGs by Regulatory Factor X transcription factors (RFX TFs), a gene family known for transcriptionally regulating ciliary genes. We identify conserved X-box motifs in the promoter regions of DYX1C1, DCDC2, and KIAA0319 and demonstrate their functionality, as well as the ability to recruit RFX TFs using reporter gene and electrophoretic mobility shift assays. Furthermore, we uncover a complex regulation pattern between RFX1, RFX2, and RFX3 and their significant effect on modifying the endogenous expression of DYX1C1 and DCDC2 in a human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1). In addition, induction of ciliogenesis increases the expression of RFX TFs and DCGs. At the protein level, we show that endogenous DYX1C1 localizes to the base of the cilium, whereas DCDC2 localizes along the entire axoneme of the cilium, thereby validating earlier localization studies using overexpression models. Our results corroborate the emerging role of DCGs in ciliary function and characterize functional noncoding elements, X-box promoter motifs, in DCG promoter regions, which thus can be targeted for mutation screening in dyslexia and ciliopathies associated with these genes.—Tammimies, K., Bieder, A., Lauter, G., Sugiaman-Trapman, D., Torchet, R., Hokkanen, M.-E., Burghoorn, J., Castrén, E., Kere, J., Tapia-Páez, I., Swoboda, P. Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor (RF) X transcription factors through X-box promoter motifs.

Transcription Factor RFX2 Is a Key Regulator of Mouse Spermiogenesis.

The regulatory factor X (RFX) family of transcription factors is crucial for ciliogenesis throughout evolution. In mice, Rfx1-4 are highly expressed in the testis where flagellated sperm are produced, but the functions of these factors in spermatogenesis remain unknown. Here, we report the production and characterization of the Rfx2 knockout mice. The male knockout mice were sterile due to the arrest of spermatogenesis at an early round spermatid step. The Rfx2-null round spermatids detached from the seminiferous tubules, forming large multinucleated giant cells that underwent apoptosis. In the mutants, formation of the flagellum was inhibited at its earliest stage. RNA-seq analysis identified a large number of cilia-related genes and testis-specific genes that were regulated by RFX2. Many of these genes were direct targets of RFX2, as revealed by chromatin immunoprecipitation-PCR assays. These findings indicate that RFX2 is a key regulator of the post-meiotic development of mouse spermatogenic cells.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Inhibits Major Histocompatibility Complex Class II Expression by Disrupting Enhanceosome Assembly through Binding with the Regulatory Factor X Complex.

Major histocompatibility complex class II (MHC-II) molecules play a central role in adaptive antiviral immunity by presenting viral peptides to CD4(+) T cells. Due to their key role in adaptive immunity, many viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), have evolved multiple strategies to inhibit the MHC-II antigen presentation pathway. The expression of MHC-II, which is controlled mainly at the level of transcription, is strictly dependent upon the binding of the class II transactivator (CIITA) to the highly conserved promoters of all MHC-II genes. The recruitment of CIITA to MHC-II promoters requires its direct interactions with a preassembled MHC-II enhanceosome consisting of cyclic AMP response element-binding protein (CREB) and nuclear factor Y (NF-Y) complex and regulatory factor X (RFX) complex proteins. Here, we show that KSHV-encoded latency-associated nuclear antigen (LANA) disrupts the association of CIITA with the MHC-II enhanceosome by binding to the components of the RFX complex. Our data show that LANA is capable of binding to all three components of the RFX complex, RFX-associated protein (RFXAP), RFX5, and RFX-associated ankyrin-containing protein (RFXANK), in vivo but binds more strongly with the RFXAP component in in vitro binding assays. Levels of MHC-II proteins were significantly reduced in KSHV-infected as well as LANA-expressing B cells. Additionally, the expression of LANA in a luciferase promoter reporter assay showed reduced HLA-DRA promoter activity in a dose-dependent manner. Chromatin immunoprecipitation assays showed that LANA binds to the MHC-II promoter along with RFX proteins and that the overexpression of LANA disrupts the association of CIITA with the MHC-II promoter. These assays led to the conclusion that the interaction of LANA with RFX proteins interferes with the recruitment of CIITA to MHC-II promoters, resulting in an inhibition of MHC-II gene expression. Thus, the data presented here identify a novel mechanism used by KSHV to downregulate the expressions of MHC-II genes. Kaposi's sarcoma-associated herpesvirus is the causative agent of multiple human malignancies. It establishes a lifelong latent infection and persists in infected cells without being detected by the host's immune surveillance system. Only a limited number of viral proteins are expressed during latency, and these proteins play a significant role in suppressing both the innate and adaptive immunities of the host. Latency-associated nuclear antigen (LANA) is one of the major proteins expressed during latent infection. Here, we show that LANA blocks MHC-II gene expression to subvert the host immune system by disrupting the MHC-II enhanceosome through binding with RFX transcription factors. Therefore, this study identifies a novel mechanism utilized by KSHV LANA to deregulate MHC-II gene expression, which is critical for CD4(+) T cell responses in order to escape host immune surveillance.

Abstract 2616: RFX1-dependent activation of SHP-1 inhibits STAT3 signaling in hepatocellular carcinoma cells

Abstract Obatoclax is under clinical trial currently. Previously, we have demonstrated that SC-2001, an obatoclax analogue, has potent anti-tumor effect in HCC cells through the SHP-1/p-STAT3 cascade. However, the molecular mechanism by which SC-2001 activates SHP-1 is still elusive. Regulatory factor X (RFX) 1 is a transcription factor that has been linked to negative regulation of tumor progression. Nevertheless, its biological function in HCC cells is far from clear. In this study, by modulating its expression, we demonstrated that RFX1 is important in regulating SHP-1 in HCC cells. Ectopic expression of RFX1 can lead to activation of SHP-1 and inhibition of colony forming in HCC cells. Importantly, in a xenograft mouse model, RFX-1 overexpression obviously inhibited the growth rate of HCC tumors, indicating its tumor suppressor role. In addition, we found that SC-2001-induced apoptosis effect is regulated by RFX1. We showed that SC-2001 enhanced RFX1 nuclear translocation and binded to SHP-1 promoter to activate SHP-1 transcription. Moreover, knockdown of RFX1 reversed the anti-tumor effect of SC-2001 in vivo. Most important, we proved that SC-2001 displayed more potent anti-tumor effect than sorafenib, a clinically approved drug for HCC, in in vitro and in vivo assays. Furthermore, combination treatment of SC-2001 and sorafenib showed synergistic effect in HCC cells. Collectively, our study confirmed that RFX1 acts as a tumor suppressor in HCC and might be a new target for HCC therapy. The findings of this study also provide a new lead compound for target therapy via the activation of the RFX1/SHP-1 pathway. (Supported by NSC-102-2325-B-010-007) Citation Format: Jung-Chen Su. RFX1-dependent activation of SHP-1 inhibits STAT3 signaling in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2616. doi:10.1158/1538-7445.AM2014-2616

MHC class I and II deficiencies

Deficiencies of MHC complex class I or II are rare primary immunodeficiencies, both of which are inherited in an autosomal recessive pattern. MHC class II deficiency is a prototype of a disease of gene regulation. Defects in transacting regulatory factors required for expression of MHC class II genes, rather than the genes themselves, are responsible for the disease phenotype. The affected genes are known to encode 4 distinct regulatory factors controlling transcription of MHC class II genes. These transacting factors are the class II transactivator and 3 subunits of regulatory factor X (RFX): RFX containing ankyrin repeats (RFXANK), the fifth member of the RFX family (RFX5), and RFX-associated protein (RFXAP). Mutations in one of each define 4 distinct complementation groups termed A, B, C, and D, respectively. MHC class I deficiency is extremely rare and has been reported in less than 30 patients worldwide. Here we review the clinical, genetic, and molecular features that characterize these primary immunodeficiencies and discuss therapy options. Beyond the description of MHC class I and II deficiencies, their discovery has fascinated scientists and clinicians because of their ability to reveal the molecular basis of MCH regulation.

RFX7 is required for the formation of cilia in the neural tube

Regulatory Factor X (RFX) transcription factors are important for development and are likely involved in the pathogenesis of serious human diseases including ciliopathies. While seven RFX genes have been identified in vertebrates and several RFX transcription factors have been reported to be regulators of ciliogenesis, the role of RFX7 in development including ciliogenesis is not known. Here we show that RFX7 in Xenopus laevis is expressed in the neural tube, eye, otic vesicles, and somites. Knockdown of RFX7 in Xenopus embryos resulted in a defect of ciliogenesis in the neural tube and failure of neural tube closure. RFX7 controlled the formation of cilia by regulating the expression of RFX4 gene, which has been reported to be required for ciliogenesis in the neural tube. Moreover, ectopic expression of Foxj1, which is a master regulator of motile cilia formation, suppressed the expression of RFX4 but not RFX7. Taken together, RFX7 plays an important role in the process of neural tube closure at the top of the molecular cascade which controls ciliogenesis in the neural tube.

Transcription Factor RFX1 Is Crucial for Maintenance of Genome Integrity in Fusarium graminearum

The survival of cellular organisms depends on the faithful replication and transmission of DNA. Regulatory factor X (RFX) transcription factors are well conserved in animals and fungi, but their functions are diverse, ranging from the DNA damage response to ciliary gene regulation. We investigated the role of the sole RFX transcription factor, RFX1, in the plant-pathogenic fungus Fusarium graminearum. Deletion of rfx1 resulted in multiple defects in hyphal growth, conidiation, virulence, and sexual development. Deletion mutants of rfx1 were more sensitive to various types of DNA damage than the wild-type strain. Septum formation was inhibited and micronuclei were produced in the rfx1 deletion mutants. The results of the neutral comet assay demonstrated that disruption of rfx1 function caused spontaneous DNA double-strand breaks (DSBs). The transcript levels of genes involved in DNA DSB repair were upregulated in the rfx1 deletion mutants. DNA DSBs produced micronuclei and delayed septum formation in F. graminearum. Green fluorescent protein (GFP)-tagged RFX1 localized in nuclei and exhibited high expression levels in growing hyphae and conidiophores, where nuclear division was actively occurring. RNA-sequencing-based transcriptomic analysis revealed that RFX1 suppressed the expression of many genes, including those required for the repair of DNA damage. Taken together, these findings indicate that the transcriptional repressor rfx1 performs crucial roles during normal cell growth by maintaining genome integrity.

The mood stabilizer valproate activates human FGF1 gene promoter through inhibiting HDAC and GSK‐3 activities

Valproic acid (VPA) is the primary mood-stabilizing drug to exert neuroprotective effects and to treat bipolar disorder in clinic. Fibroblast growth factor 1 (FGF1) has been shown to regulate cell proliferation, cell division, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) has been shown to recapitulate endogenous FGF1 gene expression and facilitates the isolation of neural stem/progenitor cells (NSPCs) from developing and adult mouse brains. In this study, we provide several lines of evidence to demonstrate the underlying mechanisms of VPA in activating FGF-1B promoter activity: (i) VPA significantly increased the FGF-1B mRNA expression and the percentage of F1BGFP(+) cells; (ii) the increase of F1BGFP expression by VPA involves changes of regulatory factor X (RFX) 1-3 transcriptional complexes and the increase of histone H3 acetylation on the 18-bp cis-element of FGF-1B promoter; (iii) treatments of other histone deacetylases (HDAC) inhibitors, sodium butyrate and trichostatin A, significantly increased the expression levels of FGF-1B, RFX2, and RFX3 transcripts; (iv) treatments of glycogen synthase kinase 3 (GSK-3) inhibitor, lithium, or GSK-3 siRNAs also significantly activated FGF-1B promoter; (v) VPA specifically enhanced neuronal differentiation in F1BGFP(+) embryonic stem cells and NSPCs rather than GFP(-) cells. This study suggested, for the first time, that VPA activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities.

RFX Transcription Factor DAF-19 Regulates 5-HT and Innate Immune Responses to Pathogenic Bacteria in Caenorhabditis elegans

In Caenorhabditis elegans the Toll-interleukin receptor domain adaptor protein TIR-1 via a conserved mitogen-activated protein kinase (MAPK) signaling cascade induces innate immunity and upregulates serotonin (5-HT) biosynthesis gene tph-1 in a pair of ADF chemosensory neurons in response to infection. Here, we identify transcription factors downstream of the TIR-1 signaling pathway. We show that common transcription factors control the innate immunity and 5-HT biosynthesis. We demonstrate that a cysteine to tyrosine substitution in an ARM motif of the HEAT/Arm repeat region of the TIR-1 protein confers TIR-1 hyperactivation, leading to constitutive tph-1 upregulation in the ADF neurons, increased expression of intestinal antimicrobial genes, and enhanced resistance to killing by the human opportunistic pathogen Pseudomonas aeruginosa PA14. A forward genetic screen for suppressors of the hyperactive TIR-1 led to the identification of DAF-19, an ortholog of regulatory factor X (RFX) transcription factors that are required for human adaptive immunity. We show that DAF-19 concerts with ATF-7, a member of the activating transcription factor (ATF)/cAMP response element-binding B (CREB) family of transcription factors, to regulate tph-1 and antimicrobial genes, reminiscent of RFX-CREB interaction in human immune cells. daf-19 mutants display heightened susceptibility to killing by PA14. Remarkably, whereas the TIR-1-MAPK-DAF-19/ATF-7 pathway in the intestinal immunity is regulated by DKF-2/protein kinase D, we found that the regulation of tph-1 expression is independent of DKF-2 but requires UNC-43/Ca2+/calmodulin-dependent protein kinase (CaMK) II. Our results suggest that pathogenic cues trigger a common core-signaling pathway via tissue-specific mechanisms and demonstrate a novel role for RFX factors in neuronal and innate immune responses to infection.

Overexpression of Drosophila Rfx3 Induces Apoptosis and Interferes with Differentiation of Photoreceptor Cells

Regulatory Factor X (RFX) is a protein containing a characteristic DNA binding domain that is called RFX domain. Members of the RFX family are known to be present in human, mouse, Drosophila, Caenorhabditis elegans, Schizosaccharomyces pombe and Saccharomyces cerevisiae. Two RFX proteins, Drosophila RFX (dRFX) and dRFX2 have been identified in Drosophila so far. It is known that dRFX is involved in the differentiation of central and peripheral nerves, while dRFX2 is essential to the cell cycle progression and it may be involved in regulation of apoptosis. A novel protein, dRFX3 carrying RFX domain was identified by a Drosophila genome database search. dRFX3 is a likely homolog to the human RFX5, because of a significant sequence similarity in RFX domains between these two proteins. Two lines of transgenic fly carrying the HA-dRFX3 cDNA were established. Adult flies expressing dRFX3 specifically in the eye and the wing imaginal discs exhibited severe rough eye and atrophied wing phenotypes, respectively. 5-Bromo-2’-deoxyuridine incorporation assays and the immunological detection using anti-Cyclin B antibody indicated that the overexpression of dRFX3 in the eye imaginal discs exerted no effect on the cell cycle progressions. On the other hand, overexpression of dRFX3 in the eye imaginal discs interfered with the differentiation of R2/R5 photoreceptor cells and also induced apoptosis. These data suggest that the dRFX3 plays a negative role for the differentiation of R2/R5 photoreceptor cells and plays a positive role in the regulation of apoptosis.

Regulatory Factor X1-induced Down-regulation of Transforming Growth Factor β2 Transcription in Human Neuroblastoma Cells

Regulatory factor X (RFX) proteins are transcription factors. Seven mammalian RFX proteins have been identified. RFX1 is the prototype RFX. However, its biological functions are not known. Here, RFX1 overexpression reduced fetal bovine serum-stimulated proliferation of SH-SY5Y cells, a human neuroblastoma cell line. This inhibition is associated with decreased transforming growth factor β2 (TGFβ2) and phospho-extracellular signal-regulated kinase (ERK). Exogenous TGFβ2 increased cell proliferation and phospho-ERK in cells overexpressing RFX1. An anti-TGFβ2 antibody and PD98059, an ERK activation inhibitor, inhibited SH-SY5Y cell proliferation. TGFβ2 promoter activity was decreased in cells overexpressing RFX1. Chromosome immunoprecipitation assay showed that RFX1 bound the TGFβ2 promoter. RFX1 down-regulation increased TGFβ2 in SH-SY5Y and HCN-1A cells, a normal human neuronal cell line. More importantly, TGFβ2 concentrations were negatively correlated with RFX1 levels in human medulloblastoma tissues with a R(2) of 0.464. These results suggest that RFX1 reduces cell proliferation through inhibiting the TGFβ2-ERK signaling pathway. RFX1 blocks TGFβ2 expression through its direct action on TGFβ2 transcription. This effect also appears in human brain tumor tissues. Because TGFβ is known to be involved in cancer development, our results provide initial evidence to suggest that RFX1 may play an important role in human tumor biology.