The transcription factor Rfx7 limits metabolism of NK cells and promotes their maintenance and immunity.

Regulatory factor X 7 (Rfx7) is an uncharacterized transcription factor belonging to a family involved in ciliogenesis and immunity. Here, we found that deletion of Rfx7 leads to a decrease in natural killer (NK) cell maintenance and immunity in vivo. Genomic approaches showed that Rfx7 coordinated a transcriptional network controlling cell metabolism. Rfx7-/- NK lymphocytes presented increased size, granularity, proliferation, and energetic state, whereas genetic reduction of mTOR activity mitigated those defects. Notably, Rfx7-deficient NK lymphocytes were rescued by interleukin 15 through engagement of the Janus kinase (Jak) pathway, thus revealing the importance of this signaling for maintenance of such spontaneously activated NK cells. Rfx7 therefore emerges as a novel transcriptional regulator of NK cell homeostasis and metabolic quiescence.

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.

Hsa_circ_0030042 Ameliorates Oxidized Low-Density Lipoprotein-Induced Endothelial Cell Injury via the MiR-616-3p/RFX7 Axis.

Atherosclerosis (AS) is a common etiology of cardiovascular disease. As an emerging functional biomarker, circular RNAs (circRNAs) are involved in various diseases, including cardiovascular disease. However, the mechanism of action of circ_0030042 in AS has not been reported.Human umbilical vein endothelial cells (HUVECs) stimulated by ox-LDL served as a cellular model of AS. Gene expression was detected using quantitative real-time polymerase chain reaction. The influence of circ_0030042 on cell viability, proliferation, and apoptosis was verified using Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, and flow cytometry assays. An enzyme-linked immunosorbent assay was performed to measure the contents of tumor necrosis factor-α, interleukin (IL) -6, and IL-1ß. Western blot assay was utilized to determine the protein levels of Bax, Bcl-2, PCNA, and regulatory factor X 7 (RFX7). The interrelationship between miR-616-3p and circ_0030042 or RFX7 was validated using dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays.The expression of circ_0030042 was downregulated in ox-LDL-induced HUVECs. It was found that overexpression of circ_0030042 facilitated cell proliferation, repressed apoptosis, and reduced the level of inflammatory factors in HUVECs. Circ_0030042 and miR-616-3p had a targeting relationship, and the miR-616-3p mimic eliminated the effects of overexpressed circ_0030042 on ox-LDL-induced HUVECs. RFX7 was a downstream gene of miR-616-3p and was lowly expressed in ox-LDL-induced HUVECs. The miR-616-3p inhibitor stimulated cell proliferation, arrested apoptosis, and caused a decline in the levels of inflammatory factors, whereas knockdown of RFX7 abolished the effects.Circ_0030042 weakened ox-LDL-induced HUVEC injury by regulating the miR-616-3p/RFX7 pathway, thereby influencing AS progression. Circ_0030042 is likely to be a potential biomarker for the future treatment of patients with AS.

RFX1-mediated CCN3 induction that may support chondrocyte survival under starved conditions.

Cellular communication network factor (CCN) family members are multifunctional matricellular proteins that manipulate and integrate extracellular signals. In our previous studies investigating the role of CCN family members in cellular metabolism, we found three members that might be under the regulation of energy metabolism. In this study, we confirmed that CCN2 and CCN3 are the only members that are tightly regulated by glycolysis in human chondrocytic cells. Interestingly, CCN3 was induced under a variety of impaired glycolytic conditions. This CCN3 induction was also observed in two breast cancer cell lines with a distinct phenotype, suggesting a basic role of CCN3 in cellular metabolism. Reporter gene assays indicated a transcriptional regulation mediated by an enhancer in the proximal promoter region. As a result of analyses in silico, we specified regulatory factor binding to the X-box 1 (RFX1) as a candidate that mediated the transcriptional activation by impaired glycolysis. Indeed, the inhibition of glycolysis induced the expression of RFX1, and RFX1 silencing nullified the CCN3 induction by impaired glycolysis. Subsequent experiments with an anti-CCN3 antibody indicated that CCN3 supported the survival of chondrocytes under impaired glycolysis. Consistent with these findings in vitro, abundant CCN3 production by chondrocytes in the deep zones of developing epiphysial cartilage, which are located far away from the synovial fluid, was confirmed in vivo. Our present study uncovered that RFX1 is the mediator that enables CCN3 induction upon cellular starvation, which may eventually assist chondrocytes in retaining their viability, even when there is an energy supply shortage.

Recruitment of the protein phosphatase-1 catalytic subunit to promoters by the dual-function transcription factor RFX1.

RFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes. The exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes.

DAF-19/RFX controls ciliogenesis and influences oxygen-induced social behaviors in Pristionchus pacificus.

Cilia are complex organelles involved in sensory perception and motility with intraflagellar transport (IFT) proteins being essential for cilia assembly and function, but little is known about cilia in an evo-devo context. For example, recent comparisons revealed conservation and divergence of IFT components in the regulation of social feeding behaviors between the nematodes Caenorhabditis elegans and Pristionchus pacificus. Here, we focus on the P. pacificus RFX transcription factor daf-19, the master regulator of ciliogenesis in C. elegans. Two CRISPR/Cas9-induced Ppa-daf-19 mutants lack ciliary structures in amphid neurons and display chemosensory defects. In contrast to IFT mutants, Ppa-daf-19 mutants do not exhibit social behavior. However, they show weak locomotive responses to shifts in oxygen concentration, suggesting partial impairment in sensing or responding to oxygen. To identify targets of Ppa-daf-19 regulation we compared the transcriptomes of Ppa-daf-19 and wild-type animals and performed a bioinformatic search for the X-box RFX binding-site across the genome. The regulatory network of Ppa-DAF-19 involves IFT genes but also many taxonomically restricted genes. We identified a conserved X-box motif as the putative binding site, which was validated for the Ppa-dyf-1 gene. Thus, Ppa-DAF-19 controls ciliogenesis, influences oxygen-induced behaviors and displays a high turnover of its regulatory network.

Transcription factor RFX1 is ubiquitinated by E3 ligase STUB1 in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease caused by complex interactions between genes and the environment. The expression level of transcription factor regulatory factor X 1 (RFX1) is reduced in T cells from SLE patients. RFX1 can regulate epigenetic modifications of CD70 and CD11a and plays an important role in the development of SLE. However, the mechanisms that mediate reduction of RFX1 in SLE are unclear. Here, we demonstrate that RFX1 protein expression can be tightly regulated by polyubiquitination-mediated proteosomal degradation via STIP1 homology and U-box containing protein 1 (STUB1). The E3 ligase STUB1 is upregulated in CD4(+)T cells of SLE patients compared to healthy subjects. Overexpression of STUB1 in CD4(+)T cells leads to upregulation of levels of CD70 and CD11a in T cells. The modulation of STUB1 activity may provide a novel therapeutic approach for SLE.

RFX1 maintains testis cord integrity by regulating the expression of Itga6 in male mouse embryos.

Formation and maintenance of testis cords during embryogenesis are essential for establishing testicular structure and function in adults. At least five genes (Wt1, Dhh, Sox8/Sox9, and Dax1) appear to be required for the maintenance of testis cord integrity in mice. Here, we report that RFX1 is specifically expressed in fetal Sertoli cells. Mouse embryos conditionally deficient in Rfx1 (Rfx1(flox/flox) , Amh-Cre) possessed disrupted testis cords, as the basal lamina lining was fragmented or completely absent in some areas of the testes. Spermatogenesis was blocked, leading to complete infertility. Expression of integrin alpha-6 was significantly decreased in Rfx1-deficient testes compared to control testes; indeed, luciferase and chromatin immunoprecipitation assays indicated that RFX1 directly activates transcription of Itga6 (the gene coding for integrin alpha-6). Taken together, RFX1 transcriptionally targets Itga6 in Sertoli cells, thereby, helping maintain the integrity of the basal lamina during testis cord development. Mol. Reprod. Dev. 83: 606-614, 2016. © 2016 Wiley Periodicals, Inc.

RFX1 regulates foam cell formation and atherosclerosis by mediating CD36 expression.

BACKGROUND AND AIMS: Atherosclerosis (AS) is a continuously low-grade inflammatory disease, and monocyte-derived macrophages play a vital role in AS pathogenesis. Regulatory factor X1 (RFX1) has been reported to participate in differentiation of various cells. Our previous report showed that RFX1 expression in CD14+ monocytes from AS patients was decreased and closely related to AS development. Macrophages mostly derive from monocytes and play an important role in AS plaque formation and stability. However, the functions of RFX1 in the formation of macrophage-derived foam cells and consequent AS development are unclear. METHODS: We explored the effects of RFX1 on oxidation low lipoprotein (ox-LDL)-stimulated foam cell formation and CD36 expression by increasing or silencing Rfx1 expression in mouse peritoneal macrophages (PMAs). The ApoE-/-Rfx1f/f or ApoE-/-Rfx1f/f Lyz2-Cre mice fed a high-fat diet for 24 weeks were used to further examine the effect of RFX1 on AS pathogenesis. We then performed dual luciferase reporter assays to study the regulation of RFX1 for CD36 transcription. RESULTS: Our results demonstrate that RFX1 expression was significantly reduced in ox-LDL induced foam cells and negatively correlated with lipid uptake in macrophages. Besides, Rfx1 deficiency in myeloid cells aggravated atherosclerotic lesions in ApoE-/- mice. Mechanistically, RFX1 inhibited CD36 expression by directly regulating CD36 transcription in macrophages. CONCLUSIONS: The reduction of RFX1 expression in macrophages is a vital determinant for foam cell formation and the initiation of AS, proving a potential novel approach for the treatment of AS disease.

Regulatory factor X1 induces macrophage M1 polarization by promoting DNA demethylation in autoimmune inflammation.

Abnormal macrophage polarization is generally present in autoimmune diseases. Overwhelming M1 macrophage activation promotes the continuous progression of inflammation, which is one of the reasons for the development of autoimmune diseases. However, the underlying mechanism is still unclear. Here we explore the function of Regulatory factor X1 (RFX1) in macrophage polarization by constructing colitis and lupus-like mouse models. Both in vivo and in vitro experiments confirmed that RFX1 can promote M1 and inhibit M2 macrophage polarization. Furthermore, we found that RFX1 promoted DNA demethylation of macrophage polarization-related genes by increasing APOBEC3A/Apobec3 expression. We identified a potential RFX1 inhibitor, adenosine diphosphate (ADP), providing a potential strategy for treating autoimmune diseases.

Disruption of the ATXN1-CIC complex reveals the role of additional nuclear ATXN1 interactors in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative disease in that it is caused by a mutation in a broadly expressed protein, ATXN1; however, only select populations of cells degenerate. The interaction of polyglutamine-expanded ATXN1 with the transcriptional repressor CIC drives cerebellar Purkinje cell pathogenesis; however, the importance of this interaction in other vulnerable cells remains unknown. Here, we mutated the 154Q knockin allele of Atxn1154Q/2Q mice to prevent the ATXN1-CIC interaction globally. This normalized genome-wide CIC binding; however, it only partially corrected transcriptional and behavioral phenotypes, suggesting the involvement of additional factors in disease pathogenesis. Using unbiased proteomics, we identified three ATXN1-interacting transcription factors: RFX1, ZBTB5, and ZKSCAN1. We observed altered expression of RFX1 and ZKSCAN1 target genes in SCA1 mice and patient-derived iNeurons, highlighting their potential contributions to disease. Together, these data underscore the complexity of mechanisms driving cellular vulnerability in SCA1.

RXR agonist, Bexarotene, effectively reduces drug resistance via regulation of RFX1 in embryonic carcinoma cells.

Aberrant expression of multidrug resistance (MDR) proteins is one of the features of cancer stem cells (CSCs) that make them escape chemotherapy. A well-orchestrated regulation of multiple MDRs by different transcription factors in cancer cells confers this drug resistance. An in silico analysis of the major MDR genes revealed a possible regulation by RFX1 and Nrf2. Previous reports also noted that Nrf2 is a positive regulator of MDR genes in NT2 cells. But we, for the first time, report that Regulatory factor X1 (RFX1), a pleiotropic transcription factor, negatively regulates the major MDR genes, Abcg2, Abcb1, Abcc1, and Abcc2, in NT2 cells. The levels of RFX1 in undifferentiated NT2 cells were found to be very low, which significantly increased upon RA-induced differentiation. Ectopic expression of RFX1 reduced the levels of transcripts corresponding to MDRs and stemness-associated genes. Interestingly, Bexarotene, an RXR agonist that acts as an inhibitor of Nrf2-ARE signaling, could increase the transcription of RFX1. Further analysis revealed that the RFX1 promoter has binding sites for RXRα, and upon Bexarotene exposure RXRα could bind and activate the RFX1 promoter. Bexarotene, alone or in combination with Cisplatin, could inhibit many cancer/CSC-associated properties in NT2 cells. Also, it significantly reduced the expression of drug resistance proteins and made the cells sensitive towards Cisplatin. Our study proves that RFX1 could be a potent molecule to target MDRs, and Bexarotene can induce RXRα-mediated RFX1 expression, therefore, would be a better chemo-assisting drug during therapy.

Dynamic nucleosome organization after fertilization reveals regulatory factors for mouse zygotic genome activation.

Chromatin remodeling is essential for epigenome reprogramming after fertilization. However, the underlying mechanisms of chromatin remodeling remain to be explored. Here, we investigated the dynamic changes in nucleosome occupancy and positioning in pronucleus-stage zygotes using ultra low-input MNase-seq. We observed distinct features of inheritance and reconstruction of nucleosome positioning in both paternal and maternal genomes. Genome-wide de novo nucleosome occupancy in the paternal genome was observed as early as 1 h after the injection of sperm into ooplasm. The nucleosome positioning pattern was continually rebuilt to form nucleosome-depleted regions (NDRs) at promoters and transcription factor (TF) binding sites with differential dynamics in paternal and maternal genomes. NDRs formed more quickly on the promoters of genes involved in zygotic genome activation (ZGA), and this formation is closely linked to histone acetylation, but not transcription elongation or DNA replication. Importantly, we found that NDR establishment on the binding motifs of specific TFs might be associated with their potential pioneer functions in ZGA. Further investigations suggested that the predicted factors MLX and RFX1 played important roles in regulating minor and major ZGA, respectively. Our data not only elucidate the nucleosome positioning dynamics in both male and female pronuclei following fertilization, but also provide an efficient method for identifying key transcription regulators during development.

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."

Induction of functional neutrophils from mouse fibroblasts by thymidine through enhancement of Tet3 activity.

Neutrophils are derived from bone marrow hematopoietic stem cells (HSCs) and are the largest population among circulating white blood cells in humans, acting as the first line of defense against invading pathogens. Whether neutrophils can be generated by transdifferentiation strategies is unknown. Here, we show that thymidine induces the conversion of mouse fibroblasts to neutrophils. Induced neutrophils (iNeus) showed antibacterial effects and did not undergo malignant transformation in vivo. Importantly, iNeu transplantation cured neutropenia in mice in vivo. Mechanistically, thymidine mediates iNeu conversion by enhancing Tet3 activity. Tet3 initiates the expression of the neutrophil fate decision factors Cebpδ and Rfx1 that drive the transdifferentiation of mouse fibroblasts to neutrophils. Therefore, the induction of functional neutrophils by chemicals may provide a potential therapeutic strategy for patients with neutropenia patients and infectious diseases.Fibroblasts; Neutrophils; Thymidine; Transdifferentiation; Tet3.

The decrease of intraflagellar transport impairs sensory perception and metabolism in ageing.

Sensory perception and metabolic homeostasis are known to deteriorate with ageing, impairing the health of aged animals, while mechanisms underlying their deterioration remain poorly understood. The potential interplay between the declining sensory perception and the impaired metabolism during ageing is also barely explored. Here, we report that the intraflagellar transport (IFT) in the cilia of sensory neurons is impaired in the aged nematode Caenorhabditis elegans due to a daf-19/RFX-modulated decrease of IFT components. We find that the reduced IFT in sensory cilia thus impairs sensory perception with ageing. Moreover, we demonstrate that whereas the IFT-dependent decrease of sensory perception in aged worms has a mild impact on the insulin/IGF-1 signalling, it remarkably suppresses AMP-activated protein kinase (AMPK) signalling across tissues. We show that upregulating daf-19/RFX effectively enhances IFT, sensory perception, AMPK activity and autophagy, promoting metabolic homeostasis and longevity. Our study determines an ageing pathway causing IFT decay and sensory perception deterioration, which in turn disrupts metabolism and healthy ageing.

KMT5A downregulation participated in High Glucose-mediated EndMT via Upregulation of ENO1 Expression in Diabetic Nephropathy.

Diabetic nephropathy (DN) has become the common and principal microvascular complication of diabetes that could lead to end-stage renal disease. It was reported endothelial-to-mesenchymal transition (EndMT) in glomeruli plays an important role in DN. Enolase1 (ENO1) and Lysine Methyltransferase 5A (KMT5A) were found to modulate epithelial-to-mesenchymal transition in some situations. In the present study, we speculated KMT5A regulates ENO1 transcript, thus participating in hyperglycemia-induced EndMT in glomeruli of DN. Our study represented vimentin, αSMA and ENO1 expression elevated, and CD31 expression decreased in glomeruli of DN participants and rats. In vitro, high glucose induced EndMT by increase of ENO1 levels. Moreover, high glucose downregulated KMT5A levels and increased regulatory factor X1 (RFX1) levels. KMT5A upregulation or si-RFX1 decreased high glucose-induced ENO1 expression and EndMT. RFX1 overexpression- or sh-KMT5A-induced EndMT was attenuated by si-ENO1. Further, the association between KMT5A and RFX1 was verified. Furthermore, histone H4 lysine20 methylation (the direct target of KMT5A) and RFX1 positioned on ENO1 promoter region. sh-KMT5A enhanced positive action of RFX1 on ENO1 promoter activity. KMT5A reduction and RFX1 upregulation were verified in glomeruli of DN patients and rats. KMT5A associated with RFX1 to modulate ENO1, thus involved in hyperglycemia-mediated EndMT in glomeruli of DN.

RFX1 and RFX3 Transcription Factors Interact with the D Sequence of Adeno-Associated Virus Inverted Terminal Repeat and Regulate AAV Transduction.

Adeno-associated virus (AAV) transduction efficiency depends on the way in which cellular proteins process viral genomes in the nucleus. In this study, we have investigated the binding of nuclear proteins to the double stranded D (dsD) sequence of the AAV inverted terminal repeat (ITRs) by electromobility shift assay. We present here several lines of evidence that transcription factors belonging to the RFX protein family bind specifically and selectively to AAV2 and AAV1 dsD sequences. Using supershift experiments, we characterize complexes containing RFX1 homodimers and RFX1/RFX3 heterodimers. Following transduction of HEK-293 cells, the AAV genome can be pulled-down by RFX1 and RFX3 antibodies. Moreover, our data suggest that RFX proteins which interact with transcriptional enhancers of several mammalian DNA viruses, can act as regulators of AAV mediated transgene expression.

RFX1 participates in doxorubicin-induced hepatitis B virus reactivation.

Cytotoxic chemotherapy drugs, including doxorubicin, can directly promote hepatitis B virus (HBV) replication, but the mechanism has not been fully clarified. This study investigated the potential mechanism underlying the cytotoxic chemotherapy-mediated direct promotion of HBV replication. We found that HBV replication and regulatory factor X box 1 gene (RFX1) expression were simultaneously promoted by doxorubicin treatment. The amount of RFX1 bound to the HBV enhancer I was significantly increased under doxorubicin treatment. Furthermore, the activity of doxorubicin in promoting HBV replication was significantly attenuated when the expression of endogenous RFX1 was knocked down, and the EP element of HBV enhancer I, an element that mediated the binding of RFX1 and HBV enhancer I, was mutated. In addition, two different sequences of the conserved EP element were found among HBV genotypes A-D, and doxorubicin could promote the replication of HBV harboring either of the conserved EP elements. Here, a novel pathway in which doxorubicin promoted HBV replication via RFX1 was identified, and it might participate in doxorubicin-induced HBV reactivation. These findings would be helpful in preventing HBV reactivation during anticancer chemotherapy.

An Expanded Role for the RFX Transcription Factor DAF-19, with Dual Functions in Ciliated and Nonciliated Neurons.

Regulatory Factor X (RFX) transcription factors (TFs) are best known for activating genes required for ciliogenesis in both vertebrates and invertebrates. In humans, eight RFX TFs have a variety of tissue-specific functions, while in the worm Caenorhabditis elegans, the sole RFX gene, daf-19, encodes a set of nested isoforms. Null alleles of daf-19 confer pleiotropic effects including altered development with a dauer constitutive phenotype, complete absence of cilia and ciliary proteins, and defects in synaptic protein maintenance. We sought to identify RFX/daf-19 target genes associated with neuronal functions other than ciliogenesis using comparative transcriptome analyses at different life stages of the worm. Subsequent characterization of gene expression patterns revealed one set of genes activated in the presence of DAF-19 in ciliated sensory neurons, whose activation requires the daf-19c isoform, also required for ciliogenesis. A second set of genes is downregulated in the presence of DAF-19, primarily in nonsensory neurons. The human orthologs of some of these neuronal genes are associated with human diseases. We report the novel finding that daf-19a is directly or indirectly responsible for downregulation of these neuronal genes in C. elegans by characterizing a new mutation affecting the daf-19a isoform (tm5562) and not associated with ciliogenesis, but which confers synaptic and behavioral defects. Thus, we have identified a new regulatory role for RFX TFs in the nervous system. The new daf-19 candidate target genes we have identified by transcriptomics will serve to uncover the molecular underpinnings of the pleiotropic effects that daf-19 exerts on nervous system function.