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.

Hypoxia-induced exosomes facilitate lung pre-metastatic niche formation in hepatocellular carcinoma through the miR-4508-RFX1-IL17A-p38 MAPK-NF-κB pathway.

Background: Hypoxia plays an important role in the lung metastasis of hepatocellular carcinoma (HCC). However, the process by which hypoxia promotes the formation of a pre-metastatic niche (PMN) and its underlying mechanism remain unclear. Methods: Exosomes derived from normoxic and hypoxic HCC cells were collected to induce fibroblast activation in vitro and PMN formation in vivo. The micro RNA (miR) profiles of the exosomes were sequenced to identify differentially expressed miRNAs. Gain- and loss-of-function analyses were performed to investigate miR-4508 function. Dual-luciferase, western blotting, and real-time reverse transcription-PCR analyses were used to identify the direct targets of miR-4508 and its downstream signaling pathways. To demonstrate the roles of hypoxic tumor-derived exosomes (H-TDEs) and miR-4508 in the lung metastasis of liver cancer, H22 tumor cells were injected through the tail vein of mice. Blood plasma-derived exosomes from patients with HCC who underwent transarterial chemoembolization (TACE) were applied to determine clinical correlations. Results: We demonstrated that H-TDEs activated lung fibroblasts and facilitated PMN formation, thereby promoting lung metastasis in mice. Screening for upregulated exosomal miRNAs revealed that miR-4508 and its target, regulatory factor X1 (RFX1), were involved in H-TDE-induced lung PMN formation. Moreover, miR-4508 was significantly upregulated in plasma exosomes derived from patients with HCC after TACE. We confirmed that the p38 MAPK-NF-κB signaling pathway is involved in RFX1 knockdown-induced fibroblast activation and PMN formation. In addition, IL17A, a downstream target of RFX1, was identified as a link between RFX1 knockdown and p38 MAPK activation in fibroblasts. Conclusion: Hypoxia enhances the release of TDEs enriched with miR-4508, thereby promoting lung PMN formation by targeting the RFX1-IL17A-p38 MAPK-NF-κB pathway. These findings highlight a novel mechanism underlying hypoxia-induced pulmonary metastasis of HCC.

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.

HLA-DRB1: A new potential prognostic factor and therapeutic target of cutaneous melanoma and an indicator of tumor microenvironment remodeling.

Cutaneous melanoma (CM) is the most common skin cancer and one of the most aggressive cancers and its incidence has risen dramatically over the past few decades. The tumor microenvironment (TME) plays a crucial role in the occurrence and development of cutaneous melanoma. Nevertheless, the dynamics modulation of the immune and stromal components in the TME is not fully understood. In this study, 471 CM samples were obtained from TCGA database, and the ratio of tumor-infiltrating immune cells (TICs) in the TME were estimated using the ESTIMATE algorithms and CIBERSORT computational method. The differently expressed genes (DEGs) were applied to GO and KEGG function enrichment analysis, establishment of protein-protein interaction (PPI) network and univariate Cox regression analysis. Subsequently, we identified a predictive factor: HLA-DRB1 (major histocompatibility complex, class II, DR beta 1) by the intersection analysis of the hub genes of PPI network and the genes associated with the prognosis of the CM patients obtained by univariate Cox regression analysis. Correlation analysis and survival analysis showed that the expression level of HLA-DRB1 was negatively correlated with the Stage of the patients while positively correlated with the survival, prognosis and TME of melanoma. The GEPIA web server and the representative immunohistochemical images of HLA-DRB1 in the normal skin tissue and melanoma tissue from the Human Protein Atlas (HPA) database were applied to validate the expression level of HLA-DRB1. CIBERSORT analysis for the ratio of TICs indicated that 9 types of TICs were positively correlated with the expression level of HLA-DRB1 and only 4 types of TICs were negatively correlated with the expression level of HLA-DRB1. These results suggested that the expression level of HLA-DRB1 may be related to the immune activity of the TME and may affect the prognosis of CM patients by changing the status of the TME.

Identification of RFX5 as prognostic biomarker and associated with immune infiltration in stomach adenocarcinoma.

BACKGROUND: Regulatory factor X (RFX) gene family is a series of encodes transcription factors with a highly conserved DNA binding domain. RFXs played a vital role in the development and progression of cancer. However, the significance of RFXs in stomach adenocarcinoma (STAD) has not been fully clarified. METHODS: Online bioinformatics tools such as GSCALite, Kaplan-Meier Plotter, TIMER, LinkedOmics were used to explore the immunomodulatory function and clinical value of RFXs in STAD. RESULTS: The mRNA level of RFX1, RFX3, RFX4, RFX5, RFX7 and RFX8 was significantly elevated in STAD tissue versus adjacent normal tissue. We also summarize the copy number variation, single nucleotide variants and drug sensitivity of RFXs in STAD. Prognostic analysis indicated that STAD patients with high RFX5 and RFX7 expression had a better overall survival, first progression, and post-progression survival. Moreover, RFX5 expression was significantly associated with the abundance of immune cells, the expression of immune biomarkers and tumor mutational burden score in STAD. Functional enrichment analysis revealed that RFX5 and its related genes were mainly involved in T cell activation, antigen receptor-mediated signaling pathway, cell adhesion molecules, and Th17 cell differentiation. Validation study further verified the expression and prognosis of RFX5 in STAD. Further univariate and multivariate analyses suggested that pathological stage and RFX5 could be a potential independent prognostic factor for STAD. CONCLUSIONS: RFX5 was a candidate prognostic biomarker and associated with immune infiltration in STAD.

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.

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.

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.

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.

Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response.

A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

RFX1 downregulation contributes to TLR4 overexpression in CD14+ monocytes via epigenetic mechanisms in coronary artery disease.

BACKGROUND: Toll-like receptor 4 (TLR4) expression is increased in activated monocytes, which play a critical role in the pathogenesis of coronary artery disease (CAD). However, the mechanism remains unclear. Regulatory factor X1 (RFX1) is a critical transcription factor regulating epigenetic modifications. In this study, we investigated whether RFX1 and epigenetic modifications mediated by RFX1 contributed to the overexpression of TLR4 in activated monocytes. RESULTS: Compared with those of the controls, the mRNA and protein expression of RFX1 were downregulated and the mRNA expression of TLR4 was upregulated in CD14+ monocytes obtained from CAD patients and CD14+ monocytes obtained from healthy controls treated with low-density lipoprotein (LDL). The mRNA expression of RFX1 was negatively correlated with the mRNA expression of TLR4 in CD14+ monocytes. RFX1 knockdown led to the overexpression of TLR4 and the activation of CD14+ monocytes. In contrast, the overexpression of RFX1 inhibited TLR4 expression and the activation of CD14+ monocytes stimulated with LDL. Moreover, TLR4 was identified as a target gene of RFX1. The results indicated that RFX1 downregulation contributed to the decreased DNA methylation and histone H3 lysine 9 trimethylation and the increased H3 and H4 acetylation in the TLR4 promoter via the lack of recruitments of DNA methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone-lysine N-methyltransferase SUV39H1 (SUV39H1), which were observed in CD14+ monocytes of CAD patients. CONCLUSIONS: Our results show that RFX1 expression deficiency leads to the overexpression of TLR4 and the activation of CD14+ monocytes in CAD patients by regulating DNA methylation and histone modifications, which highlights the vital role of RFX1 in the pathogenesis of CAD.

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.

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.

ATOH1/RFX1/RFX3 transcription factors facilitate the differentiation and characterisation of inner ear hair cell-like cells from patient-specific induced pluripotent stem cells harbouring A8344G mutation of mitochondrial DNA.

Degeneration or loss of inner ear hair cells (HCs) is irreversible and results in sensorineural hearing loss (SHL). Human-induced pluripotent stem cells (hiPSCs) have been employed in disease modelling and cell therapy. Here, we propose a transcription factor (TF)-driven approach using ATOH1 and regulatory factor of x-box (RFX) genes to generate HC-like cells from hiPSCs. Our results suggest that ATOH1/RFX1/RFX3 could significantly increase the differentiation capacity of iPSCs into MYO7AmCherry-positive cells, upregulate the mRNA expression levels of HC-related genes and promote the differentiation of HCs with more mature stereociliary bundles. To model the molecular and stereociliary structural changes involved in HC dysfunction in SHL, we further used ATOH1/RFX1/RFX3 to differentiate HC-like cells from the iPSCs from patients with myoclonus epilepsy associated with ragged-red fibres (MERRF) syndrome, which is caused by A8344G mutation of mitochondrial DNA (mtDNA), and characterised by myoclonus epilepsy, ataxia and SHL. Compared with isogenic iPSCs, MERRF-iPSCs possessed ~42-44% mtDNA with A8344G mutation and exhibited significantly elevated reactive oxygen species (ROS) production and CAT gene expression. Furthermore, MERRF-iPSC-differentiated HC-like cells exhibited significantly elevated ROS levels and MnSOD and CAT gene expression. These MERRF-HCs that had more single cilia with a shorter length could be observed only by using a non-TF method, but those with fewer stereociliary bundle-like protrusions than isogenic iPSCs-differentiated-HC-like cells could be further observed using ATOH1/RFX1/RFX3 TFs. We further analysed and compared the whole transcriptome of M1ctrl-HCs and M1-HCs after treatment with ATOH1 or ATOH1/RFX1/RFX3. We revealed that the HC-related gene transcripts in M1ctrl-iPSCs had a significantly higher tendency to be activated by ATOH1/RFX1/RFX3 than M1-iPSCs. The ATOH1/RFX1/RFX3 TF-driven approach for the differentiation of HC-like cells from iPSCs is an efficient and promising strategy for the disease modelling of SHL and can be employed in future therapeutic strategies to treat SHL patients.

IL-6/STAT3 pathway induced deficiency of RFX1 contributes to Th17-dependent autoimmune diseases via epigenetic regulation.

Epigenetic modifications affect the differentiation of T cell subsets and the pathogenesis of autoimmune diseases, but many mechanisms of epigenetic regulation of T cell differentiation are unclear. Here we show reduced expression of the transcription factor RFX1 in CD4+ T cells from patients with systemic lupus erythematosus, which leads to IL-17A overexpression through increased histone H3 acetylation and decreased DNA methylation and H3K9 tri-methylation. Conditional deletion of Rfx1 in mice exacerbates experimental autoimmune encephalomyelitis and pristane-induced lupus-like syndrome and increases induction of Th17 cells. In vitro, Rfx1 deficiency increases the differentiation of naive CD4+ T cells into Th17 cells, but this effect can be reversed by forced expression of Rfx1. Importantly, RFX1 functions downstream of STAT3 and phosphorylated STAT3 can inhibit RFX1 expression, highlighting a non-canonical pathway that regulates differentiation of Th17 cells. Collectively, our findings identify a unique role for RFX1 in Th17-related autoimmune diseases.

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.

Discovery of Novel Human Gene Regulatory Modules from Gene Co-expression and Promoter Motif Analysis.

Deciphering gene regulatory networks requires identification of gene expression modules. We describe a novel bottom-up approach to identify gene modules regulated by cis-regulatory motifs from a human gene co-expression network. Target genes of a cis-regulatory motif were identified from the network via the motif's enrichment or biased distribution towards transcription start sites in the promoters of co-expressed genes. A gene sub-network containing the target genes was extracted and used to derive gene modules. The analysis revealed known and novel gene modules regulated by the NF-Y motif. The binding of NF-Y proteins to these modules' gene promoters were verified using ENCODE ChIP-Seq data. The analyses also identified 8,048 Sp1 motif target genes, interestingly many of which were not detected by ENCODE ChIP-Seq. These target genes assemble into house-keeping, tissues-specific developmental, and immune response modules. Integration of Sp1 modules with genomic and epigenomic data indicates epigenetic control of Sp1 targets' expression in a cell/tissue specific manner. Finally, known and novel target genes and modules regulated by the YY1, RFX1, IRF1, and 34 other motifs were also identified. The study described here provides a valuable resource to understand transcriptional regulation of various human developmental, disease, or immunity pathways.

Regulatory factor X1 depresses ApoE-dependent Aß uptake by miRNA-124 in microglial response to oxidative stress.

Decreased proteolytic clearance of soluble amyloid ß (Aß) in microglia affects Aß accumulation on Alzheimer's disease progression. However, the potential molecular mechanism by which microglial Aß uptake is regulated remains unclear. In this study, we identified a microRNA, miR-124, that was down-regulated in aging with a function in regulating apolipoprotein E (ApoE)-dependent Aß uptake by targeting regulatory factor X1 (RFX1) transcripts on BV2 microglia cell. Decreased expression of miRNA-124 in BV2 cells exposed to mild hydrogen peroxide increased RFX1 protein level and decreased the expression of ApoE, a gene which has been suggested to enhance cellular Aß uptake in microglia. We also identified a miR-124 binding site in the 3'-UTR of RFX1 mRNA and a RFX1 binding site in the first intron of ApoE gene. Furthermore, interfering this signaling pathway by knocking down RFX1 significantly improved Aß uptake in BV2 cells. These data demonstrate the mechanism through which decreased miR-124 expression under oxidative stress slowed Aß uptake and suggest that RFX1 might be a target for improving Aß clearance during aging.

[Overexpression of lentivirus RFX1 and its inhibitory effect on proliferation of glioblastoma cells].

OBJECTIVE: To construct overexpression lentivirus vector for human regulatory factor X1 (RFX1) gene, and to explore its effect on proliferation of F98 cell line.
 Methods: Huamn RFX1 gene was amplified by polymerase reaction. Gene amplification products were inserted into lentivirus vector pITA, and the lentivirus vector pITA-RFX1 was constructed. The constructed vector was verified by agarose gel electrophoresis and DNA sequencing. Lentivirus vector pITA-RFX1 and virus packaging plasmids were cotransfected into 293T cells, and then transfected into F98 cells. RFX1 protein expression were detected by Western blot and laser confocal before and after transfection. Flow cytometry and cell counting kit-8 were used to detect cellular proliferation.
 Results: Agarose gel electrophoresis and DNA sequencing showed that recombinant lentivirus plasmids pITA-RFX1 were constructed successfully. After transfection of pITA-RFX1, the RFX1 protein were over-expressed, which significantly inhibited the proliferation of F98 cells.
 Conclusion: The overexpression lentivirus vector for RFX1 was constrcted successfully, and the up-regulation of RFX1 can prevent the proliferation of glioblastoma cells.

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.