RFX3

Abstract: Regulatory factor (RF)X proteins are unique transcription factors that contain a highly conserved 76-amino-acid DNA binding domain.1 This domain binds the X-box consensus sequence in promoter regions to regulate gene expression. Seven RFX proteins have been identified in mammals.2 Each of them may have different functions. For example, RFX5 has been shown to play an important role in regulating the expression of the major histocompatibility complex class II genes.3 RFX5 knockout results in bare lymphocyte syndrome in mice.4 Knockout of the RFX3 gene causes severe ciliopathies that can result in diabetes and left-right asymmetry specification.5,6 However, the biological functions of RFX1, the prototype of the RFX family, are not well known. RFX1 downregulates the proto-oncogene c-myc.7 The RFX1 gene is often epigenetically silenced due to hypermethylation in human glioblastomas, the most common and most deadly primary human brain tumors.8 RFX1 expression in the esophageal epithelium is decreased gradually, with the pathology changed from normal epithelium to esophageal adenocarcinoma.9 We recently discovered that RFX1 directly downregulates transforming growth factor (TGF)β2 in human SH-SY5Y cells, a neuroblastoma cell line. RFX1 expression was inversely correlated with TGFβ2 in human neuroblastoma tissues.10 TGFβ2 plays an important role in cell proliferation and survival.11 These results suggest that RFX1 is an important molecule that participates in the regulation of human cancer biology. Clusters of differentiation (CD)44 is a cell surface protein that works as a receptor for hyaluronan and other ligands, such as collagens.12–15 Although there is only one human CD44 gene, multiple CD44 splicing variants have been found.12,13,15 Activated CD44 can interact with growth factor receptors and the leukemia-associated Rho-guanine nucleotide exchange factor to activate Akt for cell survival, mitogen-activated protein kinase for cell proliferation, and calcium/calmodulin-dependent protein kinase type II to activate cytoskeleton for cell migration and invasion.12,13,15 CD44 has been found in most cell types under normal conditions.14 However, increased CD44 has been shown to play a critical role in the biology of many human cancers, including breast cancer and head and neck squamous cell carcinoma.12,15 A recent study has shown that CD44 is upregulated in human glioblastoma tissues.16 To determine whether RFX1 can regulate CD44 expression, we searched for a putative RFX1 binding site in the CD44 gene. We identified an excellent putative RFX1 binding site in the first exon of the CD44 gene. Thus, we hypothesized that RFX1 might directly regulate CD44 expression and that this regulation could lead to the regulation of proliferation, survival, migration, and invasion of tumor cells. In this study, we experimentally tested these hypotheses and found that RFX1 directly regulates CD44 expression to inhibit glioblastoma malignant functions.

Abstract: 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., Castren, E., Kere, J., Tapia-Paez, I., Swoboda, P. Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor (RF) X transcription factors through X-box promoter motifs.

Abstract: 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.