The RFX protein family includes members from yeast to humans, which function in various biological systems, and share a DNA-binding domain and a conserved C-terminal region. In the human transcription regulator RFX1, the conserved C terminus is an independent functional domain, which mediates dimerization and transcriptional repression. This dimerization domain has a unique ability to mediate the formation of two alternative homodimeric DNA-protein complexes, the upper of which has been linked to repression. The complex formation capacity has been localized to several different RFX1 C-terminal subregions, each of which can function independently to generate the upper complex and repress transcription, thus correlating complex formation with repression. To gain an evolutionary perspective, an examination was carried out to see whether the different properties of the RFX1 C terminus exist in the two yeast RFX proteins, which are involved in signaling pathways. Replacement of the RFX1 C terminus with those of Sak1 and Crt1, its orthologs from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, and analysis of fusions with the Gal4 DNA-binding domain, revealed that the ability to generate the two alternative complexes is conserved in the RFX family, from S. cerevisiae to man. While sharing this unique biochemical property, the three C termini differed from each other in their ability to mediate dimerization and transcriptional repression. In both functions, RFX1, Sak1, and Crt1 showed high capacity, moderate capacity, and no capacity, respectively. This comparative analysis of the RFX proteins, representing different evolutionary stages, suggests a gradual development of the conserved C terminus, from the appearance of the ancestral motif (Crt1), to the later acquisition of the dimerization/repression functions (Sak1), and finally to the enhancement of these functions to generate a domain mediating highly stable protein-protein interactions and potent transcriptional repression (RFX1) (Katan-Khaykovich, 1999).
Regulatory factor X (RFX) proteins are transcriptional activators that recognize X-boxes (DNA of the sequence 5'-GTNRCC(0-3N)RGYAAC-3', where N is any nucleotide, R is a purine and Y is a pyrimidine) using a highly conserved 76-residue DNA-binding domain (DBD). DNA-binding defects in the protein RFX5 cause bare lymphocyte syndrome or major histocompatibility antigen class II deficiency. RFX1, -2 and -3 regulate expression of other medically important gene products (for example, interleukin-5 receptor alpha chain, IL-5R alpha). Fusions of the ligand-binding domain of the oestrogen receptor with the DBD of RFX4 occur in some human breast tumors. A 1.5 Å-resolution structure is presented of two copies of the DBD of human RFX1 (hRFX1), binding cooperatively to a symmetrical X-box. hRFX1 is an unusual member of the winged-helix subfamily of helix-turn-helix proteins because it uses a beta-hairpin (or wing) to recognize DNA instead of the recognition helix typical of helix-turn-helix proteins. A new model for interactions between linker histones and DNA is proposed (Gajiwala, 2000).
RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C by unknown mechanisms. By confocal fluorescence microscopy, a nonclassical nuclear localization signal (NLS) has been identified at the extreme C-terminus. The adjacent 'acidic region', which shows no independent NLS activity, potentiates the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibits nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which is able to both mediate nuclear import of RFX1, and also inhibit its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC (Katan-Khaykovich, 2001).
Many types of sensory neurons contain modified cilia where sensory signal transduction occurs. The C. elegans gene daf-19 encodes an RFX-type transcription factor that is expressed specifically in all ciliated sensory neurons. Loss of daf-19 function causes the absence of cilia, resulting in severe sensory defects. Several genes that function in all ciliated sensory neurons have an RFX target site in their promoters and require daf-19 function. Several other genes that function in subsets of ciliated sensory neurons do not have an RFX target site and are not daf-19 dependent. These results suggest that expression of the shared components of sensory cilia is activated by daf-19, whereas cell-type-specific expression occurs independently of daf-19 (Swoboda, 2000).
Cilia and flagella are important organelles involved in diverse functions such as fluid and cell movement, sensory perception and embryonic patterning. They are devoid of protein synthesis, thus their formation and maintenance requires the movement of protein complexes from the cytoplasm into the cilium and flagellum axoneme by intraflagellar transport (IFT), a conserved process common to all ciliated or flagellated eukaryotic cells. Mutations in the Caenorhabditis elegans gene Y41g9a.1 are responsible for the ciliary defects in osm-5 mutant worms. This was confirmed by transgenic rescue of osm-5(p813) mutants using the wild-type Y41g9a.1 gene. osm-5 encodes a tetratricopeptide repeat (TPR)-containing protein that is the homolog of murine polaris (Tg737), a protein associated with cystic kidney disease and left-right axis patterning defects in the mouse. osm-5 is expressed in ciliated sensory neurons in C. elegans and its expression is regulated by DAF-19, an RFX-type transcription factor that governs the expression of other genes involved in cilia formation in the worm. Similar to murine polaris, the OSM-5 protein is found to be concentrated at the cilium base and within the cilium axoneme as shown by an OSM-5::GFP translational fusion and immunofluorescence. Furthermore, time-lapse imaging of OSM-5::GFP fusion protein shows fluorescent particle migration within the cilia. Overall, the data support a crucial role for osm-5 in a conserved ciliogenic pathway, most likely as a component of the IFT process (Haycraft, 2001).
In Xenopus neuroectoderm, posterior cells start differentiating at the end of gastrulation, while anterior cells display an extended proliferative period and undergo neurogenesis only at tailbud stage. Recent studies have identified several important components of the molecular pathways controlling posterior neurogenesis, but little is known about those controlling the timing and positioning of anterior neurogenesis. The role of Xrx1, a homeobox gene required for eye and anterior brain development, was investigated in the control of proliferation and neurogenesis of the anterior neural plate. Xrx1 is expressed in the entire proliferative region of the anterior neural plate delimited by cells expressing the neuronal determination gene X-ngnr-1, the neurogenic gene X-Delta-1, and the cell cycle inhibitor p27Xic1. Positive and negative signals position Xrx1 expression to this region. Xrx1 is activated by chordin and Hedgehog gene signaling, which induce anterior and proliferative fate, and is repressed by the differentiation-promoting activity of neurogenin and retinoic acid. Xrx1 is required for anterior neural plate proliferation and, when overexpressed, induces proliferation, inhibits X-ngnr-1, X-Delta-1 and N-tubulin and counteracts X-ngnr-1- and retinoic acid-mediated differentiation. Xrx1 does not act by increasing lateral inhibition but by inducing the antineurogenic transcriptional repressors Xhairy2 and Zic2, and by repressing p27Xic1. The effects of Xrx1 on proliferation, neurogenesis and gene expression are restricted to the most rostral region of the embryo, implicating this gene as an anterior regulator of neurogenesis responsible for the maintenance of anterior neuronal precursors in a proliferative state (Andreazzoli, 2003).
In Drosophila, prepattern genes that are expressed before the onset of neurogenesis control the region-specific activation of proneural genes. Prepattern genes include hairy and the Iroquois family homeobox genes. In vertebrates, homologs of the Iroquois genes play a similar role, functioning during early neurulation in the specification of neural precursors in the posterior neural plate. There are several similarities between the activities of Xenopus Iroquois (Xiro) genes and Xrx1, since these genes: (1) repress neuronal differentiation at early neurula; (2) do not work through lateral inhibition; (3) are repressed by X-ngnr-1 and activated by hedgehog signaling; (4) upregulate Xhairy2 and Zic2; and (5) act after neural induction and before the selection of neuronal precursor cells (Andreazzoli, 2003).
Moreover, the loss of function of Rx genes in vertebrates as well as of the Iroquois complex in Drosophila, results in the absence of the structures where these genes are normally expressed. Beside these similarities, it is worth noting that while the Iroqouis genes play a role in positioning domains of proneural gene expression, this function has not been demonstrated for the Rx genes. However, the complementary expression of Xrx1 and Xiro genes together with their similar activities suggest the existence of two gene systems, one acting in the anterior and the other in the posterior neural plate, the function of which is to control the timing and delimit the location of neuronal differentiation (Andreazzoli, 2003).
Major histocompatibility complex (MHC) class II deficiency, or bare lymphocyte syndrome (BLS), is a disease of gene regulation. Patients with BLS have been classified into at least three complementation groups (A, B, and C) believed to correspond to three distinct MHC class II regulatory genes. The elucidation of the molecular basis for this disease will thus clarify the mechanisms controlling the complex regulation of MHC class II genes. Complementation groups B and C are characterized by a lack of binding of RFX, a nuclear protein that normally binds specifically to the X box cis-acting element present in the promoters of all MHC class II genes. RFX has been purified to near homogeneity by affinity chromatography. Using an in vitro transcription system based on the HLA-DRA promoter, it has been shown that extracts from RFX-deficient cells from patients with BLS (BLS cells) in groups B and C, which are transcriptionally inactive in this assay, can be complemented to full transcriptional activity by the purified RFX. As expected, purified RFX also restores a completely normal pattern of X box-binding complexes in these mutant extracts. This provides the first direct functional evidence that RFX is an activator of MHC class II gene transcription and that its absence is indeed responsible for the regulatory defect in MHC class II gene expression in patients with BLS (Durand, 1994).
DNA methylation inhibits transcription driven by the collagen alpha2(I) promoter and the 5' end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha2(I) gene. Complex formation increases when the CpG site at +7 base pairs from the transcription start site is methylated. A RFX1-specific antibody supershifts the collagen gene DNA-protein complexes. In vitro translated RFX1 protein forms a specific complex with the collagen DNA sequence; the complex is also supershifted with the RFX1 antibody. MDBP/RFX displays a higher affinity binding to the collagen sequence if the CpG at +7 is mutated in a manner similar to TpG. This same mutation within reporter constructs inhibits transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen alpha2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region (Sengupta, 1999).
The proliferating cell nuclear antigen (PCNA) is an essential eukaryotic DNA replication factor that is transcriptionally regulated by the adenovirus oncoprotein E1A 243R. Inducibility of the human PCNA promoter by E1A 243R is conferred by the cis-acting PCNA E1A-responsive element (PERE), which associates with the ATF-1, cAMP response element-binding protein (CREB), and RFX1 transcription factors and is modulated by cellular proteins such as the coactivator CREB-binding protein (CBP) and tumor suppressor p107. RFX1 also forms a complex with sequences in the PCNA promoter of mouse and rat that share homology with the RFX1 consensus site. To explore the role of RFX1 in regulating the PCNA promoter, the effects were examined of mutations in the human PERE on RFX1 binding and gene expression. Mutations within the RFX1 consensus binding site reduces RFX1 binding, whereas mutations upstream of the site, or on its border, increases RFX1 binding. These mutations also affect the transcriptional activity of PCNA-chloramphenicol acetyltransferase reporter constructs in transient expression assays. The relative transcriptional activity of mutant PCNA promoters, both in the presence and absence of E1A 243R, is inversely related to their ability to complex with RFX1. These findings suggest that the binding of RFX1 is influenced by sequences outside its consensus binding site and that this transcription factor plays an inhibitory role in the regulation of PCNA gene expression (Liu, 1999).
Interleukin-5 (IL-5) plays a central role in the differentiation, proliferation, and functional activation of eosinophils. The specific action of IL-5 on eosinophils and hematopoietically related basophils is regulated by the restricted expression of IL-5 receptor alpha (IL-5Ralpha), a subunit of high-affinity IL-5R, on these cells. An enhancer-like cis element has been identified in the IL-5Ralpha promoter that is important for both full promoter function and lineage-specific activity. RFX2 protein specifically binds to this cis element. RFX2 belongs to the RFX DNA-binding protein family, the biological role of which remains obscure. Using an electrophoretic mobility shift assay, it has been shown that RFX1, RFX2, and RFX3 homodimers and heterodimers specifically bind to the cis element of the IL-5Ralpha promoter. The mRNA expression of RFX1, RFX2, and RFX3 is detected ubiquitously, but in transient-transfection assays, multimerized RFX binding sites in front of a basal promoter efficiently function in a tissue- and lineage-specific manner. To further investigate RFX functions on transcription, full-length and deletion mutants of RFX1 were targeted to DNA through fusion to the GAL4 DNA binding domain. Tissue- and lineage-specific transcriptional activation with the full-length RFX1 fusion plasmid on a reporter controlled by GAL4 binding sites was observed. Distinct activation and repression domains within the RFX1 protein were further mapped. These findings suggest that RFX proteins are transcription factors that contribute to the activity and lineage specificity of the IL-5Ralpha promoter by directly binding to a target cis element and cooperating with other tissue- and lineage-specific cofactors (Iwama, 1999).
Epstein-Barr virus (EBV)-induced B-cell growth transformation, a central feature of the virus' strategy for colonizing the human B-cell system, requires full virus latent gene expression and is initiated by transcription from the viral promoter Wp. Interestingly, when EBV accesses other cell types, this growth-transforming program is not activated. The present work focuses on a region of Wp which in reporter assays confers B-cell-specific activity. Bandshift studies indicate that this region contains three factor binding sites, termed sites B, C, and D, in addition to a previously characterized CREB site. Site C binds members of the ubiquitously expressed RFX family of proteins, notably RFX1, RFX3, and the associated factor MIBP1, whereas sites B and D both bind the B-cell-specific activator protein BSAP/Pax5. In reporter assays with mutant Wp constructs, the loss of factor binding to any one of these sites severely impaired promoter activity in B cells, while the wild-type promoter could be activated in non-B cells by ectopic BSAP expression. It is suggested that Wp regulation by BSAP helps to ensure the B-cell specificity of EBV's growth-transforming function (Tierney, 2000).
Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking transcription elongation at sites near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1, which acutely activates protein kinase C (PKC), decreases the levels of myc mRNA and Myc protein. The inhibition of Myc synthesis accounts for the drop in Myc protein, because PMA treatment has no effect on Myc turnover. Treatment with PMA or bryostatin 1 increases nuclear protein binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum supershifts MIE1-protein complexes. Increased MIE1 binding is independent of protein synthesis and is abolished by a selective PKC inhibitor, which also prevents the effect of PMA on myc mRNA and protein levels and Myc synthesis. PMA treatment increases RFX1 in the nuclear fraction and decreases it in the cytosol without affecting total RFX1. Transfection of HL-60 cells with myc reporter gene constructs shows that the RFX1-binding X box is required for the down-regulation of reporter gene expression by PMA. These findings suggest that nuclear translocation and binding of RFX1 to the X box cause the down-regulation of myc expression, which follows acute PKC activation in undifferentiated HL-60 cells (Chen, 2001).
Microtubule-associated protein (MAP) 1A is expressed abundantly in mature neurons and is necessary for maintenance of neuronal morphology and localization of some molecules in association with the microtubule-based cytoskeleton. Previous studies indicated that its complementary expression together with MAP1B during the nervous system development is regulated at the transcriptional level and that the mouse MAP1A gene is transcribed under the control of 5 and intronic promoters. The regulatory mechanisms that govern the neuronal cell-specific activation of the MAP1A 5 promoter have been examined. Two regulatory factor for X box (RFX) binding sites have been found in exon1 of both the mouse and human genes that are important for effective transcriptional repression observed only in non-neuronal cells by reporter assays. Among RFX transcription factor family members, RFX1 and 3 are those that mainly interact with repressive elements in vitro. Cotransfection studies indicate that RFX1, which is expressed ubiquitously, downregulates the MAP1A 5 promoter activity in non-neuronal cells. Unexpectedly, RFX3, which is abundantly expressed in neuronal cells, downregulates the transactivity as well, when RFX3 is expressed in non-neuronal cells. Neither RFX1 nor RFX3 downregulate the transactivity in neuronal cells. These results suggest that RFX1 and 3 are pivotal factors in downregulation of the MAP1A 5 promoter in non-neuronal cells. The cell type-specific downregulation, however, does not depend simply on which RFX interacts with the elements, but seems depending on underlying profound mechanisms (Nakayama, 2002).
The transcription start site of the collagen alpha2(1) gene (COL1A2) has a sequence-specific binding site for a DNA methylation-responsive binding protein called regulatory factor for X-box 1 (RFX1). RFX1 forms homodimers as well as heterodimers with RFX2 spanning the collagen transcription start site. Methylation at +7 on the coding strand increases RFX1 complex formation in gel shift assays. Methylation on the template strand, however, does not increase RFX1 complex formation. DNA from human fibroblasts contains minimal methylation on the coding strand (<4%) with variable methylation on the template strand. RFX1 acts as a repressor of collagen transcription as judged by in vitro transcription and co-transfection assays with an unmethylated collagen promoter-reporter construct. In addition, an RFX5 complex present in human fibroblasts interacts with the collagen RFX site, which is not sensitive to methylation. This is the first demonstration of RFX5 complex formation on a gene other than major histocompatibility complex (MHC) promoters. Also, RFX5 represses transcription of a collagen promoter-reporter construct in rat fibroblasts that have no detectable RFX5 complex formation or protein. RFX5 complex activates MHC II transcription by interacting with an interferon-gamma (IFN-gamma)-inducible protein, major histocompatibility class II trans-activator (CIITA). Collagen transcription is repressed by IFN-gamma in a dose-dependent manner in human but not in rat fibroblasts. IFN-gamma enhances RFX5 binding activity, and CIITA is present in the RFX5 complex of IFN-gamma-treated human fibroblasts. CIITA represses collagen gene transcription more effectively in human fibroblasts than in rat fibroblasts, suggesting that the RFX5 complex may, in part, recruit CIITA protein to the collagen transcription start site. Thus the RFX family may be important repressors of collagen gene transcription through an RFX binding site spanning the transcription start site (Sengupta, 2002).
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