InteractiveFly: GeneBrief

Rfx: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The transcription factor Rfx (Regulatory factor X) is an essential regulator of ciliated sensory neuron differentiation in Drosophila. Ciliated neurons play an important role in sensory perception. Modified cilia at dendrite endings serve as sites of sensory signal capture and transduction. Mutations are described that affect the transcription factor Rfx and genetic rescue experiments demonstrate its central role in sensory cilium differentiation. Rfx mutant flies show defects in chemosensory and mechanosensory behaviors but have normal phototaxis, consistent with Rfx expression in ciliated sensory neurons and neuronal precursors but not in photoreceptors. The mutant behavioral phenotypes are correlated with abnormal function and structure of neuronal cilia, as shown by the loss of sensory transduction and by defects in ciliary morphology and ultrastructure (Dubruille, 2002).

In multicellular organisms, sensory perception relies on cells with specialized sensory structures. In many sense organs these structures are modified cilia: vertebrate examples include the outer segments of the retinal and pineal photoreceptors, the kinocilia associated with the stereocilia of the hair cells and the multiple cilia on the sensory neurons in the main olfactory epithelium. In invertebrates, chemosensory sensilla and many mechanosensory organs, but not photoreceptors, are innervated by ciliated neurons (Dubruille, 2002).

Cilia are found in most eukaryotes except for fungi and higher plants. They are distinguished by an axoneme, a radially symmetric cytoskeleton of nine microtubule doublets and associated structures, enclosed in an extension of the plasma membrane. The presence or absence of a central microtubule pair classifies cilia into two types. Those with a central pair (9+2 configuration) usually have a propulsive function, while those without a central microtubule pair (9+0 configuration) are found on many animal cell types, where they are known as 'primary' cilia. Some 9+0 cilia [e.g. those on the mammalian embryonic node, move with a circular, whirling motion. Sensory cilia are derived from primary cilia and have been modified to varying degrees; most are probably non-motile (Dubruille, 2002).

In Drosophila, nonvisual sensory perception relies on two major classes of sense organs. Type I organs or sensilla include one or more neurons and several support cells that construct specialized sensory structures such as bristles. They include the olfactory and mechanosensory bristles, as well as chordotonal organs (internally located stretch receptors that transduce auditory or proprioceptive stimuli). Each neuron in a type I organ bears a single sensory dendrite with a modified cilium. Type II sense organs are multidendritic neurons that lack cilia and specialized support cells. Their sensitivities are not known, but they also have been suggested to function as proprioceptors or mechanoreceptors (Dubruille, 2002).

Several mutants affecting sensory perception by type I sensilla have been isolated in Drosophila in screens for loss of mechanosensation, audition or olfaction. Those that have been molecularly characterized include nompC, which encodes a member of the TRP channel superfamily, and nompA, a component of the dendritic cap that ensheaths the sensory cilium. In nompA mutants, defects in mechanosensory behavior and electrophysiology are associated with disconnection of dendritic caps from the sensory cilia. Two other mutants specifically affecting chordotonal organs, btv and tilB, have axonemal defects illustrating the importance of axoneme integrity for chordotonal organ function (Dubruille, 2002 and references therein).

Structural components of cilia such as tubulins, tektins and axonemal dynein subunits have mostly been isolated from the single-celled alga Chlamydomonas reinhardtii and from sea urchin but are highly conserved in other phyla. An intraflagellar transport (IFT) mechanism required for ciliary assembly is also widely conserved. Best characterized in Chlamydomonas, IFT is a rapid movement of particles along the axonemal microtubules of cilia and flagella. Although many individual proteins involved in cilium architecture and IFT are well described, factors that regulate and coordinate their expression are poorly understood. In C. elegans, one such factor is DAF-19, a member of the RFX family of transcription factors (Swoboda, 2000). Loss of function daf-19 mutations result in the absence of cilia in sensory neurons, the only type of ciliated structures present in the nematode. DAF-19 regulates several genes required for normal sensory cilium formation, including components of the intraflagellar transport complex: che-2, osm-1, osm-5 and osm-6 (Haycraft, 2001; Qin, 2001; Swoboda, 2000: Dubruille 2002 and references therein).

RFX transcription factors are defined by a 76 amino acid DNA-binding domain with a characteristic wing-helix structure. The yeasts S. pombe and S. cerevisiae each have a single RFX factor (Huang, 1998; Wu, 1995), while five RFX proteins have been identified in mammals. Mammalian RFX5 is essential for the transcription of MHC class II genes in the immune response (for a review, see Reith, 2001), but little is known about the cellular functions of the other mammalian RFX proteins. Two Rfx genes can be identified in Drosophila (Durand, 2000). Rfx is homologous to daf-19 and to mammalian Rfx1, Rfx2 and Rfx3, whereas the second gene shares conserved motifs with Rfx5, the most divergent mammalian Rfx. Rfx is expressed in the peripheral nervous system (PNS), in the brain and in the testis during Drosophila development (Vandaele, 2001; Dubruille, 2002).

Two Rfx mutations have been described that define a novel pseudolethal complementation group. One of these mutations, Rfx²⁵³ alters an absolutely conserved serine in the DNA-binding domain. This mutation is the first one affecting the RFX DNA-binding domain, thus confirming its importance for the biological function of RFX transcription factors. It supports predictions from crystallographic studies (Gajiwala, 2000) showing that this serine residue contacts DNA (Dubruille, 2002).

Rfx function in ciliated sensory neurons is essential for the proper signaling in type I sensory organs. Rfx mutant larvae have severe chemosensory and olfactory defects, being insensitive to attractive and repulsive odors. These sensory defects could explain the peculiar, potentially insufficient foraging and feeding behavior of the mutants, and could thus be responsible for major developmental delays, leading to lethality at significant frequencies. Moreover, consistent with their adult uncoordinated phenotype, Rfx mutant bristles show clear electrophysiological defects: they are completely unable to generate mechanoreceptor potentials or currents. Reductions in resting transepithelial potentials and transepithelial resistance are not sufficient to explain the complete loss of transduction, because mutants with Transepithelial mechanoreceptor potentials (TEPs) in the normal range still show no receptor current, and receptor currents can not be restored by voltage clamping the TEP at wild-type levels. Although recordings were limited to mechanosensory macrochaete bristles, the combination of mechanosensory and chemosensory behavioral phenotypes suggests a global defect in type I sense organ function, consistent with the pattern of Rfx expression (Dubruille, 2002).

Remarkably, the sensory defects are correlated with abnormal morphogenesis of the dendrite, which affects primarily the organization of its ciliated sensory endings. Indeed Rfx mutants show sensory dendrites that have severe cilium defects in femoral chordotonal organs as well as wing campaniform sensilla. This probably reflects a general defect in cilium assembly, since both the proximal and the distal parts of the organelle are disorganized -- that is, both rootlet apparatus and axonemal structures are absent from scolopidia in Rfx mutant antennae as observed by electron microscopy. All together, these observations show that the dendrite cilium is required for signal transduction in these neurons (Dubruille, 2002).

The observed mutant sensory defects are probably not restricted to only cilium disorganization. Indeed, electron microscopy reveals that the scolopale cell of chordotonal organs is also modified in the mutant antennae: the scolopale rods and membranes are disorganized compared with wild type. A similar effect on the sheath cell (the homolog of the scolopale cell in bristle organs) and other supporting cells could underlie the reduced epithelial resistance and TEP observed in Rfx mutant bristles. Rfx is expressed as early as the first neural precursor and is maintained throughout successive asymmetric division in the type I neuron lineage. Rfx is transiently expressed in the accessory scolopale cell (Vandaele, 2001). It could thus be necessary for proper accessory cell differentiation and in particular for cell junction formation between these cells (Dubruille, 2002).

Another observation suggesting that mutant defects are probably not exclusively restricted to the cilium is the reduced anti-HRP staining in mutant embryos. Anti-HRP has been shown to bind to neuronal membranes. It is known to recognize an epitope of carbohydrate origin, an alpha1,3-fucosylated, N-linked glycan associated with proteins such as Nervana 2, an Na⁺K⁺ ATPase expressed in all neurons, and with cell adhesion molecules, Fasciclin I and II, neurotactin and neuroglian. In Rfx mutants, either the expression of these proteins is downregulated or the relevant post-translational modification does not operate efficiently. Interestingly, a reduction in anti-HRP staining is seen in both type I and non-ciliated type II PNS neurons, even though no morphological defects of type II neurons are observed by anti-HRP labeling. Type I and type II neurons arise from common precursors expressing Rfx, but the latter lose Rfx expression upon differentiation and only type I neuron lineage express Rfx throughout embryonic development (Vandaele, 2001). Anti-HRP staining phenotype may suggest that no PNS neurons reach full terminal differentiation in Rfx mutant. Rfx transient expression in type II precursors could also be necessary for their differentiation. The anti-HRP staining phenotype could thus reflect a more general function for Rfx during neuronal differentiation (Dubruille, 2002).

Cilium defects are associated with dendrite morphogenesis defects such as incorrect positioning in the wing, swollen shape in the femur and the wing. It is not known if the cilium defect is responsible for these abnormal dendrite differentiations. The swollen dendrite could be a consequence of a defect in outward transport of intraflagellar transport, resulting in an accumulation of unassembled cilium components. The cilium defect could be responsible for incorrect dendrite positioning by disrupting close interactions between the cilium and structures of the sheath and the hair cell. Interestingly, the dendritic cap in the Johnston's chordotonal organ is not affected by cilium defects: this suggests that its production by the scolopale cell does not depend on the cilium. Analyzing Rfx regulatory pathways with the available mutants will allow these question to be clarified (Dubruille, 2002).

Cilia are assembled through a process called intraflagellar transport (IFT) first described in the unicellular green alga Chlamydomonas and highly conserved throughout evolution. osm-1, osm5 and osm-6, which encode proteins involved in IFT are under the control of daf-19, the sole RFX gene in C. elegans (Swoboda, 2000; Haycraft, 2001; Qin, 2001). osm-1 and osm-6 encode homologous proteins to Chlamydomonas IFT polypeptides p172 and p52. osm-5 encodes a protein involved in cilium assembly and is homologous to Chlamydomonas IFT88 protein and to a mouse protein called Tg737/Polaris that is required for assembly of the primary cilia in mouse kidney and node. Thus, IFT involves conserved proteins in many different organisms and plays fundamental roles in a variety of physiological functions. The conservation of function of the two invertebrate homologous genes as essential regulators of sensory ciliogenesis will allow a fruitful comparative identification of novel target genes likely to be involved in IFT (Dubruille, 2002).

In C. elegans, the only ciliated cells are the sensory neurons; sperm cells are not flagellated and do not express daf-19. Conversely, in Drosophila, spermatozoids have an unusually long flagella. Interestingly, Rfx is also expressed in spermatids but after flagellar elongation during a stage in which only a few genes have been reported to be transcribed, suggesting a different role in spermatogenesis. Rfx mutant sperm are normally elongated and motile, and preliminary electron microscopy data show no gross defects in spermatid flagellar 9+2 ultrastructure. Because Rfx mutants are too uncoordinated to mate, it is not known if mutant spermatozoids are functional in fertilization. Rfx may have a function in spermatogenesis other than ciliary assembly. This would imply a different mode of flagellar assembly in Drosophila spermatozoids that could reflect structural and functional differences between sensory cilia and sperm flagella. Rfx is also expressed in subdomain-specific regions of the brain. No ciliated cells have been described in Drosophila brain at the present time, whereas ciliated cells are present in mammalian brain. Studying Rfx function in Drosophila will thus bring important information regarding the diversification of the function of RFX transcription factors in evolution. Whether the ciliary function of RFX genes has been conserved in vertebrates remains an unanswered question and is of great interest regarding human pathologies associated with cilium defects (Dubruille, 2002).

REGULATION

Transcriptional Regulation

The gene regulatory cascade linking proneural specification with differentiation in Drosophila sensory neurons

In neurogenesis, neural cell fate specification is generally triggered by proneural transcription factors. While the role of proneural factors in fate specification is well studied, the link between neural specification and the cellular pathways that ultimately must be activated to construct specialised neurons is usually obscure. High-resolution temporal profiling of gene expression reveals the events downstream of atonal proneural gene function during the development of Drosophila chordotonal (mechanosensory) neurons. Among other findings, this reveals the onset of expression of genes required for construction of the ciliary dendrite, a key specialisation of mechanosensory neurons. It was determined that atonal activates this cellular differentiation pathway in several ways. Firstly, atonal directly regulates Rfx, a well-known highly conserved ciliogenesis transcriptional regulator. Unexpectedly, differences in Rfx regulation by proneural factors may underlie variations in ciliary dendrite specialisation in different sensory neuronal lineages. In contrast, fd3F encodes a novel forkhead family transcription factor that is exclusively expressed in differentiating chordotonal neurons. fd3F regulates genes required for specialized aspects of chordotonal dendrite physiology. In addition to these intermediate transcriptional regulators, it was shown that atonal directly regulates a novel gene, dilatory, that is directly associated with ciliogenesis during neuronal differentiation. This analysis demonstrates how early cell fate specification factors can regulate structural and physiological differentiation of neuronal cell types. It also suggests a model for how subtype differentiation in different neuronal lineages may be regulated by different proneural factors. In addition, it provides a paradigm for how transcriptional regulation may modulate the ciliogenesis pathway to give rise to structurally and functionally specialised ciliary dendrites (Cachero, 2011).

Numerous genetic and misexpression analyses in a range of organisms have shown that proneural factors influence a neuron's ultimate phenotype (including its subtype identity) at an early stage in its development. However, the nature of this influence on the cell biological processes of neuronal differentiation has remained obscure. This study bridges the gap between early specification by the proneural factor, ato, and the differentiation of Ch neurons. The current model in both Drosophila and vertebrates is that proneural factors activate two types of target gene during neural precursor specification: a common target set for shared neuronal properties and a unique target set for subtype-specific properties. The data suggest that such neuronal subtype differences are ultimately controlled by proneural factors in several ways: by the differential regulation of both specific and common intermediate transcription factors, which in turn regulate genes for aspects of neuronal structural and functional differentiation, and by direct regulation of potential differentiation genes (Cachero, 2011).

The proneural factors ato and sc commit cells to similar but distinct neural precursor fates: Ch and ES neurons are evolutionarily related cell types with similar but distinct structural and physiological properties. Notably, both are characterised by the possession of specialised ciliary-based dendrites. Thus, ciliogenesis is a key pathway that must ultimately be activated in sensory neurons subsequent to proneural factor function. However, there are important differences between the dendrites of Ch and ES neurons. Ch dendrites have a more prototypically organised axonemal structure and possess a characteristic ciliary dilation - a specialisation that separates the Ch ciliary dendrite into functionally distinct zones. Moreover, there is evidence for an active 'beat' of Ch cilia during sensory transduction. In general, ES dendrites appear reduced in structure: although a basal body and short axoneme are present, the tip of the dendrite consists of a 'tubular body' of irregularly packed microtubules. Thus the basic ciliogenesis pathway must be modulated differently in Ch and ES differentiation, and ultimately this must reflect a difference in function between ato and sc proneural factors. The ciliogenic regulator Rfx is expressed and required for both ES and Ch lineages, but it is more strongly and more persistently expressed in Ch lineages (the Ch-enriched pattern). This modulation of Rfx expression is at least partly due to differences in its regulation by proneural factors, since it appears to be a direct target of ato but not sc. This study hypothesises that differences in Rfx regulation by the proneural factors lead to differences in implementation of a core cilia biogenesis program, thereby directly linking early proneural factor function with key differences of neuronal morphology. Consistent with this idea, the data show that several known or predicted ciliogenesis genes also exhibit this Ch-enriched pattern, and some of these are predicted or known Rfx targets (Cachero, 2011).

In this view, the subtype differences between Ch and ES neurons are partly produced by quantitative differences in timing or level of expression of a common differentiation process, which ultimately depends on a qualitative difference in Rfx regulation by the proneural factors. A possible example of this is CG6129. This gene is a predicted Rfx target gene and is expressed in a Ch-enriched pattern (Laurenon, 2007). The homologous mouse protein (Rootletin) localises to the ciliary rootlet and is required for its formation. Thus Ch-enriched expression of CG6129 explains the presence of the ciliary rootlet in Ch neurons but not ES neurons. One prediction of this hypothesis is that overexpression of Rfx in ES neurons will upregulate Ch-enriched genes, and this is borne out by preliminary experiments that show an increase in CG6129 expression in ES neurons upon Rfx overexpression. It is notable that differences in IFT activity are proposed to underlie differences in ciliary morphology while RFX class factors have been associated with regulating genes for IFT in a variety of organisms. This work suggests that variations in Rfx expression level and timing should be explored as a possible factor in cilium diversity (Cachero, 2011).

fd3F fits the more conventional view of a proneural target gene that implements a subtype-specific program of differentiation. It is expressed downstream of ato uniquely in Ch neurons and regulates genes required for functional specialisation of the Ch ciliary dendrite. It is likely that Forkhead factors regulate specialisation of ciliogenesis in other organisms. In C. elegans, FKH-2 is expressed widely early in development but is also required specifically for ciliary specialisation of one type of sensory neuron. Foxj1 in mice, Xenopus, and zebrafish appears to be required for the motile cilia of the lung airway and embryonic node, but not for primary cilia. It remains to be determined whether fd3F regulates the machinery for the active beat that occurs in Ch dendrites as part of sensory transduction. Together, these studies of Rfx and fd3F extend the previously limited knowledge of the gene regulatory network underlying ciliogenesis and provide insight into how the core program may be modified to produce the highly specialised and diverse morphologies that cilia adopt for different functions (Cachero, 2011).

Previous to this study, little was known about how ato/sc proneural genes control the acquisition of Ch/ES subtype identity, except that regulation of the Cut homeodomain transcription factor is involved. Mutant and misexpression analyses show that cut is a fate selector switch for ES identity downstream of sc, but nothing is known of its mode of action or targets. Whereas Rfx and fd3F functions are likely to be confined to neuronal morphology, cut affects the identity of support cells too. As a fate switch in the entire lineage, it appears likely that cut is involved in high-level fate specification (like proneural genes) rather than regulating aspects of differentiation directly. However, it is also possible that cut may repress ciliogenesis genes in ES neurons, either directly or by repressing Rfx expression. It will be important to integrate cut into the Ch/ES gene regulatory network in the future (Cachero, 2011).

In the temporal expression profiling data, there is a steady increase in the number of known or suspected differentiation genes expressed in developing Ch cells. Many more are not expressed until after the analysis ends. Ciliogenesis is a highly intricate cellular process requiring the coordination of perhaps hundreds of genes and differences in expression onset may indicate prerequisite steps in the process of differentiation and ciliogenesis. A surprising observation was the significant number of ciliogenesis and differentiation genes that are expressed even at the earliest profiling time point. This is unexpected, since the earliest time point is predicted to be not only before differentiation but also even before cell divisions have generated the neurons. It is suggested that further analysis of expression timing may lead to insights into the cell biology of ciliogenesis. The early activation of differentiation genes may reflect the rapid pace of development in the Drosophila embryo. Thus, early expression of ciliogenesis genes may provide components that prime cells for rapid cilium assembly later once differentiation has been triggered. Along these lines, the findings mirror striking observations of retinal ganglion cells, whose rapid differentiation within 15 minutes of the exit from mitosis has been taken to imply that genes required in postmitotic cells must be transcribed before cell division. A more intriguing possibility is that early expression reflects an orderly time course for ciliogenesis that begins many hours before the final cell division. For example, unc is thought to be required for the conversion of the mitotic centriole to ciliogenic basal body, but this stuyd found that the mRNA and fusion protein are expressed even in SOPs, several cell divisions before terminal differentiation. Interestingly, in mammals newly replicated centrioles mature over two cell cycles. It is conceivable that the sensory neuron basal body might similarly need time to mature (Cachero, 2011).

Since Rfx and some ciliogenesis genes are expressed in SOPs, what prevents ciliogenesis from being activated in the non-neuronal support cells? One possibility would be an extension of model recently proposed for the generation of support cell differences, in which Notch signalling between daughter cells confines the function of genes to one branch of the lineage. This would predict that ciliogenesis genes and/or Rfx are Notch target genes. Another possibility is that some of the gene products are asymmetrically segregated. Thirdly, ciliogenesis may not be triggered until one or more key gene products are produced in the neuronal cell (Cachero, 2011).

As a corollary, it will be important to explore further the gene regulatory network underlying the temporal and cell-type differences in ciliogenesis genes. Some early expressed differentiation genes are known or predicted Rfx targets (Laurenon, 2007). This gives a rationale for the early regulation of Rfx by ato in Ch lineages. However, in both C. elegans and D. melanogaster, Rfx regulates only a subset of ciliogenesis genes (notably, it does not regulate IFT-A genes). Further studies on ato target genes and the ciliogenesis regulatory network in sensory neurons will identify other important regulators. It remains to be determined how many differentiation genes are, like dila, direct targets of ato. Interestingly, vertebrate proneural factors are hypothesised to regulate directly the transition from cycling neural progenitor (or neural stem cell) to postmitotic differentiating neuron. Perhaps ato has retained some part of an ancestral proneural factor function in direct regulation of terminal differentiation despite the subsequent evolution of SOPs that must undergo several divisions before differentiating (Cachero, 2011).

Targets of Activity

Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species

Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, novel genes were identified regulated by dRFX in Drosophila. A subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. An X-box consensus sequence was designated (GYTRYY N1-3 RRHRAC) and a genome wide computer screen was carried out to identify novel genes under RFX control. 412 genes were identified that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. 25 of these 83 genes were analyzed, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. It is concluded that this X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. A highly valuable Drosophila cilia and basal body dataset was established. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-Biedl syndrome (Laurençon, 2007).

Ciliogenic RFX regulatory networks are conserved between C. elegans and D. melanogaster. Based on these first observations, the genomic screens that were conducted combined with functional and in vivo gene analyses led to the identification of at least 11 novel genes that had never been described as RFX targets in any biological model. In addition, this screen allowed identification of at least two novel genes specifically expressed in ciliated sensory neurons in Drosophila that are potentially involved in sensory ciliogenesis. These results validate the accuracy of the screens. This work thus provides a new set of candidate genes for further functional studies in ciliogenesis (Laurençon, 2007).

The presence of a conserved X-box upstream of genes in both D. melanogaster and D. pseudoobscura is thus a good prognostic factor to predict novel dRFX target genes. The genome of both Drosophila species was screened for the presence of X-boxes. All possible matches were sought to a defined motif sequence using a Perl based algorithm. The most degenerated consensus RYYNYY N1-3 RRNRAC found 50,000 hits throughout the entire genome of D. melanogaster and, therefore, could not be used within the experimental framework. Five different more restricted consensus motifs were chosed that cover X-boxes of the entire set of known target genes at the time. Four (RYYVYY N1-3 RRHRAC, GYTNYY N1-3 RRNRAC, GYTDYY N1-3 RRNRAC, GYTRYY N1-3 RRHRAC) were searched in a 1 kb window upstream of the ATG, and the less degenerated one, RTNRCC N1-3 RGYAAC, in a 3 kb window (Laurençon, 2007).

Under these conditions, 4,726 non-redundant genes in D. melanogaster and 3,848 in D. pseudoobscura with an X-box upstream of the start codon were selected. Based on a best hit reciprocal search between the two coding sequence (CDS) lists, 1,462 homologous genes were identified having an X-box in their 5' region in both species. This first set of 1,462 genes was further restricted by selecting only genes that share an X-box with no more than 4 bases different (out of the 12 nucleotides recognized by the protein on either side of the spacer) between each species and in a conserved position upstream of the ATG (500 bp difference at most). The list was thus restricted to a subset of 412 genes. An even more restricted subset of genes was selected using the X-box motif GYTRYY N1-3 RRHRAC, which was found upstream of most known target RFX genes at the beginning of this work, leading to a list of 83 genes. Indeed, among the identified dRFX target genes for which a conserved X box was found in both Drosophila species, the highest percentage of target genes (50%, 8 out of 16) was found in this list of 83 genes. The remaining 50% of known RFX target genes were not selected by the X-box screen and thus represent false negatives (Laurençon, 2007).

The Drosophila genome wide X-box screen led to the identification of 83 X-box genes among which 11 novel RFX targets were identified. Combined with the genes identified by comparisons to C. elegans or to other genomic studies in Drosophila, 35 genes regulated by dRFX are reported in this study. Most of these genes can be classified based on their described function. Many of the RFX target genes are involved in IFT, which is necessary for cilium assembly and function. Remarkably, a second class of genes regulated by dRFX includes all the Drosophila homologs of Bardet-Biedl Syndrome (BBS) genes. Similarly, most C. elegans BBS genes are regulated by DAF-19. This strong dependence of BBS genes on RFX control may thus be conserved in mammals. Hence, RFX proteins may be involved in BBS in humans. Interestingly, two of the three Drosophila genes coding for proteins with B9 domains are also controlled by dRFX (tectonic, CG14870). One human B9 domain protein, MKS1, is known to be involved in the human Meckel-Gruber syndrome. The molecular function of this domain is unknown and work in Drosophila suggested that these two B9 domain containing proteins are likely involved in ciliogenesis. Several of the novel dRFX target genes that were identified in this study encode known components of the ciliary axoneme and associated structures, such as axonemal dyneins or rootletin. Other genes encode different types of proteins likely involved in sensory transduction (CG4536/osm-9/TRPV4 or MIP-T3). A last class includes genes for which the function is either not described or poorly understood, such as CG31036 and CG13125. However, functional studies strongly suggest that they are also probably involved in sensory ciliogenesis in Drosophila as well. Thus, RFX target genes play various roles in ciliary structure and function and the X-box search strategy has proven to be useful to identify novel ciliogenic genes (Laurençon, 2007).

This full set of dRFX target genes in Drosophila is of crucial importance, since it is now possible to more precisely define X-box sequences and the promoter context required for dRFX control. This will be particularly useful for further database mining of dRFX target genes in Drosophila. In fact, several genes that are under dRFX control, for example CG4525, CG17599) for which an X-box can be identified did not come out in the whole genome X-box screen. Several reasons can explain this result. First, homologs were not all annotated in CDS listings that were available at the time of the search (for example, CG18631, CG9595, nompB in D. pseudoobsura). Second, annotation of both Drosophila databases is incomplete, as sometimes the start codon is not properly defined for all genes. The X-box search algorithm keeps only genes for which the X-box match is upstream of the ATG. For example, for CG15666/GA13881, it was clearly predicted that the correct ATG should be considered 75 bp downstream of the currently defined ATG, based on evolutionarily conserved sequences. This definition clearly excludes the homologous genes CG15666 and GA13881 from the dataset. However, the X-box consensus cannot define a clearly conserved X-box match in the two Drosophila species for genes that appear to be down-regulated in a dRfx mutant, while several individual X-boxes are found separately in each organism. This could either reflect that these genes are not direct dRFX targets but are shut down by a feedback control loop that is not dependent on a X-box motif, or that the X-box is only loosely conserved in some promoter contexts. Notably, homologs of these genes in C. elegans are under RFX (DAF-19) control and have a well defined X-box (for example, CG9333/che-2, CG13691/bbs-8), which argues in favor of the second possibility. Interestingly, the expression levels were also quantified in control and dRfx deficient Drosophila of several genes of the DCBB dataset that did not come out of the X-box genome-wide motif search. It allowed identification of several novel genes that are indeed down-regulated in dRfx mutants, but for which no conserved X-box can be recognized based on the initial consensus motif. Altogether, these observations clearly highlight the difficulties encountered in motif definition in promoters. Similar conclusions were deduced from a parallel approach performed in C. elegans, which has led to the identification of several novel DAF-19 target genes. Interestingly, in that study the in silico search was associated with microarray analysis of transcripts in wild-type and daf-19 mutant worms. The in silico search allowed the identification of 93 X-box genes. Yet, among the 466 genes that were shown to be down-regulated at least two-fold in microarray hybridization experiments, only 25 were also represented in the 93 in silico X-box gene list. Thus, in silico searches on isolated motifs are likely hampered by a high level of false negatives. In order to improve the screening efficiency, the use of combinatorial motif searches would probably greatly enhance the accuracy of the screen as proposed by other studies. Even though, since conserved X-boxes that were identified are rarely associated with highly conserved surrounding sequences, it is reasonable to assume that other conserved nearby motifs, still to be identified, could help to discriminate between false positives and false negatives (Laurençon, 2007).

35 genes were identified that are transcriptionally down-regulated in dRfx mutants. RFX regulatory networks are conserved between C. elegans and Drosophila as most of the genes controlled by DAF-19 in C. elegans are also under dRFX control in D. melanogaster. Interestingly, the results show that only certain subsets of ciliogenic genes are regulated by RFX proteins. For example, in assay conditions all the genes known to be involved in IFT-A complexes are not regulated by dRFX, whereas all IFT-B homologous proteins are regulated by dRFX. In addition, retrograde motors are also regulated by dRFX (CG15148/btv and CG3769), whereas anterograde motors seem not to be. Indeed, in addition to CG10642/KIF3A, the main described anterograde motor in several organisms, it was shown that two other kinesin subunits, CG17461/Kif3C/osm-3 and CG7293/Klp68D, are invariantly expressed in wild-type and dRfx-deficient Drosophila. It is also interesting to note that all the BBS gene homologs in D. melanogaster are under dRFX control (Laurençon, 2007).

The biological significance of these observations is unclear. It could reflect the fact that IFT-B proteins, BBS proteins and the dyneins involved in IFT are dedicated to ciliogenesis and, therefore, need to be turned on concomitantly only when the cilium is formed, whereas IFT-A complexes or anterograde transport kinesin II share more complex regulatory controls as they might be necessary also for other cellular functions. This is the case for kinesin II motors, but does not seem to be true for IFT-A complexes as these proteins are proposed to be specific for ciliated organisms. In C. elegans, the ciliary IFT machinery works in modular fashion, and it is tempting to speculate that RFX-dependent proteins could be involved in specialized ciliogenic transport modules (Laurençon, 2007).

Genes necessary for centriole biogenesis or replication, such as the recently described DSas-6, DSas-4 or sak genes are not present in the screen and no conserved X-box can be found upstream of these genes. Thus, dRFX does not seem to regulate centriole biogenesis and appears to be restricted to cilia assembly only (Laurençon, 2007).

To find which transcription factors are responsible for governing other sets of ciliary proteins will certainly be one track to follow. Based on the data, it would be of particular interest to compare promoter sequences of genes, either regulated by dRFX, or not. It may allow discovery og novel regulatory motifs and protein modules that are necessary to coordinate ciliogenesis control. So far, only a few transcription factors have been shown to be involved in the control of ciliogenesis: the RFX proteins, Foxj1, and HNF1-beta. However, these have no obvious homologs in Drosophila. Thus, this work strongly suggests that novel transcription factors necessary for ciliogenesis still need to be discovered (Laurençon, 2007).

Some of the novel RFX target genes found in Drosophila were unexpected. For example, several proteins were identified that are proposed to be involved in flagella or cilia motility, such as dynein heavy chains (CG17150/Dhc93AB). Recently, a CG13125 homolog has also been shown to function as a motility factor in T. brucei (TbCMF46). Sensory cilia are thought not to be motile in general. However, it has been shown that Drosophila chordotonal neurons of the antenna generate motion that depends on the integrity of proteins encoded by genes such as CG15148/btv (cytoplasmic dynein heavy chain) or CG14620/tilB (LRRC6 homolog), described to affect the axonemal structure. In addition, cilia of the chordotonal neurons of the grasshopper bend upon vibration stimulation. Thus, proteins involved in axonemal motility might be important for motion generation of the cilium in response to mechanical stimulation. It will be of high interest to determine whether flies defective in these 'motility' genes are affected in hearing and, more specifically, in the motility of the mechanosensory cilium that amplifies hearing vibrations. Interestingly, CG13125/TbCMF46 does not seem to be expressed in fly testis, where the spermatozoa are the only cell type with a motile flagellum in flies. This suggests that like CG15148/btv, CG13125/TbCMF46 function could be restricted to the sensory cilium and, more specifically, in allowing these cilia to mechanically respond to auditory vibrations. Thus, the data suggest that in the fly, possible axonemal motility could be regulated by different subsets of proteins in sperm flagella and in mechanosensory cilia. This is of particular interest with regard to hearing in mammals, which is dependent on hair cell motility. It will be very interesting to determine whether the CG13125/TbCMF46 homolog in mammals does have a specific function in those cell types (Laurençon, 2007).

Three genes (CG6054/Su(fu), CG13415/Cby, CG33038/Ext(2)) were identified that are known to be involved in the hedgehog or wingless signaling pathways in Drosophila. Su(fu) and Ext(2) are involved in the Hedgehog pathway and Su(fu) is localized to cilia in mammalian cells. However, Su(fu) and Ext(2) do not appear to be under dRfx control according to real-time PCR quantification results and may be false positives in the screen. This result argues in favor of the generally accepted observation that the Hedgehog signaling pathway does not seem to depend on ciliogenic proteins in Drosophila. Only Chibby (Cby) is statistically down-regulated two-fold in a dRfx deficient background. Cby was isolated in a two-hybrid screen for armadillo/beta-catenin interactors. RNAi knock-down of Cby in Drosophila embryos leads to ectopic activation of the wingless pathway. Cby is also described to antagonize the Wnt/beta-catenin pathway in mammalian cells. However, the expression pattern of Cby in Drosophila is not documented, so it is not known if the variations of expression observed in the dRfx deficient background are connected to dRfx expression and, thus, if it is biologically significant (Laurençon, 2007).

Among the 83 genes with conserved X-boxes between D. melanogaster and D. pseudoobscura, several genes were hardly detectable by quantitative RT-PCR. Hence it was not possible to determine by this approach if they are under dRFX control. This could reflect that these genes are expressed only in a subset of sensory neurons and, thus, difficult to detect by quantitative RT-PCR. Nevertheless, several genes are interesting as potential ciliogenic or RFX target genes. For example, CG14079 is homologous to a mouse protein that appears to be specific to testis. CG11356 is homologous to mammalian arl13, which has just been isolated in an ethyl-nitroso-urea screen for neural tube defects in mouse. Indeed, mutation of arl13 affects ciliary architecture and Sonic-Hedgehog signaling in mouse. This gene, CG11356, was not found in any previous ciliogenesis study, again illustrating the accuracy of the screen. Functional studies in Drosophila will be of particular importance to demonstrate the role of this gene in sensory ciliogenesis (Laurençon, 2007).

This study has identified more than 30 dRFX target genes in Drosophila by exploiting the efficiency of the X-box promoter motif search by using two divergent Drosophila species in a comparative approach. These full sets of RFX dependent or independent ciliary genes are of particular importance for studies of X-box promoter motifs and associated promoter contexts in Drosophila. More remarkably, the screen allowed the identification of at least two novel genes specific to sensory ciliary architecture in D. melanogaster and provides several new RFX target gene candidates potentially involved in ciliogenesis. This is of particular importance with regard to the growing number of human diseases that are being associated with ciliary defects (Laurençon, 2007).

DEVELOPMENTAL BIOLOGY

Embryonic

Rfx expression was analyzed during embryogenesis. Rfx mRNAs are detected only in the peripheral nervous system and in the brain of the embryo (Durand, 2000).

Drosophila Rfx immunoreactivity is distributed in type I sensory neuron lineage of the peripheral nervous system throughout Drosophila development and thus represents the first described type I lineage characteristic marker in Drosophila. In addition, Rfx is also detected in the brain throughout development and in spermatids in adult flies (Vandaele, 2001).

In embryos, Rfx is first detected at the onset of segmentation in the sensory organ precursors (SOPs) located in the gnathal segment and in the two first SOPs of each thoracic and abdominal hemisegments described as the A (anterior) and the P (posterior) SOPs. At stages 12-14 of embryogenesis, when A and P SOPs have generated new precursors by division or recruitment, Rfx is detected in these secondary precursors. At stage 15, Rfx is found predominantly in nuclei of all chordotonal (ch) and external sensory (es) organ neurons, and, at a lower level, in the accessory sister cells resulting from the last asymmetric division of the precursors. At stage 16 of embryogenesis, Rfx progressively disappears in the accessory sister cells and is only maintained in neuron nuclei at the end of embryogenesis. These results are in agreement with Rfx mRNA expression described previously (Durand, 2000) and provide new important information on Rfx stability. Whereas Rfx messenger RNAs disappear in all cells of the peripheral nervous system except ch neurons at the end of embryogenesis, the protein still persists in es neurons. Two types of sensory neurons can be distinguished in Drosophila: (1) type I neurons that innervate es and ch organs bear a ciliated sensory process at the tip of a unique dendrite and are surrounded by specialized supporting cells, and (2) type II neurons are nonciliated multidendritic neurons. In the embryo, both types of neurons are also characterized by their relative position and shape. Using an antibody that labels all sensory neurons (mAb 22C10), Rfx is found to be restricted to type I neurons of the thoracic and abdominal segments. In the head, Rfx is detected in all sensory neurons, except Bolwig's organ, which represents the future larval visual organ (Vandaele, 2001).

An accumulation of Rfx in the brain is also observed starting from stage 12 of embryogenesis in two small bilateral cell clusters in the procephalic neurogenic region. From stage 14 to 17 of embryonic development, Rfx persists in a small number of cells abutting the optic lobe invagination. Rfx is detected in the brain throughout larval and pupal development in a restricted number of cells in each brain lobe (Vandaele, 2001).

Larvae and Pupae

During larval and pupal life, Rfx is absent in early SOPs of the imaginal discs, but appears later in development, i.e. after puparium formation (APF). Rfx is found in all adult type I sensory organ lineage analyzed (antennae, leg and wing discs). No expression is detected in the eye disc. Expression is first detected in the leg discs, at the beginning of eversion, in the innermost SOPs of the femoral ch organ, that are ready to differentiate. Similarly, Rfx is detected in the Johnston organs of the second antennal segment of the antennal discs only once they are fully everted (around 24 h APF) (Vandaele, 2001).

Finally, Rfx is very transiently present in adult males during spermatogenesis in syncitial bundles of 64 spermatids. According to their shape and position within the testis lumen, these spermatids are in the elongation phase of their flagellum. The protein is not detectable in spherical spermatid nuclei nor in late condensing spermatid nuclei. Rfx was not detected in the female germline and in other tissues of whole stained dissected larvae (Vandaele, 2001).

EFFECTS OF MUTATION

Rfx is located on the third chromosome at 86A1-2 and is flanked by two genes transcribed on the opposite strand: jumeau (also described as Domina) and CG17100. CG17100 has been suggested to be affected in stich1 mutants. Available deficiencies were investigated in the 86A-B domain that would uncover Rfx. Df(3R)hth uncovers Rfx, as visualized by polytene chromosome in situ hybridization with a Rfx probe. Moreover, no trace of RFX protein is detected in Df(3R)hth homozygous embryo tissues by immunostaining. All together with complementation results these data indicate that Df(3R)hth is a deficiency uncovering the entire 85F-86C region (Dubruille, 2002).

A P-element inserted 5' of Rfx (A143.1F3) was mobilized to generate more aberrations affecting the Rfx locus. Several chromosomes were selected by molecular screening, including Rfx⁴⁹, a small deficiency uncovering the 5' end of Rfx. Molecular characterization of Rfx⁴⁹ breakpoints revealed that the deletion does not uncover the adjacent jumu gene. Indeed, Rfx⁴⁹ complements the lethal phenotype of all jumu lethal alleles tested. Moreover, no RFX immunostaining was found in the peripheral nervous system of Rfx⁴⁹ mutant embryos compared with wild-type embryos (Vandaele, 2001; Dubruille, 2002).

Complementation analysis with Rfx⁴⁹ and Df(3R)hth chromosomes identifies two complementation groups in the 86A1-2 region that have been identified as jumu and Rfx. All stich1 alleles fail to complement jumu loss of function alleles. S143702 is actually a deficiency uncovering both jumu and Rfx loci. All together these results imply that stich1 mutations affect the jumu gene but not CG17100 (Dubruille, 2002).

The more distal breakpoint of Rfx⁴⁹ maps 100 bp upstream of the 5' end of CG17100. It is therefore theoretically possible that Rfx⁴⁹ also affects this gene. An EMS non-complementation screen was undertaken to isolate other Rfx alleles. The Rfx²⁵³ chromosome was isolated that defines the same complementation group as Rfx⁴⁹. Sequencing analysis revealed a mutation causing an amino acid substitution, S⁴³⁵F, in the DNA-binding domain. Serine at position 65 of the DBD is replaced by a phenylalanine. Remarkably, the mutated serine is absolutely conserved in all RFX proteins described from yeast to mammals and, in a crystallographic structure of the mammalian RFX1 DBD, is located in the 'wing' loop that mediates most DNA contacts (Gajiwala, 2000). Electromobility shift assay confirmed that Rfx²⁵³ is no longer able to bind its target sequence. Thus, based on its lack of DNA binding, Rfx²⁵³ represents a hypomorphic or null allele of Rfx (Dubruille, 2002).

Rfx is necessary for olfactory and gustatory perception in Drosophila larvae. Under standard rearing conditions, most Rfx⁴⁹ and Rfx²⁵³ homozygotes die as larvae. Mutant pupae could not be observed on the walls of the culture vials, regardless of allelic combination. However, in uncrowded conditions, a small proportion of homozygous pupae were detected on vials, indicating that a few larvae can pupate. Approximately 15% of first instar larvae give rise to pupae. Mutant larvae lag their heterologous siblings by more than 3 days in development to pupae. Close observation of larvae show a peculiar foraging behavior with weak food digging efficiency. This could result from an altered peripheral sensitivity, a hypothesis that was suggested by the fact that Rfx is expressed in sensory neurons during embryogenesis (Vandaele, 2000). The gustatory and olfactory-driven behavior of mutant larvae was therefore investigated (Dubruille, 2002).

An olfactory test was performed using two pure odors (butanol and n-octyl-acetate) that induce characteristic attractive and repulsive responses. Whereas control larvae responded efficiently, mutant larvae showed no sensitivity to either odor. Also, repulsion was assayed by high NaCl concentrations: while control larvae are repulsed by concentrated salt, mutant larvae do not avoid this medium, showing that Rfx mutant larvae are defective in gustatory chemotaxis as well. To demonstrate that these responses are not due to a defect in the larval motility, phototaxis behavior was tested. This is based on the finding that Rfx is not expressed in the Bolwig organs, the larval visual system (Vandaele, 2001). Rfx mutant larvae should therefore not be affected in visual perception. This was clearly demonstrated by phototaxis behavioral assay. The phototaxis behaviors of Rfx mutant and control larvae are identical, demonstrating that Rfx mutants are not affected in their capacity to move. These data led to the conclusion that Rfx is necessary for olfactory and gustatory perception in Drosophila larvae (Dubruille, 2002).

Emerging adults are not able to walk and fly and thus stick to the medium and cannot be retrieved in culture vials. In order to recover adult mutants, third instar larvae and pupae were selected and raised until hatching on wet filter paper. The 15% to 30% of Rfx⁴⁹/Rfx²⁵³ or S143702/Rfx²⁵³ adults that emerge from pupae, all present a characteristic uncoordinated phenotype. Mutant flies cannot stand or walk: legs are crossed and wings are held up. However, mutants do react to light stimulation by moving legs and wings in an effort to right themselves and walk, but uncoordination limits their movement to a few steps or a flip from one side to another. This transient activity indicates that general neuromuscular excitability is retained. To ascertain that Rfx is responsible for the observed phenotype, an Rfx cDNA was expressed in all neurons in Rfx⁴⁹/Rfx²⁵³ mutants. The transgene expression indeed completely rescued the Rfx⁴⁹/Rfx²⁵³ uncoordinated and low viability phenotypes (Dubruille, 2002).

The uncoordination of Rfx mutant flies is similar to the phenotype of mutants with defects in mechanosensory transduction. Type I external mechanosensory bristles include three support cells and one neuron. The innermost support cell (sheath cell) forms a tubular extension around the sensory process and produces an extracellular dendritic cap that covers the cilium tip. The two outer support cells (shaft and socket cells) construct the cuticular bristle socket and shaft and in mature organs generate the receptor lymph. To test if Rfx mutants also have defects in mechanosensory bristle electrophysiology, transepithelial potentials (TEPs) and currents were recorded from macrochaete bristles. Unstimulated wild-type bristles have a positive TEP, due to electrogenic K⁺ transport by sensory support cells into the restricted apical extracellular space. This creates the electrical driving force for a receptor current that is triggered by bristle deflection: current flows from the apical extracellular space into the sensory neuron, reducing the TEP and depolarizing the neuron so that it fires a series of action potentials. The response adapts to a sustained deflection (Dubruille, 2002).

The average resting TEP in Rfx mutants (12±9 mV) is significantly lower than in their heterozygous sibs (61±4 mV), although the mutant and control distributions overlap. More strikingly, no mechanically elicited changes in TEP were detected in any Rfx mutant bristle, even in bristles with TEP in the normal range. Holding the TEP constant while stimulating the bristle enables a mechanoreceptor current to be recorded. No receptor currents were elicited by mechanical stimulation in Rfx mutants, even when the TEP was clamped at a large positive value (+100 mV). Finally, no action potentials were observed in mutant neurons. Thus, the adult uncoordination phenotype probably reflects a failure of bristle mechanotransduction. Maintenance of the TEP requires both active ion transport by sensory support cells, and high cuticular and transepithelial resistance. The resistance at Rfx mutant bristles was found to be, on average, one-third that at control bristles, suggesting some reduction in the integrity of the intercellular junctions connecting the neuron and the surrounding support cells (Dubruille, 2002).

Most mechanosensory mutations affect chordotonal organs as well as bristles. Chordotonal organs are type I stretch receptors that transduce proprioceptive and auditory stimuli. To test chordotonal organ operation, extracellular sound-elicited potentials were recorded from the antennal nerve projecting from the auditory chordotonal organ (Johnston's organ) in wild-type and mutant antennae. Johnston's organ comprises over 100 individual sensory units or scolopidia located in the second antennal segment, that normally respond when distal antennal segments are vibrated by near-field sound. A series of sound pulses elicited a corresponding response from Rfx⁴⁹/TM6B heterozygotes, but Rfx⁴⁹/Rfx²⁵³ mutants showed no response to stimuli of the same or greater amplitude, indicating a complete loss of auditory chordotonal organ function (Dubruille, 2002).

Taken together, the broad spectrum of behavioral and electrophysiological defects suggest that all type I organs are affected in Rfx mutants. This idea was further pursued using light and electron microscopy (Dubruille, 2002).

To examine the morphology of sensory neurons in Rfx mutants, a series of molecular markers for the peripheral nervous system (PNS) was used. With the 22C10 antibody, which stains a microtubule-associated protein in all sensory neurons, no differences were observed between mutant and wild-type embryos in the number and position of sensory neurons. By contrast, the anti-HRP antibody, which stains all neuron surfaces in wild-type embryos, revealed a very weak staining of the PNS in the mutant embryos regardless of the Rfx allelic combination used. An identical weak anti-HRP staining of sensory neurons was also reported for S143702 embryos (a deficiency mutation uncovering Rfx), confirming that this phenotype is specifically due to loss of Rfx function (Dubruille, 2002).

Anti-HRP staining in mutant embryos is, however, strong enough to visualize a specific dendrite defect, particularly visible in the lateral chordotonal organ clusters (1ch5). Indeed, this array of chordotonal organs is disorganized in Rfx embryos. Most dendrites are shorter, and vary in length within a cluster. The cilia present in wild type or control embryos are always either shorter or missing in Rfx mutants (Dubruille, 2002).

To visualize neuronal dendrites in more detail, mutant flies were engineered expressing a membrane-localized GFP in all neurons. Focus was placed on two types of adult sensory organs: campaniform sensilla of the wing and chordotonal organs of the femur (Dubruille, 2002).

Campaniform sensilla of the wing are composed of a single neuron and three accessory cells. The neuronal cell body and the dendrite are situated in the same focal plane and are thus easily visible when marked with GFP. The cilium at the tip of the dendrite is located exactly under the dome-shaped sensilla in wild-type or in rescued wings. In Rfx mutant wings, the dendrite does not reach the dome and the cilium is either absent, or shorter (Dubruille, 2002).

In the femoral chordotonal organ, each scolopidium is innervated by two ciliated neurons. Instead of external cuticular structures, a rigid scolopale structure encloses the ciliary outer segments. By confocal microscopy analysis of GFP distribution, three ciliary regions can be distinguished. These regions correlate with an ultrastuctural organization that has been described by transmission electron microscopy: the axoneme, the ciliary dilation and the tubular body enclosing the tubular bundle (a dense array of microtubules). These subregions are clearly distinguished in control or rescued Rfx mutant flies. By contrast, the cilium is always shorter and even absent in mutant flies. When present, no clear cilium subregions can be distinguished indicating that the architecture of the cilium is very disorganized. Moreover, the inner dendritic segment appears swollen. These defects are specifically rescued when the Rfx cDNA is expressed in all neurons, thus demonstrating that Rfx expression in the neuron is necessary for differentiation of the ciliated sensory ending in adult sense organs (Dubruille, 2002).

Investigation of the ultrastructure of sensitive neurons was carried out on the Johnston's organ in the second antennal segment. Each scolopidium of the Johnston's organ includes two neurons, a scolopale cell and an elongated conical dendritic cap that encloses the distal tips of the two cilia. The tips of a pair of cilia are attached via an extracellular cap to a distal attachment cell (cap cell), and their bases are anchored within the neurons by a long axial filament that extends through the neuronal dendrites and cell bodies. Cross-sections performed at different proximodistal positions reveal the three different typical ultrastructural subregions of chordotonal cilia. The sensory cilium is composed of 9+0 microtubule doublets. The ciliary dilation contains a paracrystalline inclusion and the tubular body is composed of microtubule arrays that are not assembled in a stereotyped fashion as in the cilium. The two cilia are surrounded by electron dense structures (scolopale rods) produced by the scolopale cell. The cilium at the tip of the dendrite contacts an extracellular matrix (dendritic cap) (Dubruille, 2002).

Examination of sectioned antennae of Rfx mutants by light microscopy showed no gross defects in Johnston's organ, but the ultrastructure of individual scolopidia was profoundly altered. Sections of several scolopidia at different proximodistal positions clearly show the 9+0 axonemal structure in wild-type antennae, but no axoneme profiles were found in serial sections of Rfx²⁵³/Rfx⁴⁹ antennae. In longitudinal sections the anchoring axial filament can be clearly observed in wild type, but not in mutant scolopidia. Its absence indicates that Rfx mutant defects extend beyond the cilium proper and affect associated structures in the neuron. In addition, a non-neuronal defect is noted: the electron dense scolopale rods enclosing the cilium are not as regularly arrayed as in wild-type flies. By contrast, the dendritic cap, also produced by the scolopale cell appears equally well organized in wild type and in Rfx mutants. These results demonstrate that Rfx is necessary for proper cilium assembly in chordotonal organs (Dubruille, 2002).

EVOLUTIONARY HOMOLOGS

RFX structural studies

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).

Regulatory Factor X (RFX)-mediated transcriptional rewiring of ciliary genes in animals

Cilia were present in the last eukaryotic common ancestor (LECA) and were retained by most organisms spanning all extant eukaryotic lineages, including organisms in the Unikonta (Amoebozoa, fungi, choanoflagellates, and animals), Archaeplastida, Excavata, Chromalveolata, and Rhizaria. Cilia have been lost secondarily from several groups of organisms, most notably in derived fungi (unikonts) and flowering plants (nonunikonts). In certain animals, including humans, ciliary gene regulation is mediated by Regulatory Factor X (RFX) transcription factors (TFs). RFX TFs bind X-box promoter motifs and thereby positively regulate >50 ciliary genes. Though RFX-mediated ciliary gene regulation has been studied in several bilaterian animals, little is known about the evolutionary conservation of ciliary gene regulation. This study explored the evolutionary relationships between RFX TFs and cilia. By sampling the genome sequences of >120 eukaryotic organisms, this study shows that RFX TFs are exclusively found in unikont organisms (whether ciliated or not), but are completely absent from the genome sequences of all nonunikont organisms (again, whether ciliated or not). Sampling the promoter sequences of 12 highly conserved ciliary genes from 23 diverse unikont and nonunikont organisms further revealed that phylogenetic footprints of X-box promoter motif sequences are found exclusively in ciliary genes of certain animals. Thus, there is no correlation between cilia/ciliary genes and the presence or absence of RFX TFs and X-box promoter motifs in nonanimal unikont and in nonunikont organisms. These data suggest that RFX TFs originated early in the unikont lineage, distinctly after cilia evolved (see Schematic diagram depicting the evolutionary conservation of cilia, RFX TFs, and the control of ciliary genes by RFX TFs). The evolutionary model that best explains these observations indicates that the transcriptional rewiring of many ciliary genes by RFX TFs occurred early in the animal lineage (Piasecki, 2010)

C. elegans daf-19 encodes an RFX-type transcription factor

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).

Xenopus Xrx1

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).

Mammalian RFX transcription factors

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).

REFERENCES

Search PubMed for articles about Drosophila Rfx

Andreazzoli, M., et al. (2003). Xrx1 controls proliferation and neurogenesis in Xenopus anterior neural plate. Development 130: 5143-5155. 1297534

Cachero, S., et al. (2011). The gene regulatory cascade linking proneural specification with differentiation in Drosophila sensory neurons. PLoS Biol. 9(1): e1000568. PubMed Citation: 21283833

Chen, L., et al. (2000). Activation of protein kinase C induces nuclear translocation of RFX1 and down-regulates c-myc via an intron 1 X box in undifferentiated leukemia HL-60 cells. J. Biol. Chem. 275(41): 32227-33. 10918054

Dubruille, R., et al. (2002). Drosophila Regulatory factor X is necessary for ciliated sensory neuron differentiation. Development 129: 5487-5498. 12403718

Durand, B., Kobr, M., Reith, W. and Mach, B. (2002). Functional complementation of major histocompatibility complex class II regulatory mutants by the purified X-box-binding protein RFX. Mol. Cell Biol. 14(10): 6839-47. 7935401

Durand, B., Vandaele, C., Spencer, D., Pantalacci, S. and Couble, P. (2000). Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors. Gene 246(1-2): 285-93. 10767550

Gajiwala, K. S., et al. (2000). Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding. Nature 403(6772): 916-21. 10706293

Haycraft, C. J., Swoboda, P., Taulman, P. D., Thomas, J. H. and Yoder, B. K. (2001). The C. elegans homolog of the murine cystic kidney disease gene Tg737 functions in a ciliogenic pathway and is disrupted in osm-5 mutant worms. Development 128: 1493-1505. 11290289

Huang, M., Zhou, Z. and Elledge, S. J. (1998). The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell 94: 595-605. 9741624

Iwama, A., et al. (1999). Dimeric RFX proteins contribute to the activity and lineage specificity of the interleukin-5 receptor alpha promoter through activation and repression domains. Mol. Cell. Biol. 19(6): 3940-50. 10330134

Katan-Khaykovich, Y., Spiegel, I. and Shaul, Y. (1999). The dimerization/repression domain of RFX1 is related to a conserved region of its yeast homologues Crt1 and Sak1: a new function for an ancient motif. J. Mol. Biol. 294(1): 121-37. 10556033

Katan-Khaykovich, Y. and Shaul, Y. (2001). Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism. Eur. J. Biochem. 268(10): 3108-16 . 11358531

Laurençon, A., et al. (2007). Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species. Genome Biol. 8(9): R195. PubMed Citation: 17875208

Liu, M., Lee, B. H. and Mathews, M. B. (1999). Involvement of RFX1 protein in the regulation of the human proliferating cell nuclear antigen promoter. J. Biol. Chem. 274(22): 15433-9. 10336433

Nakayama, A., et al. (2003). Role for RFX transcription factors in non-neuronal cell-specific inactivation of the microtubule-associated protein (MAP) 1A promoter. J. Biol. Chem. 278(1): 233-40. 12411430

Piasecki, B. P., Burghoorn, J. and Swoboda, P. (2010). Regulatory Factor X (RFX)-mediated transcriptional rewiring of ciliary genes in animals. Proc. Natl. Acad. Sci. 107: 12969-12974. PubMed Citation: 20615967

Qin, H., Rosenbaum, J. L. and Barr, M. M. (2001). An autosomal recessive polycystic kidney disease gene homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons. Curr. Biol. 11: 457-461. 11301258

Reith, W. and Mach, B. (2001). The bare lymphocyte syndrome and the regulation of MHC expression. Annu. Rev. Immunol. 19: 331-373. 11244040

Sengupta, P. K., Ehrlich, M. and Smith, B. D. (1999). A methylation-responsive MDBP/RFX site is in the first exon of the collagen alpha2(I) promoter. J. Biol. Chem. 274(51): 36649-55. 10593968

Sengupta, P. K., Fargo, J. and Smith, B. D. (2002). The RFX family interacts at the collagen (COL1A2) start site and represses transcription. J. Biol. Chem. 277(28): 24926-37 . 11986307

Swoboda, P., Adler, H. T. and Thomas, J. H. (2000). The RFX-type transcription factor DAF-19 regulates sensory neuron cilium formation in C. elegans. Mol. Cell 5: 411-421. 10882127

Tierney, R., et al. (2000). The Epstein-Barr virus promoter initiating B-cell transformation is activated by RFX proteins and the B-cell-specific activator protein BSAP/Pax5. J. Virol. 74(22): 10458-67 . 11044090

Wu, S. Y. and McLeod, M. (1995). The sak1+ gene of Schizosaccharomyces pombe encodes an RFX family DNA-binding protein that positively regulates cyclic AMP-dependant protein kinase mediated exit from the mitotic cycle. Mol. Cell. Biol. 15: 1479-1488. 7862141

Vandaele, C., Coulon-Bublex, M., Couble, P. and Durand, B. (2001). Drosophila regulatory factor X is an embryonic type I sensory neuron marker also expressed in spermatids and in the brain of Drosophila. Mech. Dev. 103: 159-162 . 11335126

date revised: 30 December 2012

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