The in vivo dissection of direct RFX-target gene promoters in C. elegans reveals a novel cis-regulatory element, the C-box

Abstract

At the core of the primary transcriptional network regulating ciliary gene expression in Caenorhabditis elegans sensory neurons is the RFX/DAF-19 transcription factor, which binds and thereby positively regulates 13-15 bp X-box promoter motifs found in the cis-regulatory regions of many ciliary genes. However, the variable expression of direct RFX-target genes in various sets of ciliated sensory neurons (CSNs) occurs through as of yet uncharacterized mechanisms. In this study the cis-regulatory regions of 41 direct RFX-target genes are compared using in vivo genetic analyses and computational comparisons of orthologous nematode sequences. We find that neither the proximity to the translational start site nor the exact sequence composition of the X-box promoter motif of the respective ciliary gene can explain the variation in expression patterns observed among different direct RFX-target genes. Instead, a novel enhancer element appears to co-regulate ciliary genes in a DAF-19 dependent manner. This cytosine- and thymidine-rich sequence, the C-box, was found in the cis-regulatory regions in close proximity to the respective X-box motif for 84% of the most broadly expressed direct RFX-target genes sampled in this study. Molecular characterization confirmed that these 8-11 bp C-box sequences act as strong enhancer elements for direct RFX-target genes. An artificial promoter containing only an X-box promoter motif and two of the C-box enhancer elements was able to drive strong expression of a GFP reporter construct in many C. elegans CSNs. These data provide a much-improved understanding of how direct RFX-target genes are differentially regulated in C. elegans and will provide a molecular model for uncovering the transcriptional network mediating ciliary gene expression in animals.

Switching on cilia: transcriptional networks regulating ciliogenesis

“…GTYNCY-AT-RGNAAC) to which RFX dimers make contacts on opposing sides of the DNA (Burghoorn et al, 2012;Efimenko et al, 2005;Gajiwala et al, 2000;Laurençon et al, 2007;Swoboda et al, 2000). The dimer combinations identified for Rfx1-Rfx4 include all homodimers and various heterodimers, supporting the notion that DNA binding and the subsequent transactivation of target genes occurs in a coordinated and closely interdependent fashion (Iwama et al, 1999;Morotomi-Yano et al, 2002;Reith et al, 1994a).…”

“…Although these 60 sensory neurons are clearly present in daf-19 mutant animals, they entirely lack sensory cilia, indicating that DAF-19 is necessary for cilia formation. Furthermore, the transcriptional activation of genes encoding IFT subunit genes, such as che-2, osm-1 and osm-6, and of many other ciliary genes requires DAF-19 function mediated via functional X-box elements in the promoters of these genes (Burghoorn et al, 2012). Therefore, in C. elegans, DAF-19 is the central regulator of ciliogenesis and is specifically required during late differentiation (Senti and Swoboda, 2008;Swoboda et al, 2000).…”

“…DYF-17, B9 and BBS proteins), the transition zone (NPH-1 and NPH-4) and the axoneme (DYF-1) fall into this category, as do components of the IFT machinery (e.g. IFT88/OSM-5, IFT172/OSM-1 and XBX-1) (Ansley et al, 2003;Ashique et al, 2009;Burghoorn et al, 2012;Efimenko et al, 2005;Haycraft et al, 2001;Ou et al, 2005;Phirke et al, 2011;Schafer et al, 2003;Signor et al, 1999;Williams et al, 2008;Winkelbauer et al, 2005 and Drosophila Nan and Iav) (Burghoorn et al, 2012;Dwyer et al, 1998;Efimenko et al, 2005;Newton et al, 2012). RFX factors are thought to orchestrate ciliary differentiation programs after a cell has become committed towards a particular fate.…”

“…Strikingly, however, in both invertebrates and the vertebrates, several transcription factors are also regulated via conserved X-box motifs (e.g. Rax, Zic1, Zic3, Msx3 and nuclear hormone receptors such as nhr-44, nhr-45 and nhr-120), suggesting that besides playing an essential role in promoting ciliogenesis, the RFX factors could also be directly involved in the specification of the ciliated cell types (Burghoorn et al, 2012;Efimenko et al, 2005;Zhang et al, 2006), a hypothesis that clearly requires further exploration. Given this possibility, the cell and tissue-patterning defects that occur in the absence of proper Rfx gene function may not solely be the outcome of perturbed signaling pathways triggered by ciliary abnormalities.…”

Contrôle transcriptionnel des gènes ciliaires

“…Ainsi, il a été montré que les différents gènes requis pour l'assemblage de tous les cils chez C. elegans possèdent dans leur promoteur, en plus d'un motif de liaison des protéines RFX, un motif conservé appelé boîte C, qui est requis pour l'activation de ces gènes. Le (ou les) facteur qui pourrait contrôler l'expression des gènes ciliaires en utilisant ce motif reste néanmoins inconnu [36]. Toujours chez C. elegans, il a été montré que la protéine RFX (DAF-19) induit l'expression d'un autre facteur de transcription, FKH-2 (forkhead-2), spécifique d'un sous-type de cellule ciliée et qui lui confère son identité [37].…”