Identification and characterization of novel human tissue-specific RFX transcription factors

Abstract

Background: Five regulatory factor X (RFX) transcription factors (TFs)-RFX1-5-have been previously characterized in the human genome, which have been demonstrated to be critical for development and are associated with an expanding list of serious human disease conditions including major histocompatibility (MHC) class II deficiency and ciliaophathies.

Results: In this study, we have identified two additional RFX genes-RFX6 and RFX7-in the current human genome sequences. Both RFX6 and RFX7 are demonstrated to be winged-helix TFs and have well conserved RFX DNA binding domains (DBDs), which are also found in winged-helix TFs RFX1-5. Phylogenetic analysis suggests that the RFX family in the human genome has undergone at least three gene duplications in evolution and the seven human RFX genes can be clearly categorized into three subgroups: (1) RFX1-3, (2) RFX4 and RFX6, and (3) RFX5 and RFX7. Our functional genomics analysis suggests that RFX6 and RFX7 have distinct expression profiles. RFX6 is expressed almost exclusively in the pancreatic islets, while RFX7 has high ubiquitous expression in nearly all tissues examined, particularly in various brain tissues.

Conclusion: The identification and further characterization of these two novel RFX genes hold promise for gaining critical insight into development and many disease conditions in mammals, potentially leading to identification of disease genes and biomarkers.

Figure 1

Mammalian RFX DBDs are highly conserved. DBDs from six mammalian RFX genes were aligned using ClustalW. The conservation of amino acid is depicted by a color gradient from the color yellow, which indicates low conservation, to red, which indicates high conservation. Nine residues that make direct or water-mediated DNA contacts are indicated with arrow heads. The species names included in this figure are abbreviated. They are: Mus–mouse (Mus musculus); Rno–Rat (Rattus norvegicus); Cfa–dog (Canis familiaris); Ptr–chimpanzee (Pan troglodytes); Mmu–monkey (Macaca mulatta) and Hsa–human (Homo sapiens).

Figure 2

Functional domains in the known and novel human RFX genes. The functional domains, AD, DBD, B, C, and D are indicated using color-coded boxes. Genes are represented using horizontal lines, which are proportional to the protein lengths. The domain lengths and positions are also proportional to their actual lengths. The graphs are aligned based on the position of the DBDs.

Figure 3

RFX interactome. Circles depict gene products and lines depict protein-protein interactions. The interactions between RFX6 and its direct interactors were obtained using yeast-two-hybrid method in a large-scale human protein-protein interaction study [34]. Additional interactions were constructed by Rhodes et al[46]. The network was generated using program available at the HiMap website http://www.himap.org/[46].

Figure 4

Phylogenetic analysis of mammalian RFX genes. This phylogenetic tree was constructed based on DBDs of RFX genes for six mammalian species and C. elegans using yeast RFX gene product Crt1 as the out-group. The phylogenetic tree was bootstrapped for 100 times with the numbers at each internal node being the bootstrap values. Each ortholog group is colored differently. The species names included in this figure are abbreviated. They are: Mus–mouse (Mus musculus); Rno–Rat (Rattus norvegicus); Cfa–dog (Canis familiaris); Ptr–chimpanzee (Pan troglodytes); Mmu–monkey (Macaca mulatta) and Hsa–human (Homo sapiens).

Figure 5

Relative expression of human RFX genes revealed by SAGE. Original SAGE libraries were generated by the Mouse Atlas Project [37]. X-axis shows different tissue types, while Y-axis shows relative SAGE tag frequency.