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BMC Evolutionary Biology

BioMed Central

Open Access

Research article

Identification and characterization of novel human tissue-specific
RFX transcription factors
Syed Aftab, Lucie Semenec, Jeffrey Shih-Chieh Chu and Nansheng Chen*
Address: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada
Email: Syed Aftab - saaftab@sfu.ca; Lucie Semenec - lucie.semenec@gmail.com; Jeffrey Shih-Chieh Chu - jeff.sc.chu@gmail.com;
Nansheng Chen* - chenn@sfu.ca
* Corresponding author

Published: 1 August 2008
BMC Evolutionary Biology 2008, 8:226

doi:10.1186/1471-2148-8-226

Received: 1 April 2008
Accepted: 1 August 2008

This article is available from: http://www.biomedcentral.com/1471-2148/8/226
© 2008 Aftab et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Background
The regulatory factor X (RFX) gene family transcription
factors (TFs) were first detected in mammals as the regulatory factor that binds to a conserved cis-regulatory element
called the X-box motif about 20 years ago [1]. The X-box
motifs, which are typically 14-mer DNA sequences, were
initially identified as a result of alignment and inspection
of the promoter regions of major histocompatibility complex (MHC) class II genes for conserved DNA elements
[2,3]. Further investigations revealed that the X-box motif
is highly conserved in the promoter regions of various

MHC class II genes [4]. The first RFX gene (RFX1) was later
characterized as a candidate major histocompatibility
complex (MHC) class II promoter binding protein [5].
RFX1 was later found to function also as a transactivator
of the hepatitis B virus enhancer [6]. Subsequent studies
revealed that RFX1 is not alone. Instead, it became the
founding member of a novel family of homodimeric and
heterodimeric DNA-binding proteins, which also
includes RFX2 and RFX3 [7]. More members of this gene
family were subsequently identified. A fourth RFX gene
(RFX4) was discovered in a human breast tumor tissue [8]
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BMC Evolutionary Biology 2008, 8:226

and the fifth, RFX5, was identified as a DNA-binding regulatory factor that is mutated in primary MHC class II
deficiency (bare lymphocyte syndrome, BLS) [9]. The
identification of RFX1-5 and RFX genes in other genomes
including the genomes of lower eukaryote species Saccharomyces cerevisiae [10] and Schizosaccharomyces pombe [11],
and higher eukaryote species the nematode Caenorhabdits
elegans [12] helped understand both the evolution of the
RFX gene family and the DNA binding domains [13].
Notably, while previous studies reported five RFX genes
(RFX1-5) in human, only one RFX gene has been identified in most invertebrate animals and yeast. In contrast,
the fruit fly (Drosophila melanogaster) genome has been
found to have two RFX genes, dRFX [14] and dRFX2 [15].
All of these RFX genes are transcription factors possessing
a novel and highly conserved DNA binding domain
(DBD) called RFX DNA binding domain [13], the defining feature of all members belonging to the RFX gene family, suggesting that these RFX TFs all bind to the X-box
motifs.
In addition to the defining DBD domains in all of these
RFX genes, most of these previously identified RFX genes
also contain other conserved domains including B, C, and
D domains [13]. The D domain is also called the dimerization domain [13]. The B and C domains also play a role
in dimerization and are thus called the extended dimerization domains [16]. Another important domain found
in many members of the RFX family is the RFX activation
domain (AD). For instance, RFX1 contains a well defined
AD [16]. However, AD is not found in many other members of the RFX family including the human RFX5 and C.
elegans DAF-19 [13]. Outside of these conserved domains,
RFX genes from different species or even from same species show little similarity in other regions, which is quite
consistent with their diverse functions and distinct expression profiles.
In humans, RFX1 is primarily found in the brain with high
expression in cerebral cortex and Purkinje cells [17]. RFX2
[18] and RFX4 [19] are found to be heavily expressed in
the testis. RFX4 is also expressed in the brain [20]. RFX3 is
expressed in ciliated cells and is required for growth and
function of cilia including pancreatic endocrine cells [21],
ependymal cells [22], and neuronal cells [23]. RFX3-deficient mice show left-right (L-R) asymmetry defects [23],
developmental defect, diabetes [21], and congenital
hydrocephalus in mice [22]. RFX5 is the most extensively
studied RFX gene so far primarily since it serves as a transcription activator of the clinically important MHC II
genes [24] and mediates a enhanceosome formation,
which results in a complex containing RFXANK (also
known as RFX-B), RFXAP, CREB, and CIITA [25]. Mutation in any one of these complex members leads to bare
lymphocyte syndrome (BLS) [25]. In C.elegans and S.cere-

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visae only one copy of the RFX gene exists. In C. elegans it
is called DAF-19 and in S.cerevisae it is called Crt1. DAF19 is involved in regulation of sensory neuron cilium
whereas Crt-1 is involved in regulating DNA replication
and damage checkpoint pathways [10,12]. In D.melanogaster, two of RFX genes have been identified, one is
called dRFX and the other is called dRFX2. dRFX is
expressed in the spermatid and brain and is necessary for
ciliated sensory neuron differentiation [14,26]. dRFX2 has
not been studied extensively and as such its function in
Drosophila still remains unclear; however, there is evidence suggesting that dRFX2 plays a role in cell-cycle of
the eye imaginal discs [15].
In this project, we have identified and characterized two
novel RFX genes in genomes of human and many other
mammals, which have now been sequenced, annotated,
and analyzed.

Results and discussions
With the current version of the human genome [27,28],
we explored whether additional members of the RFX TF
family could be identified and characterized in the human
genome. We applied a Hidden Markov Model (HMM)
based search method [29] and used DBD domain
sequences of known human RFX TFs to search the entire
human proteome. In addition to retrieving all known
human RFX genes–RFX1-5, we identified two additional
genes in the human genome that contain well conserved
RFX DBDs. These two genes were previously assigned as
RFXDC1 and RFXDC2 by the HUGO Gene Nomenclature
Committee (HGNC, http://www.genenames.org/); this
nomenclature was based solely on an initial bioinformatic analyses. There are no previous publications
describing these two genes. Here, we demonstrate that
these two genes are also RFX gene family members closely
related to RFX1-5, and our phylogenetic analysis suggests
two separate recent gene duplications leading to the generation of these two genes. Thus, we proposed new gene
nomenclature of RFX6 and RFX7 (Table 1), respectively.
Our proposal has been accepted by the HGNC.
Because all known human RFX genes–RFX1-5–are well
conserved and have been identified in other mammalian
genomes, we hypothesized that orthologs of RFX6 and
RFX7 also exist in other mammalian genomes. As
expected, we have retrieved all seven RFX genes in the
genomes of five other mammalian species including
chimpanzee (Pan troglodytes), monkey (Macaca mulatta),
dog (Canis familiaris), mouse (Mus musculus), and rat (Rattus norvegicus) with only one exception. In the rat genome,
all except RFX2 were found despite extensive searches
(Additional file 1). Most identified RFX genes are
expressed and their transcripts can be found in existing
EST libraries. Interestingly, existing EST evidence suggests

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Table 1: Names and Protein ID of Representative RFX genes.

Gene
names

Accession
Number
(RefSeq)

ESEMBL
protein ID

Genomic coordinates

Protein
lengths

Number of
exons

Number of
isoforms

chromosome
RFX1

NM_002918

RFX2

NM_000635

RFX3

NM_134428

RFX4

NM_213594

RFX5

NM_000449

RFX6

NM_173560

RFX7

NM_022841

ENSP000002
54325
ENSP000003
06335
ENSP000003
71434
ENSP000003
50552
ENSP000003
57864
ENSP000003
32208
ENSP000003
73793

start

end

strand

19

13933353

13978097

-1

979

21

1

19

5944175

6061554

-1

723

18

2

9

3208297

3515983

-1

749

18

8

12

105501163 105680710

1

744

18

4

1

149581060 149586457

-1

616

11

3

6

117305068 117351384

1

928

19

2

15

54166958

-1

1281

7

1

that RFX6 and RFX7 have no or very few alternative isoforms similar to RFX1. In contrast, RFX2-4 usually have
more alternative isoforms (Additional file 1).
To confirm that the two novel human RFX genes–RFX6
and RFX7 are indeed RFX TFs, we further examined their
DBDs by aligning them with DBDs from RFX1-5 protein
sequences. As expected, the DBDs of RFX6 and RFX7 align
well with those of RFX1-5 (Figure 1). RFX TFs belong to
the winged-helix family of DNA binding proteins because
their DBDs are related in structure and function to the
helix-turn-helix bacterial transcriptional regulatory proteins [30]. DBDs from RFX6 and RFX7 each contain one
wing (W1), which is the same as DBDs from RFX1-5. W1
interacts with the major groove and another conserved
fold H3 (helix 3) interacts with the minor groove of DNA.
In particular, the nine residues in DBDs (Figure 1, indicated with arrow heads) that make direct or water-mediated DNA contacts [31] are almost entirely conserved in
RFX6 and RFX7 (Figure 1) with a couple of minor exceptions. Of the nine residues, the human RFX7 DBD has two
residues different from most of the other RFX DBDs. The
first different residue is the first of the nine indicated residues. It is Lys in RFX7 DBD and RFX5 DBD, compared to
Arg in DBDs of other RFX genes. Thus this difference is
shared with the RFX5 DBD. The other different residue is
the third of the nine residues. It is Lys in RFX7, compared
to Arg at this site for DBDs of all other RFX genes. Because
both Lys and Arg are basic amino acids, such substitutions
are not expected to have dramatic impacts on the binding
between the DBDs and their cognate binding sites. This
high degree of conservation suggests that RFX6 and RFX7
may bind to similar if not identical cis-regulatory elements, i.e., the X-box motif [1]. Hence RFX6 and RFX7 are

54222377

new members of the human RFX gene family with conserved DBDs.
In addition to the highly conserved DBDs, other domains
including ADs, B, C, and D domains (also known as
dimerization domain) [13] have been described in
human RFX1-3 (Figure 2). Among these functional
domains, ADs have been identified in RFX1-3. However,
ADs have not been identified RFX4-5. The B and C
domains, which are usually called extended dimerization
domains, play supporting roles in dimerization [16]. B, C,
and D domains have also been identified in RFX4 but are
missing from RFX5. Using InterProScan [32] and HMMER
[29], we have found that RFX6 possesses B, C, and D
domains, but not AD (Figure 2). The motif composition
of RFX6 is similar to RFX4, which also has B, C, and D
domains but lacks AD. In contrast, we failed to identify B,
C, and D domains or AD in RFX7. None of these domains
can be found in RFX5 as well. Because these C-terminal
domains–B, C, and D domains–have been shown to
mediate dimerization as well as transcriptional repression
[33], RFX6, which contains B, C, D domain, and RFX7,
which does not possess B, C, or D domains, may therefore
play different role in transcriptional regulation.
Characterization of the functional domain composition
of RFX genes will provide insights into how different RFX
TFs function. In particular, how do RFX6 and RFX7, as
well as RFX4 and RFX5, function in transcription considering that they do not have identified ADs? There are two
possible mechanisms. First, because RFX TFs are known to
form dimers and bind to same or similar binding sites
(the X-box motifs) in DNA [31], they may function
together with RFX genes (RFX1-3) that do have ADs.

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Figure 1
Mammalian RFX DBDs are highly conserved
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).

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Figure 2
Functional domains in the known and novel human RFX genes
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.

Examination of a recently available proteome-scale map
of the human protein-protein interaction network [34],
which was constructed using yeast-two-hybrid technique,
has shown that RFX6 and RFX1-4 interact with each other
and also interact with many other genes (Figure 3). RFX6
interacts directly with RFX2 and RFX3, the latter of which
has been shown to be expressed and to function in the
pancreas [21], as well as many other tissues. The interaction between RFX6 and other RFX TFs provides further
supporting evidence that RFX6 is indeed a member of the
RFX gene family. Interactions between RFX7 and other
genes were not observed, which is likely due to the incomplete coverage of the human protein-protein interactions
analyzed in this study. Second, RFX TFs may function by
interacting with many other non-RFX TFs. For example, it
has been demonstrated that mammalian RFX 5 forms a
complex ("enhanceosome") with RFXANK (also known
as RFX-B), RFXAP, CREB, and CIITA to regulate expression
of MHC class II genes [25]. Notably, all of the five genes
shown to interact with RFX6 (DTX1, DTX2, FHL3, CCNK,
and SS18L1) (Figure 3) except only one–SS18L1–are also
putative TFs.
To explore the relationship between RFX6 and RFX7 and
the known RFX family members RFX1-5, we have constructed a phylogenetic tree that contains all mammalian
RFX genes described above (Additional file 1, Figure 1), as
well as C. elegans RFX gene daf-19 product DAF-19 [12],
which has been extensively studied, for comparison. We
used the DBD sequence of the yeast Saccharomyces cerevisiae RFX gene Crt-1[10] as an out group in the phylogenetic tree construction. From the phylogenetic tree (Figure
4), all seven genes show perfect one-to-one orthologous
relationships between different mammalian genomes. It

is clear that the seven mammalian RFX genes fall into
three subgroups (Figure 4). The first subgroup contains
RFX1-3; the second RFX4 and RFX6; while the third RFX5
and RFX7. It is likely that RFX4 and RFX6 resulted from
one gene duplication that predated the split of these
mammalian species, while RFX5 and RFX7 resulted from
another similar independent duplication. This hypothesis
is generally consistent with the gene models of these RFX
genes (Additional file 2). RFX6 has 19 exons, which is
similar to the number of exons contained in RFX4 (18
exons); while RFX7 has 6 exons, which is similar to the
number of exons contained in RFX5 (9 exons). The C. elegans RFX gene, DAF-19 clusters together with RFX1-3
genes, supporting a previously proposed hypothesis that
the divergence of the subgroup RFX1-3 from other two
subgroups likely predated the divergence between mammals and the nematodes [13]. This hypothesis predicts
that C. elegans should have orthologous RFX TFs to RFX47 [35]. However, only one C. elegans RFX gene–daf-19–has
been reported so far and our extensive search has concluded that daf-19 is the only RFX TF in C. elegans. One
possible explanation is that additional RFX TFs were lost
in evolution. Alternatively, RFX4-7 may have undergone
positive selection in mammals to accommodate additional functional complexity in mammalian gene regulation, while RFX1-3 and daf-19 remained highly conserved
due to purifying evolution. Interestingly, although the
phylogenetic tree was constructed based only on DBDs,
the grouping of these mammalian RFX genes is also consistent with the composition of other conserved domains.
In particular, RFX1-3 all contain DBDs, ADs, Bs, Cs and
Ds, while RFX4 and RFX6 have all of these domains except
ADs, and RFX5 and RFX7 have only DBDs (Figures 2 and
4).

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Figure 3
RFX interactome
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].
To gain insight into the function of these two newly identified RFX genes, we explored the expression profiles of
RFX6 and RFX7 and compared them to those of RFX1-5.
We analyzed two independent datasets. First, we searched
the
dbEST
database
in
genBank
http://
www.ncbi.nlm.nih.gov/dbEST/[36] to examine which
EST libraries express transcripts of these RFX genes. The
results indicate that the expression profile of RFX1-5
matches well with previously published data (see INTRODUCTION): RFX1 is found in many different tissue types
including white blood cells, heart, eye, testis, and cancerous cell; RFX2 appears to be expressed in testis and brain;
RFX3 appears to be expressed in the placenta and brain
(i.e., medulla); RFX4 is found in the brain, as well as in
testis as RFX2; and RFX5 expression has been observed in
various different tissues including thymus, T-cells, kidney,

brain, and lymph. The consistency of expression for RFX15 obtained from the dbEST database with previous observations suggests that dbEST provides good estimations of
RFX genes' expression profiles. Using the same method,
we found that RFX6 is primarily expressed in pancreas,
with minor expression in liver, while RFX7 is widely and
heavily expressed in many different tissue types including
kidney (tumor tissues), thymus, brain, and placenta.
Second, to gain a quantitative understanding of the
expression of RFX genes, we took advantage of the recent
availability of serial analysis of gene expression (SAGE)
libraries constructed by the Mouse Atlas of Gene Expression Project http://www.mouseatlas.org/[37]. To start
with, we tested the hypothesis that the expression of
mouse RFX TFs approximates the expression of human

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Figure 4
Phylogenetic analysis of mammalian RFX genes
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).

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expression profiles of human RFX genes obtained from
the dbEST database analysis, as well as previous publications about human RFX gene expressions (Figure 5). In
contrast to all other RFX genes–RFX1-5 and RFX7, which
are heavily expressed in the brain, RFX6 is clearly absent
from all types of brain tissues (Figure 5). RFX6 is primarily
found in the pancreas (Figure 5) which is consistent with
results obtained from analyzing dbEST. Low level expression of RFX6 is found in liver (also detected in dbEST) and
RFX TFs. We analyzed 196 mouse SAGE libraries, each of
which was produced by using a RNA library prepared
from different tissue types (some of which are duplicates).
Different SAGE libraries contain slightly different number
of total SAGE tags. To ensure that SAGE tags and tag
counts were comparable between different SAGE libraries
all the libraries were normalized to 1,000,000 SAGE tags.
Qualitatively, expression profiles of mouse RFX genes
obtained from SAGE analysis are consistent with the

1000
160
140
120
100
80
60
40
20
0
0

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BMC Evolutionary Biology 2008, 8:226

Figure expression of human RFX genes revealed by SAGE
Relative 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.

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heart. In addition to the high tissue-specificity, RFX6 has
the lowest overall expression level among all seven RFX
genes, suggesting that RFX6 may be under tighter regulatory control. In contrast, RFX7 has the highest relative
expression level among all seven mouse RFX genes. Similar to RFX1 and RFX5, RFX7 is found in essentially all
types of tissues that were examined (Figure 5).
Examining additional gene expression databases, including publicly available Genomics Institute of the Novartis
Research Foundation (GNF) Gene Expression Database
http://symatlas.gnf.org/SymAtlas/, revealed very similar
results.

Conclusion
Our results show that we have identified two novel RFX
genes in the human genome, RFX6 and RFX7, thus
expanding the human RFX gene family from five members (RFX1-5) to seven members (RFX1-7). In addition to
their possession of highly conserved DBDs, RFX6 and
RFX7 show similarity to known human RFX TFs in their
functional domains. In particular, RFX6 and RFX4 all have
B, C, and D domains, while RFX7 and RFX5 only have
DBDs. Studies carried out over the past 20 years have
demonstrated that RFX1-5 are critical for development
and many additional biological processes and play an
important role in various devastating disease conditions.
For example, RFX3-deficient mice show left-right (L-R)
asymmetry defects [23], developmental defects, diabetes
[21], and congenital hydrocephalus [22]. RFX3 may regulate the transcription of many genes that, when mutated,
cause cilia defects and many disease conditions collectively called ciliopathies [38]. Many known ciliopathy
genes, including Bardet-Biedle syndrome (BBS) genes, are
well conserved and the transcription of their C. elegans
orthologs are regulated by the only RFX gene in C. elegans–
DAF-19 [12,39-41]. Mutation in any one of the RFX5
enhanceosome members–RFXANK, RFXAP, CREB, and
CIITA–leads to bare lymphocyte syndrome (BLS) [25]. We
hypothesize that RFX6 and RFX7 are equally important as
RFX1-5. The fact that RFX6 is primarily expressed in pancreatic tissues and is expressed at a low level compared to
all other RFX genes (Figure 5) is particularly interesting.
RFX6 may function as a key component of a transcriptional regulatory complex that regulates pancreas development and function.

Methods
Data source and data mining
Gene sets were obtained from the FTP site of the
ENSEMBL
database
http://www.ensembl.org/
index.html[42]. In this project, the genomes of six mammals were analyzed. They are human (Homo sapiens,
NCBI36.44), chimpanzee (Pan troglodytes, CHIMP2.1.44),
dog (Canis familiaris, BROADD2.44), monkey (Macaca

http://www.biomedcentral.com/1471-2148/8/226

mulatta,
MMUL_1.44),
mouse
(Mus
musculus,
NCBIM36.44), and rat (Rattus norvegicus, RGSC3.4.44).
DBD sequences in human RFX1-5 were manually identified and extracted to a file. The sequences were aligned
using ClustalW [43]. The alignment was used as input to
the profile building program hmmbuild, which is a program in the HMMER package http://hmmer.jane
lia.org[29]. The resulting profile was used for searching
curated proteomes of the six mammals described above
using hmmsearch, another program in the HMMER package.
Gene model improvement
All RFX genes except one–dog (Cfa) RFX7–show good
alignment with their corresponding orthologs. Dog RFX7
gene is truncated at the N-terminus, missing 37 residues
compared to other RFX7 genes. We attempted to use
to
GeneWise
http://www.ebi.ac.uk/Wise2/[44,45]
remodel this RFX gene. Using human (Hsa) RFX7 as the
reference protein sequence and GeneWise, we recovered
the missing residues. However, the first codon so identified was not the typical Met. Extending the coding
sequence upstream did not help. This is likely due to a
sequencing error.
Protein domain analysis
We retrieved DBDs and ADs from RFX genes using InterProScan (version 4.3.1) [32]. To identify B, C, D domains,
we used the HMMER program [29] as described above.
Briefly, for HMMER searches, we used sequences of B, C,
and D domains from known RFX genes (RFX1-3) to generate profiles for these domains respectively. We then
searched for candidate B, C, and D domains in RFX6 and
RFX7 using these profiles.
RFX interactome network analysis
Data were obtained at the HiMAP http://www.himap.org/
database [46] following online search instructions. All
types of interactions were selected for searching. All seven
interactions between RFX6 and other genes (DAT1, DTX2,
FHL3, SS18L1, CCNK, RFX2, and RFX3) were previously
reported by Rual et al[34].
Sequence alignment and phylogenetic analysis
Multiple-sequence alignment was carried out using the
program ClustalW (version 1.83) [43]. Phylogenetic tree
construction was performed using PHYLIP http://evolu
tion.genetics.washington.edu/phylip.html
(Version
3.66). Briefly, sequence alignment in PHYLIP format was
first created using ClustalW (Version1.83) [43]. The alignment was used as input for PHYLIP. Programs utilized in
the PHYLIP, in their respective order, were seqboot, protdist, neighbor, and consense. The phylogenetic tree file
was visualized using Tree View http://taxonomy.zool
ogy.gla.ac.uk/rod/treeview.html.

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BMC Evolutionary Biology 2008, 8:226

Expression profile of mammalian RFX genes using
ESTs and SAGE libraries
The EST database from NCBI was used to perform tblastn.
The queries used for this tblastn were RFX1-7 of H. sapiens,
M. musculus, and R. norvegicus. Hits with identity greater
than or equal to 95% were selected.

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6.

7.

Authors' contributions
NS conceived of the study, participated in experimental
design. SA, LS and JSCC carried out the analysis. SA and
NS wrote the manuscript. All authors read and approved
the final manuscript.

Additional material
Additional File 1
Gene names and Protein ID of mammalian RFX genes.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712148-8-226-S1.doc]

Additional File 2
Gene models of human RFX genes, including RFX1-5 and newly identified RFX6-7. (a) Exon-intron structures of human RFX genes. Exons are
represented using boxes, while introns are represented using lines. Both
exons and introns shown in this panel are proportional to their real
lengths. (b) Illustration of exon-intron structures of human RFX-genes. In
this panel, while exons are proportional to their real lengths, for better visualization, introns are represented using lines of same lengths, regardless
of their real lengths.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712148-8-226-S2.pdf]

8.
9.

10.
11.

12.
13.
14.

15.
16.
17.
18.

Acknowledgements
This project was supported by an NSERC Discovery Award to NC. SA is
supported by a NSERC USRA. LS is a Pacific Century Graduate Scholar.
JSCC is supported by a Hemingway Nelson Architects Graduate Scholarship and a Weyerhaeuser Molecular Biology Graduate Scholarship. NC is
also a Michael Smith Foundation for Health Research Scholar. We thank
Drs. Robert Johnsen and Maja Tarailo for critical reading of the manuscript
and insightful suggestions.

19.

20.

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