©
1996 Oxford University Press
803-807 Footnote
RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic
kingdom
RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom
Patrick
Emery
,
Bénédicte
Durand
,
Bernard
Mach
and
Walter
Reith*
Department of Genetics and Microbiology, University of Geneva Medical School,
Centre Médical Universtaire, 1211
Geneva
4,
Switzerland
Received November 27, 1995;
Revised and Accepted January 19, 1996
ABSTRACT
Until recently, the RFX family of DNA binding proteins consisted exclusively of
four mammalian members (RFX1-RFX4) characterized by a novel highly conserved DNA binding domain.
Strong conservation of this DNA binding domain precluded a precise definition
of the motif required for DNA binding. In addition, the biological systems in
which these RFX proteins are implicated remained obscure. The recent
identification of four new RFX genes has now shed light on the evolutionary
conservation of the RFX family, contributed greatly to a detailed
characterization of the RFX DNA binding motif, and provided clear evidence for
the function of some of the RFX proteins. RFX proteins have been conserved
throughout evolution in a wide variety of species, including
Saccharomyces cerevisiae
,
Schizosaccharomyces pombe
,
Caenorhabditis elegans
, mouse and man. The characteristic RFX DNA binding motif has been recruited
into otherwise very divergent regulatory factors functioning in a diverse
spectrum of unrelated systems, including regulation of the mitotic cell cycle
in fission yeast, the control of the immune response in mammals, and infection
by human hepatitis B virus.
The RFX family was first defined by the identification, in man and mouse, of three highly homologous site-specific DNA-binding proteins called RFX1, RFX2 and RFX3 (
1
,
2
) (Fig.
1
). These proteins were found to share a novel 76 amino acid DNA binding domain
that was called the RFX DNA binding motif (
1
,
2
). The sequence of a fourth member of the RFX family (RFX4) was subsequently
found fused to the oestrogen receptor in two aberrant cDNA clones derived from
a human breast tumour (
2
,
3
) (Fig.
1
). Until recently, these four mammalian proteins were the only ones in which the
RFX DNA binding motif had been identified. Moreover, the strong homology
existing between their DNA binding domains (Fig.
2
, 76-96% in pairwise comparisons) revealed little about the nature of this
novel DNA-binding motif. Finally, although RFX1 was clearly shown to be a cellular
transactivator that is used by the highly pathogenic hepatitis B virus (
4
,
5
), little was known about the cellular functions of RFX proteins; candidate
genes that may be controlled by RFX1 are the c-myc gene (
6
) and the ribosomal protein L30 gene (
7
), while for RFX2, RFX3 and RFX4 no potential target genes have yet been
reported. In the past year, however, four additional members of the RFX family
have been identified. These are RFX5 in man and mouse (
8
), sak1 in
Schizosaccharomyces pombe
(
9
), a gene (ScRFX) in
Saccharomyces cerevisiae
and a gene (CeRFX) in
Caenorhabditis elegans
(Fig.
1
). The identification of these RFX genes has permitted a precise definition of
the consensus motif for this novel DNA binding domain, and has shed new light
on the evolutionary conservation and functional importance of RFX proteins in a
surprisingly diverse range of biological systems.
Figure 1
.
Schematic representation of the RFX1-5 and sak1 proteins, and of the open reading frames corresponding to
ScRFX and CeRFX. The RFX DNA binding domain motif (DBD) is indicated in red.
The conserved regions A, B and C are indicated in blue. The dimerization domain
homology (D) is indicated in green. Regions rich in proline (P), glutamine (Q)
or acidic amino acids (DE) are indicated in yellow. The RFX4 DNA binding domain
was identified at the C-terminus of two aberrant oestrogen receptor (ER) cDNA clones: the ER
portion of these clones is indicated in grey. ScRFX represents a single 811 aa
open reading frame. The DNA binding domain of CeRFX is contained in two
adjacent exons separated by a 66 bp intron. The B, C and D regions of CeRFX are
present on a single exon situated ~500 bp downstream of the two DNA binding domain exons. Where known, the
size of the proteins is indicated. For ScRFX the size is predicted from the
open reading frame present in the genomic sequence. ScRFX (genome sequencing
identification name L9470.18) and CeRFX (genome sequencing identification name
F33H1.1) were identified by a database search using the BLAST server (16). The
MmRFX5 sequence is unpublished. The EMBL accession numbers are HsRFX1 (A20492),
MmRFX1 (X76088), HsRFX2 (X76091), MmRFX2 (X76089), HsRFX3 (X76092), MmRFX3
(X76090), HsRFX4 (M69296), HsRFX5 (X85786), sak1 (U19978), CeRFX (Z48783) and
ScRFX (U17246).
Figure 2
.
Conservation of
the DNA binding domains of human (Hs) and mouse (Mm) RFX1-5, and homologous sequences found in the
S.pombe
gene sak1, the
S.cerevisiae
gene ScRFX and the
C.elegans
gene CeRFX. (
A
) Amino acid sequence alignment of the DNA binding domains. The consensus
sequence is given below. Invariant residues are highlighted in red and similar
residues conserved in all sequences are highlighted in blue. (
B
) The percent homology between the DNA binding domains is given for all pairwise
alignments. Similar residues were considered to be W/F/Y, K/R, D/E, M/F/I/L/V,
S/T/A and N/Q.
RFX5 is the 75 kDa subunit of a nuclear complex called RFX (
8
,
10
-
12
). This RFX complex is a transcription factor that is essential and highly
specific for the expression of MHC class II genes, the Ii chain gene and DM
genes (
8
,
10
-
12
). These genes play a key role in the immune system because they control the
presentation of foreign antigenic peptides to CD4+ helper T lymphocytes. RFX5
is thus a crucial regulator of the immune response. Mutations disrupting the
human RFX5 gene (
8
) result in MHC class II deficiency (also referred to as the bare lymphocyte
syndrome), a debilitating primary immunodeficiency that is due to a complete
absence of MHC class II gene transcription in all cell types and tissues
(reviewed in refs
12
-
14
).
Sak1 is the first member of the RFX family discovered in a non-mammalian organism. It was isolated on the basis of its ability to
suppress mutants of the cAMP dependent protein kinase (cAPK) pathway in
S.pombe
(
9
). Sak1 is an essential regulatory gene in the life cycle of
S.pombe
. It appears to function downstream of cAPK to allow cells to exit the mitotic
cycle and enter either stationary phase or the pathway leading to sexual
differentiation. The target genes of sak1 are not known.
The genes, ScRFX and CeRFX, are of unknown function, present in the genomes of
S.cerevisiae
and
C.elegans
, respectively. We identified these genes in the EMBL data library by means of a
search for homology with the RFX DNA binding motif. ScRFX corresponds to an 811
amino acid open reading frame containing a centrally placed RFX DNA binding
motif (Fig.
1
). Three exons of the CeRFX gene were identified (Fig.
1
); the RFX DNA binding motif is contained within two exons separated by a small
66 bp intron, and a third exon was localized by virtue of the fact that it
contains additional regions homologous to RFX1-3 (see below).
Figure 3
.
Conservation of
the dimerization (D) domains. The amino acid sequence alignement (
A
) and the percent homology in all pairwise comparisons (
B
) are shown as in Figure 2.
2
). These four new motifs are sufficiently divergent among themselves and with
the previously known RFX1-4 sequences to permit a detailed characterization of the RFX DNA binding
domain (Fig.
2
). The consensus sequence for this domain (Fig.
2
A) shows no significant homology to any other known DNA binding motif. It is
characterized by 20 invariant and 12 similar amino acids. Among these, aromatic
residues (W, F, Y), basic residues (K, R), hydrophobic residues (I, L, V) and
four glycine residues are particularly prominent. The majority of the conserved
amino acids, particularly the invariant residues, are clustered in the C-terminal half of the domain, which consequently exhibits an overall
homology that is considerably greater than that observed in the N-terminal half.
Despite the strong conservation of the DNA binding motif, RFX proteins are
likely to have different target site specificities. This is already clear for
the RFX proteins for which target site specificity has been analysed in detail.
Optimal target sites for the three most closely related proteins, RFX1-3, are inverted repeats, referred to as either EF-C or MDBP motifs, that are present in several cellular genes as
well as in the enhancers of polyomavirus, cytomegalovirus and hepatitis B virus
(see refs
2
,
4
and references therein). On the other hand, RFX1-3 bind with lower affinity the X box motifs of MHC class II promoters,
sequences that do not show a perfect match to the consensus EF-C/MDBP site (
2
,
15
). The opposite is observed for the complex of which RFX5 is a subunit. The
optimal known target sites for this complex are the MHC class II X box motifs
rather than EF-C/MDBP sites (
11
). In addition, RFX1-3 complexes exhibit the peculiar and characteristic feature of being able
to bind to certain EF-C/MDBP sites only when they contain methylated CpG dinucleotides (see refs
2
,
4
and references therein). This dependence on methylation of certain target sites
is not observed for the complex containing RFX5 (
11
).
RFX1-3 bind DNA as homo- or heterodimeric complexes. The domain responsible for
dimerization has been mapped to a conserved C-terminal region in RFX1-3 (Fig.
1
, region D) (
1
,
2
). Two of the new RFX genes, sak1 (
9
) and CeRFX, contain a region exhibiting significant homology to this dimerization domain (Fig.
3
), suggesting that they also bind as homo- or heterodimers. The dimerization domain of RFX proteins consists of two
strongly conserved subregions separated by a region that is more divergent and
variable in length (Fig.
3
A). The two conserved subregions exhibit no significant homology to any other
known proteins, and do not contain any motifs known thus far to be implicated
in dimerization or protein-protein interactions. In particular, no potential coiled coil structure
is evident. Thus, like the DNA binding domain of RFX proteins, the dimerization
domain appears to represent a novel motif. Surprisingly, although RFX5 is known
to be a subunit of a multimeric complex, it does not contain a sequence showing
clear homology to the dimerization domain of RFX1-3 (
8
).
Outside of the DNA binding and dimerization domain, two other features
characterize RFX proteins. (i) Of three additional homologous regions (A, B and
C) first identified in RFX1-3 (
2
), two (B and C) have also been maintained in other members of the family (Fig.
1
). Region B is present in sak1, ScRFX and CeRFX (Fig.
4
), and region C is present in sak1 and CeRFX (Fig.
4
). Regions B and C have been conserved both in their sequence and in their
positions relative to the DNA binding and dimerization domains (Fig.
1
). (ii) RFX1-3, RFX5, sak1 and ScRFX all contain regions that share no sequence
homology but are rich in proline, glutamine or acidic amino acids (Fig.
1
). These features are characteristic of transcription activation domains,
suggesting that RFX proteins are transcription factors. This has in fact been
demonstrated to be the case for RFX1 (
4
) and RFX5 (
8
,
11
).
Figure 4
.
Conservation of
region B (
A
and
B
) and C (
C
and
D
). The amino acid sequence alignment (A and C) and the percent homology in all
pairwise comparisons (B and D) are shown as in Figure 2.
Figure 5
.
The phylogenetic tree of the RFX DNA binding domain sequences was generated
with the clustalW program (17) which uses the neighbour joining method (18).
The horizontal branch lengths (indicated above the branches) are proportional
to the percent divergence. The reproducibility of the tree was determined using
1000 bootstrap replicas. The percent of replicas containing a given branch is
indicated below the branches.
5
). Together with the presence and extent of homology of the other conserved regions, the analysis of the
phylogenetic tree raises a number of interesting issues. First, it shows that
the mammalian RFX1-3 genes form a subfamily of recently diverged genes, which is consistent
with the high conservation observed in the A, B, C and D regions. Secondly,
among the novel RFX genes, CeRFX appears to be the closest relative of the RFX1-3 subfamily. This is evident both from the sequence of its DNA binding
domain and from the strong conservation in the B, C and D regions.
Surprisingly, CeRFX seems to have diverged more recently than the two mammalian
RFX4 and RFX5 genes. This implies that duplication events leading to a
multigene family must have preceded the emergence of vertebrates, and indicates that invertebrates such as
C.elegans
should have additional RFX genes, possibly homologues of RFX4 and RFX5.
Thirdly, the DNA binding domains of the two yeast genes, which diverge most
from those of RFX1-3, are nevertheless associated with regions retaining homology to the B,
C or D regions, suggesting that these regions were present in an ancestral
gene. Finally, perhaps the most intriguing finding is that RFX5 is predicted,
on the basis of its strong divergence, to be a very ancient member of the
family (Fig.
5
), yet has acquired a highly specialized role in a system that has evolved only
relatively recently, namely the mammalian immune system. It should be mentioned
however that the phylogenetic tree was made under the assumption that all the
genes in the family diverged at identical rates. Consequently, if RFX5 has
diverged more rapidly than the other RFX genes, it may well have emerged more
recently than predicted by the phylogenetic tree.
In conclusion, RFX proteins constitute a family of DNA binding proteins that has
been conserved in
S.cerevisiae
,
S.pombe
,
C.elegans
, mouse and man, and must thus have appeared over a billion years ago. RFX
proteins are characterized by several highly conserved features, among which
the most prominent are novel DNA binding and dimerization motifs that are
clearly distinct from those found in other known DNA binding proteins. Finally,
RFX proteins function as regulatory factors in a wide variety of unrelated
systems, including regulation of the mitotic cell cycle in fission yeast, the
control of the immune response in mammals, and infection by human hepatitis B
virus. It thus appears likely that the RFX family will turn out to be as
widespread and functionally important as the well known zinc finger,
homeodomain, basic-leucine-zipper and basic-helix-loop-helix families of DNA binding proteins.
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*
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