From the Division of Experimental Therapeutics, and
Department of Immunology, University of Toronto, University Health
Network, Toronto General Research Institute, Toronto General Hospital,
Toronto, Ontario M5G 2C4, Canada, the Departments of
§ Microbiology and Immunology and of
Biochemistry,
University of Western Ontario, London, Ontario N6A 5C1, Canada, and
** Robarts Research Institute,
London, Ontario N6G 2V4, Canada
Received for publication, November 8, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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The chemokine receptors CCR8 and CX3CR1 are key
players in adaptive immunity and are co-receptors for human
immunodeficiency virus. We describe here the genomic organization and
evolutionary history of both of these genes. CX3CR1 has
three promoters that transcribe three separate exons that are spliced
with a fourth exon containing the coding region. CCR8 has
two promoters. One promoter produces a transcript of two spliced exons,
and the other promoter transcribes an exon containing the coding region
and lacks introns. We analyzed these promoters in the context of a luciferase reporter and identified several positive and negative regulatory elements. Identification of the genomic organization of
these genes in mouse demonstrates a similar organization for CCR8, but mouse CX3CR1 lacks two of the human
promoters and has an additional mouse-specific promoter that
transcribes only the exon containing the coding region and therefore
resembles the organization of the human and mouse CCR8
genes. We also identify two nontranscribed regions that are highly
conserved between human and mouse CX3CR1 containing
possible regulatory elements. Examination of the CX3CR1 and
CCR8 genes and surrounding genomic regions indicates that
these genes are the result of the duplication of an ancestral gene
prior to the divergence of teleost fish. We characterize single
nucleotide polymorphisms in the promoters of human CCR8 and
CX3CR1 and establish linkage relationships between
CX3CR1 promoter polymorphisms and two previously described
CX3CR1 coding polymorphisms associated with human
immunodeficiency virus disease progression and arteriosclerosis susceptibility.
Chemokines (chemotactic cytokines) are an extensive family of
homologous proteins initially described for their ability to control
leukocyte chemotaxis. Their role in immune biology has expanded to
include secretagogue, angiostatic, and angiogenic functions as well as
modulators of hematopoiesis and possibly organogenesis (1). Chemokines
share several functional properties and are identified and classified
on a structural basis (1-3). The CC and CXC are the two largest
subfamilies, containing four conserved cysteine residues, and can be
differentiated by the presence of intervening amino acid between the
first and second conserved cysteines (1, 2). Lymphotactin- The receptors for chemokines are members of the serpentine,
heterotrimeric G-protein coupled, seven-transmembrane-spanning receptor
superfamily (1, 2). Each chemokine receptor has a distinct set of
chemokine agonists and is expressed on a specific subset of leukocytes.
The array of chemokine receptors displayed on a leukocyte surface
regulates their chemotactic responsiveness (1, 2, 4). Chemokines and
their respective receptors orchestrate the immune response by
regulating leukocyte infiltration and subsequent leukocyte effector
functions as well as leukocyte emigration and homing to secondary
lymphoid organs.
CX3CR1 is the receptor of the chemokine fractalkine (CX3CL1), only one
of two chemokines present in both a membrane-bound and a soluble form
following proteolytic cleavage (5). CX3CR1/fractalkine have been
demonstrated to function in inflammatory responses and in particular
appear to be linked to Th1 adaptive immune responses (6). CX3CR1 serves
a unique role in microglia chemotaxis and communication with neurons in
the brain in addition to its role in immune cell migration (7). CX3CR1
can also be antagonized by vMIP-VMIPII from human herpesvirus 8 (8).
Furthermore, like many other chemokine receptors, CX3CR1 is a
coreceptor for HIV1 entry
(9). In fact, HIV infection leads to dramatic increase in the
expression of CX3CR1 in lymphoid tissues (plasma cells and
dendritic cells) and a corresponding increase in CX3CR1
expression on CD4 T-cells (10). Recently, a number of coding
polymorphisms have been characterized in CX3CR1 and shown to
alter susceptibility to both HIV infection and arteriosclerosis
(11-15). The two mutations are a valine to isoleucine substitution at
position 249 (V249I) and a threonine to methionine substitution at
position 280 (T280M). These two polymorphisms are in complete linkage
disequilibrium, so that all chromosomes possessing the
Met280 mutation also have the Ile249 mutation.
The Ile249 mutation, however, also exists together with the
common Thr280 form of the receptor. The polymorphism
leading to a threonine to methionine substitution at position 280 has
been shown to increase susceptibility to HIV in French Caucasians and
to have a modest delay in AIDS onset and all-cause death in North
American Caucasians (11, 13, 14). The Ile249 mutation, on
the other hand, is associated with decreased susceptibility to
arteriosclerosis (12, 15).
CCR8 is the chemokine receptor for I309 (CCL1) and is also a coreceptor
for HIV (16-20). Increased expression of CCR8 correlates with
activated Th2 T cells, although the receptor plays a role in the
trafficking of other cell types such as monocytes and endothelial cells
(21-24). Deletion of CCR8 in mice leads to impaired TH2
responses and decreased eosinophil recruitment (25). As with CX3CR1,
vMIP-VMIPII is also an antagonist to CCR8, as is MC148, a
chemokine-like molecule from Molluscum
contagiosum virus (26, 27). vMIP-I, another chemokine from
human herpesvirus 8, on the other hand, can function as a full CCR8
agonist (27). Like many chemokine receptors, both CCR8 and
CX3CR1 are located on human chromosome 3.
Whereas chemokine receptors have been identified for a number of years,
relatively little work has been done in examining their gene structure
and promoter function. CXCR1, -2, and
-4 as well as CCR2, -3, and
-5 have been examined (28-33), but the more than 15 other
members have yet to be analyzed. Furthermore, it is unclear how
chemokine receptor gene structure is conserved between species. Mouse
models are commonly used to study gene function due to the ease with
which knockouts and transgenic animals can be created. However,
translating the data from mouse models to human clinical applications
can be difficult, particularly in regard to gene regulation.
In this study, we have determined the gene structure and identified the
functional promoter elements in both human genes for CX3CR1
and CCR8. In addition, we have identified several noncoding polymorphisms and examined their linkage to other known mutations in
the CX3CR1 and CCR8 genes. Furthermore, a
comparative analysis between human and mouse genes shows that the gene
structure for CCR8 is conserved between mouse and human, but
the gene structure for CX3CR1 has undergone significant
mutations since the divergence of primates and rodents. Human and mouse
CX3CR1 genes also possess conserved regions within the
promoter and intronic elements. This form of multigene interspecies
comparison provides insight not only into the evolutionary history of
gene families but aids in the identification of conserved regulator
elements and potential differences in gene regulation.
Phylogeny of Chemokines and Chemokine Receptors--
The amino
acid sequence of human chemokines receptors along with three receptors
we identified from the fugu genomics project (available on the World
Wide Web at www.hgmp.mrc.ac.uk, scaffold M001015) were aligned using
ClustalW (34), and the alignment was edited by hand. Consensus trees
were generated using the protein parsimony method with a bootstrap
value of 1000 using the PHYLIP package (40).
Screening and Sequencing of Human CCR8 and CX3CR1 Genes--
To
obtain a human genomic clone containing the CCR8 and
CX3CR1 genes, primers to human CCR8-specific
primers (sense, atgccgtgtatgccctaaag; antisense, actttcacagctctcccta)
were submitted to Incyte Genomics (Palo Alto, CA) for PCR
screening of their human bacterial artificial chromosome (BAC) library.
Two overlapping clones were obtained (Incyte reference numbers 21533 and 21534). The entire 21534 BAC clone was sequenced using the GPS-1
genome priming system (New England Biolabs, Beverly, MA). Custom
software was developed to prejoin GPS-1 pairs, and then contigs were
assembled in the Sequencher DNA sequencing software (Gene Codes, Ann
Arbor, MI). The BAC vector sequence was removed, and the resultant
86,172-bp sequence was entered into GenBankTM under
ascension number AY016370.
RACE Analysis--
Five prime RACE analysis of the human and
mouse CCR8 and CX3CR1 transcripts was performed
using the 5'/3'-RACE Kit (Roche Molecular Biochemicals) according to
the recommended procedure using the following primers in order: human
CCR8, aggttcaagaggtatacatc, ctcagcttcttgcagaccac,
tttcccagaagactgaatac; human CX3CR1, gcacatggcattgtggaggc, ctacaaacagcagatcagac, gacactcttgggcttcttgc; mouse CCR8,
aagatggccagatacagcat, ggtagtagtcggtcatcgtg, atccatcgaggcaggaagac; mouse
CX3CR1, ttccggctgttggtgagagc, cggacaggaagatggttcca,
ttctctagatccagttcagg. The resulting PCR products were purified using
the GeneClean III system (Qbiogene, Carlsbad, CA) and cloned into the
pGEM-T cloning vector (Promega, Madison, WI) and subsequently
sequenced. RNA was purified for the RACE from human peripheral blood
mononuclear cells and mouse spleen (for CX3CR1) and thymus
(for CCR8) using the Trizol reagent according to the
manufacturer's recommendations. For each gene, three independent RACE
reactions were performed, and results were compiled.
Polymorphism Analysis--
We screened for polymorphisms by
sequencing the genomes of 10 clinically normal individuals. Overlapping
fragments of the CX3CR1 promoter were amplified using
primers caagtctgccaacttaaacgc and atgctcttcacttggtccatct (514-bp
product), gagatgtcagctcatccttcaa and atcactgtccagggcgtc (490-bp
product), gcagagaaatgagagtgtggtg and atgatcatgtggctgcactc (463-bp
product) and caatatttgaacttagcattgagg and ctgcctcagcctcccaagta (395-bp
product). Exon 1 of CX3CR1 was amplified with primers
cactggccattctccacc and gagagaacccctggaggg (89-bp product). Overlapping
fragments of the CCR8 promoter were amplified using primers
acaggtgacagcaggcaagtg and cccagcaatgcagaactgtga (850-bp product),
tagtccgttgtcacactgcta and gttatggaagtcctagcagac (909-bp product),
agaaaatctaggtagacaacacag and ttacttcttgtgctagacctctgaa (650-bp
product), tttgagacagagtctcactctg and tgactatttggagtgggtgagac (699-bp
product), and aaagtgctaggattacaggcatgag and tagtagtagtcggtcactgttgtca (301-bp product). PCRs were performed for 30 amplification cycles at an
annealing temperature of 57 °C for each reaction. Purified amplification fragments were sequenced using an Applied Biosystems Prism 377 Automated DNA Sequencer, and electropheretograms were interpreted using Sequence Navigator software. Genotypes of all CCR8 and CX3CR1 polymorphisms were determined
from electropheretogram tracings of direct sequencing experiments in
samples of clinical normal unrelated subjects from Caucasian, African,
South Asian, Chinese, Inuit, and Native American backgrounds (60 subjects in each group). We also genotyped two previously reported
nonsynonymous coding polymorphisms in CX3CR1, namely V249I
and T280M (14), to determine linkage disequilibrium relationships with
the CX3CR1 promoter polymorphisms. Promoter Analysis--
Fragments of the human CCR8 P1
and P2 and CXC3CR1 P1 and P3 putative promoters were cloned
into the luciferase-containing pGL3-Basic vector (Promega) using the
following primers: CCR8 P1 sense primers
( Comparison between Species--
The mouse genomic sequences for
the CX3CR1 and CCR8 genes were obtained from the
mouse genome data base of the Celera Discovery System (Celera Genomics,
Rockville, MD), reference GA_x6K02T2NQG0:3500001.4000000. The
chromosomal regions were compared with each other with the Blast2Sequences program (37). Individual exons and promoter regions
were also compared using the water program from the European Molecular Biology Open Software Suite (38). Potential transcription factor binding sites were identified using Matinspector Professional software (36).
The Phylogenetic Relationship of CCR8 and CX3CR1 to the Chemokine
Receptors on Chromosome 3--
Despite broad classification of
chemokine receptors based upon their ligand affinity, the relationship
of the receptors based on amino acid composition clusters receptors
into three broad phylogenetic trees. The vast majority of chemokine
receptors fall within a large cluster on chromosome 3 (Fig.
1). This group is composed of XCR1,
CX3CR1, CCR1, CCR2, CCR3, CCR4, CCR5, CCR8, CCRL2, and CCBP2. Further
subclassification indicates that CCR1 and CCR3 are most closely
related, as is CCR2 with CCR5 and CX3CR1 with CCR8.
CCR8 and CX3CR1 Are Closely Linked on Chromosome 3--
To further
determine the fine mapping and genomic organization of CCR8 and CX3CR1,
we screened a human genomic library by PCR and obtained two overlapping
BAC clones containing both the CX3CR1 and CCR8
genes. Sequencing revealed that the two genes are in a head to head
orientation and their coding regions are separated by 66 kb
(GenBankTM accession number AY016370). Within the context
of the entire human chromosome 3, the CDS for CX3CR1 begins
6.3 megabase pairs after CCR4, and the CDS for
CCR8 begins 6.5 megabase pairs before CCR9.
Chemokine receptors on chromosome 3 are thus organized into three
distinct clusters. CCR1, -3, -2, and
-5 and CCRL2 constitute one minicluster all
located within just over 200 kb flanked by the genes vascular
endothelial growth factor receptor and lactotransferrin.
This is interesting, since CCR1 and CCR3, located
beside each other, are most closely related (80% AA similarity,
63% AA identity), as are CCR2 and CCR5 (82% AA
similarity, 72% AA identity). CX3CR1 and CCR8
are similarly situated side by side and are most homologous to each
other (61% AA similarity, 40% AA identity) (Fig. 1). It would appear
that these "pairs" of genes have arisen by gene duplication.
Gene Structure of CCR8 and CX3CR1--
We next determined the exon
intron structure of the CX3CR1 and CCR8 genes. We
performed 5'-RACE on human peripheral blood mononuclear cells for both
receptors. In the case of CCR8, we identified two species of
transcripts, indicating two likely initiation sites and promoters (Fig.
2). The most abundant transcript begins at position Analysis of the Promoters--
To determine the functional
organization of the putative promoter elements identified above, we
created constructs for both putative CCR8 promoters as well
as the first and third promoters of CX3CR1 containing
sequentially longer fragments up to
The CCR8 P1 promoter shows no appreciable activity in THP-1
or Jurkat cells. This may indicate that this promoter is only active in
other cell types or that there are positive regulatory elements in the
intron or at a significant distance from the start of transcription.
The CCR8 P2 promoter, however, shows a strong positive
element from 0 to Identification and Characterization of Polymorphisms--
Single
nucleotide polymorphisms in CX3CR1 have been shown to alter
susceptibility to both HIV infection and arteriosclerosis (11-15). To
further characterize single nucleotide polymorphisms within the genes
for CX3CR1 and CCR8, we screened both genes
within the
Two common nonsynonymous coding polymorphisms of human
CX3CR1, namely V249I and T280M, were previously shown to
result in altered ligand affinity and expression (12). Contrary to a
previous study (14), we found both polymorphisms in all ethnic groups examined, although to varying degrees (Fig.
4D). In our samples, we found
strong linkage disequilibrium between genotypes of these two coding
SNPs across all ethnic groups. Since the most common chromosomal
haplotype defined by CX3CR1 promoter and coding SNPs was
different for each ethnic group (Fig. 4B), we performed a preliminary analysis of pairwise linkage disequilibrium relationships to determine whether there were differences between ethnic groups. Overall, there was weak linkage disequilibrium between the T280M and
Comparison of the Human Gene Structure with Mouse--
Since mice
are frequently used as immunological models of human diseases, we
examined the structures of the CCR8 and CX3CR1 mouse genes. We performed 5'-RACE on RNA from mouse spleen cells for
CX3CR1 and thymocytes for CCR8 and mapped the
corresponding 5' transcripts onto the murine genomic fragment
identified in the Celera Mouse Genome Database (Fig.
5). The gene structure of CCR8
appears to be similar between humans and mice. Both have two promoters,
where the first promoter initiates transcripts with a single intron and
the second promoter commencing just prior to the coding sequence and
lacking any introns. Sequence comparison of human and mouse exon 1 shows a substantial amount of sequence identity of 79.5% (Fig.
6).
The human and mouse CX3CR1 genes contain several differences
and similarities. Murine CX3CR1 has two putative promoters,
with the transcript from first promoter containing a single intron and
the transcript of the second promoter containing no introns. This
structure is similar to the structure of the mouse and human CCR8 genes and differs from human CX3CR1, which
contains three putative promoters that all produce transcripts with a
single intron. Comparison of the genomic sequences also revealed two conserved nonrepetitive and noncoding elements in the human and mouse
CX3CR1 genes. The first conserved region (Fig. 6, alignment 1) is located upstream of the third human and first mouse putative promoters. This region also overlaps with a positive regulatory region
identified in our promoter analysis (Fig. 3). The second conserved
region is contained within intron 3 in humans and intron 1 in mice.
Furthermore, a region within exon 3 in human CX3CR1 has an
83% sequence identity with a region in exon 1 of mouse CX3CR1.
The CX3CR1/CCR8 Duplication Predates the
Fish/Mammal Division--
To date, a small number of
chemokine receptors, namely CXCR1 and -4 and CCR1, -7, and -9, have
been described in various fish species. The majority of receptors,
however, do not currently have a fish homologue. Recently, the draft
genome of the pufferfish Fugu rubpribes, a teleost fish, has
been released. This species appears to be unique in that whereas it
maintains the same number of genes as similar species, the genome is
only one-eighth the size. We screened the pufferfish genome and
identified three putative chemokine receptors present within a single
contig (Fig. 6). Whereas direct comparison with other receptors fails
to give an unambiguous identity, phylogenetic analysis suggests that
the three receptors represent CX3CR1, CCR8, and CCR2, respectively
(Fig. 1). Interestingly, none of the receptors appear to possess the
entire coding sequence on a single exon as is the case for their
mammalian counterparts. Whether this is the result of the process by
which fugu compacts its genome or is indicative of these genes being
converted to pseudogenes has yet to be determined. Nevertheless, this
appears to be a primitive locus corresponding to at least part of the primary chemokine cluster on chromosome 3 and indicates that the CX3CR1/CCR8 duplication predates the division of
mammals and fish.
In summary, CCR8 and CX3CR1 appear to have been
duplicated prior to the branching of mammals and fish. Throughout
evolution, the human and mouse genes have maintained many similar
features both within coding and noncoding regions, although
differences, such as the loss of one promoter and the addition of two
others in human CX3CR1, have resulted and are likely to
contribute to the interspecies variation in gene regulation. Both genes
possess coding and promoter polymorphisms that may alter susceptibility to various diseases.
In the present study, we have determined the human and mouse
genomic organization of the genes for CX3CR1 and
CCR8. In humans and mice, these two genes are located within
66 and 24 kb of each other, respectively, in a head to head fashion. In
pufferfish, the genes are located 4 kb from each other in a tail
to head manner (Fig. 6). Based on amino acid composition, CX3CR1 and
CCR8 are more closely related to each other than they are to any other chemokine receptor. The close proximity and high amino acid identity indicates that these genes probably arose through a duplication event
prior to the divergence between fish and mammals. Structurally, the
gene for mouse CCR8 is similar to the structure of the mouse gene for CX3CR1 and the human gene for CCR8. This
suggests that the duplication event encompassed a region larger than
the coding sequence alone and included additional promoter elements. In
fact, the entire chemokine receptor cluster on chromosome three appears to be a result of sequential duplication events. The premise that at
least some of these duplications include more than just the coding
region of genes is supported by the fact that several other genes
appear to have also been duplicated within the cluster, including a
series of krueppel-related C2H2-type zinc finger proteins, elongation
factors, keratin genes, ribosomal proteins, and WD domain-containing
proteins interspaced within the chemokine receptors (data not
shown) in both mice and humans.
Despite the close amino acid homology of CX3CR1 and CCR8, the ligands
for these receptors are phylogenically different. This raises
interesting questions about the evolution of chemokine receptors and
their ligands. External binding domains of the CX3CR1 and CCR8
receptors are conserved, since both can bind vMIP-VMIPII; the
receptors have obviously diverged to accommodate binding of very
different ligands. CCR8 binds CCL1 (I309), and CX3CR1 binds CX3CL1
(fractalkine). Phylogenetic chemokine analysis shows that these two
chemokines fall within different subtrees (data not shown). This
probably indicates that chemokine receptor evolution through
duplication does not parallel the duplication of the chemokines themselves and that duplicated receptors undergo
significant changes in their ligand binding properties. Since the
majority of the receptor is not directly involved in ligand binding,
these nonbinding regions provide a more accurate evolutionary history
of the molecule. The extracellular binding portions of divergent
receptors may have convergently evolved in some cases to bind similar
ligands. For example, CXCR3 resembles two other receptors more closely than CXCR4, but SDF1 and ITAC (ligands for the two respectively) are
most closely related to each other.
Of the chemokine receptor genes examined in detail, multiple promoters
have been found in all cases (28-33). In fact, the 5'-untranslated regions of chemotactic receptor genes tend to be heterogeneous with
multiple exons often accompanied by alternate splicing.
CXCR2, for example, has 12 alternately spliced first exons
(29, 32). CCR5 has three alternately spliced exons
transcribed by two promoters preceding the fourth exon that contains
the entire coding region (28). Typically, the coding sequencing is
contained in a single exon, although the receptor CCR7 is coded
for in multiple exons (6). Our work confirms this trend, showing that
CCR8 and CX3CR1 possess multiple promoters in
both humans and mice, although there appear to be significant
differences between mouse and human CX3CR1. Mouse
CX3CR1 as well as mouse CCR8 and human
CCR8 all have promoters (P2 promoters) that produce
transcripts containing the coding sequence (and flanking 5'- and
3'-untranslated regions) but contain no introns. This is not the case
in human CX3CR1. The corresponding promoter appears to have
been lost following the divergence between human and mouse lineages.
However, the P3 region and third exon of human CX3CR1 are
highly homologous to the P1 region and first exon of mouse
CX3CR1. Human CX3CR1 possesses two additional
promoters (P1 and P2) and exons located upstream of P3. These promoters are not present in mouse CX3CR1, mouse CCR8, or
human CCR8 genes. It is possible, however, that the
transcripts we used to define the promoter regions for each of these
genes do not represent the full complement of transcripts, and cryptic
promoters may be functional in alternate tissues.
This raises some interesting questions in regard to gene regulation.
Our data would suggest similarities and differences in the regulation
of human CX3CR1 and mouse CX3CR1. Whereas the
homologous promoter/exon between human and mouse CX3CR1 may
provide for an overlapping function, the distinct promoters in both
species may contribute to species-specific roles for this gene.
Interestingly, Garin et al. (41) utilized our initial report
(GenBankTM accession number AY016370) of sequencing the
CX3CR1 locus, defining the CX3CR1 P1 and P3
promoters, to identify three CX3CR1 promoters. They were
able to confirm our location of the P1 and P3 promoters but
additionally identified a unique third promoter distinct from the P2
promoter we report in the present study. They did not examine the
organization of CCR8 or SNPs located within the promoter
regions of either gene.
We also observed an additional highly conserved region (33 bases, 94%
conserved) within the large intron of human and mouse CX3CR1. This region does not appear to be a repetitive
element as determined by RepeatMasker (available on the Internet at
ftp.genome.washington.edu/RM/RepeatMasker.html).
Neither of these conserved regions within CX3CR1 have
reported gene polymorphisms, either from our observations or data
present in the NCBI dbSNP or Celera databases. Nevertheless, we found eight polymorphisms within the first putative promoter of
CX3CR1. To date, two identified human CX3CR1
coding polymorphisms have been implicated in altered expression and
function of the receptor, namely the V249I and T280M mutations
(11-14). Defining the individual contributions of these polymorphisms
is complicated because they are in linkage disequilibrium, and
homozygotes for both Ile249 and Thr280 are
rare. Nevertheless, initial studies suggest that the Ile249
polymorphism correlates with a 35% decrease in cell surface receptor number (12) and the Met280 polymorphism is associated with
decreased affinity for the CX3CL1 ligand (14). The Ile249
has been associated with decreased availability of fractalkine binding
sites, although the number of binding sites is highly variable between
individuals with the same genotype. It is also unclear whether this SNP
is fully responsible for the observed phenotype or whether it is linked
to another causal factor. Hudson and colleagues (39) identified a
polymorphism within the promoter region of human insulin-like growth
factor-binding protein-3 that is responsible for altered gene
expression. In our present study, we characterized nine additional
polymorphisms in the CX3CR1 gene and expanded the scope of
the T280M and V249I mutations, which were previously demonstrated
mainly in Caucasian samples, to include several different ethnic
groups. Two promoter SNPs, namely Similarly, we have identified five polymorphisms within the first
putative promoter of human CCR8, although whether any of these serve a regulatory function has yet to be determined. In addition
to our data, Celera has described a G/C SNP at amino acid 27 that leads
to a glycine to alanine substitution. Whether this mutation has a
resulting phenotype has yet to be determined.
Chemokine receptors have evolved into extremely specific effectors of
cellular activation and migration. Our study examines the evolution of
the CCR8 and CX3CR1 receptors by examining both the macroscopic and microscopic events that shaped the evolution of the
receptor. We report that these genes arose through a duplication event
prior to the branching of fish and mammal lineages and that this
duplication included both coding and noncoding regions. It is likely
that prior to the division of human and mouse, both receptors contained
two promoters, the first producing a transcript with a single intron
and the second producing a transcript consisting of the coding exon
exclusively. The CX3CR1 receptor, however, appears to have
undergone subsequent evolution in the human, leading to the addition of
two other noncoding exons and the loss of the P2 promoter in mice.
Despite this, nontranscribed regions in both the promoter (P3 human, P1
mouse) and intron have persisted to the present day, highlighting both
the overlapping function of these receptors and their interspecies
diversity. Within humans alone, polymorphisms within the receptor lead
to altered phenotypes and changes in disease susceptibility. Within
different racial groups, the distribution and linkage of these
polymorphisms has led to interethnic diversity that may well contribute
to a RACE-specific impact of a polymorphism block defined by all SNPs
rather than a single polymorphism in isolation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/XCL1 and
lymphotactin-2/XCL2 are the sole C chemokines and lack the first and
third cysteines of the four-cysteine motif (1). Fractalkine/CX3CL1 is
the only member of the CX3C subfamily and has three intervening amino
acids between the first and second conserved cysteines.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 analysis
was used to assess deviation of genotype frequencies from
Hardy-Weinberg expectations. Linkage disequilibrium between markers was
estimated using a maximal likelihood method with p value
adjusted for multiple comparisons (35).
199gctgccttggtaccgtgtcctcacatggcc,
299aaacaacaggtacctgttttcccacagttc,
399aaacaacaggtacctgttttcccacagttc,
519atggaggaggtaccgtcctggtataaattg,
1423tgccgaggtatgaattcaggtaccagcaga), CCR8 P1
antisense primer tgatcctcactcgagtgcagctcaatgagc, CCR8 P2
sense primers (
46gaggacacggtaccgtccataacatccccc,
96aatagtcaggtacccaccggaggttgagca,
196cctcccaaggtaccaggattacaggcatga,
296catgcccgggtaccttttgtatttttagta,
396agtggtgtggtaccggctcacagcaacctc,
501gggccctgggtacctaacctcatttaacct,
1312gaacattaggtacctctctattgcatcaga), CCR8 P2
antisense primer aatccatcctcgaggcgggacctggtcaca, CX3CR1 P1
sense primers (
52agcacggtaccaagtgagaaggggcgggccgt,
105ggaaggtacctgccacgggcacaggcatgttgg,
206gcatgggtacctgtgcttgctagcagctgtggaac,
303ctcacggtaccacaggtatctatccaagccatg,
403tgacaggtacctcagggccaaataaccgacttgc,
505ctttctggtaccatgagtgtgtcaagaatgcaaagca,
1308aggctgaggtaccaggatcgttcgagc), CX3CR1 P1
antisense primer cccactcgagattctccacccagaagccagagagctc, CX3CR1 P3 sense primers
(
51cttacaggaaggtacctactattggctaactctgcaa,
120ttggcaggtacccctccaccctggagtatctga,
222tgcccccccggtaccaggttggggtaagtggcacctc,
328gggcagggtacctgagggccccatgagctct,
403tgggacggtaccttgaggagaagagctgtgtgtcccca,
498catatcaggtacctaagaatttcattaaaatgca,
1619gacttggtacccaggaagagctctctg), and CX3CR1
P3 antisense primer ttccactcgagacgagacgatctgccagtcagccaccc. All sense
primers contain an XhoI restriction site, and antisense primers contain a KpnI restriction site. Polymerase chain
reaction was performed using a PTC-150 Minicycler (MJ Research Inc.,
Waltham, MA) for 30 cycles with 95 °C denaturation 1 min, 57 °C
annealing 45 s, and 72 °C elongation 1 min 45 s. PCR
products were digested with XhoI and KpnI (New
England Biolabs, Beverly, MA) and ligated into the pGL3-Basic vector
(Promega), and sequence was verified. THP1 and Jurkat cells were
cultured in complete RPMI 1640 with 10% fetal calf serum (Sigma). For
transfections of CX3CR1 constructs, cells were spun down at
1600 rpm and resuspended in RPMI 1640 to a concentration of 20 × 106 cells/ml. 750 µl of resuspended cells were added to
micropulser cuvettes (Bio-Rad catalog no. 165-2089), and 15 µg of
construct were added. Cells were electroporated at 200 V and 975 microfarads for THP1 cells and 400 V and 975 microfarads for Jurkat
cells, using a Bio-Rad gene pulser II, and added to 3 ml of 37 °C
complete RPMI medium in a 0.1-cm six-well plate (VWR, West Chester,
PA). Cells were then incubated at 37 °C for 48 h. After
incubation, luciferase activity was measured using the Bright-Glo
luciferase assay system (Promega) according to the manufacturer's
directions. All transfections were performed four times, and triplicate
samples were measured for each replicant. CCR8 construct
transfections for THP1 cells were performed as above, whereas
transfections into Jurkat cells were performed as follows. Jurkat cells
were spun down at 1500 rpm and resuspended in Opti-MEM I reduced serum medium (Invitrogen) to a concentration of 10 × 106
cells/ml. 200 µl of resuspended cells were added to each well of a
six-well plate. 3 µg of DNA construct in 500 µl of Opti-MEM and 24 µl of GenePORTER reagent (GenePORTER Transfection Reagent, Gene
Therapy Systems, Inc., San Diego, CA) in 500 µl of Opti-MEM were
premixed and incubated at room temperature for 30 min before the
complexes were added to the cells. Cells were then incubated at
37 °C for 4 h, followed by the addition of BoosterExpress
reagent number 1 at a 1:50 dilution in Opti-MEM medium. Cells were then returned to the incubator for 48 h at 37 °C. After incubation, luciferase activity was measured using the Bright-Glo luciferase assay
system (Promega) according to the manufacturer's direction. All
transfections were performed in triplicate samples for each construct.
Potential transcription factor binding sites were identified using
Matinspector Professional software (36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
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Fig. 1.
Phylogeny of chemokine receptors. Known
human chemokine receptors and three putative fugu chemokine receptors
were aligned using ClustalW and a consensus tree generated by the
protein parsimony method with a bootstrap score of 1000.
2626 relative to the start codon for CCR8 and
has a single intron. The other transcript species begins at position
48 and has no intron. Similarly, three species of transcripts were
identified for CX3CR1, indicating three putative promoters commencing at positions P1 (
15,191), P2 (
13,851), and P3 (
13,532) relative to the start codon. All three transcript species possess a
single intron. The third species of transcript is most abundant followed by the first and second species, respectively. They all possess unique first exons but share exon 4 that contains the entirety
of the coding region.
View larger version (17K):
[in a new window]
Fig. 2.
Gene structure of human CCR8
and CX3CR1 chemokine receptor genes.
5'-RACE analysis was performed for human CCR8 and
CX3CR1 receptors in peripheral blood mononuclear cells, and
gene structure was determined by comparison with genomic sequence.
Position numbers are relative to the start codons of the genes. RACE
products occurring more than once are indicated with a multiplier
showing the number of times that particular product was identified. *,
the most commonly found transcript.
500 bases from transcription
start and a long promoter fragment (>1000 bp) cloned in front of a
luciferase reporter gene lacking promoter or enhancer elements.
Constructs were transfected into THP-1 and Jurkat cells, and promoter
activity was measured (Fig. 3). THP-1 cells are mononuclear in nature, most closely resembling monocytes, whereas Jurkat cells most closely resemble T-cells. CX3CR1
P2 was omitted from the analysis due to the closeness of the third promoter (300 bp) and the low frequency of P2 transcript. The P1
promoter of CX3CR1, regions from
105 to
206 and >
505,
appear to contain positive promoter elements, whereas the region from
303 to
403 appears to contain a negative regulatory element. The
third promoter contains positive elements in the
51 to
120 and
120 to
222 regions and negative elements in the
222 to
328
region. There also exist cell-specific differences in promoter expression. In particular, the
403 to
498 region of the
CX3CR1 P3 promoter has a positive regulatory effect in THP-1
cells and a negative regulatory effect in Jurkat cells.
View larger version (34K):
[in a new window]
Fig. 3.
Promoter analysis of human CCR8
and CX3CR1 genes. Promoter fragments of
human CX3CR1 P1 and P3 promoters and CCR8 P1 and
P2 promoters were cloned into an enhancerless luciferase reporter
vector and transfected into THP1 cells or Jurkat cells, and activity
was measured. Results are reported as the -fold increase over
transfection of the luciferase vector alone ± S.E. from a
representative experiment. Putative transcription factor binding sites
are also indicated.
46 with a negative element between
46 and
96.
The
196 to
296 region has a positive regulatory element in THP1
cells alone. The
196 to
296 region has a negative regulatory effect
in THP1 cells but a positive regulatory effect in Jurkat cells and may
be involved in cell-specific expression.
15,192 to
17,242 regions, relative to the start codon,
of human CX3CR1 and
16 to
4677 of human CCR8
(Table I). We identified nine additional
polymorphisms within the CX3CR1 gene. Eight of these nine
polymorphisms are located within the first putative promoter, with the
ninth polymorphism being present within the first exon of the gene.
Three of these polymorphisms have been described in the Celera data
base, with one of them being present within the NCBI SNP data base as
well. Within the human CCR8 gene, we identified four
polymorphisms within the first promoter, one of which has been
identified in the Celera data base and one within the second promoter
(Table I). The
40T/G polymorphism in the second promoter lies within
the
46 promoter construct (Fig. 3) that contains positive regulatory
elements.
Single nucleotide polymorphisms in the CX3CR1 and CCR8 genes
16290T
C SNP and between V249I and the
15854A
G SNP (Fig.
4A). However, there was a unique linkage relationship in Caucasians, with moderately strong linkage disequilibrium between both
the CX3CR1 V249I and T280M SNPs and the
15430G
C SNP
(Fig. 4C). This was not seen in other ethnic groups. Such
interethnic differences were also seen for linkage relationships
between CCR8 SNPs (data not shown). This preliminary
analysis indicates that there are interethnic differences in linkage
relationships involving functional SNPs in the CX3CR1
protein sequence and promoter. These genomic differences may
contribute to interethnic disparities in associations with biochemical
and/or clinical phenotypes. These preliminary findings suggest that
future studies of genotype-phenotype association should have
determinations of all possible SNPs at these loci. The functional
consequences due to genomic differences at either CX3CR1 or
CCR8 may be related to the ethnic-specific intact complement
or haplotype block defined by all SNPs rather than to the putative
functional consequence of any single SNP in isolation.
View larger version (38K):
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Fig. 4.
Analysis of human CX3CR1
single nucleotide polymorphisms. Seven SNPs were identified
in the human CX3CR1 P1 promoter, and one was identified in
the human CX3CR1 first exon in addition to the previously described
T280M and V249I coding polymorphisms. A, linkage
disequilibrium between CX3CR1 SNPs was estimated using a
maximal likelihood method with p value adjusted for multiple
comparisons. B, most commonly found alleles in Caucasian,
African, South Asian, Chinese, Inuit, and Native ethnic groups.
C, linkage disequilibrium between CX3CR1 SNPs in
the Caucasian population alone. D, frequency of
Ile249 and Met280 coding polymorphisms in
multiple ethnic groups. 60 subjects were studied for each ethnic group.
*, p < 0.05, **, p < 0.0014.
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Fig. 5.
Gene structure of murine CCR8
and CXC3CR1 chemokine receptor genes.
5'-RACE analysis was performed for mouse CCR8 and
CX3CR1 in thymocytes and splenocytes, respectively. Gene
structure was determined by comparison of RACE sequences with genomic
sequence. Position numbers are relative to the start codons of the
genes. RACE products occurring more than once are indicated with a
multiplier showing the number of times that particular product was
identified.
View larger version (29K):
[in a new window]
Fig. 6.
Interspecies comparison of the
CX3CR1 and CCR8 genes in humans,
mice, and pufferfish (fugu). In humans and mice, the
CX3CR1 and CCR8 genes are located in a head to
head fashion in their respective genomes. Their order relative to other
genes in the cluster is also shown. Sequence comparison was performed
using the Blast2Sequences program between the corresponding genomic
regions premasked for repetitive elements. Four regions of homology
outside of the coding sequences were identified: 1) first exon of
CCR8 in both human and mouse genes, 2) the P3 promoter
region of the human CX3CR1 gene and the P1 promoter region
of the mouse CX3CR1 gene, 3) third and first exons of the
human and mouse CX3CR1 genes, respectively, and 4) an
intronic element conserved between human and mouse CX3CR1.
For conserved sequences that are nontranscribed, putative transcription
factor binding sites shared between mouse and human sequences are
shown. We also identified a pufferfish genomic contig containing three
putative chemokine receptors. Numbering in all cases is relative to the
start of the indicated genomic contig.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
16290T
C and
15854A
G are
in weak linkage disequilibrium with the T280M and V249I SNPs,
respectively. Furthermore, the
15430G
C SNP is in moderately
strong linkage disequilibrium with both coding SNPs. Whether these
promoter SNPs influence association with phenotype is unknown, but the
interethnic differences in linkage disequilibrium suggest that
preliminary association studies of CX3CR1 SNPs and both HIV
and arteriosclerosis should be expanded to ethnic groups other than Caucasian.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Joseph Andrews for technical support along with the support of the London Regional Genomics Institute.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Heart and Stroke Foundation of Canada, Genome Canada, the Canadian Institutes of Health Research (CIHR), and a special award from the Robarts Research Institute (London, Ontario, Canada).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY016370.
¶ A CIHR scholar.
To whom correspondence should be addressed: Division of
Experimental Therapeutics, Toronto General Hospital, 200 Elizabeth St.,
MBRC5R425, Toronto, Ontario M5G 2C4, Canada. Tel.: 416-340-4800 (ext.
6984); E-mail: dkelvin@uhnres.utoronto.ca.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211422200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HIV, human immunodeficiency virus; BAC, bacterial artificial chromosome; RACE, rapid amplification of cDNA ends; AA, amino acid; contig, group of overlapping clones; SNP, single nucleotide polymorphism.
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REFERENCES |
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