(Received for publication, August 18, 1994)
From the
The human Fc receptor with low affinity for IgG (FcRIII,
CD16) is encoded by two nearly identical genes, Fc
RIII-A and
Fc
RIII-B, resulting in tissue-specific expression of alternative
membrane-anchored isoforms. The transmembrane CD16 receptor forms a
heteromeric structure with the Fc
RI (
) and/or CD3 (
)
subunits on the surface of activated monocytes/macrophages, NK cells,
and a subset of T cells. The expression of the
glycosylphosphatidylinositol-anchored CD16 isoform encoded by the
Fc
RIII-B gene is restricted to polymorphonuclear leukocytes and
can be induced by Me
SO differentiation of HL60 cells. We
have isolated and sequenced genomic clones of the human Fc
RIII-A
and Fc
RIII-B genes, located their transcription initiation sites,
identified a different organization of their 5` regions, and
demonstrated four distinct classes of Fc
RIII-A transcripts
(a1-a4) compared with a single class of Fc
RIII-Bb1
transcripts. Both CD16 promoters (positions -198 to -10)
lack the classical ``TATA'' positioning consensus sequence
but confer transcriptional activity when coupled to the human lysozyme
enhancer. Both promoters also display different tissue-specific
transcriptional activities reflecting the expected gene expression of
Fc
RIII-A and Fc
RIII-B in NK cells versus polymorphonuclear leukocytes. Within the -198/-10
fragments, the sequences of the two CD16 genes have been identified to
differ in 10 positions. It is suggested that these nucleotide
differences might contribute to cell type-specific transcription of
Fc
RIII genes.
Human leukocyte receptors for the Fc fragment of IgG (FcR)
play an important role in immune responses as they link the humoral
immune system with cellular effector functions. Fc
receptors
represent a family of cell surface glycoproteins mediating clearance
and phagocytosis of immune complexes, various forms of
antibody-dependent cellular cytotoxicity (ADCC)
and release
of inflammatory cytokines (for review, see (1) ). Fc
receptors have been divided into three classes, namely a high affinity
class (Fc
RI) and two low affinity classes (Fc
RII and
Fc
RIII). All are members of the Ig gene superfamily. Within each
group, multiple subtypes have been identified by molecular cloning.
Structurally conserved extracellular domains are linked to divergent
transmembrane and intracellular domains. Distinctive Fc
receptor
genes in addition to differently spliced messages from the same Fc
receptor gene are expressed on different cell types in a
tissue-specific manner generating diverse cellular responses upon
interaction with IgG immune complexes (for review, see (2) ).
The low affinity receptor FcRIII (CD16) is a 50-80-kDa
membrane glycoprotein(3) . At least two isoforms encoded by two
distinct genes, Fc
RIII-A and Fc
RIII-B, have been
identified(4) . An allotypic polymorphism, NA1 and NA2, has
been described to be associated with the Fc
RIII-B receptor
isoform. Fc
RIII-A is a transmembrane protein expressed on
activated monocytes/macrophages, NK cells, and a subset of T
cells(5, 6, 7) . In contrast, Fc
RIII-B
is a glycosylphosphatidylinositol (GPI)-anchored protein that is
constitutively expressed by neutrophils and after
-interferon
(IFN-
) stimulation by
eosinophils(4, 8, 9) . In addition,
Fc
RIII-A-specific transcripts can be induced in growth-arrested
human mesangial cells by IFN-
(10) . While the GPI
Fc
RIII-B isoform is expressed on the cell surface without other
subunits, efficient expression of the transmembrane Fc
RIII-A
requires the presence of the Fc
RI
subunit and is also
enhanced by co-expression of CD3
, a subunit of the T cell
receptors' complex (11, 12, 13) .
Through their differential requirements for these associated
``trigger'' molecules, the Fc
RIII isoforms mediate
different functions(14) . The Fc
RIII-A transmembrane
receptor on NK cells mediates ADCC, where it represents the only
Fc
R(5) . The GPI-linked Fc
RIII-B receptor on
polymorphonuclear leukocytes is certainly involved in cell activation,
but its detailed role is still controversial(15, 16) .
The FcRIII receptor appears to be under complex control. First,
it is highly tissue-specific and is expressed on granulocytes as well
as on activated monocytes/macrophages, NK cells, and some cytolytic T
cell subsets. Second, only granulocytes use the GPI-linked
Fc
RIII-B isoform encoded by a second gene. Third, the
Fc
RIII-B isoform can be induced during Me
SO-initiated
granulocytic differentiation of the HL60 human promyelocytic cell line.
Fourth, depending on the cell type, both isoforms can be up-regulated
by IFN-
.
In this study we report the cloning of both
FcRIII-A and Fc
RIII-B gene promoters. Sequence analysis of
all the cloned transcription initiation sites demonstrates structural
heterogeneity through the use of alternative 5`-UT/S1 exons from the
Fc
RIII-A but not from the Fc
RIII-B gene. We have defined the
5` sequences necessary for promoter activity by transient transfection
experiments with different parts of the Fc
RIII-A and Fc
RIII-B
promoters in different cell lines. Both promoters (positions -198
to -10) displayed different tissue-specific transcriptional
activities when coupled to a heterologous enhancer. These different
tissue specificities are suggested to be due to nucleotide differences
of the Fc
RIII-A and Fc
RIII-B genes identified at 10 positions
in the -198/-10 region.
Figure 2:
Panel A, structure of the human
FcRIII cDNA pGP5. The exon organization derived from the gene map
is drawn to scale (1977 bp), 5` to 3`, left to right.
Exon-intron boundaries are indicated by verticalsolidlines. The signal sequences (S), extracellular
domains (EC), transmembrane domain (TM), cytoplasmic
region (C), and 3`-untranslated region (UT) are
shown. The restriction endonuclease sites and nucleotide map units are
designated for BglII (Bg), EcoRI (E), HindIII (H), KpnI (K), and SalI (S). The fragments of pGP5
utilized as hybridization probes are depicted (80 = BamHI-BglII, 178 = BglII-SalI, 380 = SalI-KpnI, 601 = KpnI-EcoRI, 670 = EcoRI-HindIII). Panel B, structural
organization of the two genes for human Fc
RIII A and B. The
exon-intron organization is presented to scale as a linear map, 5` to
3`, left to right. Exon locations (5` S1, S2,
EC1, EC2, TM/C 3`) are boxed. Below the map, the
two selected genomic clones
32 and
31 for human Fc
RIII
genes A and B are shown. Restriction enzyme sites for BamHI (B), EcoRI (E), HindIII (H), and SalI (S) are indicated by verticallines. C, sequencing strategy of
the 5` end of genes A and B. The restriction maps of the subcloned 5`
ends of both genes (p32 = 5` end of Fc
RIII-A; p31 =
5` end of NA2-Fc
RIII-B) are designated. Restriction sites for ApaI (A), BamHI (B), BstYI (Bs), EcoRI (E), EcoRV (RV), SacI (S) and SphI (Sp) are indicated by verticallines. The
sequencing strategy is indicated by arrows below the
maps.
Figure 1:
Southern
blots of human genomic DNA identify differences among the FcRIII-A
and the NA1 and NA2 alleles of the Fc
RIII-B genes. Genomic DNAs isolated from peripheral blood lymphocytes from
individuals of a different phenotype (designated NA2, NA1, NA1NA2) were
used. Panel A, digestion was performed with BamHI (B), HindIII (H), EcoRI (E), or with the combination of two enzymes (HB) and (EB). The digested DNA was electrophoresed in an agarose gel,
transferred to Hybond-N, and hybridized with the 5` 80 bp containing BamHI-BglII fragment of cDNA pGP5 (Fig. 2A). The NA2 homozygote donor demonstrates a
second BamHI restriction fragment not observed in the absence
of the NA2 allele. PanelB, digestion was performed
with TaqI. After electrophoresis and transfer to nylon, the
blot was hybridized with an EC1 oligonucleotide. Rehybridization was
performed with the 30-mer oligonucleotide complementary to S1 sequences
used by primer extension analysis (see ``Materials and
Methods'').
Figure 3:
Nucleotide sequence comparison of
5`-flanking regions derived from the human FcRIII genes A and B.
The HindIII-XhoI fragment containing the 5`-flanking
region and the first three coding exons for gene A are in uppercase letters; the translated amino acid sequence is written in the three-letter code. Nucleotide sequences shown at the right are numbered starting from the ATG translation-initiation codon
designated as +1. If nucleotide sequences for gene B differ from
gene A, they are given below those of Fc
RIII-A. Dashed lines indicate nucleotide identity to gene A. Base pair
substitutions are shown by the nucleotide change; nucleotide deletions
are marked by asterisks.
Figure 5:
Mapping the transcription-initiation sites
of the human FcRIII A and B mRNA. a, schematic
presentation of the promoter region of the Fc
RIII genes A and B,
the two
P-labeled antisense RNA probes, and the
oligonucleotide primer used for RNase protection and primer extension
assays, respectively. The sequences of genes A and B containing
transcription-initiation sites are shown below the schematic diagram.
The transcription-initiation sites are indicated by vertical
bars; the height of each bar is roughly proportional to the amount
of mRNA starting at a particular site, as estimated from the RNase
protection experiments or primer extension assay. b, primer
extension of the human Fc
RIII A and B RNAs: 50 µg of total
RNA from PMN and negative control tRNA (not shown) and 10 µg of
poly(A)
RNA from NK cells were hybridized with a
5`-end labeled synthetic 30-mer oligonucleotide complementary to
nucleotides +11 to +40 in exon S1 followed by extension using
reverse transcriptase. Sizes of reverse transcribed products were
determined by comparison with HaeIII digested pBR322 DNA
fragments of known molecular weights as shown on the right.
The numbering of the primer-extended products indicate their distance
to the ATG codon designated as +1. The localization of the major
RNA species relative to the ATG codon is indicated on the left for the Fc
RIII-Aa1 and Fc
RIII-Bb1 transcripts. The 167-,
156-, 153-, and 540-nucleotide bands represent the Fc
RIII-Aa2/a3
and the potential Fc
RIII-Aa4 transcripts. c, RNase
protection assay of the 5` portion of Fc
RIII A and B RNAs: 30
µg of total RNA from negative control tRNA and from PMN and NK
cells were hybridized to the riboprobe synthesized from the gene
A-derived BamHI-EcoNI fragment or from the gene
B-derived BstYI-EcoNI fragment. Sizes of protected
fragments were determined by comparison with a sequencing reaction run
in parallel. The negative numbering of the multiple
transcription-initiation sites indicate their distance to the ATG codon
designated as +1. Using the III-B riboprobe, the 380-nucleotide
Fc
RIII-Aa4 protected fragment observed in NK cells with the III-A
riboprobe is converted into four smaller bands, as indicated by the arrows. The protected band at -45 represents the splice
site used by the Fc
RIII-Aa2 transcript, as highlighted by the asterisks.
The restriction pattern of TaqI after hybridization with oligonucleotides specific for
the two Exons S1 and EC1 distinguishes between the FcRIII-A and
the NA1 or NA2 Fc
RIII-B genes. Irrespective of the phenotype used, TaqI demonstrated two restriction fragments of about 6 kb
(Fc
RIII-A, from 0.2 kb downstream to 5.8 kb upstream of the ATG)
and about 2 kb (NA1/NA2-Fc
RIII-B, from 0.2 kb downstream to 1.8
upstream of the ATG) (Fig. 1B). The location of these TaqI sites within the first intron and the 5`-flanking regions
of the Fc
RIII-A and the two alleles of the Fc
RIII-B genes
were observed to be different from the corresponding region presented
earlier(4) .
The nucleotide sequences
beginning at the HindIII sites 1817 and 1821 bp upstream and
ending at different XhoI sites 1115 and 1216 bp downstream of
the first ATG present in the pGP5 cDNA sequence were determined for the
FcRIII-A and Fc
RIII-B genes (Fig. 3). The XhoI sites are located at positions within the EC1 exon as
expected from cDNA sequence analysis of NA1 or NA2 Fc
RIII-B and
Fc
RIII-A
transcripts(4, 23, 30, 31) .
Comparison of the sequences indicates only slight differences. A total
of 58 substitutions and 16 deletions are detected within the first two
introns and the flanking region. From the 38 substitutions of the
flanking region, 26 are found within the first 500 bp upstream of the
ATG codon. The deletion of an 8-bp sequence TGGAGCCT at position
-880 in the Fc
RIII-A gene changes the 3-fold repeating
sequence GGAGCCCT present at the same position within the Fc
RIII-B
gene. The genomic sequences upstream from the ATG codon lack the common
CAAT and TATA promoter elements at their characteristic positions. In
case of the Fc
RIII-B gene, a pyrimidine-rich initiator (Inr)
sequence is present (32) . To identify putative regulatory DNA
elements important for the different cell type specificities of the
Fc
RIII-A and Fc
RIII-B genes, the 1.8-kb flanking regions were
analyzed by the TFD (version 7.3, September 1993) data base. A total of
599 transcription factor consensus sites for Fc
RIII-A and of 583
for Fc
RIII-B was observed. No mismatch to the consensus was
allowed during this search. 39 consensus sites are differentially
distributed between the Fc
RIII-A and Fc
RIII-B genes, 13 of
them are located near the Fc
RIII-Aa1 and Fc
RIII-Bb1
transcription start sites (see below) (see Fig. 7).
Figure 7:
Different distribution of putative
transcription factor binding sites to the FcRIII-A and
Fc
RIII-B promoters. Shown are the sequences of each
-198/-10 gene promoter. The positions of all nucleotide
differences are indicated. All transcription factor consensus sites are boxed.
Comparison with promoter sequences of other Fc receptor genes
reveals a significant conservation in one region of these promoters. We
termed this region the FcR motif. The FcR motif has the sequence
TTCCTTCCTCTTTT homologous to the PU-box and is found in the human
FcRI-A/-B/-C, Fc
RII-A, and the mouse Fc
RI, Fc
RIII
genes within the first 100 bp of the
ATG(33, 34, 35, 36) . A similar FcR
motif is also present in the human Fc
RIII-A/-B (positions
-80 to -67, Fig. 3) and the rat and mouse Fc
RI
genes(37, 38) .
Figure 4:
Structure of the 5`-end of the human
FcRIII-A and Fc
RIII-B genes and nucleotide sequence
comparison of distinct transcripts derived from the two Fc
RIII
genes. a, 5`-end exon-intron organization of the two genes for
Fc
RIII. Exon locations (5`-UT, 5`-UT/S1, S2, EC1) are indicated by boxes. There are multiple transcription initiation sites shown
by the arrows. The positions of the start sites cloned by
RACE/PCR are indicated as the distance to the ATG codon. The distinct
transcripts are designated as b1 in the case of the
Fc
RIII-B gene, and as a1, a2, a3, and a4 in the case of the Fc
RIII-A gene. Positions
-113/-111/-107 represent the main starting site used
by Fc
RIII-Bb1 transcripts. Use of the more 5`-sites at
-860/-849 by the Fc
RIII-Aa2 and Fc
RIII-Aa3
transcripts are associated with alternative splicing of the 3` end of
the first intron, as indicated by the lines below the
Fc
RIII-A gene. A Fc
RIII-Aa4 cDNA clone starting at -333
is also shown, but it should be noted that it does not represent a
full-length a4 transcript. b, nucleotide sequence comparison
of distinct transcripts derived from the two Fc
RIII genes.
RACE/PCR products resulting from amplification with a reverse
complement EC1 primer, followed by a SalI/BglII
digest were cloned into pBluescript KS+, as described under ``Material and Methods.'' Sequence analysis was
performed with the oligonucleotide reverse complementary to the underlined nucleotides. Four types of transcripts originating
from the Fc
RIII-A gene and one transcript type originating from
the Fc
RIII-B gene can be distinguished. The transcript names are
listed at the left. With the exception of the Fc
RIII-Aa4
transcript, all types of transcripts (a1, a2, a3, and b1) are
associated with multiple initiation sites. For reasons of clarity,
these sites are not presented here but are shown under a (see
above). The positions from the ATG codon (typed in boldface)
of the most 5`-site used by each type of transcripts and from the
5`-UT/5`UT-S1 exon borders of the Fc
RIII-Aa2/a3 transcripts are
shown above the respective nucleotide
sequence.
In order to verify the
heterogeneity of the cloned transcription-initiation sites from the
different types of FcRIII-A and Fc
RIII-B transcripts,
primer-extension analysis and RNase protection experiments were
performed. The results obtained by both methods are summarized in Fig. 5a (lowerpart). Due to the absence of a
single defined major transcription initiation site common to both
genes, the numbers of nucleotide positions in the 5`-flanking regions
were assigned on the basis of the first nucleotide of the ATG
translation start codon as +1. Therefore, the size of the observed
protected bands or reverse transcribed products is shown by negative
numbering estimated by their distance to the ATG codon. For
primer-extension analysis, the
P-end labeled
oligonucleotide reverse complementary to nucleotides +11 to
+40 of both genes (Fig. 5a) was used, as described
under ``Materials and Methods.'' By using RNA from
NK cells and PMN, multiple bands were observed that were absent with
yeast tRNA. The starting sites at 20, 27, and 33 bp upstream of the
first ATG codon (Fig. 5b) were used equally by
Fc
RIII-Aa1 in NK cells and Fc
RIII-Bb1 in PMN. In NK cells,
the bands corresponding to the putative -45, -54, -63
transcripts were hardly detectable compared with PMN. Compared with the
RACE/PCR data, the 167-, 156-, and 153-nucleotide reverse transcribed
products represented the Fc
RIII-Aa2/a3 transcripts (Fig. 4b). In the RNase protection experiments shown in Fig. 5c, two Fc
RIII-A and Fc
RIII-B specific
riboprobes, from -348 to +60 relative to ATG, as diagrammed
at the top of Fig. 5a were used. Consistent with the
primer extension data, RNase protection located the
transcription-initiation sites common to both genes at -19,
-27, -33 but with different intensities to -45,
-54, -63 upstream of the first ATG codon (Fig. 5c). In NK cells, the protected fragment at
-45 is much more prominent than the corresponding reverse
transcribed product from the same position, as indicated by the asterisks in Fig. 5c compared with Fig. 5B. Therefore, this band represents the frequently
used splice site found in the cloned Fc
RIII-Aa2 transcripts (Fig. 4b).
Transcripts at positions
-77/-81 and clustering around -113 were observed by
both methods in PMN but not in NK cells (Fig. 5, b and c). The cluster is the main FcRIII-Bb1 transcription
initiation site not only in PMN but also in
Me
SO-differentiated HL60 cells (data not shown).
Immediately upstream, there is a region with homology to the functional
active interferon responsive region of the Fc
RI receptor
gene(39) . Not only in NK cells but also in activated monocytes
and the Fc
RIII-A positive T cell clone 1B3(7) , the
transcripts from -77/-81 were not initiated (data not
shown). All of these cell types express the Fc
RIII-A gene-encoded
isoform of the receptor. A sequence closely resembling the initiator
element (Inr) encompasses the transcription start site at
-77/-81. At position -75 within this Inr motif, a
nucleotide exchange T to C was detected between the Fc
RIII-A and
Fc
RIII-B genes.
A major protected fragment of about 380
nucleotides corresponding to a putative FcRIII-Aa4 -338
transcript is seen in NK cells only using the Fc
RIII-A derived
riboprobe (Fig. 5c, closedarrow).
After hybridization with the Fc
RIII-B derived riboprobe, this
specific band was converted to four main protected fragments of 236,
204, 151, and 137 nucleotides caused by improper
Fc
RIII-A:Fc
RIII-B RNA:RNA pairing recognized by RNaseA (Fig. 5c, open arrows). This pattern of protected
fragments in NK cells was also observed by using a Fc
RIII-B
riboprobe from -707 to +60 relative to ATG (data not shown).
Reverse transcriptase PCR analysis with the primer extension
oligonucleotide and a second primer complementary to positions
-300/-280 but not to -370/-350 yields a
Fc
RIII-Aa4 product of the expected size in NK cells, which is
absent in PMN (data not shown). Results of primer extension did not
predict a start site at -338 but showed a 540-nucleotide extended
product in NK cells (Fig. 5b, openarrow). Therefore, it is very likely that the position at
-338 represents a Fc
RIII-Aa4 specific splice acceptor site.
Figure 6:
Cell type specific activity of the
FcRIII A and B promoters after transfection into different cell
lines. a, genomic material of the Fc
RIII A and B genes
extending to the upstream positions -33, -198, and
-1817/-1821 from the ATG codon were cloned into the
luciferase reporter plasmid pLuc (left part). Transfections
were performed using electroporation of logarithmically growing U937,
HL60, YT, and Jurkat cells as described under ``Materials
and Methods.'' 20 h after electroporation, cell extracts were
prepared and assayed for luciferase activity in a luminometer. An SV40
luciferase chimeric gene was used as a positive control to monitor the
efficiency of transfection, as indicated by the gray bars. White
bars represent activities from constructs containing promoter
sequences of the Fc
RIII-A gene, dark bars represent
results from the B gene. At least five independent experiments were
performed with each construct. The promoter activities of a
representative experiment measured in relative light units (RLU) are shown in the right part for all cell types
analyzed. b, YT, HL60, and U937 cells were transfected with
the 198 promoter fragment of the Fc
RIII-A and Fc
RIII-B genes
in the presence or absence of the heterologous human lysozyme enhancer
cloned 3` to the luciferase reporter gene into the same plasmid. Dark and white bars represent luciferase activities
dependent on Fc
RIII-B and Fc
RIII-A gene sequences,
respectively. Results are depicted as described
above
In the promyelocytic HL60 cells, the complete 5`-flanking region of
the FcRIII-B gene, pIII-B(-1821)Luc, shows a strong promoter
activity compared with a reduced activity using the same fragment of
the Fc
RIII-A gene (Fig. 6a). Similar results are
obtained using HL60 cells expressing Fc
RIII-B after
Me
SO treatment (data not shown). Using the promonocytic
U937 cells, results are less pronounced, and without a heterologous
enhancer only basal and almost equal levels of transcription can be
observed (see below). In contrast, in the NK-like YT cell line, the
Fc
RIII-A gene construct pIII-A(-1817)Luc is much more active than
the corresponding fragment of the Fc
RIII-B gene (Fig. 6a). Thus, the differential promoter activities
of the two 1.8-kb 5`-flanking regions in the YT and HL60 ±
Me
SO cells reflect the expected gene activities of
Fc
RIII-A and Fc
RIII-B in NK cells and PMN, respectively. The
198-bp promoter fragments of gene A termed pIII-A(-198)Luc and of
gene B termed pIII-B(-198)Luc, each containing all respective
starting sites of the Fc
RIII-Aa1 and Fc
RIII-Bb1 transcripts (Fig. 4a) conferred only a reduced activity compared
with the complete 1.8-kb sequences. This indicates that additional more
distal located elements enhance the activity of the 198-bp
Fc
RIII-A and Fc
RIII-B promoters. The activities of these two
198-bp promoters were also stimulated in U937, HL60, and YT cells
through the heterologous, human lysozyme enhancer within the combined
pIII-A(-198) + (LysE)Luc and pIII-B(-198) +
(LysE)Luc constructs (Fig. 6b). In YT cells, the
enhancer-dependent stimulation was selective for the Fc
RIII-A
198-bp promoter inducing a differential activity specific for NK cells.
On the other hand, a selective enhancement of the Fc
RIII-B
promoter was found in the myeloid U937 and HL60 cells. Therefore,
sequences that direct a different cell type specificity of
Fc
RIII-Aa1 versus Fc
RIII-Bb1 transcription are
located to the first 198 bp from the ATG codon within the respective
Fc
RIII-A and Fc
RIII-B promoters.
These studies establish the initial characterization of the
FcRIII-A and Fc
RIII-B genes with emphasis on the structure of
their promoter regions. Both genes have nearly identical restriction
maps but can be distinguished through different gene-specifically
associated TaqI restriction fragments. Differences in the
location of the TaqI sites within the 5`-flanking regions of
the Fc
RIII-A and Fc
RIII-B genes proved to be useful in
cloning the respective gene promoter regions. Here, we demonstrate
differences in the sites of transcription initiation as well as in the
5` end gene organization of both genes. Regulatory regions within their
5`-flanking sequences contributing to the cell type specificity of
Fc
RIII-A and Fc
RIII-B gene expression were identified.
DNA
sequence analysis of the 1.8-kb FcRIII-A and Fc
RIII-B
promoters reveals an overall identity of >95%. Most of the
nucleotide differences between Fc
RIII-A and Fc
RIII-B are
within the first 500 bp of the ATG. From the substitutions identified
between Fc
RIII-A and Fc
RIII-B, 11 generate the dinucleotide
CpG, a candidate for methylation. In the murine system, thymoma cells
express the low affinity receptors Fc
RII and Fc
RIII only
after pretreatment with 5`-azacytidine, indicating a possible role for
CpG methylation in controlling Fc
gene
activity(40, 41) . The methylation status of the
transcriptionally active region of the human Fc
RIII-A and
Fc
RIII-B receptors is currently under investigation.
Differential experimental strategies were necessary to map the
transcription initiation sites, to identify a more 5` end heterogeneity
in FcRIII-A mRNA than in Fc
RIII-B mRNA, and to determine at
least four distinct types of Fc
RIII-A transcripts, a1-a4.
The 5` end of the Fc
RIII-B gene is organized in a single 5`-UT/S1
exon continuous with the ATG codon. The 5` end of the Fc
RIII-A
gene can be encoded by alternative 5`-UT and 5`-UT/S1 exons. Therefore,
compared with the organization of other human Fc
receptor genes,
the 5` end of Fc
RIII-B is similar to that of
Fc
RI(39) , whereas the 5` end of Fc
RIII-A is related
to Fc
RII-A(34) . At least four distinct types of
Fc
RIII-A transcripts could be identified, distinguished by 5`-UT
ends of different sizes and sequences. Each type of transcripts uses
multiple starting sites. Fc
RIII-Aa1 transcripts start from
-33, -27, and -20 upstream from the ATG. These three
sites are also used by the Fc
RIII-Bb1 transcripts. Initiation of
Fc
RIII-Aa2 occurs at -860 and -849 in a discrete 5`-UT
exon. The sequence is colinear with Fc
RIII-A genomic sequence
until a 5`-GT splice site (position -794) and continues through a
3`-AG splice site (position -45) into the 5`-UT/S1 exon. The
related type of Fc
RIII-Aa3 transcripts uses an alternative
3`-consensus AG splice site located at -63. The protection
pattern of RNA obtained from NK cells with different riboprobes
demonstrates an Fc
RIII-Aa4 transcript at about -338 from
ATG. Results of primer extension did not predict a start site at
-338 but showed a 540-nucleotide extended product. A potential
intron 3`-splice acceptor site is present near the -338 site.
These observations are indicative for the existence of another discrete
exon mapped further upstream, which is encoded only by the
Fc
RIII-Aa4. The a4 transcript derived by RACE/PCR cloning from NK
cells shows a continuous sequence from -333 to the ATG identical
with Fc
RIII-A gene sequences. Northern blot analysis with
exon-specific Fc
RIII cDNA probes derived from pGP5 (Fig. 2A) demonstrates no variation in molecular
weight; only a single 2.2-kb Fc
RIII-A mRNA is detectable.
Therefore, we speculate that the elongation of the 5` end has to be
compensated by a shortening at another region within the
Fc
RIII-Aa4 transcript. It will be necessary to obtain full-length
cDNA clones corresponding to Fc
RIII-Aa4.
Two main differences
have been observed in the initiation of FcRIII-Aa1 and
Fc
RIII-Bb1 transcripts. First, the major transcription initiation
sites of Fc
RIII-Bb1 in freshly isolated PMN and in
PMN-differentiated HL60 cells (data not shown) are clustered around
-113 from ATG and are absent in Fc
RIII-Aa1 expressing NK
cells. Immediately upstream there is a region of homology to the
functional active interferon responsive region present within the
promoter of the high affinity Fc
RI receptor gene (Fig. 7)(39) . INF
-induced Fc
RIII-B
transcription in eosinophils and INF
-induced Fc
RIII-A
transcription in glomerular mesangial cells has been
described(9, 10) . Second, Fc
RIII-Bb1, but not
Fc
RIII-Aa1 transcripts, can be initiated at -77 and
-81 from ATG mediated by a pyrimidine-rich initiator (Inr) of the
sequence TACTCCCT also found within the adenovirus major late promoter
(TFII-I-ML-Inr2)(42) . At position -75 within this Inr
motif, a nucleotide exchange of C to T was detected between the
Fc
RIII-B and Fc
RIII-A genes resulting in the alteration of
the Inr consensus YAYTCYYY to YAYTTYYY(42) . Based on recent in vitro findings, this alteration by itself cannot be
sufficient for a nonfunctional initiator in case of
Fc
RIII-A(43) . The factor TFII-I is binding not only to
some Inr elements but also to upstream USF (E box) sites. It was also
suggested that TFII-I and USF interact cooperatively at both Inr and E
box sites(42) . In this respect, it is interesting to note that
a potential USF binding site of the sequence GGCAGGTGAC is present at
position -122 within the Fc
RIII-B gene (44) . This E
box is mutated by substituting the CA to a TG at position
-120/-119 in case of Fc
RIII-A. Therefore, it might be
possible that the mutations both at positions -120/-119 and
-75 are necessary to account for the observation that the
-77/-81 transcriptional start site is not utilized in the
Fc
RIII-A gene.
FcRIII-A and Fc
RIII-B do not possess
typical TATA and CAAT boxes, a finding consistent with the presence of
multiple initiation sites. Most but not all of the different initiated
Fc
RIII-Aa1 and Fc
RIII-Bb1 transcripts can be explained by the
presence of the potential interferon responsive region and the
Inr/E-box sequences. Several putative binding sites of transcription
factors are differentially distributed between the Fc
RIII-A and
Fc
RIII-B gene promoters as marked in Fig. 7. A Sp1
consensus sequence at -97(45) , two AP-2 sites at
-98 and -181(46) , one AP-1 site at
-155(47) , one CDP (CCAAT displacement protein) site at
-125(48) , one NF-
B site at -182(44) ,
an Ets-1 element at -78(44) , and a potential site for
the ets family member PU.1 (GAGGAA) in inverse orientation at
-76 (49) are present in case of Fc
RIII-A only. PU.1
binds to the PU box and is expressed in myeloid cells like U937 and
HL60 (49) but not in NK cells and the YT cell line (data not
shown). The sequence around the putative PU.1 site (TTACTTCCTCCTGT) in
the Fc
RIII-A gene is nearly identical to a 14-bp promoter element
TTCCTTCCTCTTTT conserved among the human Fc
RI-A/-B/-C,
Fc
RII-A, and the mouse Fc
RI, Fc
RIII
genes(33, 34, 35, 36) . A similar
region of homology (TTCCTTCTCCTTYG) is also present in the rat and
mouse Fc
RI genes (37, 38) . A common feature of
the Fc receptor genes is the location of this conserved element, the
FcR motif, centered at the transcription initiation units within the
first 100 bp from the ATG codon. This FcR motif has been characterized
to be the DNA target of myeloid specific activators in the Fc
RI-A
gene(50) . Preliminary analysis suggests an interaction of
nuclear proteins from myeloid but not NK cells with an oligonucleotide
representing the Fc
RIII-A promoter region from bp -95 to
-54 containing the putative PU.1 site (data not shown).
Interestingly, the myeloid-specific shifted complex can be competed
with this FcR motif. Thus, we speculate that the conserved FcR motif
most likely recognized by PU.1 might be involved in the expression of
Fc receptors in myeloid cells.
Cells expressing the FcRIII-A or
Fc
RIII-B receptor, namely culture-activated monocytes,
granulocytes, and NK cells are refractory to transfection. Therefore,
we have utilized the NK cell-like YT line, the promonocytic U937 cells,
and the promyelocytic HL60
cells(21, 18, 19) . These cells are easy to
transfect, but they present a premature state of Fc
RIII-A and
Fc
RIII-B expression. The NK-like YT cells have been described to
express Fc
RIII only at low levels.
U937 and HL60 cells
are normally negative for Fc
RIII-A or Fc
RIII-B expression.
However, it should be noted that the expression of the Fc
RIII-B
receptor can be induced after granulocytic differentiation of HL60
cells by Me
SO(20) . Sequences of both genes from
positions -198 to -10 encoding for all of the mapped and
cloned initiation sites of Fc
RIII-Aa1 and Fc
RIII-Bb1
transcripts behave like a typical promoter in these cells: they direct
transcription in an orientation-dependent manner (data not shown) and
are stimulated by a heterologous enhancer (Fig. 6b).
Furthermore, using YT and HL60 cells, the -198/-10
promoters are stimulated in a cell type-specific manner by the human
lysozyme enhancer as well as with homologous sequences up to about 1800
bp. For example in the NK-like YT cells, the Fc
RIII-A promoter is
much more active. In contrast, in HL60 cells, the Fc
RIII-B
promoter is most active. In accordance with this observation,
granulocytic differentiation of HL60 cells using Me
SO
results in Fc
RIII-B specific promoter activity (not shown). This
differential activity agrees to the in vivo specificity of the
complete Fc
RIII-A and Fc
RIII-B genes in NK cells versus PMN. Thus, important tissue-specific elements are located in the
-198/-10 regions. A total of 10 nucleotide exchanges
predicted by sequence analysis exists within the Fc
RIII-A and
Fc
RIII-B -198/-10 promoters. 8 of the 10 nucleotide
differences are of special interest able to generate different
consensus sites for well characterized transcription factors
(summarized in Fig. 7). Preliminary analysis suggests that cell
type specificity cannot simply be mapped and attributed to just one
nucleotide difference only. (
)For both cell type
specificities, several elements are likely to work together in a
cooperative fashion. Therefore, different combinations of mutated
Fc
RIII-A and Fc
RIII-B hybrid promoters need to be constructed
and tested for their ability to change the promoter activities and
specificities. With HL60 and YT cells, there is now an excellent model
available to identify and characterize mutations sufficient and
necessary for expression of Fc
RIII-A and Fc
RIII-B in NK cells versus PMN, respectively.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z46222 [GenBank]and Z46223[GenBank].