| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322;
Division of Molecular Biology, Department of Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The conserved S, X (comprising X1 and X2), and Y box DNA sequence regulatory elements that have been identified in the proximal promoter of MHC class II, class I, and their accessory genes are bound by several DNA-binding protein complexes (reviewed in Refs. 3 , 6 , 7 , and 11, 12, 13, 14). These include RFX, X2BP (cAMP response element binding protein; CREB), and NF-Y, which bind cooperatively to the X1 box (8, 15), X2 box (8, 9, 16, 17), and Y box (18, 19), respectively. Together these proteins are engaged in the formation of a highly stable multiprotein DNA complex (20, 21, 22, 23, 24). Although individual binding of RFX or X2BP to some of the isotypic MHC class II-conserved elements has been shown to be weak or absent (8, 22), the RFX/X2BP/NF-Y complex is formed on X-Y DNA of all MHC class II isotypic promoters (22). In a similar fashion, multiprotein complex formation also occurs on X-Y DNA of the MHC class I and ß2-microglobulin (ß2m) promoters.6 This complex formation allows the MHC class II isotypes to be regulated in a coordinate fashion. The lack of MHC class II expression and the reduction of MHC class I expression in BLS patient-derived cell lines with defects in one of the RFX subunits is attributed to the failure to assemble this complex in vivo (25, 26).6
Formation of this multiprotein complex on X-Y DNA is not sufficient for
activation of MHC class II genes. Activation requires the activity of
the MHC class II transactivator (CIITA) (27). CIITA is
believed to act as a transcriptional coactivator through
protein/protein interactions with the RFX complex and as such connects
the MHC-specific transcription complex with the basal transcription
initiation apparatus (28, 29, 30). CIITA has strong
transactivation properties that are mediated through its acidic
NH2-terminal domain (31, 32).
Whereas CIITA is critical for expression of MHC class II genes, CIITA
augments IFN-
induction of MHC class I and
ß2m genes (8, 9, 10).
Four distinct genetic complementation groups have been described in BLS (A–D) (33), and the genes affected in these complementation groups have been identified. BLS patients of complementation group A are defective in CIITA (27, 34). The genes affected in groups B, C, and D all encode subunits of the heterotrimeric phosphoprotein RFX (35). BLS groups B, C, and D are defective in RFX-B (also referred to as RFXANK) (36, 37), RFX5 (38, 39), and RFXAP (40, 41), respectively. RFX-B is the smallest (33 kDa) of the three subunits that constitute the RFX complex (36, 37). Despite evidence demonstrating that RFX-B interacts with DNA (42), the protein has no known DNA binding domain (36, 37). RFX-B contains three ankyrin repeats that may be important in the formation of the RFX complex and the assembly of the multiprotein complex involving RFX, X2BP, and NF-Y on X-Y DNA. Alternatively, these ankyrin repeats may mediate interactions of the multiprotein/DNA complex with the MHC CIITA.
Here we describe the molecular characterization of BLS patients that were found to belong to BLS complementation group B. The defects in RFX-B resulted in absence of MHC class II expression and reduced levels of MHC class I expression. Expression of exogenous CIITA, in the absence of wild-type RFX-B, resulted in the induction of appreciable amounts of HLA-DR and also of HLA-DP in RFX-B-deficient fibroblasts. Exogenous CIITA alone was able to transactivate the MHC class I and the ß2m promoter in RFX-B-deficient fibroblasts as well. These results reveal that certain mutations in RFX-B can function in CIITA-mediated transactivation of MHC class II, class I, and ß2m promoters and that CIITA overexpression may override the deficiency of BLS group B patients.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
BLS patient EBA was 8 mo of age at the time he was referred to the Leiden University Medical Center for allogenic bone marrow transplantation (43). Patient FZA was 14 years of age at the time of cell sampling. FACS analysis revealed that <1% of his lymphocytes stained weakly positive for HLA-DR. Patient EBA was characterized by oligotyping as DR7/DR11, and patient FZA was characterized by oligotyping as DR4/CDR10. Primary fibroblasts from a skin biopsy of the two patients were first transformed with an SV40 ori plasmid and subsequently stably cotransfected with pCMVEBNA and pRSVneo (44). SV40-transformed fibroblasts derived from BLS group C patients OSE (DR17/DR4) and SSI (DR7/DR10) and BLS group D patient ABI (DR16/DR7) were described previously (44, 45, 46, 47). SV40-transformed JVH (DR17/DR11) and ABL fibroblasts (DR1/DR15) were derived from BLS-group A and B patients, respectively (48). The BLS group B patient-derived B cell line Ramia was described previously (48). Fibroblasts and B lymphoblastoid cell lines were grown in Iscove’s modified DMEM (Life Technologies, Paisley, U.K.) supplemented with 10% (v/v) FCS (Life Technologies) penicillin (100 IU/ml), and streptomycin (100 µg/ml).
Transient heterokaryon analysis
The generation and analysis of transient fibroblast homo- and
heterokaryons were essentially as described before (44).
Upon treatment of fibroblasts with polyethylene glycol 4000, cells were
cultured in the absence or presence of 500 U/ml of IFN- for 48
h. RNA was isolated from the cells and subjected to RT-PCR using
HLA-DRB haplotype-specific oligonucleotides (located in exon 2) as 5'
primers and a generic HLA-DRB oligonucleotide (located in exon 3) as
the 3' primer. The RT-PCR products were size-fractionated on an 1%
agarose gel, transferred to Hybond N+ membranes
(Amersham, Little Chalfont, U.K.), and hybridized with biotin-labeled
HLA-DRB haplotype-specific probes. The generic 3' primer as well as the
5' primers and biotin-labeled hybridization oligonucleotides and the
critical washing temperatures were described before
(44, 45, 46). To assure that the quality and the amount
of the various cDNAs were similar, GAPDH-specific RT-PCR and
hybridization was performed as described previously
(44).
RNA and DNA hybridization analysis
For Figs. 1
and
2, total cellular RNA was prepared using
RNAzolB (Cinna/Biotecx Laboratories, Houston, TX) following the
manufacturer’s instructions. Twenty micrograms of total RNA was
separated on an 1.2% agarose gel containing 2.2 M formaldehyde,
transferred to a Hybond N membrane (Amersham), and hybridized using
probes that were labeled with [32P]dCTP by
random priming (DuPont-NEN, Brussels, Belgium). Transfer and
hybridization were performed according to the instructions of the
manufacturer of the membranes. The human cDNA probes for MHC class I,
MHC class II, invariant chain (Ii), ß2m, and
GAPDH were described before (43, 44, 45, 46). For Fig. 7, RNA was
extracted from cells using the RNeasy kit (Qiagen, Valencia, CA). Total
RNA (10 µg) was used for Northern blot analysis. Random primed RFX-B
cDNA (Rediprime II; Amersham) spanning exons 3–10 was used as a
probe, and hybridization was conducted in Ultrahyb (Ambion, Austin,
TX). The blots were reprobed for GAPDH to verify equal loading of RNA.
Southern blot analysis of RT-PCR products was conducted as described
(49) using an end labeled primer as probe. Primers
corresponding to exons 4 and 5 were 5'-CCACCACTCTCACCAACCGG and
5'-CTGTCCATCCACCAGCTCGCAGCACAG, respectively.
|
|
The following plasmids were used in this study: pREP4-CIITA
(9, 31), pSVK-FLAG-CIITA (50), pRFX-B
(37), pRFX-B
5 (37), and pRFX5 (created by
inserting the RFX5 cDNA into pcDNA3.1). The base vectors for the above
plasmids, pREP4 (Invitrogen, Carlsbad, CA), pcDNA3.1 (Invitrogen), and
pSVK (Pharmacia, Uppsala, Sweden) were used as controls. The promoter
reporter constructs pGL3-B250,
pGL3-ß2m1, pGL3-Ii,
pDRCAT, and pDRCATwt have been described previously (8, 9, 31, 46). Stable cell lines were generated in fibroblasts by the
calcium phosphate precipitation method (51) or
electroporation (31) with the indicated plasmid and were
selected in either 50 µg/ml G418 (Life Technologies) or 50 µg/ml
hygromycin (Boehringer Mannheim, Mannheim, Germany) for an average of 2
wk. Resistant colonies were pooled and used for analysis of MHC surface
expression.
Promoter activity assays
For luciferase containing reporter plasmids, in each of four wells of a six-well plate, 1.5 x 105 fibroblast cells were transfected by the calcium phosphate method, with a DNA mix containing 1 µg firefly luciferase pGL3 reporter plasmid (pGL3-DRA, pGL3-B250, pGL3-ß2m, or pGL3-Ii), 1 µg Renilla luciferase pRL-TK control plasmid (Promega, Madison, WI), and 1 µg of pREP4 or 0.5 µg of pREP4-CIITA. Cells were harvested 3 days after transfection. Luciferase activity was determined using the dual-luciferase reporter assay system (Promega) and a luminometer (Tropix, Bedford, MA).
For chloramphenicol acetyltransferase (CAT)-based reporter constructions, cells were cotransfected with 10 µg pRFX-B, 10 µg pSVK-FLAG-CIITA; 10 µg pDRCATwt, a reporter that contains the SXY box of class II promoter (52); and 0.5 µg of pGL3-promoter vector (Promega), which encodes the luciferase gene driven by a constitutive promoter (SV40). Control transfections were conducted similarly with the pDRCAT reporter that does not contain the SXY box of class II promoter (52). Cell lysates were prepared 72 h posttransfection, and 5% of the lysate was analyzed for expression of luciferase product using the Luciferase Assay System (Promega). The remaining sample was analyzed for CAT protein using an ELISA (Boehringer Mannheim) according to the manufacturer’s instructions. The data were normalized to the expression of luciferase.
Flow cytometric analysis
To measure MHC class II and class I surface expression, cells were stained with mAbs against the HLA-DR backbone (B8.11.2) (53), -DQ backbone (SPV-L3) (54), -DP backbone (B7/21) (55), or MHC class I (WT6/32) and FITC-conjugated goat anti-mouse IgG (Becton Dickinson, Mountain View, CA). Cells stained with the corresponding conjugated or unconjugated Ig isotype were used as controls. In some experiments, the Abs described in Nagarajan et al. (37) were used. Approximately 5000 cells were analyzed on a FACScan flow cytometer (Becton Dickinson) in each assay.
RT-PCR
PolyA RNA was made using PolyA Tract mRNA isolation system (Promega). Reverse transcriptase reactions were conducted using Superscript II (Life Technologies), and PCR was conducted using native PfuI polymerase (Stratagene, La Jolla, CA). The RFX-B cDNA from exons 2–10 was amplified using primers with restriction site overhangs: 5'-CTAGTCTAGACAGATCGCTGAGGGTCCG and 5'-CCGGAATTCCGGCAGGCGGCCTTCACTC. Thirty cycles of 30 s at 95°C, 30 s at 55°C, 1.0 min at 72°C, and a final extension at 72°C for 7 min, were used to amplify the RFX-B cDNA for all RT-PCR samples. GAPDH was amplified from the same reverse transcription reaction as a control, using the primers 5'-CCATGGGGAAGGTGAGGTAGGATC and 5'-GAGGAGTGGGTG TCGCTGTTGATC.
RFX-B cDNAs obtained by RT-PCR from patient RNAs were cloned into the pCRBlunt vector (Invitrogen) for sequencing and also cloned into the mammalian expression vector pcDNA3.1- at XbaI/EcoRI sites.
Genomic DNA PCR and analysis of homozygosity
Genomic DNA from the cell lines was made as described in Ausubel et al. (56). PCR of the RFX-B gene consisted of 30 cycles of 10 s at 92°C, 30 s at 60°C, and 2 min at 68°C. The last cycle was extended for 7 min. DNA fragments encoding exons 4–8 were amplified using the primers, 5'-CCACCACTCTCACCAACCGG and 5'-CGCATTTCACGTGGTTCCCGCG-3'. To analyze the mutations in FZA and family members, exon 8 was amplified using primers corresponding to introns 7 and 8: 5'-GAACTGCGCTGCGATGGCAGATG and 5'-GCAGGCAGGGACTACCTGCTCC, respectively. The PCR products were purified and sequenced. To detect homozygosity in EBA, PCR was conducted using Taq polymerase and primer sets specific for the mutated or wild-type sequence in exon 5. A wild-type forward primer located in exon 4, 5'-CCACCACTCTCACCAACCGG, was used with either the wild type 5'-GCTCCTTCAGCTGGTCCAGCTC or mutated 5'-GCTCCTTCAGCTGGTCCAGCTA reverse primers.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Patients FZA and EBA presented themselves with an apparent loss of
MHC class II surface expression on lymphocytes, although in the case of
FZA <1% of the lymphocytes stained weakly for HLA-DR. Immortalized
fibroblast cell lines were established from skin biopsies as previously
described (44). To examine the expression characteristics
of MHC class II (HLA-DRA), MHC class I, Ii, and
ß2m genes in fibroblasts derived from BLS
patients FZA and EBA, RNA was isolated from untreated and
IFN--treated cells and subjected to Northern blot analysis (Fig. 1
).
Compared with a normal control fibroblast cell line WSI, no HLA-DRA
mRNA was detected upon induction of FZA and EBA fibroblasts with
IFN-. Furthermore, Ii expression both in FZA and EBA could not be
detected upon treatment with IFN-. The amount of constitutively
expressed MHC class I and ß2m transcripts were
greatly reduced. The expression characteristics of MHC and accessory
genes in FZA and EBA is similar to our previous observations in other
BLS patients that contain defects in the RFX5 and RFXAP subunits of RFX
(3, 8, 44, 45, 46).
FZA and EBA belong to BLS complementation group B
Somatic cell fusion experiments were performed to determine the
complementation group assignment of FZA and EBA. FZA fibroblasts were
fused with fibroblasts derived from BLS patients JVH, ABL, SSI, and
ABI, which were representative for complementation groups A, B, C, and
D, respectively. The transient heterokaryons were analyzed for
restoration of MHC class II expression by RT-PCR and Southern blotting
for HLA-DRB. As shown in Fig. 2, A and B, both
FZA and EBA were complemented by JVH, SSI, and ABI, but not by ABL and
EBA. These results demonstrate that both FZA and EBA belong to
complementation group B and suggest that they contain defects in the
RFX-B subunit of the RFX complex (36, 37).
|
IFN- induces MHC class II genes through the induction and
expression of the transactivator CIITA. Expression of exogenous
CIITA via plasmid transfection results in the induction of MHC class II
expression of all three isotypes in many cell types (47, 57). To determine whether overexpression of CIITA could result
in MHC class II expression, EBA fibroblasts were stably transfected
with pREP4-CIITA. Following hygromycin selection, these cells were
found to express HLA-DR and HLA-DP, and to a lesser extent HLA-DQ (Fig. 3). Expression of all three MHC class II
isotypes in the absence of functional RFX has not been observed in
fibroblast cells with defects in RFX5 or RFXAP (46, 47).
CIITA-transfected RFX5 or RFXAP-deficient cells were found to
express HLA-DR (results not shown; see Refs. 46 and
47). In addition, these CIITA-transfected EBA cells also
displayed a moderate increase in MHC class I cell-surface expression,
which was also not noted in CIITA-transfected RFX5 and RFXAP-deficient
fibroblasts.
|
To further investigate the role of RFX-B in constitutive and
CIITA-mediated transactivation of MHC class II, MHC class I, and
accessory genes, transient transfection experiments were performed
using FZA and EBA fibroblasts with either HLA-DRA promoter-driven CAT
reporter constructions or HLA-B7, ß2m, and Ii
promoter-driven luciferase reporter constructs. For comparison to FZA
and EBA, another BLS group B cell line, Ramia, was also analyzed. Ramia
cells are EBV-transformed B lymphocytes and like FZA and EBA are
negative for class II expression (48). Transient
cotransfection of Ramia cells with the HLA-DRA reporter or a control
reporter, an RFX-B expression plasmid or a plasmid expressing a splice
variant of RFX-B, RFXB5, showed that RFX-B but not the control
vectors could complement the defect in Ramia cells (Fig. 4A). RFX-B5 expression
showed a slight rescue of class II expression. Because Ramia cells are
B cells, exogenous CIITA expression was not required. FZA and EBA
fibroblasts were similarly cotransfected; however, in these experiments
a CIITA expression vector was required for expression. Similar to the
flow cytometry experiments in Fig. 3, transfection of the CIITA
expression vector alone results in a moderate level of expression (Fig. 4A). As with Ramia cells, transfection of RFX-B rescues the
class II defect. Moreover, transfection of the RFX5 subunit in FZA
fibroblasts did not result in rescue, demonstrating the specificity of
the complementation.
|
Expression of RFX-B leads to surface expression of MHC class II in both Ramia B cells and FZA fibroblasts
Stable cell lines expressing RFX-B were created to determine
whether RFX-B could fully complement the defect of the endogenous MHC
class II genes in both Ramia and FZA cells. Flow cytometric analysis of
vector-transfected or RFX-B-transfected cell lines showed that RFX-B
could complement all three MHC class II isotypes (Fig. 5). FZA cells coexpressing both CIITA and
RFX-B showed enhanced expression of MHC class II compared with
CIITA-expressing cells. Thus, mutations in the RFX-B gene are likely
responsible for the lack of class II expression in these cells.
|
The above results could suggest that the mutant RFX-B alleles
produce proteins that could provide some function in the presence of
excess CIITA. Alternatively, excess CIITA may be sufficient to drive
class II expression in the absence of functional RFX-B protein. To
begin to distinguish between these hypotheses, the nature of the defect
and the genotypes of these cell lines were determined. Previous
analysis of RFX-B-deficient cells identified two mutant alleles, both
of which affected the splicing of exon 6. One allele contained a 26-bp
deletion in a region encompassing the splice acceptor site of exon 6
(36, 37). The second allele identified was a 56-bp
deletion of the DNA encompassing the splice donor site 5' to exon 6
(36) Both of these mutations cause a frame shift and a
premature stop codon. To determine the mutations in each of these cell
lines, RFX-B cDNA from each of the cell lines was generated by RT-PCR;
however, as discussed below, full-length RT-PCR products were obtained
from FZA only. Shorter transcripts were obtained from EBA and Ramia.
The RT-PCR products were sequenced after cloning into PCR blunt vector.
Both FZA and EBA DNA sequences each contained a single base pair
substitution (Fig. 6A). FZA
contained a T 
C transition in exon 8, resulting in a
Leu195 Pro substitution. This mutation alters
a conserved leucine residue in the third ankyrin repeat of the RFX-B
protein. Sequence analysis of EBA’s RT-PCR product showed that it
corresponded to the sequence of RFX-B5 as observed in wild-type Raji
cells. This suggested that EBA could either contain a splice
acceptor/donor mutation flanking exon 5 or a point mutation in exon 5
that could result in an unstable full-length transcript. To delineate
the defect in EBA, genomic DNA was isolated from EBA cells and exons
4–8 of RFX-B gene were amplified by PCR. A transversion, which changed
GAG (Glu103) TAG (amb) was found in exon 5,
thereby encoding a truncated protein. The mutation in Ramia was found
to be the same 26-bp deletion described above (Fig. 6A). A
smaller RT-PCR product, lacking both exons 5 and 6, was also found in
Ramia cells.
|
B). Wild-type DNA
(Raji) and EBA DNA were analyzed in parallel. The results show that
each of FZA’s parents carries one wild-type and one mutant allele and
that FZA is homozygous for the point mutation. To determine whether EBA
was homozygous for the nonsense codon, wild-type and mutation-specific
PCR primers were generated and used to amplify genomic DNA by PCR (Fig. 6C). The results showed that EBA DNA was amplified by the
mutation-specific primer only. Control reactions included wild-type DNA
from Raji cells and DNA from FZA, which is wild type at this position.
Thus, both FZA and EBA are homozygous for their mutations. Ramia was
also found to be homozygous by Southern blot analysis (data not shown),
as described in Nagarajan et al. (37). RNA analysis of the group B cell lines shows distinct expression patterns
The 26-bp deletion mutation that is also found in Ramia cells was
shown previously to result in destabilization and loss of the RFX-B
mRNA. To determine whether the same were true for the FZA and EBA
mutations, RNA analyses by Northern blotting and RT-PCR were performed
(Fig. 7). Northern blot analysis of
Ramia, FZA, and EBA showed two patterns. Ramia and EBA were found to
express undetectable and very low levels of RFX-B mRNA by Northern
blot, respectively, whereas, FZA expressed wild-type RFX-B mRNA levels
(Fig. 7A). RT-PCR of exons 2–10 was conducted to determine
whether RNA could be detected in EBA cells (Fig. 7B). RT-PCR
of RFX-B from the wild-type Raji cells shows both the full-length RFX-B
band (upper) and the minor splice variant, RFX-B5 (lower band). FZA
produces an identical pattern to the wild type. Surprisingly, EBA
displays only the lower band. To demonstrate that this lower band was
in fact the splice variant, a Southern blot was performed on this gel
using probes specific for exon 5, which is missing in the splice
variant, and exon 4, which is present in the splice variant. The result
showed that EBA expresses the splice variant, but not the full-length
mRNA that would contain the nonsense codon. As described above, this
result was verified by sequencing the RT-PCR product. Thus, by
expressing the splice variant, EBA cells can bypass the nonsense codon
generated by the mutation. However, this transcript is not stable in
cells and is expressed at very low levels, suggesting that if any
protein is synthesized it would be low in abundance.
RFX-BFZA can provide a low level of expression
Because FZA produces normal RFX-B mRNA levels, it is possible that
the RFX-BFZA protein can stimulate a low level of
MHC class II expression. Similarly, the RFX-B5 splice variant, which
is found in EBA, may also be able to provide some level of MHC class II
expression. To assay these variants, the RFX-BFZA
mRNA and the splice variant were cloned into the mammalian expression
vector pcDNA3.1. Ramia cells, which are deficient for both the
wild-type and splice variant, were cotransfected with the above
expression vectors and a DRA promoter-dependent reporter construct
(pDRCATwt) or its control vector (pDRCAT). The results showed that the
splice variant could provide a small increase in expression of the MHC
class II promoter-dependent reporter over the background associated
with this vector (Fig. 8). However,
RFX-BFZA overexpression resulted in almost
one-third of the wild-type activity. Thus, the ability to transactivate
MHC class II expression by the mutant proteins may be partially
achieved when the levels of CIITA reach a threshold level, such as that
in normal B lymphocytes, or through the overexpression of CIITA from an
exogenous promoter.
|
As stated above, Ramia cells produce an unstable RFX-B mRNA that
is not detectable by Northern analysis. Thus, these cells are most
likely devoid of RFX-B protein. However, Ramia cells do express normal
B cell levels of CIITA. Thus, this cell line could serve as a model for
overexpression of CIITA in the absence of RFX-B. Therefore, to
determine whether CIITA could transactivate in the absence of RFX-B, a
CIITA expression vector was transfected into Ramia cells (Fig. 9). This resulted in a 4- to 5-fold
increase in the activity of the cotransfected DRA reporter. As above,
transfection of the RFX-B expression vector yielded a 12-fold increase
in expression. Interesting, expression of both RFX-B and CIITA vectors
resulted in a 26-fold increase, suggesting that CIITA expression was
limiting in these cells. Moreover, mRNA from wild-type RFX-B or the
mutant alleles expressed in FZA or EBA were not found to be induced by
treatment of the cells with IFN- or in cells transfected with CIITA
(data not shown). These experiments eliminate the possibility that
CIITA or IFN- treatment might contribute to class II expression
through an increase in RFX-B levels. Thus, these data demonstrate that
CIITA overexpression can partially override the RFX-B defect and
provide a mechanism by which EBA cells can express class I, class II,
and ß2m genes following CIITA transfection but
not following IFN- treatment.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and 8.
The partial restoration of MHC class II, class I, Ii, and
ß2m expression following overexpression of
CIITA in the FZA and EBA fibroblasts suggests that the mutant RFX-B
proteins are either partially active or that the CIITA overexpression
can compensate in some manner for a mutated RFX-B protein. The data
argue for a combination of these two view points. Unlike RFX-B mRNA in
EBA and Ramia, RFX-BFZA mRNA is expressed at
normal levels and is not rapidly degraded. The fact that one-third of
wild-type activity is observed when the RFX-BFZA
mRNA in Ramia cells suggests that the mutant protein is produced. The
development of an antiserum to RFX-B will allow confirmation of this
prediction. Thus, with regard to RFX-BFZA, it is
likely that the protein is partially functional. The
RFX-BFZA mutation changes
Leu195 Pro in the third ankyrin repeat of the
RFX-B protein. These ankyrin repeats are conserved domains that are
involved in protein-protein interactions and have 
helical
structures. The proline substitution in RFX-BFZA
may affect the helix within the third repeat. We have recently
found that the third ankyrin repeat is required for association of
RFX-B with the other two RFX subunits, RFX-5 and RFXAP (DeSandro,
Nagarajan, and Boss, unpublished data). It is not known at the current
time whether FZA mutation specifically affects the interactions between
the three subunits, although this is a likely prediction from our
current data.
The EBA mutation provides an opportunity to examine the role of the
RFX-B splice variant. The stop codon caused by the single base pair
mutation results in a frame shift and an unstable mRNA for the
full-length transcript. However, because the splice variant
fortuitously removes the exon encoding the mutation, it is unaffected
by the mutation. A low level of the splice variant mRNA can be detected
by overexposure of a Northern blot. RT-PCR readily detects this
transcript as shown in Fig. 7. Expression of the splice variant mRNA
does not normally rescue RFX function or MHC class II expression. This
could be due to instability of the RFX-B RNA or to the protein that it
encodes. However, in the presence of excess CIITA, a low but
significant level of MHC class II and an increase in MHC class I
expression can be detected in EBA cells. Additionally, overexpression
of the splice variant in several of the experiments presented showed a
low level of MHC class II expression. One interpretation of this result
is that the low level of splice variant-generated protein may function
in these assays. In contrast, the mutation in Ramia, which results in a
frame shift in exon 6 and unstable mRNA, is completely devoid of RFX
activity. The splice variant would also be affected by this mutation as
well, as it includes exon 6. Thus, these three mutations may be grouped
into three categories with regard to the transcriptional potential of
RFX-B: partial activity in FZA, low activity in EBA, and no activity in
Ramia.
The finding that IFN- treatment of FZA and EBA cells did not induce
MHC class I or II expression but that CIITA expression in these cell
lines did was intriguing and suggested that CIITA may partially
override the RFX-B defect. This hypothesis was tested by transfecting
CIITA into Ramia cells, which lack RFX-B. The results showed a 4- to
5-fold increase in expression of the MHC class II reporter gene and
demonstrated that CIITA could compensate for the RFX-B defect. We have
found in other experiments that CIITA expression from the pcDNA3.1
vector provides at least a 3-fold increase in the level of CIITA mRNA
in transfected cells as compared with IFN--treated cells (data not
shown). Additionally, in stable cell lines, CIITA is constitutively
expressed and always present, allowing accumulation of CIITA protein.
Thus, the effect is likely to be greater in the EBA and FZA cell lines
stably transfected with CIITA.
What is CIITA’s role in these experiments? It is possible that CIITA
stabilizes weak interactions at the class I or even class II promoters.
There is evidence to suggest that CIITA expression could enhance the in
vivo footprint in cells that are normally class II negative
(58, 59, 60). Thus, in the case of the EBA and Ramia RFX-B
alleles, CIITA may stabilize the promoter complex in the absence of a
functional RFX-B protein. In doing so, CIITA would then be able to
activate transcription. The FZA allele is expressed and may provide
partial activity to the RFX complex, as indicated by partial rescue of
Ramia cells. Because MHC class I genes use other elements for their
expression and induction by IFN-, which are located upstream to the
SXY motifs, it is possible that these elements provide additional
stability or aid in the assembly of the SXY-specific factors when CIITA
is expressed. Thus, deficiencies in RFX-B may not be sufficient to
prevent transcription of MHC class II genes under all conditions.
Therefore, it is not surprising that some of the RFX-B-deficient
patients have lived into adulthood. The analysis of the three patient
cell lines in this report provide evidence for single base pair
substitution within an important regulatory element. Thus, this
analysis questions whether there are other alleles for complementation
group B. It is possible that such mutations may be plentiful in humans
and that there is allowance at this locus and protein structure for
expression and a viable immune system.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 U.M.N. and A.P. contributed equally to the content of this manuscript.
3 J.M.B. ans P.vD.E. contributed equally to the supervision of this manuscript.
4 Address correspondence and reprint requests to Dr. Jeremy M. Boss, Department of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322. E-mail address:
5 Abbreviations used in this paper: BLS, bare lymphocyte syndrome; CREB, cAMP response element binding protein; ß2m, ß2-microglobulin; CIITA, class II transactivator; Ii, invariant chain; CAT, chloramphenicol acetyltransferase.
6 Gobin, S. J. P., M. Van Zutphen, S. D. Westerheide, J. M. Boss, and P. J. van den Elsen. MHC gene transactivation through the SXY regulatory module is controlled by a specific enhanceosome. Submitted for publication.
Received for publication October 5, 1999. Accepted for publication January 19, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is crucial for two routes of IFN-induced MHC class I transactivation: the ISRE-mediate route and a novel pathway involving CIITA. Immunity 6:601.[Medline]
encoded by recombinant vaccinia virus. J. Immunol. 143:623.[Abstract]
mediated by the transactivator gene CIITA. Science 265:106.: similarities and distinctions with respect to HLA-DR constitutive B cells. J. Immunol. 156:4254.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  M. Krawczyk, K. Masternak, M. Zufferey, E. Barras, and W. Reith New Functions of the Major Histocompatibility Complex Class II-Specific Transcription Factor RFXANK Revealed by a High-Resolution Mutagenesis Study Mol. Cell. Biol., October 1, 2005; 25(19): 8607 - 8618. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xu, L. Wang, G. Buttice, P. K. Sengupta, and B. D. Smith Interferon {gamma} Repression of Collagen (COL1A2) Transcription Is Mediated by the RFX5 Complex J. Biol. Chem., December 5, 2003; 278(49): 49134 - 49144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. K. Howcroft, A. Raval, J. D. Weissman, A. Gegonne, and D. S. Singer Distinct Transcriptional Pathways Regulate Basal and Activated Major Histocompatibility Complex Class I Expression Mol. Cell. Biol., May 15, 2003; 23(10): 3377 - 3391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. P. Gobin, M. van Zutphen, S. D. Westerheide, J. M. Boss, and P. J. van den Elsen The MHC-Specific Enhanceosome and Its Role in MHC Class I and {beta}2-Microglobulin Gene Transactivation J. Immunol., November 1, 2001; 167(9): 5175 - 5184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Nekrep, M. Geyer, N. Jabrane-Ferrat, and B. M. Peterlin Analysis of Ankyrin Repeats Reveals How a Single Point Mutation in RFXANK Results in Bare Lymphocyte Syndrome Mol. Cell. Biol., August 15, 2001; 21(16): 5566 - 5576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.-M. Lennon-Dumenil, M.-R. Barbouche, J. Vedrenne, T. Prod'Homme, M. Bejaoui, S. Ghariani, D. Charron, M. Fellous, K. Dellagi, and C. Alcaide-Loridan Uncoordinated HLA-D Gene Expression in a RFXANK-Defective Patient with MHC Class II Deficiency J. Immunol., May 1, 2001; 166(9): 5681 - 5687. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. DeSandro, U. M. Nagarajan, and J. M. Boss Associations and Interactions between Bare Lymphocyte Syndrome Factors Mol. Cell. Biol., September 1, 2000; 20(17): 6587 - 6599. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
