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-Induced p67phox and gp91phox Expression1 ,2
urleen B. Wallace Tumor Institute, Department of Hematology and Oncology and the Comprehensive Cancer Center, University of Alabama, Birmingham, and The Birmingham Veterans Administration Hospital, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induces transcription of the genes encoding
p67phox (the NCF2 gene) and
gp91phox (the CYBB gene) during
monocyte differentiation, and also in mature monocytes. In these
studies, we identify an NCF2 cis element, necessary for
IFN--induced p67phox expression, and
determine that this element is activated by cooperation between the
transcription factors PU.1, IFN regulatory factor 1 (IRF1), and the IFN
consensus-binding protein (ICSBP). Previously, we identified a
CYBB cis element, necessary for IFN--induced
gp91phox expression, and also activated by this
transcription factor combination. In these investigations, we determine
that recruitment of a coactivator protein, CBP (the CREBbinding
protein), to the CYBB or NCF2 promoter is the
molecular mechanism of transcriptional activation by PU.1, IRF1, and
ICSBP. Also, we determine that the multiprotein interaction of CBP with
PU.1, IRF1, and ICSBP requires either the CYBB- or
NCF2--binding site. Because IFN- induces simultaneous
expression of p67phox and
gp91phox, these investigations identify a
molecular event that coordinates oxidase gene transcription during the
inflammatory response. Also, these investigations identify CBP
recruitment by cooperation between PU.1, IRF1, and ICSBP as a novel
molecular mechanism for IFN--induced activation of myeloid genes
that are involved in the system of host defense. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the immune response, IFN- induces monocyte differentiation of
committed progenitors, and also increases oxidase gene transcription in
mature monocytes (6, 7, 8). IFN- treatment of myeloid cell
lines increases gp91phox protein expression by
24 to 48 h (9). Consistent with this, CYBB
transcription increases in both ex vivo monocytes and myeloid cell
lines by 24 h of IFN- treatment (8, 10). In
contrast, p22phox mRNA is abundant in monocytes
and is minimally altered by IFN- (8). Since
p22phox protein stability depends on
gp91phox, the two proteins increase in parallel
in IFN--treated myeloid cells (10). IFN- increases
p47phox protein in myeloid cell lines within
2 h and, in monocytes, cooperates with other inflammatory
mediators to rapidly increase NCF1 transcription
(9). In contrast, IFN--induced
p67phox expression follows the same time course
as gp91phox (9, 10). Therefore, the
rate-limiting components for increased oxidase activity, in response to
IFN-, are gp91phox and
p67phox (9). Synchronous expression
of gp91phox and p67phox
suggests that common factors may regulate IFN--induced
CYBB and NCF2 transcription.
In these studies, we compare CYBB and NCF2 cis
elements to investigate the mechanism of IFN- induced, coordinate
gp91phox and p67phox
expression. Previously, we described a CYBB cis element,
necessary for IFN--induced gp91phox
expression, referred to as the HAF1-binding site (11). In
vivo, mutation of the HAF1-binding site results in chronic
granulomatous disease, a disorder of host defense (12, 13). We demonstrated that the HAF1-binding element is activated
by cooperation between PU.1, and the IFN regulatory factors (IRFs),
IRF1 and ICSBP (14).
Since NCF2 regulatory elements had not been previously identified, we began the current investigations by comparing the NCF2 and CYBB sequences. We identified 8 bp in NCF2 intron 1, identical to the CYBB HAF1-binding site. NCF2 exon 1 contains only 5' untranslated region (5'-UTR),4 and several different p67phox transcripts have been identified that vary in 5'-UTR length (3, 15). Some of these transcripts appear to initiate in intron 1 (3), suggesting intron 1 might be involved in NCF2 regulation. Therefore, we investigated the functional significance of the NCF2 intron 1 sequence with homology to the CYBB HAF1-binding site. We also investigated whether this NCF2 sequence interacts with the same transcription factors as the CYBB HAF1-binding site.
PU.1 has been hypothesized to activate TATA-less promoters by
interacting with TBP and TFIID, however, the mechanism of PU.1
transcriptional activation for specific target genes is generally
unknown (16). Similarly, IRF1 and ICSBP are necessary for
myeloid differentiation (17, 18), but the mechanism of
IFN--induced gene transcription by IRF1 and ICSBP is unknown. Some
transcription factors interact with transcriptional coactivator
proteins, which subsequently activate the transcriptional apparatus
(19, 20). CBP is a transcriptional coactivator involved in
regulation of IFN--inducible genes. CBP interacts with ets1 to
activate the APN1 gene (21), and with CIITA to
activate the MHC class II gene (22). CBP also interacts
with Stat1 (19) and IRF3 (23), but genuine
target genes for these interactions are unknown. To better understand
myeloid gene transcription, we also investigated whether the mechanism
of IFN--induced NCF2 and CYBB transcription is
recruitment of CBP by cooperation between PU.1, IRF1, and ICSBP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genomic clones and reporter constructs. NCF2 genomic clone C3, containing 4.5 kb of 5' flank, was obtained from T. Leto (National Institutes of Health, Bethesda, MD) (15). Sequence of the clone was determined by standard dideoxy techniques. Genomic fragments for reporter gene constructs were generated using endogenous restriction sites, or with Pfu DNA polymerase (Stratagene, LaJolla, CA). Mutant NCF2 sequences were generated by Quickswitch site-directed mutagenesis (Stratagene). Mutant and wild-type CYBB promoter sequences have been previously described (11, 24). Genomic sequences were subcloned into the reporter gene vector pCATE (Promega, Madison, WI). Artificial promoter/reporter constructs were generated as previously described (14), in the minimal promoter/reporter vector, p-TATACAT (25) (obtained from Dr. A. Kraft, University of Colorado, Denver, CO). Constructs were generated with one or five copies (in the forward orientation) of NCF2 intron 1 sequence from 160 to 190 bp 5' of the ATG (the five-copy construct is referred to as p-ncf2hafTATACAT). Constructs with four copies of the CYBB -32- to -69-bp sequence have been previously described (p-cybbhafTATACAT) (14).
Plasmids for protein expression.
The cDNA for human PU.1 was obtained from M. Klemsz (Indiana
University, Indianapolis, IN) and subcloned in to the mammalian
expression vector pSR
(14, 26). The human ICSBP cDNA,
obtained from B-Z. Levi (Technicon, Haifa, Israel), and the human IRF1
cDNA, obtained from R. Pine (New York University Medical Center, New
York, NY), were subcloned into the mammalian expression vector pcDNAamp
(Invitrogen, San Diego, CA) (14). The viral E1a
oncoprotein cDNA in pcDNA3 (Invitrogen), and the cDNA for the murine
CREB-binding protein (CBP) in pRSV, were obtained from T. Gabig
(Indiana University).
Oligonucleotides
Oligonucleotides were synthesized by the Core Facility of the
Comprehensive Cancer Center, University of Alabama, Birmingham.
Oligonucleotides: CYBB promoter -32 to -69 bp (cybbhaf)
(11); 5'-ctgctgttttcatttcctcattggaagaagaagcatag-3', CGD
bp -57 promoter mutant -32 to -69 bp (cybbhafmut) (12);
5'-ctgctgttttcctttcctcattggaagaagaagcatag-3', NCF2 intron 1
from 160 to 190 bp 5' of the ATG (ncf2haf);
5'-ccaaaaggtgggacatttcctgtggatttgc-3', NCF2 intron 1 bp
179 mutant sequence (ncf2hafmut);
5'-ccaaaaggtgggacctttcctgtggatttgc-3'; CCAAT box from the globin
gene (urccaat) (24);
5'-ccgggctccgcgccagccaatgagcgccgcgg-3'.
Cell culture
All cell lines were of human origin. The epithelial carcinoma
line HeLa (27) was obtained from American Type Culture
Collection (Manassas, VA). The myelomonocytic cell line U937
(28) was obtained from Andrew Kraft (University of
Colorado). Cell lines were maintained and differentiated as described
(14). U937 cells were treated with 200 or 1000 U/ml human
rIFN- (Boerhinger Mannheim, Indianapolis, IN).
EMSA
Nuclear extract proteins were prepared by the method of Dignam
(29) with protease and phosphatase inhibitors, as
described (14). Oligonucleotide probes were prepared, and
EMSA and Ab supershift assays were performed, as described
(14). Antiserum to PU.1 was obtained from M. Klemsz
(Indiana University, Indianapolis, IN) (30). IRF2 and
ICSBP Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-IRF1 serum (whole protein) was provided by Richard Pine (Public
Health Research Institute, New York, NY) (31). Antisera to
ICSBP peptides (anti-310, -311, and -312) were provided by S. Vogel
(Uniformed Services University of the Health Sciences, Bethesda, MD)
(32). Anti-PIP serum was a gift from M. Atchison
(University of Pennsylvania, Philadelphia, PA). Anti-p48-ISGF3 serum
was provided by D. Levy (New York University, New York, NY).
Transient transfection and reporter gene assays
Cells were transfected by electroporation as described
(14). U937 cells were transfected with 50 µg of
pCATE constructs and 15 µg p-CMV/ß-galactosidase
(Clontech, Palo Alto, CA). HeLa cells were transfected with 15 µg of
pCATE constructs and 5 µg p-CMV/ß-galactosidase. In
other experiments, U937 cells were transfected with 70 µg of
p-TATACAT, p-ncf2hafTATACAT, or p-cybbhafTATACAT; 30 µg of pSR or
PU.1/pSR; 30 µg of pcDNAamp or IRF1/pcDNAamp or ICSBP/pcDNAamp; or
15 µg each of IRF1/pcDNAamp and ICSBP/pcDNAamp, and 15 µg
p-CMV/ß-galactosidase. In some experiments, U937 cells
were also transfected with 30 µg of E1a/pcDNA3 or pcDNA3, or 30 µg
of CBP/pRSV, or pRSV. HeLa cells were transfected with 20 µg of
p-TATACAT or p-ncf2hafTATACAT; 10 µg of pSR or PU.1/pSR; 10
µg or pcDNAamp or IRF1/pcDNAamp or ICSBP/pcDNAamp; or 5 µg each of
IRF1/pcDNAamp and ICSBP/pcDNAamp, and 5 µg p-CMV/ß-galactosidase.
Transfectants were incubated for 24 h at 37°C, 5%
CO2, followed by 24 h with or without IFN- (200 or
1000 U/ml). Preparation of cell extracts, ß-galactosidase and
chloramphenicol acetyltransferase assays (CAT) assays were performed as
described (33, 34).
Northern blots and primer extension assay
Total cellular RNA was extracted (35) from U937
cells, with or without 24 h of IFN- treatment. Northern blots
were performed with 20 µg of RNA, as described (10). For
some primer extension experiments, RNA was extracted 48 h after
transfection with pCATE constructs containing NCF2 sequences
(or empty vector control), with 24 h of IFN- treatment. Primer
extension was performed, as described (36). Primer for
endogenous p67phox message was the first 21 bp
of the cDNA-coding sequence (3), and reporter gene
transcript primer was the first 21 bp of the CAT cDNA-coding sequence.
The size of reverse transcripts was determined by comparison with a
sequencing reaction of either the genomic clone, or the reporter gene,
using the same primer.
Immunoprecipitation
Immunoprecipitation experiments were performed with 30 µg of nuclear proteins in 200 µl of HN buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 0.5% nonidet P-40, and with protease and phosphatase inhibitors, as described (14). Nuclear proteins were incubated with either 1 µl of ICSBP antiserum (Santa Cruz Biotechnology) or preimmune serum, with or without 20 ng of double-stranded synthetic oligonucleotides (cybbhaf, ncf2haf, urccaat) for 4 h at 4°C followed by 1-h incubation with 15 µl of 50% staphylococcal protein A-Sepharose bead slurry. Beads were washed with 1 ml of HN buffer; proteins were eluted in SDS sample buffer, separated on 8% SDS-PAGE, and transferred to nitrocellulose. Blots were serially probed with Abs to CBP (sc-20, Santa Cruz), IRF1, PU.1 (Santa Cruz Biotechnology), and anti-ICSBP and detected by chemiluminescence, according to the manufacturer’s instructions (Amersham, Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced p67phox expression
Expression of p67phox and
gp91phox increases during IFN--induced
monocyte differentiation, and in mature ex vivo monocytes stimulated
with IFN- (3, 8, 10). U937 cells are committed to
monocyte/macrophage differentiation (28), and have minimal
gp91phox and p67phox
expression (Fig. 1
). In response to
48 h of treatment with IFN-, U937 cells undergo monocytic
differentiation, and demonstrate respiratory burst activity upon
stimulation with PMA (37). Since IFN- treatment of U937
cells increases abundance of p67phox and
gp91phox mRNA (Fig. 1), we employed this line as
a model for IFN--inducible NCF2 and CYBB
transcription.
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A) and CYBB
promoter elements involved in IFN--induced
gp91phox expression. Similar to the previously
reported NCF2 genomic organization (15), we
determined that 1.0 kb of NCF2 5' flank includes: 480 bp 5'
to exon 1, a 70-bp first exon, a 469-bp first intron, and 30 bp of 5'
untranslated from exon 2 (Fig. 2B). Several different
p67phox transcripts were previously identified,
varying only in the length of 5'-UTR (3, 15). The lengths
of these 5'-UTRs suggested that some transcripts initiated in intron 1.
However, there is no TATA box consensus in either intron 1 or 5' of
exon 1, although there are two Inr consensus sequences
(38), representing possible transcription initiation sites
(534 or 570 bp 5' of the ATG). Interestingly, the NCF2
intron 1 sequence includes 8 bp identical to a CYBB promoter
element that is activated by PU.1, IRF1, and ICSBP (173 to 180 bp 5' of
the ATG) (Fig. 2C) (14).
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A). After 24 h of
IFN- treatment, these transcripts increased in abundance, and an
additional transcript with 100 bp of 5'-UTR appeared. This result was
at slight variance with the transcripts previously demonstrated by
other investigators by RT-PCR with HL60 mRNA (with 24 or 68 bp of
5'-UTR). Transcripts with 58 or 100 bp of 5'-UTR would overlap Inr
sequences, unless the transcripts initiate in intron 1. To determine if
any transcripts initiate in intron 1, U937 cells were transfected with
CAT reporter constructs containing either NCF2 intron 1
+ 33 bp of exon 2 (referred to as INT1NCF2), or 480 bp 5' of
exon 1 + exon 1 + INT1 (referred to as -480 +
INT1NCF2), or pCATE empty vector control (Fig. 2
B). RNA was extracted from IFN--treated transfectants
and analyzed by primer extension. Reporter transcripts with 42 or 58 bp
of 5'-UTR were detected in RNA from U937 transfectants with the
INT1NCF2 construct, and transcripts with 42, 58, or 100 bp
of 5'-UTR from -480 + INT1NCF2 construct transfectants
(Fig. 3B), suggesting that transcripts with 42 or 58 bp of
5'-UTR initiate in intron 1.
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-induced p67phox expression, U937 cells
were transfected with NCF2/reporter constructs, and reporter
gene expression was determined with or without IFN- treatment. U937
cells were transfected with the INT1NCF2 construct, or
the -480 + INT1NCF2 construct, or a construct
containing exon 1 + 480 bp 5' of exon 1 without intron 1
(-480NCF2), or constructs containing the proximal 50 or 100
bp of intron 1 (100INT1NCF2 or 50INT1NCF2,
respectively). Results are expressed as percentage of increase in CAT
activity relative to empty vector control transfectants, which are
considered to have 0% CAT activity. In non-IFN--treated U937
transfectants, all of the constructs, except for 50INT1NCF2,
exhibit modest reporter gene expression (Fig. 4A). Therefore, constructs
containing either NCF2 intron 1 or sequence 5' to exon 1
demonstrate promoter activity in U937 cells.
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significantly increases reporter expression from
U937 transfectants with either the INT1NCF2 construct
(p = 0.010, n = 6) or the
-480 + INT1NCF2 construct (p =
0.026, n = 6). Reporter expression from IFN--treated
transfectants with these two constructs is not significantly different
(p = 0.74, n = 6) (Fig. 4A). In contrast, IFN- incubation does not significantly
increase reporter gene expression from U937 transfectants with the
-480NCF2 construct, which does not include intron 1
(p = 0.19, n = 6), or with
control vector. Therefore, NCF2 intron 1 is necessary for
IFN--inducible expression in U937 transfectants. In contrast, HeLa
cells transfected with these NCF2 constructs do not have a
significant CAT activity, with or without IFN- treatment, suggesting
lineage specificity of the NCF2 elements
(INT1NCF2: 5.5 ± 30.6%, -480 +
INT1NCF2: 2.0 ± 22.5%, and -480NCF2:
34.0 ± 65.6%, n = 4).
To determine if the 8-bp NCF2 intron 1 sequence, homologous
to a CYBB promoter element, is involved in IFN--inducible
p67phox expression, U937 cells were transfected
with mutant NCF2/reporter constructs. We previously
demonstrated that mutation of bp -57 in the CYBB promoter
abolishes IFN--inducible reporter expression from myeloid
transfectants with CYBB promoter constructs (mutant and
wild-type constructs: -470(hafmut)CYBB and
-470CYBB, respectively) (11).
CYBB bp -57 is within the 8 bp identical to the
NCF2 intron 1 sequence, and a homologous NCF2
mutation changes bp 179 (5' of the ATG) from A to C (Fig. 2C). This mutation was introduced into the
INT1NCF2 and the -480 + INT1NCF2 constructs
(INT1(hafmut)NCF2 and 480 +
INT1(hafmut)NCF2, respectively) and these constructs were
transfected into U937 cells.
Mutation of bp -57 in the CYBB promoter results in loss of
IFN--induced reporter gene expression in U937 cells transfected with
the -470(hafmut)CYBB construct (Fig. 4B). This
result is consistent with our previous results in PLB985 stable
transfectants with the same mutant and wild-type CYBB
promoter sequences in different reporter vector (11).
Additionally, the -57-bp mutation abolishes basal reporter expression
from the CYBB construct, which our previous system of stable
transfectants, assayed with and without IFN- treatment
(11), had not allowed us to demonstrate.
Introduction of intron 1 bp 179 mutation results in loss of
IFN--induced reporter gene expression in U937 transfectants with
either the INT1(hafmut)NCF2, or -480 +
INT1(hafmut)NCF2 construct (inhibition of
IFN--induced reporter expression was statistically significant,
p < 0.05, n = 6, for both
mutant vs wild-type construct pairs) (Fig. 4B). However,
mutation of NCF2 intron 1 bp 179 does not significantly
affect basal reporter gene expression from U937 transfectants with the
mutant INT1NCF2, or -480 + INT1NCF2
construct in comparison with wild-type construct transfectants
(p = 0.122 or p = 0.750,
n = 6, respectively) (Fig. 4B).
An IFN--inducible NCF2 intron 1 element interacts
with PU.1, IRF1, and ICSBP
We previously demonstrated that the -52- to -62-bp
CYBB promoter sequence interacts with the transcription
factors PU.1, IRF1, and ICSBP, in vitro and in transfection experiments
(14). EMSA were preformed to determine if the homologous
NCF2 intron 1 sequence (173- to 180-bp 5' of the ATG)
interacts with the same factors. In EMSA with a -32- to -69-bp
CYBB probe (the cybbhaf probe), U937 nuclear proteins
generate several specific protein complexes, which are unchanged by
IFN- treatment: the HAF1 complex; a heterodimer of PU.1 and IRF1 or
ICSBP, and the HAF1a complex; a heterotrimer of PU.1, IRF1, and ICSBP,
and the HAF1a complex; a heterotrimer of PU.1, IRF1, and ICSBP
(14). Mutation of bp -57 (the cybbhafmut probe) abolishes
binding of both the HAF1 and the HAF1a complexes (11, 12, 14).
EMSA were performed with U937 nuclear proteins, the cybbhaf probe, and
oligonucleotide competitors representing wild-type NCF2
intron 1 (bp 160 to 190 5' of the ATG), or bp 179 mutant
NCF2 intron 1 sequence (ncf2haf and ncf2hafmut,
respectively). Consistent with the homology between the CYBB
and NCF2 sequences, binding of the HAF1 and HAF1a complexes
to the cybbhaf probe is efficiently competed for by ncf2haf
oligonucleotide, but not ncf2hafmut oligonucleotide (Fig. 5A). Neither ncf2
oligonucleotide competes for binding of a complex that represents CP1
interaction with the CCAAT sequence in the cybbhaf probe
(11). The reciprocal experiments were performed with the
ncf2haf probe. In EMSA with nuclear proteins from U937 cells, with or
without IFN- treatment, the ncf2haf probe generates two complexes of
similar mobilities to the HAF1 and HAF1a complexes (Fig. 5B). Binding of these complexes to the ncf2haf probe is
competed for by ncf2haf and cybbhaf oligonucleotides, but not by
ncf2hafmut or cybbhafmut oligonucleotides (Fig. 5C).
Identical results were obtained in EMSA with nuclear proteins from U937
cells with or without IFN- treatment (data not shown).
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D). Identical results were obtained in EMSA with nuclear
proteins from IFN- treated U937 cells (data not shown). Binding of
the HAF1a complex to the cybbhaf probe is completely disrupted by Ab to
either IRF1 or ICSBP, but that complete disruption of the HAF1 complex
requires Abs to both IRF1 and ICSBP (14). Consistent with
these results, in EMSA with U937 nuclear proteins and the ncf2haf
probe, the complex with the same mobility as HAF1a is completely
disrupted by Ab to either IRF1 or ICSBP (Fig. 5E). And,
binding of the complex with the same mobility as HAF1 is partly
disrupted by Ab to IRF1 or ICSBP, and completely disrupted by the two
Abs together (Fig. 5E). These results were the same with
nuclear proteins from IFN- treated U937 cells (not shown).
However, interpretation of these experiments is complicated by binding
of a protein complex to the ncf2haf probe, which has mobility
intermediate to the HAF1 and HAF1a complexes, and is unmasked by
disruption of these complexes by Abs to PU.1, IRF1, or ICSBP. This
intermediate mobility complex is not competed for by homologous ncf2haf
oligonucleotide, and appears to represent nonspecific protein binding
(Fig. 5, C and E).
To determine if PU.1, IRF1, and ICSBP functionally activate
transcription from the NCF2 intron 1 sequence, U937 cells
were cotransfected with an artificial promoter construct containing
five copies of the NCF2 160- to 190-bp intron 1 sequence, a
minimal promoter and CAT reporter (p-ncf2hafTATACAT), and vectors to
overexpress PU.1, IRF1, and ICSBP. Results are expressed as percentage
of increase in absolute CAT activity relative to control vector
transfectants, which are considered to have 0% CAT activity. In
comparison with control p-TATACAT transfectants, U937 transfectants
with p-ncf2hafTATACAT have significantly increased reporter gene
expression (Fig. 5F). Cotransfection of U937 cells with
p-ncf2hafTATACAT and vectors to overexpress PU.1, IRF1, or ICSBP, alone
or in any combination of two, does not further increase reporter
expression. However, cotransfection of U937 cells with
p-ncf2hafTATACAT, PU.1, IRF1, and ICSBP results in significantly more
reporter gene expression than transfectants with the same reporter
construct, PU.1 and equivalent amounts of either IRF1 or ICSBP (p
= 0.025 for PU.1 + IRF1 vs PU.1, IRF1, ICSBP, and
p = 0.019 for PU.1 + ICSBP vs PU.1, IRF1, ICSBP,
n = 8) (Fig. 5F).
PU.1, IRF1, and ICSBP also synergistically activate a construct with one copy of the NCF2 intron 1 sequence, suggesting that the three proteins interact with one sequence, not between sequences (data not shown). However, overexpression of PU.1, IRF1, and ICSBP does not alter reporter gene expression from control p-TATACAT transfectants. And, in HeLa transfectants, the p-ncf2hafTATACAT construct does not have significantly different reporter gene activity than p-TATACAT, with or without PU.1, IRF1, and ICSBP overexpression (-0.2 ± 5.6%, n = 3).
IFN- treatment of U937 cells (200 U/ml) transfected with
p-ncf2hafTATACAT results in a significant increase in reporter gene
expression in comparison with untreated controls (125 ± 20%,
p < 0.05, n = 3). However, treatment
of U937 cells cotransfected with p-ncf2hafTATACAT, PU.1, IRF1, and
ICSBP with IFN- (200 U/ml) did not significantly alter reporter gene
expression in comparison with untreated transfectants
(p = 0.23, n = 3), consistent
with our previous results with p-cybbTATACAT transfectants
(14). However, treatment of the U937 transfectants with
1000 U/ml of IFN- significantly increases reporter gene activity
from the p-ncf2hafTATACAT and p-cybbhafTATACAT constructs,
with and without overexpression of PU.1, IRF1, and ICSBP (Fig. 5G, p < 0.01 for all combinations,
n = 3). The IFN--increased reporter gene activity in
U937 transfectants with p-ncf2TATACAT is not significantly different
than with p-cybbhafTATACAT transfectants, without
(p = 0.124), or with PU.1, IRF1, and ICSBP
(p = 0.136).
CBP is necessary for IFN--induced transcription of the
NCF2 and CYBB genes
Interaction of ets and IRF transcription factors with CBP
increases transcription of some genes (21, 22). Therefore,
we investigated whether CBP interacts with PU.1, IRF1, and ICSBP to
increase CYBB or NCF2 transcription. U937 cells
were cotransfected with CYBB or NCF2/reporter
constructs, and a vector to overexpress either the adenovirus early
gene product, E1a (39), or CBP. E1a interacts with a CBP
protein-protein interaction domain, blocking interaction with other
proteins, and specifically inhibiting CBP (39). In U937
transfectants with either the INT1NCF2, the -480 +
INT1NCF2, or the -470CYBB construct,
overexpression of E1a significantly decreases basal reporter
expression, and completely abolishes IFN--induced reporter
expression (p < 0.05, n = 6)
(Fig. 6A). Consistent with
these results, U937 cells cotransfected with these NCF2/ or
CYBB/constructs, and a vector to overexpress CBP,
demonstrate significantly increased reporter gene expression in
comparison with control expression vector transfectants (Fig. 6A) (INT1NCF2; p = 0.046,
n = 6, -470CYBB; p = 0.005,
n = 6). Overexpression of CBP also increases reporter
gene expression from these constructs in IFN--treated U937
transfectants (p = 0.062, n = 6
and p = 0.049, n = 6, respectively).
Neither E1a nor CBP overexpression alters CAT activity from U937
transfectants with pCATE empty reporter vector.
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B). Overexpression of E1a or
CBP does not effect CAT activity from U937 transfectants with control
p-TATACAT.
We investigated the possibility that CBP is a
component of the complex formed by interaction of
PU.1, IRF1, and ICSBP with the homologous CYBB or NCF2
cis elements. In EMSA with U937 nuclear proteins and either the
cybbhaf or ncf2haf probe, binding of the HAF1a complex is specifically
disrupted by a CBP-specific Ab (Fig. 6C). Results were
identical in EMSA with nuclear proteins from IFN--treated U937 cells
(not shown). To determine whether DNA binding is required for CBP to
associate with PU.1, IRF1, and ICSBP, immunoprecipitation experiments
were performed. Nuclear proteins from U937 cells were
immunoprecipitated with ICSBP Ab and either cybbhaf, ncf2haf or
irrelevant oligonucleotide, under nondenaturing conditions. Anti-ICSBP
immunoprecipitates were analyzed by Western blot. IRF1 and PU.1
coimmunoprecipitate with ICSBP, independent of a specific
oligonucleotide-binding site (Fig. 6D) (14, 40). In contrast, CBP coimmunoprecipitates with ICSBP with
either the cybbhaf or ncf2haf oligonucleotide, but not an irrelevant
oligonucleotide (Fig. 6D), or HAF1 site mutant
oligonucleotides (data not shown). Identical results were obtained with
nuclear proteins from IFN--treated U937 cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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augments respiratory
burst oxidase activity by two mechanisms: increased transcription of
oxidase component genes in mature myeloid cells, and increased monocyte
differentiation of committed progenitors (6, 7, 8, 9). In either
monocytes or myeloid cell lines, IFN- induces simultaneous
expression of gp91phox and
p67phox (9, 10). Although gene
expression in myeloid lines may not exactly parallel transcription
during normal differentiation, these findings suggest
that IFN- induces coordinate gp91phox and
p67phox expression during the immune response.
Also, IFN--induced gp91phox and
p67phox expression is rate limiting for
increased respiratory burst activity in myeloid cells (9).
Therefore, transcription factors regulating CYBB and
NCF2 transcription also regulate IFN--induced respiratory
burst activity at the molecular level.
In these investigations, we hypothesized that common trans
factors interact with homologous cis elements to coordinate
CYBB and NCF2 transcription. Consistent with
this, we identify a cis element in the NCF2 gene,
necessary for IFN--induced p67phox
expression, and homologous to a CYBB element, necessary for
IFN--induced gp91phox expression (the
HAF1-binding element). We demonstrate that this NCF2 element
is activated by cooperation between PU.1, IRF1, and ICSBP; the same
factors activating the CYBB HAF1 element. Therefore, these
investigations provide the first identification of a regulatory element
in the NCF2 gene and describe a novel molecular mechanism
for coordinate transcription of two myeloid-specific genes during the
immune response. Since the CYBB and NCF2 genes
encode rate-limiting oxidase components, the current investigations
identify interaction of PU.1, IRF1, and ICSBP as a molecular regulator
of IFN--induced respiratory burst activity.
These investigations provide novel insight into gene transcription by PU.1. Our investigations indicate that PU.1 recruits IRF1 and ICSBP to activate the CYBB and NCF2 genes. In these studies, we determine that PU.1, IRF1, and ICSBP recruit CBP, a transcriptional coactivator, to the proximal CYBB and NCF2 promoters. We demonstrate that inhibition of CBP blocks the ability of PU.1, IRF1, and ICSBP to activate CYBB and NCF2 transcription, indicating that CBP recruitment is the mechanism of transcriptional activation by these factors. Therefore, these studies describe the mechanism by which PU.1 activates transcription of two target genes, suggesting a model for PU.1 activation of multiple genes transcribed during mid/late myeloid differentiation.
Similarly, these investigations address the mechanism of gene
transcription by IRF proteins. IRF3 has been demonstrated to interact
with CBP and activate artificial promoter constructs with ISRE
consensus sites (23). However, our investigation
identifies genuine target genes for IFN--induced IRF/IRF/CBP
interaction. Our investigations also suggest a mechanism for the
ability of ICSBP to activate transcription under some conditions, and
repress under others (40). If ICSBP activates
transcription by recruiting CBP, ICSBP repression may occur under
conditions of corepressor recruitment. ICSBP phosphorylation, induced
by IFN-, may determine differential coactivator and corepressor
recruitment.
CBP is hypothesized to increase transcription by bringing histone
acetyltransferase (HAT) activity to the proximal promoter
(20). Acetylation of DNA-bound histones by CBP or proteins
recruited by CBP permits access of the transcriptional apparatus to the
start site. It will be of interest to determine if HAT activity of the
HAF1a complexes is altered by IFN-. Despite the requirement for
PU.1/IRF1/ICSBP/CBP interaction for IFN--induced CYBB and
NCF2 transcription, IFN- does not alter in vitro
interaction of these proteins. This may be similar to CBP interaction
with Stat3 during neural differentiation. The Stat3/CBP complex is
present in undifferentiated cells, but undergoes
differentiation-dependent interaction with another coactivator protein,
Smad1 (41). Identification of other coactivators, or
corepressors that interact with the PU.1/IRF1/ICSBP/CBP complex will
provide further information about gene transcription during monocyte
differentiation and the immune response.
We found that CBP association with PU.1, IRF1, and ICSBP requires the DNA-binding site from the CYBB or NCF2 gene. Although ICSBP coprecipitates IRF1 or PU.1 without a DNA-binding site (14, 40), the three proteins may not exist as a heterotrimer in the absence of DNA binding. If CBP interacts with domains from more than one of these proteins, conformational changes in PU.1, IRF1, or ICSBP, induced by assembly on DNA, might be necessary to bring domains into a proximity permitting CBP interaction. This mechanism would confer more specificity for transcriptional activation than the interaction of CBP with a single transcription factor.
We identified NCF2 transcription start sites in intron 1,
and 5' of exon 1. Since these transcripts all have the same coding
sequence, the functional significance of alternative
transcription start sites is not obvious. The 5'- most IFN--induced
transcript includes 70 bp from exon 1, differing from the 35-bp exon 1
transcript detected by other investigators in DMSO-treated HL60 cells
(15). Since IFN--treated U937 cells are monocytoid, and
DMSO-treated HL60 cells are neutrophil like, these results suggest that
different regulatory elements may be used in monocytes and in
neutrophils.
Mutation of the HAF1-like binding site in NCF2 intron 1
abolishes IFN--induced expression, similar to the CYBB
HAF1 element (11). However, unlike the CYBB
element, mutation of this NCF2 element does not decrease
basal transcription. Therefore, our results are consistent with the
role of PU.1 in oxidase gene expression, previously suggested by murine
gene disruption experiments (42). PU.1 knockout mice lack
gp91phox expression, but express
p67phox (42); however, it has not
been determined if PU.1 knockout abolishes induction of oxidase gene
expression by inflammatory mediators. Such investigations would further
clarify the essential molecular mechanisms that regulate various stages
of hemopoiesis and the inflammatory response.
In EMSA, more NCF2 intron 1 oligonucleotide is required to compete for HAF1 and HAF1a binding to the CYBB probe than the amount of CYBB competitor required in the reciprocal experiment (200x vs 50x molar excess). This suggests that sequences outside of the 8 identical bp lower-binding affinity to the NCF2 sequence, in comparison with the CYBB sequence. PU.1 monomer binding to the NCF2 probe is variably detected, although PU.1 monomer consistently binds the CYBB probe (14). However, 50-fold more CYBB probe is shifted into the HAF1 and HAF1a complexes than is shifted by PU.1 monomer (14). Given the decreased affinity of the NCF2 probe for these complexes, PU.1 monomer binding may be below the level of detection. Our findings do not exclude the possibility of other proteins, including other ets or IRF proteins, interacting with one or both of these elements. Which proteins interact with the elements under different conditions may be determined by differentiation state, cell lineage, or stimulation by inflammatory mediators.
Cooperative interaction of PU.1, IRF1, and ICSBP with both the
CYBB and NCF2 genes suggests that this
combination of factors may regulate other IFN--inducible myeloid
genes. Although p47phox expression requires PU.1
interaction with an NCF1 promoter element (43),
this PU.1 box is not homologous to the HAF1-binding sites. Given the
difference in the timing of IFN--induced
p47phox expression, in comparison with
gp91phox and p67phox
expression, perhaps this is not unexpected. However, it will be of
interest to determine if other IFN--inducible myeloid genes are
activated by cooperative recruitment of CBP by PU.1, IRF1, and ICSBP.
Also, determining if these proteins are also involved in
CYBB and NCF2 transcription in response to other
differentiating agents, such as retinoic acid or vitamin D, will
provide further insights into molecular regulation of myeloid
differentiation and the immune response.
|
Footnotes |
|---|
2 Sequenced regions of NCF2 are available from GenBank
(accession numbers U00776 and M32011).
3 Address correspondence and reprint requests to Dr. Elizabeth A. Eklund, Lurleen B. Wallace Tumor Institute, Department of Hematology and Oncology and the Comprehensive Cancer Center, University of Alabama, Birmingham, and The Birmingham Veterans Administration Hospital, Birmingham, AL 35294. E-mail address:
4 Abbreviations used in this paper: 5'-UTR, 5' untranslated region; IRF, interferon-regulatory factor; ICSBP, interferon consensus sequence-binding protein; CBP, CREB-binding protein; HAF1, hemopoiesis-associated factor 1; CGD, chronic granulomatous disease; CAT, chloramphenicol acetyltransferase; HAT, histone acetyltransferase; PIP, PU.1 interacting protein.
Received for publication May 14, 1999. Accepted for publication September 13, 1999.
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E. A. Eklund, I. Goldenberg, Y. Lu, J. Andrejic, and R. Kakar SHP1 Protein-tyrosine Phosphatase Regulates HoxA10 DNA Binding and Transcriptional Repression Activity in Undifferentiated Myeloid Cells J. Biol. Chem., September 20, 2002; 277(39): 36878 - 36888. [Abstract] [Full Text] [PDF]
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D. Meraro, M. Gleit-Kielmanowicz, H. Hauser, and B.-Z. Levi IFN-Stimulated Gene 15 Is Synergistically Activated Through Interactions Between the Myelocyte/Lymphocyte-Specific Transcription Factors, PU.1, IFN Regulatory Factor-8/IFN Consensus Sequence Binding Protein, and IFN Regulatory Factor-4: Characterization of a New Subtype of IFN-Stimulated Response Element J. Immunol., June 15, 2002; 168(12): 6224 - 6231. [Abstract] [Full Text] [PDF]
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S.-L. Li, A. J. Valente, M. Qiang, W. Schlegel, M. Gamez, and R. A. Clark Multiple PU.1 sites cooperate in the regulation of p40phox transcription during granulocytic differentiation of myeloid cells Blood, May 29, 2002; 99(12): 4578 - 4587. [Abstract] [Full Text] [PDF]
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V. S. Carl, K. Brown-Steinke, M. J. H. Nicklin, and M. F. Smith Jr. Toll-like Receptor 2 and 4 (TLR2 and TLR4) Agonists Differentially Regulate Secretory Interleukin-1 Receptor Antagonist Gene Expression in Macrophages J. Biol. Chem., May 10, 2002; 277(20): 17448 - 17456. [Abstract] [Full Text] [PDF]
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A. Kumatori, D. Yang, S. Suzuki, and M. Nakamura Cooperation of STAT-1 and IRF-1 in Interferon-gamma -induced Transcription of the gp91phox Gene J. Biol. Chem., March 8, 2002; 277(11): 9103 - 9111. [Abstract] [Full Text] [PDF]
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K. A. Gauss, P. L. Bunger, and M. T. Quinn AP-1 is essential for p67phox promoter activity J. Leukoc. Biol., January 1, 2002; 71(1): 163 - 172. [Abstract] [Full Text] [PDF]
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S.-L. Li, A. J. Valente, L. Wang, M. J. Gamez, and R. A. Clark Transcriptional Regulation of the p67phox Gene. ROLE OF AP-1 IN CONCERT WITH MYELOID-SPECIFIC TRANSCRIPTION FACTORS J. Biol. Chem., October 12, 2001; 276(42): 39368 - 39378. [Abstract] [Full Text] [PDF]
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M. Deng and G. Q. Daley Expression of interferon consensus sequence binding protein induces potent immunity against BCR/ABL-induced leukemia Blood, June 1, 2001; 97(11): 3491 - 3497. [Abstract] [Full Text] [PDF]
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 S. A. K. Merediz, M. Schmidt, G. J. Hoppe, J. Alfken, D. Meraro, B.-Z. Levi, A. Neubauer, and B. Wittig Cloning of an interferon regulatory factor 2 isoform with different regulatory ability Nucleic Acids Res., November 1, 2000; 28(21): 4219 - 4224. [Abstract] [Full Text] [PDF]
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H. Cohen, A. Azriel, T. Cohen, D. Meraro, S. Hashmueli, D. Bech-Otschir, R. Kraft, W. Dubiel, and B.-Z. Levi Interaction between Interferon Consensus Sequence-binding Protein and COP9/Signalosome Subunit CSN2 (Trip15). A POSSIBLE LINK BETWEEN INTERFERON REGULATORY FACTOR SIGNALING AND THE COP9/SIGNALOSOME J. Biol. Chem., December 8, 2000; 275(50): 39081 - 39089. [Abstract] [Full Text] [PDF]
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B. Kautz, R. Kakar, E. David, and E. A. Eklund SHP1 Protein-tyrosine Phosphatase Inhibits gp91PHOX and p67PHOX Expression by Inhibiting Interaction of PU.1, IRF1, Interferon Consensus Sequence-binding Protein, and CREB-binding Protein with Homologous Cis Elements in the CYBB and NCF2 Genes J. Biol. Chem., October 5, 2001; 276(41): 37868 - 37878. [Abstract] [Full Text] [PDF]
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E. A. Eklund, A. Jalava, and R. Kakar Tyrosine Phosphorylation of HoxA10 Decreases DNA Binding and Transcriptional Repression during Interferon gamma -induced Differentiation of Myeloid Leukemia Cell Lines J. Biol. Chem., June 23, 2000; 275(26): 20117 - 20126. [Abstract] [Full Text] [PDF]
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