BACKGROUND
mir- <dig> is one of the most extensively studied mirs due to its involvement in immune cell development and function and in various diseases  <cit> .
increased mir- <dig> expression is typically seen following the treatment of cells with proinflammatory signaling molecules like lipopolysaccharide , interleukins, interferons, and tumor necrosis factor  <cit> .
mice with a knockout of mir- <dig> have dysfunctional lymphocytes and dendritic cells  <cit> .
increased mir- <dig> expression is also observed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis  <cit> , and in many b- and t-lymphomas and leukemias  <cit> .
transgenic mice with tissue-specific expression of mir- <dig> can develop hematopoietic malignancies  <cit> .
the mature form of mir- <dig> is encoded by the mir155hg gene   <cit> .
although mir155hg encodes two mirs , mir155-5p is the more commonly studied product, and herein will be referred to as mir- <dig>  mir- <dig> is excised from an exon of its pre-mirna precursor by dicer and then loaded into the risc complex  <cit> .
this mir-155/risc complex can then bind to mrna transcripts with mir- <dig> target sequences in their 3`-utrs.
mir-155-regulated transcripts include ones encoding pu.
<dig>  aid, ikkϵ, c/ebpβ, socs <dig>  mitf, and fadd  <cit> , and mir- <dig> has been shown to decrease translation of these target mrnas.
to date, the majority of evidence that transcription factor nf-κb activates mir- <dig> expression is circumstantial, extrapolated from the positive correlation between high nuclear nf-κb activity and increased mir- <dig> expression in both normal lymphoid cells and various cancers, including many hematopoietic malignancies  <cit> .
several studies have set out to identify the cis regulatory elements in the mir155hg promoter, producing mixed and sometimes conflicting results.
one study claims that ebv-induced mir155hg expression is primarily regulated by an ap- <dig> site found  <dig> nt upstream of the transcription start site   <cit> , while another concludes that ebv induces mir155hg through two nf-κb sites over  <dig> nt upstream of the tss  <cit> .
a third study suggests that nf-κb up-regulates ap- <dig> components, which then bind to the ap- <dig> site  to up-regulate mir- <dig> expression  <cit> .
thus, no study has convincingly established mir155hg as a direct target of nf-κb.
in this report, we show that nf-κb p50/ <dig> can directly bind to and activate the human mir155hg proximal promoter through a site approximately  <dig> nt upstream of the mir155hg tss.
furthermore, we demonstrate that mir155hg/mir- <dig> expression and p50/p <dig> binding to this site are rapidly induced after treatment of the b-lymphoma cell line bjab with the nf-κb inducer lps.
lastly, in a panel of b-lymphoma cell lines, nuclear nf-κb protein levels generally correlate with increased mir- <dig> expression.
the results of this study show that mir- <dig> expression can be directly controlled by nf-κb through a site in the mir155hg proximal promoter.
methods
plasmids
the firefly luciferase reporter plasmid containing  <dig> nt region upstream of the mir155hg tss pwt-mir155hg), the - <dig> κb-site mutant plasmid , and the ap-1mut-mir155hg were gifts of eric flemington and have been described previously  <cit> .
the - <dig>  and - <dig>  mutants were created by insertion of an xhoi site in the middle of the given κb site, as in -1150mut-mir155hg .
reporter plasmids with truncations at - <dig> and - <dig> were created by amplifying the relevant regions of the mit155hg promoter  and subcloning them into nhei/hindiii-digested pwt-mir155hg.
pcdna-flag-p <dig> and pcdna-flag-p <dig> were created by subcloning pcr-amplified sequences of each into the xhoi site of pcdna-flag.
pcdna-rel has been described previously  <cit> .
cell culture
the cell lines used in this study were a <dig> human embryonic kidney cells, cos- <dig> monkey kidney cells, 3t <dig> and 3t3nfkb1-/-nfkb2-/-relb-/-crel-/-  mouse fibroblast cells, and the following human b-lymphoma cell lines: gcb diffuse large b-cell lymphoma bjab, sudhl <dig>  and pfeiffer  <cit> ; follicular lymphoma bl <dig>  <cit> ; burkitt’s lymphoma daudi and ramos  <cit> ; hodgkin lymphoma kmh <dig> and l <dig>  <cit> ; lmp1+ b-lymphoblastoid ib <dig>  <cit> .
all cell lines were cultured in dulbecco’s modified eagle’s medium  or rpmi medium  <dig>  supplemented with 10-20% heat-inactivated fetal bovine serum   as previously described  <cit> .
for transfections, a <dig> and cos- <dig> cells were seeded in 24-well, 35-mm, or 60-mm tissue culture dishes such that they were approximately 60% confluent on the following day when transfections were performed using polyethylenimine    <cit> .
on the day of transfection, dna:pei mixtures of 1: <dig>  and 1: <dig>  were incubated for 15 min at room temperature , then added to cells, and incubated overnight.
for transfections of 3t <dig> and 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- cells, the cells were seeded in a 24-well dish at a density of  <dig>  ×  <dig> on the day of transfection.
prior to plating cells, transfection mixtures containing a plasmid dna:enhancer ratio of 1: <dig>  were incubated for 5 min at rt and these mixtures were incubated for 30 min at rt containing a dna:effectene ratio of 1: <dig>  .
after the 30 min incubation, cells were plated, the transfection mixtures were immediately added to each well, and cells were incubated overnight.
for both pei and effectene transfections, the day after transfection the transfection media was replaced with fresh dmem containing 10% fbs.
cells were harvested and lysed 24 h later for the appropriate assay.
western blotting
western blotting was performed as described previously  <cit> .
whole cell extracts were prepared in at buffer  or nuclear extracts were prepared as described below for emsas.
samples were boiled for 10 min in sds sample buffer .
samples containing equal amounts of protein were separated on sds-polyacrylamide gels and transferred to nitrocellulose membranes .
the following primary antisera were used: p <dig> ; parp ; β-tubulin ; flag ; rel ; and p <dig> .
nitrocellulose filters were incubated with primary antiserum for  <dig> or 18 h at room temperature or 4°c, respectively.
the appropriate horseradish peroxidase-labeled secondary antiserum was added and immunoreactive proteins were detected with the supersignal dura west extended duration substrate chemiluminescence detection system .
luciferase reporter assays
luciferase reporter assays were performed using the dual-luciferase assay system  as previously described  <cit> .
cos- <dig>  3t <dig>  and 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- cells were plated in 24-well dishes and co-transfected with  <dig>  μg of the appropriate pgl3-reporter plasmid, 15–30 ng of the transfection normalization plasmid rsv-renilla-luciferase , and  <dig>  μg of pcdna-rel or flag-tagged versions of pcdna-p <dig>  -p <dig>  or vector alone.
two days later, cells were lysed, firefly and renilla luciferase activities were determined, and values were normalized to the relevant vector control .
real-time quantitative pcr
total rna was isolated using trizol  and real-time quantitative pcr  was performed as described previously  <cit>  with the primer sets listed in additional file 1: table s <dig>  qpcr for mature mir- <dig> and 5s ribosomal rna was performed by first reverse transcribing the rna with specific stem-loop primers that contain an annealing site for the universal reverse primer  using the taqman mirna reverse transcriptase kit  as described previously  <cit> .
mature mir- <dig> and 5s sequences were then amplified using a forward-specific primer and a universal reverse primer .
the qpcr reaction was performed using the 7900ht fast real-time pcr system  with the power sybr green pcr master mix .
quantification of mir- <dig> by qpcr was performed three times with triplicate samples and values were normalized to the 5s values.
the final values represent relative expression  as compared to untreated control samples  and ramos cells .
for quantification of the unspliced form of mir155hg, total rna was isolated as above and whole cell cdna preps were made using m-mlv reverse transcriptase .
unspliced mir155hg and gapdh cdnas were amplified using specific forward and reverse primers , and qpcr was performed as above.
electrophoretic mobility shift assays
emsas were performed using 10– <dig> μg of nuclear extract prepared from a <dig> cells transfected with flag-tagged versions of pcdna, pcdna-p <dig>  or pcdna-p <dig>  or from bjab cells treated with lps, generally as described previously  <cit> .
nuclear extracts were incubated with 2 μg poly, and 32p-labeled mir155hg- <dig> or mhc <dig> nf-κb site probes  in binding buffer  in a final reaction volume of 50 μl.
dna-binding reactions were carried out for 30 min at room temperature.
supershifts were performed using two p <dig> antibodies  or normal rabbit serum, which were incubated with the protein-dna complexes for an additional 1 h on ice.
samples were resolved on 5% non-denaturing polyacrylamide gels.
gels were dried and protein-dna complexes were detected by autoradiography.
prediction of transcription factor binding sites in the mir155hg promoter
prediction of nf-κb transcription factor binding sites in the human mir155hg promoter was performed using the online prediction models promo  <cit>  and the transcriptional regulatory element database   <cit> .
the two sites with high scores in both programs---the previously described - <dig> site  <cit>  and the - <dig> site---as well as the - <dig> site that is conserved in the mouse mir155hg upstream region were selected for future experiments.
statistical analysis
standard error was calculated for all data.
p-values were calculated using an anova test.
RESULTS
lps treatment of bjab cells induces p <dig> nuclear translocation, increased mir155hg/mir- <dig> transcript expression, and increased dna binding to a site in the mir155hg proximal promoter
mir- <dig> has often been shown to be up-regulated by inducers of nf-κb activity  <cit> .
to determine whether mir155hg is an early response gene in lps-induced nf-κb signaling, we first measured the kinetics of p <dig> nuclear translocation following lps treatment.
bjab cells, which contain low levels of nuclear p <dig>  were treated with  <dig> μg/ml lps for increasing times up to 60 min and the levels of nuclear p <dig> were measured by western blotting .
within 15 min of lps treatment, p <dig> started to increase in the nucleus, and by 60 min, there was a substantial amount of p <dig> in the nucleus.
as controls, we show that parp is present in nuclear extracts, β-tubulin  is absent from nuclear extracts, and that p <dig>  parp and β-tubulin are present in bjab whole cell extracts.
to determine whether nuclear translocation of p <dig> coincided with increased expression of mir155hg/mir- <dig>  bjab cells were treated with lps for times up to 60 min as in figure 1a, and qpcr was performed for the unspliced mir155hg transcript and for the mature form of mir- <dig>  to ensure that lps was inducing increased levels of newly transcribed mir155hg rna, we measured the unspliced mir155hg transcript using one primer located in exon  <dig> and one in intron  <dig> of the mir155hg gene.
after 60 min of lps treatment, unspliced mir155hg was increased by approximately four-fold as compared to untreated cells .
additionally, in both bjab and ib <dig> cells , treatment with lps for 24 h resulted in 5-fold and  <dig> -fold increases in mature mir- <dig> levels, respectively .
of note, ib <dig> cells have ~ <dig> times more mir- <dig> in untreated cells as compared to untreated bjab cells, showing that induction of mir- <dig> expression is not strictly dependent on the resting levels of mir- <dig>
to assess the dna-binding ability of lps-induced nuclear p <dig>  an emsa was performed to measure the amount of nf-κb heterodimer p50/p <dig> that bound to a specific site in the mir155hg promoter .
as shown in figure 1d, nuclear extracts from untreated control bjab cells contain high levels of the nf-κb p50/p <dig> homodimer that can bind to the - <dig> probe.
following treatment of bjab cells with lps for 60 min, a new dna-protein complex appeared that migrated slightly slower than the p50/p <dig> homodimer band.
this new, higher band co-migrated with a dna-protein complex from extracts of a <dig> cells transfected with pcdna-flag versions of both p <dig> and p <dig> .
additionally, this new band was supershifted with p <dig> antibody, but not with pre-immune serum .
moreover, the appearance of the p50/p <dig> dna-binding activity coincides with the increased nuclear localization of p <dig> .
taken together, these results suggest that lps induces the nuclear translocation of an nf-κb p50/p <dig> heterodimer that can bind to a site in the mir155hg promoter.
the mir155hg promoter is selectively activated by p65
the  <dig> nt region upstream of the mir155hg tss contains three predicted nf-κb binding sites : one previously reported at - <dig>  <cit>  and two sites at - <dig> and - <dig> .
to assess the ability of nf-κb to activate transcription from the mir155hg promoter, cos- <dig> cells were co-transfected with the luciferase reporter plasmid wt-mir155hg  and expression plasmids for nf-κb proteins p <dig>  rel , or p <dig>  or with an empty vector control.
in these assays, p <dig> activated the mir155hg promoter approximately 17-fold as compared to the empty vector control.
rel and p <dig> had little to no effect on wt-mir155hg reporter gene expression .
in a separate experiment, cos- <dig> cells were transfected with the same pcdna-based plasmids, and western blotting of whole cell extracts confirmed enhanced expression of p <dig>  rel, and p <dig> .
an nf-κb p65-responsive site in the mir155hg promoter is located at -178 bp
to identify the p65-responsive region in the mir155hg promoter, 5’ truncation mutants of the upstream sequences in the wt-mir155hg reporter plasmid were created at - <dig> and - <dig> nt upstream of the tss .
the - <dig> truncation removes the - <dig> κb site and leaves the - <dig> and - <dig> sites intact, while the - <dig> truncation removes all three predicted nf-κb binding sites.
reporter gene assays showed that the - <dig> truncation retained the ability to be activated by p <dig>  whereas the - <dig> truncation showed a complete loss of p65-enhanced reporter activity .
these results indicate that the p65-responsive region lies between nt  <dig> and  <dig> upstream of the mir155hg transcription start site.
to identify the p65-responsive site located between - <dig> and - <dig>  we assessed the ability of p <dig> to increase transcription of mir155hg luciferase reporter plasmids with mutations in predicted nf-κb sites at - <dig>  or - <dig> .
as a control, we also investigated the effect of p <dig> on a reporter with a mutation at - <dig> .
p <dig> enhanced transcription from the - <dig> and - <dig> mutant site reporters to approximately the same extent as seen with the wt-mir155hg plasmid .
in contrast, the - <dig> mutant site reporter was not activated by p <dig> .
because nf-κb has been reported to up-regulate components of the ap- <dig> transcription factor, which could then have effects on mir155hg expression, we determined whether p <dig> could increase transcription of a mir155hg reporter containing a mutation of the ap- <dig> site   <cit> .
p <dig> enhanced ap-1mut-mir155hg reporter gene expression to an even greater extent than wt-mir155hg reporter , showing that the ap- <dig> site is not required for p65-based activation of the mir155hg promoter.
the p50/p <dig> heterodimer binds to the - <dig> site in the mir155hg promoter to activate transcription
to further investigate the ability of p <dig> to bind the - <dig> site in the mir155hg promoter, an emsa was performed using a 32p-labeled probe containing the - <dig> site and over-expressed forms of p <dig> and p <dig>  for this experiment, flag-tagged versions of p <dig> and p <dig> were expressed individually or together in a <dig> cells.
western blotting with flag, p <dig>  and p <dig> antibodies was first done on whole cell extracts to confirm over-expression of each protein after transfection .
the extracts from a <dig> cells expressing p <dig>  p <dig>  and p50/p <dig> were then used in an emsa with the - <dig> site probe .
p <dig> alone did not detectably bind to the - <dig> site , whereas p <dig> alone showed strong binding to the - <dig> site .
using extracts from cells co-transfected with p <dig> and p <dig>  a band larger than the p <dig> homodimer band appeared, indicating that the p50/p <dig> heterodimer can bind to this site .
this band was competed with 50x cold probe .
in addition, two anti-p <dig> antibodies supershifted the band that was slightly larger than the p <dig> alone band .
the p <dig> antibodies did not shift the p <dig> homodimer band , and no supershift was seen with pre-immune serum .
because p <dig> alone could not bind to the - <dig> site probe, we wanted to make sure that the a <dig> cell-expressed p <dig> protein was able to bind dna.
therefore, an emsa was performed with a consensus nf-κb site from the mhc <dig> enhancer, which we have previously shown can be bound by p <dig> and p <dig>  <cit> .
as shown in figure 3c, p <dig> and p <dig>  can individually bind the mhc <dig> κb-site probe.
when the p <dig> and p <dig> plasmids were co-transfected, a strong emsa band appeared that was slightly smaller than p <dig> alone and slightly larger than p <dig> alone , which is consistent with the migration of a p50/p <dig> heterodimer complex.
because p <dig> alone did not bind to the - <dig> site , but transfection of p <dig> into cos- <dig> cells requires this site for induction of mir155hg promoter expression , we hypothesized that transfected p <dig> was forming heterodimers with endogenous p <dig>  to drive expression of the mir155hg promoter in the reporter plasmid.
therefore, we predicted that activation of the mir155hg reporter by p <dig> would be reduced in cells lacking other nf-κb family members.
to test this prediction, we compared the ability of p <dig> to activate the mir155hg reporter in wild-type mouse 3t <dig> cells and 3t <dig> cells lacking all nf-κb family members except p <dig> .
both cell types were co-transfected with the wt-mir155hg reporter and expression plasmids for flag-p <dig>  flag-p <dig>  or the empty vector control.
in wild-type 3t <dig> cells, p <dig> activated the mir155hg promoter approximately  <dig> -fold as compared to the empty vector control, whereas in 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- cells, p <dig> only minimally activated luciferase activity .
transfection of p <dig> alone did not activate the mir155hg reporter in either cell type.
these results show that the mir155hg promoter can be activated by p <dig> in a cell type other than cos- <dig> cells, and that the ability of p <dig> to fully activate the mir155hg promoter requires other endogenous nf-κb family members.
activation of the nf-κb signaling pathway generally correlates with mir- <dig> expression in a panel of human b-lymphoma cell lines
given that increased nf-κb activity correlates with increased mir- <dig> expression and that the - <dig> κb site can be bound by the p50/p <dig> heterodimer, we hypothesized that b-lymphoma cell lines with high mir- <dig> would also have high nuclear p <dig> levels in their resting state whereas b-lymphoma cell lines expressing low levels of mir- <dig> would have low levels of nuclear p <dig>  nine b-lymphoma cell lines with known nf-κb activity levels were chosen .
first, mir- <dig> levels were measured by qpcr from all cells.
the three cell lines with high nf-κb activity  had substantially more mir- <dig> than the six cell lines with low nf-κb activity .
nuclear extracts were then prepared from these nine cell lines and analyzed for expression of p <dig>  rel, and parp  by western blotting .
two of the three cell lines with high nf-κb activity and high mir- <dig> expression, namely ib <dig> and kmh <dig>  showed the highest levels of nuclear p65; the third high nf-κb/high mir <dig> cell line, l <dig>  did not have high levels of nuclear p <dig>  however, l <dig> cells, as well as ib <dig> and kmh <dig> cells, have high levels of nuclear rel.
among the six cell lines with low nf-κb/low mir <dig>  all had low levels of nuclear p <dig> and five of six had low levels of nuclear rel; only sudhl <dig> cells had high nuclear rel .
these results show that increased nuclear nf-κb protein  generally correlates with high mir- <dig> expression.
discussion
this report demonstrates that mir155hg proximal promoter region can serve as a direct target for activation by nf-κb.
reporter gene assays identified an nf-κb p50/p65-responsive site located ~ <dig> nt upstream of the mir155hg tss.
moreover, mir155hg pre-mrna expression and p50/p <dig> binding to the - <dig> site can be induced by lps treatment in bjab cells.
lastly, mir- <dig> expression generally correlates with levels of nuclear nf-κb proteins  in a panel of human b-lymphoma cell lines.
we report that lps rapidly induces both mir155hg pre-mrna  and mature mir- <dig>  in bjab cells.
the use of one intron and one exon primer to measure mir155hg pre-mrna levels by qpcr suggests that lps induces new transcription of mir155hg, which then leads to increased levels of mature mir- <dig>  and that, for example, induction of mir- <dig> levels is not due to a post-transcriptional process.
this finding is consistent with our previous report  <cit>  that lps induces mature mir155hg mrna  and mir- <dig> in bjab cells.
taken together with our findings that lps treatment of bjab cells induces nuclear translocation of p <dig>  and binding of p50/p <dig> to the - <dig> site in the mir155hg promoter , and that p50/p <dig> can activate the mir155hg promoter in reporter assays , these results strongly suggest that lps induces transcription of the mir155hg promoter through binding of induced nf-κb p50/p <dig> to the - <dig> site, which ultimately results in increased levels of mir- <dig>
although some qpcr guidelines prescribe the use of multiple reference genes for qpcr analyses  <cit> , we have not done that.
nevertheless, we think it likely that lps does induce mir155hg transcription and mir- <dig> levels given that we have used different normalization transcripts and different rt-pcr approaches to measure the distinct mir155gh-generated rnas, and all methods yielded similar results.
first, gapdh was used as the reference transcript for measuring mir155hg pre-mrna levels  by qpcr .
second, c-rel and gapdh transcripts were not affected by lps treatment in bjab cells where induction of the mature mir155hg transcript was measured by conventional rt-pcr using two exon primers  <cit> .
third, 5s rna was used to normalize mir- <dig> levels using a small rna qpcr method .
in addition, as measured by northern blotting, it was previously shown that lps does not change the levels of gapdh mrna  <cit> .
details of the methods we used to isolate and process mrna and to perform qpcr experiments are included with additional file 1: table s <dig> and additional file 4: table s <dig>
transcriptional regulation of mir- <dig> has been widely regarded to be an nf-κb-dependent process, although no compelling evidence showing a direct and transcriptionally functional interaction of nf-κb with the promoter/enhancer region of mir155hg has been previously produced.
two studies  <cit>  used reporter gene analyses to identify cis elements in the mir155hg promoter that are required for transcriptional activation by the ebv lmp <dig> protein, which is an inducer of both nf-κb and ap- <dig>  <cit> .
one study  <cit>  claimed that a conserved ap- <dig> site  <dig> nt upstream of the mir155hg tss is required for high level mir- <dig> expression in ebv+ b-lymphoblast cell line jy.
a second report  <cit>  showed that the ability of the ebv lmp <dig> protein to enhance expression from the mir155hg promoter in reporter assays in primary mouse embryo fibroblasts requires two nf-κb binding sites located more than  <dig> nt upstream of the tss.
in contrast, we saw no effect of mutation of the - <dig> site  on p65-enhanced activation of the mir155hg promoter in cos- <dig> cells.
it is not clear whether the difference between those two studies and our study is due to the cell types used in the reporter assays or the method used to enhance nf-κb activity .
based on protein-binding microarrays using recombinant proteins  <cit> , the - <dig> site  is not predicted to be a strong p <dig> binding site, although it is a reasonably good p <dig> binding site due primarily to the g-rich nature of the 5’ half-site.
consistent with those data, we have found that p <dig> homodimers readily bind the - <dig> site, whereas p <dig> homodimers do not .
thus, in cells it is likely that p50/p <dig> homodimers strongly bind the - <dig> site to repress transcription in the absence of nf-κb p50/p <dig> activating signals.
that p50/p <dig> homodimers can repress transcription from the mir155hg promoter is consistent with our finding that high levels of transfected p <dig> alone reduce expression of the mir155hg reporter in both wild-type and nfkb1-/-nfkb2-/-relb-/-crel-/- 3t <dig> cells .
once nf-κb is activated, p50/p <dig> heterodimers likely bind to the - <dig> site, a process that is facilitated by the high affinity of p <dig> for the 5’ half-site.
other promoters containing sites that selectivity bind p50/p <dig> heterodimers include those of the pro-il1β, icam- <dig>  and cd <dig> genes  <cit> .
among aggressive b-cell lymphomas such as dlbcls, there is a positive correlation between high nf-κb activity and high mir- <dig> expression  <cit> .
moreover, treatment of some b-lymphoma cell lines with nf-κb signaling inhibitors results in reduced mir- <dig> expression  <cit> .
in our study, three dlbcl cell lines known to have high nf-κb activity also exhibited high expression of mir- <dig> .
ib <dig> and kmh <dig> cells have high nuclear p <dig> and rel, whereas l <dig> cells have high nuclear rel but low nuclear p <dig> .
similar to l <dig> cells, rc-k <dig> cells have inactivating mutations in iκbα, high mir- <dig> expression, and exceptionally high levels of nuclear rel   <cit> .
moreover, over-expression of an oncogenically activated mutant rel in bjab cells results in increased expression of mir155hg <cit> .
sudhl <dig> cells are unusual in that they have relatively high levels of nuclear rel protein , but express low levels of mir- <dig>  and have an overall low nf-κb gene expression profile  <cit> .
thus, nuclear rel may contribute to increased mir- <dig> expression in some  b-lymphoma cells.
nevertheless, rel did not activate the proximal mir155hg promoter in cos- <dig> cell reporter gene assays .
therefore, there may be rel-dependent sites outside of the 1494 bp fragment analyzed here that can regulate mir155hg expression.
alternatively, rel may interact with factors in some b-lymphoma cells  to enhance mir155hg expression through its proximal promoter.
we used the proximal upstream  region of the mir155hg promoter to investigate the regulation of mir- <dig> expression by nf-κb.
although only approximately 20-30% of induced p <dig> binding sites are located within ~ <dig> kbp of transcription start sites  <cit> , approximately 50-60% of the coactivator p300/cbp binding is located within proximal promoters of actively transcribed genes  <cit> , suggesting that much of the biologically relevant transcription-inducing p <dig> binding occurs in sequences proximal to the 5’ end of genes.
nevertheless, it has been reported that 28-40% of induced p <dig> binding sites are located in introns of known genes  <cit> .
indeed, the two introns of the human mir155hg gene contain several predicted nf-κb sites .
one predicted site in intron  <dig> and three in intron  <dig> have scores for nf-κb binding that are as high as the - <dig> site that we have described.
thus, it is possible that there are other p <dig>  sites that contribute to nf-κb-dependent regulation of mir155hg/mir- <dig> expression.
moreover, in the mouse mir155hg promoter there does not appear to be an nf-κb site that corresponds to the - <dig> site , suggesting that there may be differences in the regulation of mir- <dig> expression by nf-κb between the two species.
CONCLUSIONS
the results presented herein show that nf-κb p50/p <dig> can directly activate the human mir155hg promoter, consistent with the many studies that have reported a correlation between high or induced nf-κb activity and increased expression of mir- <dig> .
due to the contributions of mir- <dig> to immune cell function and malignancy, understanding how mir155hg expression is controlled could lead to new therapeutic approaches.
abbreviations
bic: b-cell integration cluster; dlbcl: diffuse large b-cell lymphoma; dmem: dulbecco’s modified eagle’s medium; emsa: electrophoretic mobility shift assay; fbs: fetal bovine serum; kbp: kilobase pairs; lps: lipopolysaccharide; mir: microrna; nt: nucleotides; pcr: polymerase chain reaction; pei: polyethylenimine; rel: human c-rel transcription factor; tss: transcription start site.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
rct planned and carried out the majority of the experiments.
iv performed tissue culture experiments and reporter gene assays.
rct and tdg participated in the design of the study and wrote the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1: table s1
primers used and their sequences.
click here for file
additional file 2: figure s1
truncation mutants of the mir155hg promoter were made via pcr, resulting in the creation of -530-mir155hg and -91-mir155hg luciferase reporter plasmids.
reporter assays were performed in cos- <dig> cells transfected with the pgl3-based -1494 bp mir155hg promoter reported plasmid  or with indicated truncation mutant and with pcdna vector control or pcdna-flag-p <dig>  values were normalized to the pcdna control .
the * indicates p <  <dig> .
click here for file
additional file 3: figure s2
reporter assays were performed in cos- <dig> cells transfected with the pgl3-based wild-type mir155hg promoter reported plasmid  or an ap- <dig> mutant  with either pcdna alone or pcdna-p <dig>  values for each transfection were normalized to rsv-renilla and then normalized to pcdna alone .
click here for file
additional file 4: table s2
miqe checklist for qpcr methods and analysis.
click here for file
acknowledgements
we thank eric flemington  for the wild-type and the - <dig> κb site and ap- <dig> site mutant mir155hg luciferase reporter plasmids, nancy rice  for anti-p <dig> antiserum, alexander hoffmann  for the 3t <dig> and 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- cells, and alan yeo  <cit>  for pcdna-flag-p <dig> and pcdna-flag-p <dig> plasmids.
we also thank susan kandarian, robert jackman, trevor siggers, frank naya, david waxman, and gerald denis  for helpful suggestions and discussions.
this research was supported by the national institutes of health .
rct was supported by an nhlbi hematology training grant .

 mir- is one of the most extensively studied mirs due to its involvement in immune cell development and function and in various diseases. increased mir- expression is typically seen following the treatment of cells with proinflammatory signaling molecules like lipopolysaccharide, interleukins, interferons, and tumor necrosis factor. mice with a knockout of mir- have dysfunctional lymphocytes and dendritic cells. increased mir- expression is also observed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and in many b- and t-lymphomas and leukemias. transgenic mice with tissue-specific expression of mir- can develop hematopoietic malignancies. the mature form of mir- is encoded by the mir155hg gene. although mir155hg encodes two mirs, mir155-5p is the more commonly studied product, and herein will be referred to as mir- mir- is excised from an exon of its pre-mirna precursor by dicer and then loaded into the risc complex. this mir-155/risc complex can then bind to mrna transcripts with mir- target sequences in their 3`-utrs. mir-155-regulated transcripts include ones encoding pu. aid, ikkϵ, c/ebpβ, socs mitf, and fadd, and mir- has been shown to decrease translation of these target mrnas. to date, the majority of evidence that transcription factor nf-κb activates mir- expression is circumstantial, extrapolated from the positive correlation between high nuclear nf-κb activity and increased mir- expression in both normal lymphoid cells and various cancers, including many hematopoietic malignancies. several studies have set out to identify the cis regulatory elements in the mir155hg promoter, producing mixed and sometimes conflicting results. one study claims that ebv-induced mir155hg expression is primarily regulated by an ap- site found nt upstream of the transcription start site, while another concludes that ebv induces mir155hg through two nf-κb sites over nt upstream of the tss. a third study suggests that nf-κb up-regulates ap- components, which then bind to the ap- site to up-regulate mir- expression. thus, no study has convincingly established mir155hg as a direct target of nf-κb. in this report, we show that nf-κb p50/ can directly bind to and activate the human mir155hg proximal promoter through a site approximately nt upstream of the mir155hg tss. furthermore, we demonstrate that mir155hg/mir- expression and p50/p binding to this site are rapidly induced after treatment of the b-lymphoma cell line bjab with the nf-κb inducer lps. lastly, in a panel of b-lymphoma cell lines, nuclear nf-κb protein levels generally correlate with increased mir- expression. the results of this study show that mir- expression can be directly controlled by nf-κb through a site in the mir155hg proximal promoter. methods plasmids the firefly luciferase reporter plasmid containing nt region upstream of the mir155hg tss pwt-mir155hg), the - κb-site mutant plasmid, and the ap-1mut-mir155hg were gifts of eric flemington and have been described previously. the - and - mutants were created by insertion of an xhoi site in the middle of the given κb site, as in -1150mut-mir155hg. reporter plasmids with truncations at - and - were created by amplifying the relevant regions of the mit155hg promoter and subcloning them into nhei/hindiii-digested pwt-mir155hg. pcdna-flag-p and pcdna-flag-p were created by subcloning pcr-amplified sequences of each into the xhoi site of pcdna-flag. pcdna-rel has been described previously. cell culture the cell lines used in this study were a human embryonic kidney cells, cos- monkey kidney cells, 3t and 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- mouse fibroblast cells, and the following human b-lymphoma cell lines: gcb diffuse large b-cell lymphoma bjab, sudhl and pfeiffer; follicular lymphoma bl; burkitt’s lymphoma daudi and ramos; hodgkin lymphoma kmh and l; lmp1+ b-lymphoblastoid ib. all cell lines were cultured in dulbecco’s modified eagle’s medium or rpmi medium supplemented with 10-20% heat-inactivated fetal bovine serum as previously described. for transfections, a and cos- cells were seeded in 24-well, 35-mm, or 60-mm tissue culture dishes such that they were approximately 60% confluent on the following day when transfections were performed using polyethylenimine. on the day of transfection, dna:pei mixtures of 1: and 1: were incubated for 15 min at room temperature, then added to cells, and incubated overnight. for transfections of 3t and 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- cells, the cells were seeded in a 24-well dish at a density of × on the day of transfection. prior to plating cells, transfection mixtures containing a plasmid dna:enhancer ratio of 1: were incubated for 5 min at rt and these mixtures were incubated for 30 min at rt containing a dna:effectene ratio of 1:. after the 30 min incubation, cells were plated, the transfection mixtures were immediately added to each well, and cells were incubated overnight. for both pei and effectene transfections, the day after transfection the transfection media was replaced with fresh dmem containing 10% fbs. cells were harvested and lysed 24 h later for the appropriate assay. western blotting western blotting was performed as described previously. whole cell extracts were prepared in at buffer or nuclear extracts were prepared as described below for emsas. samples were boiled for 10 min in sds sample buffer. samples containing equal amounts of protein were separated on sds-polyacrylamide gels and transferred to nitrocellulose membranes. the following primary antisera were used: p; parp; β-tubulin; flag; rel; and p. nitrocellulose filters were incubated with primary antiserum for or 18 h at room temperature or 4°c, respectively. the appropriate horseradish peroxidase-labeled secondary antiserum was added and immunoreactive proteins were detected with the supersignal dura west extended duration substrate chemiluminescence detection system. luciferase reporter assays luciferase reporter assays were performed using the dual-luciferase assay system as previously described. cos- 3t and 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- cells were plated in 24-well dishes and co-transfected with  μg of the appropriate pgl3-reporter plasmid, 15–30 ng of the transfection normalization plasmid rsv-renilla-luciferase, and  μg of pcdna-rel or flag-tagged versions of pcdna-p -p or vector alone. two days later, cells were lysed, firefly and renilla luciferase activities were determined, and values were normalized to the relevant vector control. real-time quantitative pcr total rna was isolated using trizol and real-time quantitative pcr was performed as described previously with the primer sets listed in additional file 1: table s qpcr for mature mir- and 5s ribosomal rna was performed by first reverse transcribing the rna with specific stem-loop primers that contain an annealing site for the universal reverse primer using the taqman mirna reverse transcriptase kit as described previously. mature mir- and 5s sequences were then amplified using a forward-specific primer and a universal reverse primer. the qpcr reaction was performed using the 7900ht fast real-time pcr system with the power sybr green pcr master mix. quantification of mir- by qpcr was performed three times with triplicate samples and values were normalized to the 5s values. the final values represent relative expression as compared to untreated control samples and ramos cells. for quantification of the unspliced form of mir155hg, total rna was isolated as above and whole cell cdna preps were made using m-mlv reverse transcriptase. unspliced mir155hg and gapdh cdnas were amplified using specific forward and reverse primers, and qpcr was performed as above. electrophoretic mobility shift assays emsas were performed using 10– μg of nuclear extract prepared from a cells transfected with flag-tagged versions of pcdna, pcdna-p or pcdna-p or from bjab cells treated with lps, generally as described previously. nuclear extracts were incubated with 2 μg poly, and 32p-labeled mir155hg- or mhc nf-κb site probes in binding buffer in a final reaction volume of 50 μl. dna-binding reactions were carried out for 30 min at room temperature. supershifts were performed using two p antibodies or normal rabbit serum, which were incubated with the protein-dna complexes for an additional 1 h on ice. samples were resolved on 5% non-denaturing polyacrylamide gels. gels were dried and protein-dna complexes were detected by autoradiography. prediction of transcription factor binding sites in the mir155hg promoter prediction of nf-κb transcription factor binding sites in the human mir155hg promoter was performed using the online prediction models promo and the transcriptional regulatory element database. the two sites with high scores in both programs---the previously described - site and the - site---as well as the - site that is conserved in the mouse mir155hg upstream region were selected for future experiments. statistical analysis standard error was calculated for all data. p-values were calculated using an anova test.  lps treatment of bjab cells induces p nuclear translocation, increased mir155hg/mir- transcript expression, and increased dna binding to a site in the mir155hg proximal promoter mir- has often been shown to be up-regulated by inducers of nf-κb activity. to determine whether mir155hg is an early response gene in lps-induced nf-κb signaling, we first measured the kinetics of p nuclear translocation following lps treatment. bjab cells, which contain low levels of nuclear p were treated with μg/ml lps for increasing times up to 60 min and the levels of nuclear p were measured by western blotting. within 15 min of lps treatment, p started to increase in the nucleus, and by 60 min, there was a substantial amount of p in the nucleus. as controls, we show that parp is present in nuclear extracts, β-tubulin is absent from nuclear extracts, and that p parp and β-tubulin are present in bjab whole cell extracts. to determine whether nuclear translocation of p coincided with increased expression of mir155hg/mir- bjab cells were treated with lps for times up to 60 min as in figure 1a, and qpcr was performed for the unspliced mir155hg transcript and for the mature form of mir- to ensure that lps was inducing increased levels of newly transcribed mir155hg rna, we measured the unspliced mir155hg transcript using one primer located in exon and one in intron of the mir155hg gene. after 60 min of lps treatment, unspliced mir155hg was increased by approximately four-fold as compared to untreated cells. additionally, in both bjab and ib cells, treatment with lps for 24 h resulted in 5-fold and -fold increases in mature mir- levels, respectively. of note, ib cells have ~ times more mir- in untreated cells as compared to untreated bjab cells, showing that induction of mir- expression is not strictly dependent on the resting levels of mir- to assess the dna-binding ability of lps-induced nuclear p an emsa was performed to measure the amount of nf-κb heterodimer p50/p that bound to a specific site in the mir155hg promoter. as shown in figure 1d, nuclear extracts from untreated control bjab cells contain high levels of the nf-κb p50/p homodimer that can bind to the - probe. following treatment of bjab cells with lps for 60 min, a new dna-protein complex appeared that migrated slightly slower than the p50/p homodimer band. this new, higher band co-migrated with a dna-protein complex from extracts of a cells transfected with pcdna-flag versions of both p and p. additionally, this new band was supershifted with p antibody, but not with pre-immune serum. moreover, the appearance of the p50/p dna-binding activity coincides with the increased nuclear localization of p. taken together, these results suggest that lps induces the nuclear translocation of an nf-κb p50/p heterodimer that can bind to a site in the mir155hg promoter. the mir155hg promoter is selectively activated by p65 the nt region upstream of the mir155hg tss contains three predicted nf-κb binding sites: one previously reported at - and two sites at - and -. to assess the ability of nf-κb to activate transcription from the mir155hg promoter, cos- cells were co-transfected with the luciferase reporter plasmid wt-mir155hg and expression plasmids for nf-κb proteins p rel, or p or with an empty vector control. in these assays, p activated the mir155hg promoter approximately 17-fold as compared to the empty vector control. rel and p had little to no effect on wt-mir155hg reporter gene expression. in a separate experiment, cos- cells were transfected with the same pcdna-based plasmids, and western blotting of whole cell extracts confirmed enhanced expression of p rel, and p. an nf-κb p65-responsive site in the mir155hg promoter is located at -178 bp to identify the p65-responsive region in the mir155hg promoter, 5’ truncation mutants of the upstream sequences in the wt-mir155hg reporter plasmid were created at - and - nt upstream of the tss. the - truncation removes the - κb site and leaves the - and - sites intact, while the - truncation removes all three predicted nf-κb binding sites. reporter gene assays showed that the - truncation retained the ability to be activated by p whereas the - truncation showed a complete loss of p65-enhanced reporter activity. these results indicate that the p65-responsive region lies between nt and upstream of the mir155hg transcription start site. to identify the p65-responsive site located between - and - we assessed the ability of p to increase transcription of mir155hg luciferase reporter plasmids with mutations in predicted nf-κb sites at - or -. as a control, we also investigated the effect of p on a reporter with a mutation at -. p enhanced transcription from the - and - mutant site reporters to approximately the same extent as seen with the wt-mir155hg plasmid. in contrast, the - mutant site reporter was not activated by p. because nf-κb has been reported to up-regulate components of the ap- transcription factor, which could then have effects on mir155hg expression, we determined whether p could increase transcription of a mir155hg reporter containing a mutation of the ap- site. p enhanced ap-1mut-mir155hg reporter gene expression to an even greater extent than wt-mir155hg reporter, showing that the ap- site is not required for p65-based activation of the mir155hg promoter. the p50/p heterodimer binds to the - site in the mir155hg promoter to activate transcription to further investigate the ability of p to bind the - site in the mir155hg promoter, an emsa was performed using a 32p-labeled probe containing the - site and over-expressed forms of p and p for this experiment, flag-tagged versions of p and p were expressed individually or together in a cells. western blotting with flag, p and p antibodies was first done on whole cell extracts to confirm over-expression of each protein after transfection. the extracts from a cells expressing p p and p50/p were then used in an emsa with the - site probe. p alone did not detectably bind to the - site, whereas p alone showed strong binding to the - site. using extracts from cells co-transfected with p and p a band larger than the p homodimer band appeared, indicating that the p50/p heterodimer can bind to this site. this band was competed with 50x cold probe. in addition, two anti-p antibodies supershifted the band that was slightly larger than the p alone band. the p antibodies did not shift the p homodimer band, and no supershift was seen with pre-immune serum. because p alone could not bind to the - site probe, we wanted to make sure that the a cell-expressed p protein was able to bind dna. therefore, an emsa was performed with a consensus nf-κb site from the mhc enhancer, which we have previously shown can be bound by p and p. as shown in figure 3c, p and p can individually bind the mhc κb-site probe. when the p and p plasmids were co-transfected, a strong emsa band appeared that was slightly smaller than p alone and slightly larger than p alone, which is consistent with the migration of a p50/p heterodimer complex. because p alone did not bind to the - site, but transfection of p into cos- cells requires this site for induction of mir155hg promoter expression, we hypothesized that transfected p was forming heterodimers with endogenous p to drive expression of the mir155hg promoter in the reporter plasmid. therefore, we predicted that activation of the mir155hg reporter by p would be reduced in cells lacking other nf-κb family members. to test this prediction, we compared the ability of p to activate the mir155hg reporter in wild-type mouse 3t cells and 3t cells lacking all nf-κb family members except p. both cell types were co-transfected with the wt-mir155hg reporter and expression plasmids for flag-p flag-p or the empty vector control. in wild-type 3t cells, p activated the mir155hg promoter approximately -fold as compared to the empty vector control, whereas in 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- cells, p only minimally activated luciferase activity. transfection of p alone did not activate the mir155hg reporter in either cell type. these results show that the mir155hg promoter can be activated by p in a cell type other than cos- cells, and that the ability of p to fully activate the mir155hg promoter requires other endogenous nf-κb family members. activation of the nf-κb signaling pathway generally correlates with mir- expression in a panel of human b-lymphoma cell lines given that increased nf-κb activity correlates with increased mir- expression and that the - κb site can be bound by the p50/p heterodimer, we hypothesized that b-lymphoma cell lines with high mir- would also have high nuclear p levels in their resting state whereas b-lymphoma cell lines expressing low levels of mir- would have low levels of nuclear p nine b-lymphoma cell lines with known nf-κb activity levels were chosen. first, mir- levels were measured by qpcr from all cells. the three cell lines with high nf-κb activity had substantially more mir- than the six cell lines with low nf-κb activity. nuclear extracts were then prepared from these nine cell lines and analyzed for expression of p rel, and parp by western blotting. two of the three cell lines with high nf-κb activity and high mir- expression, namely ib and kmh showed the highest levels of nuclear p65; the third high nf-κb/high mir cell line, l did not have high levels of nuclear p however, l cells, as well as ib and kmh cells, have high levels of nuclear rel. among the six cell lines with low nf-κb/low mir all had low levels of nuclear p and five of six had low levels of nuclear rel; only sudhl cells had high nuclear rel. these results show that increased nuclear nf-κb protein generally correlates with high mir- expression. discussion this report demonstrates that mir155hg proximal promoter region can serve as a direct target for activation by nf-κb. reporter gene assays identified an nf-κb p50/p65-responsive site located ~ nt upstream of the mir155hg tss. moreover, mir155hg pre-mrna expression and p50/p binding to the - site can be induced by lps treatment in bjab cells. lastly, mir- expression generally correlates with levels of nuclear nf-κb proteins in a panel of human b-lymphoma cell lines. we report that lps rapidly induces both mir155hg pre-mrna and mature mir- in bjab cells. the use of one intron and one exon primer to measure mir155hg pre-mrna levels by qpcr suggests that lps induces new transcription of mir155hg, which then leads to increased levels of mature mir- and that, for example, induction of mir- levels is not due to a post-transcriptional process. this finding is consistent with our previous report that lps induces mature mir155hg mrna and mir- in bjab cells. taken together with our findings that lps treatment of bjab cells induces nuclear translocation of p and binding of p50/p to the - site in the mir155hg promoter, and that p50/p can activate the mir155hg promoter in reporter assays, these results strongly suggest that lps induces transcription of the mir155hg promoter through binding of induced nf-κb p50/p to the - site, which ultimately results in increased levels of mir- although some qpcr guidelines prescribe the use of multiple reference genes for qpcr analyses, we have not done that. nevertheless, we think it likely that lps does induce mir155hg transcription and mir- levels given that we have used different normalization transcripts and different rt-pcr approaches to measure the distinct mir155gh-generated rnas, and all methods yielded similar results. first, gapdh was used as the reference transcript for measuring mir155hg pre-mrna levels by qpcr. second, c-rel and gapdh transcripts were not affected by lps treatment in bjab cells where induction of the mature mir155hg transcript was measured by conventional rt-pcr using two exon primers. third, 5s rna was used to normalize mir- levels using a small rna qpcr method. in addition, as measured by northern blotting, it was previously shown that lps does not change the levels of gapdh mrna. details of the methods we used to isolate and process mrna and to perform qpcr experiments are included with additional file 1: table s and additional file 4: table s transcriptional regulation of mir- has been widely regarded to be an nf-κb-dependent process, although no compelling evidence showing a direct and transcriptionally functional interaction of nf-κb with the promoter/enhancer region of mir155hg has been previously produced. two studies used reporter gene analyses to identify cis elements in the mir155hg promoter that are required for transcriptional activation by the ebv lmp protein, which is an inducer of both nf-κb and ap-. one study claimed that a conserved ap- site nt upstream of the mir155hg tss is required for high level mir- expression in ebv+ b-lymphoblast cell line jy. a second report showed that the ability of the ebv lmp protein to enhance expression from the mir155hg promoter in reporter assays in primary mouse embryo fibroblasts requires two nf-κb binding sites located more than nt upstream of the tss. in contrast, we saw no effect of mutation of the - site on p65-enhanced activation of the mir155hg promoter in cos- cells. it is not clear whether the difference between those two studies and our study is due to the cell types used in the reporter assays or the method used to enhance nf-κb activity. based on protein-binding microarrays using recombinant proteins, the - site is not predicted to be a strong p binding site, although it is a reasonably good p binding site due primarily to the g-rich nature of the 5’ half-site. consistent with those data, we have found that p homodimers readily bind the - site, whereas p homodimers do not. thus, in cells it is likely that p50/p homodimers strongly bind the - site to repress transcription in the absence of nf-κb p50/p activating signals. that p50/p homodimers can repress transcription from the mir155hg promoter is consistent with our finding that high levels of transfected p alone reduce expression of the mir155hg reporter in both wild-type and nfkb1-/-nfkb2-/-relb-/-crel-/- 3t cells. once nf-κb is activated, p50/p heterodimers likely bind to the - site, a process that is facilitated by the high affinity of p for the 5’ half-site. other promoters containing sites that selectivity bind p50/p heterodimers include those of the pro-il1β, icam- and cd genes. among aggressive b-cell lymphomas such as dlbcls, there is a positive correlation between high nf-κb activity and high mir- expression. moreover, treatment of some b-lymphoma cell lines with nf-κb signaling inhibitors results in reduced mir- expression. in our study, three dlbcl cell lines known to have high nf-κb activity also exhibited high expression of mir-. ib and kmh cells have high nuclear p and rel, whereas l cells have high nuclear rel but low nuclear p. similar to l cells, rc-k cells have inactivating mutations in iκbα, high mir- expression, and exceptionally high levels of nuclear rel. moreover, over-expression of an oncogenically activated mutant rel in bjab cells results in increased expression of mir155hg. sudhl cells are unusual in that they have relatively high levels of nuclear rel protein, but express low levels of mir- and have an overall low nf-κb gene expression profile. thus, nuclear rel may contribute to increased mir- expression in some b-lymphoma cells. nevertheless, rel did not activate the proximal mir155hg promoter in cos- cell reporter gene assays. therefore, there may be rel-dependent sites outside of the 1494 bp fragment analyzed here that can regulate mir155hg expression. alternatively, rel may interact with factors in some b-lymphoma cells to enhance mir155hg expression through its proximal promoter. we used the proximal upstream region of the mir155hg promoter to investigate the regulation of mir- expression by nf-κb. although only approximately 20-30% of induced p binding sites are located within ~ kbp of transcription start sites, approximately 50-60% of the coactivator p300/cbp binding is located within proximal promoters of actively transcribed genes, suggesting that much of the biologically relevant transcription-inducing p binding occurs in sequences proximal to the 5’ end of genes. nevertheless, it has been reported that 28-40% of induced p binding sites are located in introns of known genes. indeed, the two introns of the human mir155hg gene contain several predicted nf-κb sites. one predicted site in intron and three in intron have scores for nf-κb binding that are as high as the - site that we have described. thus, it is possible that there are other p sites that contribute to nf-κb-dependent regulation of mir155hg/mir- expression. moreover, in the mouse mir155hg promoter there does not appear to be an nf-κb site that corresponds to the - site, suggesting that there may be differences in the regulation of mir- expression by nf-κb between the two species.  the results presented herein show that nf-κb p50/p can directly activate the human mir155hg promoter, consistent with the many studies that have reported a correlation between high or induced nf-κb activity and increased expression of mir-. due to the contributions of mir- to immune cell function and malignancy, understanding how mir155hg expression is controlled could lead to new therapeutic approaches. abbreviations bic: b-cell integration cluster; dlbcl: diffuse large b-cell lymphoma; dmem: dulbecco’s modified eagle’s medium; emsa: electrophoretic mobility shift assay; fbs: fetal bovine serum; kbp: kilobase pairs; lps: lipopolysaccharide; mir: microrna; nt: nucleotides; pcr: polymerase chain reaction; pei: polyethylenimine; rel: human c-rel transcription factor; tss: transcription start site. competing interests the authors declare that they have no competing interests. authors’ contributions rct planned and carried out the majority of the experiments. iv performed tissue culture experiments and reporter gene assays. rct and tdg participated in the design of the study and wrote the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1: table s1 primers used and their sequences. click here for file additional file 2: figure s1 truncation mutants of the mir155hg promoter were made via pcr, resulting in the creation of -530-mir155hg and -91-mir155hg luciferase reporter plasmids. reporter assays were performed in cos- cells transfected with the pgl3-based -1494 bp mir155hg promoter reported plasmid or with indicated truncation mutant and with pcdna vector control or pcdna-flag-p values were normalized to the pcdna control. the * indicates p < . click here for file additional file 3: figure s2 reporter assays were performed in cos- cells transfected with the pgl3-based wild-type mir155hg promoter reported plasmid or an ap- mutant with either pcdna alone or pcdna-p values for each transfection were normalized to rsv-renilla and then normalized to pcdna alone. click here for file additional file 4: table s2 miqe checklist for qpcr methods and analysis. click here for file acknowledgements we thank eric flemington for the wild-type and the - κb site and ap- site mutant mir155hg luciferase reporter plasmids, nancy rice for anti-p antiserum, alexander hoffmann for the 3t and 3t3nfkb1-/-nfkb2-/-relb-/-crel-/- cells, and alan yeo for pcdna-flag-p and pcdna-flag-p plasmids. we also thank susan kandarian, robert jackman, trevor siggers, frank naya, david waxman, and gerald denis for helpful suggestions and discussions. this research was supported by the national institutes of health. rct was supported by an nhlbi hematology training grant.
mir155hg has been proposed to be a target gene for transcription factor nf-κb largely due to the positive correlation between high nuclear nf-κb activity and increased mir- expression following treatment with nf-κb inducers or in subsets of hematopoietic cancers. this report shows that induction of nf-κb activity rapidly leads to increased levels of both primary mir155hg mrna and mature mir- transcripts. we have mapped an nf-κb-responsive element to a position approximately nt upstream of the mir155hg transcription start site. nevertheless, direct regulation of the human mir155hg promoter by nf-κb has not been convincingly demonstrated previously. microrna- is the diced product of the mir155hg gene. moreover, the levels of mir- in nine human b-lymphoma cell lines generally correlate with increased nuclear nf-κb proteins.
 microrna- is the diced product of the mir155hg gene. mir- regulates the expression of many immune-specific transcripts, is overexpressed in many human lymphomas, and has oncogenic activity in mouse transgenic models. mir155hg has been proposed to be a target gene for transcription factor nf-κb largely due to the positive correlation between high nuclear nf-κb activity and increased mir- expression following treatment with nf-κb inducers or in subsets of hematopoietic cancers. nevertheless, direct regulation of the human mir155hg promoter by nf-κb has not been convincingly demonstrated previously.  this report shows that induction of nf-κb activity rapidly leads to increased levels of both primary mir155hg mrna and mature mir- transcripts. we have mapped an nf-κb-responsive element to a position approximately nt upstream of the mir155hg transcription start site. the - site is specifically bound by the nf-κb p50/p heterodimer and is required for p65-induced reporter gene activation. moreover, the levels of mir- in nine human b-lymphoma cell lines generally correlate with increased nuclear nf-κb proteins.  overall, the identification of an nf-κb-responsive site in the mir155hg proximal promoter suggests that mir155hg is a direct nf-κb target gene in vivo. understanding nf-κb-mediated regulation of mir- could lead to improved immune cell-related diagnostic tools and targeted therapies. mir-155mir155hgbicnf-kappabpromotertranscriptional activation 