the production of nitric oxide ( no ) occurs during innate immune responses to all classes of pathogens ( bogdan , 2001 ) .
the molecule has direct antimicrobial activity , contributes to cell signaling , and regulates cell survival ( bogdan , 2001 ; zwaferink et  al . , 2008 ) .
inducible nitric oxide synthase ( inos ) , the enzyme encoded by the nos2 gene and responsible for no production during infection , is synthesized de novo as a response to the recognition of microbial molecular patterns .
studies with bacterial lipopolysacharide ( lps ) or with pathogen - infected murine cells showed that full transcriptional induction of nos2 and of no production occurs only after synthesis of type i interferons ( ifn - i ) and signaling through the janus kinase ( jak)-stat pathway ( bogdan , 2001 ; gao et  al . , 1998 ) .
type ii ifn ( ifn- ) , produced by natural killer ( nk ) and t  cells , also enhances mouse nos2 induction by lps in a manner requiring stat1 activation by the ifn- receptor complex ( ifngr [ meraz et  al . ,
the published work suggests that ifn receptor - activated stats cooperate with non - ifn signals in the transcriptional regulation of nos2 .
previous analyses of the murine nos2 promoter revealed an ifn response region and binding sites for nf-b ( kleinert et  al . , 2003 ) .
the ifn response region contains binding sites for stat1 dimer ( gamma ifn - activated site , gas [ xie et  al . , 1993 ] ) and interferon regulatory factors ( irf [ kamijo et  al . , 1994 ; spink and evans , 1997 ] ) .
ifn- signaling leads to the formation of stat1 homodimers and irf1 , both of which were shown to be essential for nos2 induction by ifn-/lps ( kamijo et  al . , 1994 ; meraz et  al . ,
ifn - i causes formation of both stat1 dimers and the isgf3 complex , which comprise a stat1/stat2/irf9 heterotrimer ( darnell , 1997 ; schindler et  al . , 2007 ) .
it is unclear which of these complexes contributes to inos regulation by ifn - i and whether ifn - i , like ifn- , stimulate nos2 transcription with strong dependence on irf1 or other irf family members .
the analysis of signals received by the nos2 promoter directly from pattern recognition receptors emphasizes the role of nf-b .
two sites for the transcription factor were identified ( kleinert et  al . , 2003 ; lowenstein et  al . , 1993 ; xie et  al . ,
particularly the binding element proximal to the transcription start proved essential for the activity of the transfected promoter .
listeria monocytogenes is a gram - positive bacterial pathogen replicating in the cytoplasm of mammalian host cells .
it is recognized by a variety of different pattern recognition receptors including toll - like receptors and nod - like receptors ( tlr and nlr , respectively ) ( edelson and unanue , 2002 ; herskovits et  al . , 2007 ) .
in murine bone marrow - derived macrophages , a hitherto unknown cytoplasmic receptor initiates signaling to the ifn - i genes and subsequent release of ifn - i from the infected cells ( stetson and medzhitov , 2006 ; stockinger et  al . , 2004 ) .
exclusion of l. monocytogenes from the cytoplasm , e.g. , by mutation of its major virulence factor listeriolysin o , completely abrogates the ability to stimulate ifn - i production ( stockinger et  al . , 2002 ) . as with lps ,
transcriptional induction of the nos2 promoter was strongly diminished when either ifn - i production or signaling were disrupted ( stockinger et  al . , 2004 ) .
to continue this work , we now asked the question why the nos2 gene , unlike classical ifn - i - stimulated genes ( isgs ) or nf-b target genes , requires input from both stats and signals derived directly from pattern recognition receptors for maximal transcriptional induction . combining an examination of transcription factor and signaling requirements for transcriptional induction with an analysis of transcription factor binding to the nos2 promoter in  situ ,
we conclude that nf-b enhances carboxy - terminal domain ( ctd ) phosphorylation of rna pol ii , after recruitment of the enzyme by stats .
as discussed above , the innate immune response to l. monocytogenes results initially from plasma membrane and endosomal pattern recognition during entry and from cytoplasmic sensing after cytoplasmic escape .
the nos2 gene is paradigmatic for a large group of genes coregulated by pattern recognition receptors and ifn - i ( doyle et  al . , 2002 ;
2002 ) . to  test whether ifn - i synthesis was the only essential signal for nos2 induction derived from the cytoplasmic signaling , the two recognition phases were separated by treating macrophages with heat - killed l. monocytogenes ( hkl ) and with ifn- either separately or together .
heat - killed listeria are confined to phagosomes and can not stimulate the cytoplasmic signal required for ifn - i production .
hkl and ifn- alone were poor inducers of inos mrna synthesis ( figure  1 ) .
by contrast , both signals together synergized to produce the full - blown inos synthesis seen with viable l. monocytogenes .
this result suggests that cytoplasmic signaling can indeed be recapitulated by providing ifn - i .
in addition , it provides a valuable experimental tool to separate effects of non - ifn - i and ifn - i signals on the nos2 promoter and to study each independently from the other . in agreement with ifn - i synthesis preceding nos2 transcription ,
the kinetics of mrna synthesis after infection with viable l. monocytogenes were delayed compared to the simultaneous treatment with hkl and ifn-. many genes expressed in macrophages infected with l.  monocytogenes were found in a microarray experiment to display a pattern of regulation resembling that of the nos2 gene .
38 genes showing the strongest synergy effect between ifn- alone and the additional presence of l. monocytogenes - derived signals are shown in figure  s1 available online . to examine transcription factor requirements for transcriptional induction of the nos2 gene , we used bone marrow - derived macrophages from either wild - type or gene - targeted mice and infected them with l. monocytogenes ( figure  2 ) .
as expected , nos2 expression required signaling through both the ifn and nf-b pathways as deletion of either the stat1 or rela ( nf-b p65 ) genes strongly suppressed inos mrna induction in infected macrophages ( figure  2a ) .
more refined analyses confirmed the importance of the ifn - i receptor ( ifnar1 mice ) and the nf-b pathway ( rela and ikbkb 
  mice , deficient for nf-b p65 and the ikk kinase , respectively ) and established the importance of the isgf3 subunits stat1 , stat2 , and irf9 ( figure  2b ) . the diminished nos2 expression observed upon interference with
nf - kb signaling was not due to reduced ifn - i production as shown by the fact that addition of exogenous ifn- did not rescue this effect .
use of macrophages derived from mice expressing stat1 mutated at its s727 phosphorylation site ( stat1s727a ) showed that phosphorylation of stat1 at s727 , important for full transcriptional induction of some ifn--induced genes ( varinou et  al . , 2003 ) ,
this contrasts with the reduced induction of nos2 by ifn- early after treatment in stat1s727a - expressing macrophages ( varinou et  al . , 2003 ) . in further distinction from the ifn- response ,
two additional members of the irf family , irf3 and irf7 , are active in l. monocytogenes - infected macrophages ( stockinger et  al . , 2009 ) .
irf3 deficiency reduced nos2 induction , but the defect could be rescued by the addition of ifn- , suggesting that it resulted from reduced ifn- synthesis , but not from a direct effect on the nos2 gene .
the data suggest that ifn - i participate in nos2 regulation during l. monocytogenes infection by deploying the isgf3 complex , but not the ancillary activity of irfs .
the low levels of inos expression seen after treatment up to 6 hr with ifn- alone ( figure  1 ) were strongly reduced in mice unable to form isgf3 ( data not shown ) .
interestingly , this differs from the regulation of nos2 mrna during the late stage of the ifn - i response , which has been shown to be independent of stat1 ( plumlee et  al . ,
a distinguishing feature of typical ifn - i - induced genes is that a deacetylation step is required for transcriptional induction , which can be inhibited with the histone deacetylase ( hdac ) inhibitor trichostatin a ( tsa [ nusinzon and horvath , 2005 ] ) .
induced synthesis of nos2 mrna was tsa sensitive , suggesting that the activity of stats on the nos2 promoter abides by the same rules in this regard as the transcriptional activation of classical isgs .
map kinase pathways downstream of pattern recognition receptors ( targeting erk , jnk , and p38mapk ) were probed by pharmacological inhibition .
none of the inhibitory drugs produced a significant reduction of l. monocytogenes - induced nos2 expression ( data not shown ) . in summary
, the data from figures 1 and 2 suggest that isgf3 is the main signal derived from cytoplasmic signaling , recapitulated by the addition of exogeneous ifn- , and that nf-b is the major signal stimulated by hkl , provided by plasma membrane and/or endosomal pattern recognition receptors for nos2 induction .
association of the isgf3 complex with nos2 chromatin was examined with antibodies against stat1 and stat2 for chip .
amplification by polymerase chain reaction ( pcr ) was performed to reveal binding to the promoter - proximal region containing the essential nf-b site as well as the more distal promoter containing the ifn response region and a second potential binding site for nf-b ( figure  3a ) . treating macrophages simultaneously with hkl and ifn- stimulated binding of the isgf3 subunits stat1 and stat2 with indistinguishable kinetics ( figure  3b ) .
the same observation was made for the nf-b subunits p50 and p65 with the notable exception that a reduction of constitutive p50 binding at the earliest time point after stimulation and preceding the phase of increased promoter binding was reproducibly observed .
this finding is consistent with the reported negative regulation of nf-b target genes by p50 homodimers in resting cells ( zhong et  al .
nf-b association was found exclusively with the promoter - proximal , essential site , whereas no evidence for binding to the distal site was obtained .
as expected , stat binding was caused by treatment with ifn - i alone , whereas nf-b binding occurred after exposure to hkl ( data not shown ) .
consistently , infection with viable l.  monocytogenes resulted in similar kinetics of nf-b p65 binding , but stat1 association now required prior ifn - i synthesis and was therefore delayed by about 2 hr compared to direct stimulation with ifn - i ( figure  3c ) .
the simultaneous presence of these proteins was further examined via a chip - re - chip procedure ( figure  3d ) .
it  confirmed that after both ifn- treatment and infection with l. monocytogenes , stat1 could be reprecipitated from a stat2 chip with the expected difference in binding kinetics ( see figure  1 ) .
synergistic enhancement of transcriptional activation by isgf3 and nf-b might result from an interaction in the recruitment of histone acetylases to the nos2 promoter .
acetylation of histone h4 at the proximal and distal promoter elements was assessed . to correct for histone eviction ,
increases of histone acetylation are often rather subtle , so we verified significance and quantified our data by using a q - pcr protocol .
black bars represent amplification of the distal promoter and white bars amplification of the proximal promoter .
combined treatment of macrophages with ifn - i and hkl produced an increase of histone acetylation at both the proximal and distal promoter locations ( figure  4a ) .
treatment with ifn - i alone led to an increase of h4 acetylation almost exclusively at the distal ifn response region ( figure  4b ) .
conversely , hkl treatment alone caused an increase in h4 acetylation predominantly at the proximal nf-b element ( figure  4c ) .
our findings suggest that isgf3 and nf-b indeed cooperate in producing hyperacetylated nos2 promoter chromatin , but that their histone acetyl transferase ( hat)-recruiting activities show no signs of functional interdependence .
pol ii can be bound to transcription start sites in a poised state ( adelman et  al . , 2009 ;
alternatively , the enzyme is recruited in response to the stimulus of gene activation ( adelman et  al . , 2009 ) .
to determine which situation applies to the macrophage nos2 gene , we analyzed pol ii association by chip .
as shown in figure  5a , infection with l. monocytogenes strongly increased pol ii binding , suggesting that it occurs by regulated recruitment .
surprisingly , treatment with ifn - i alone also stimulated binding of pol ii ( figure  5b ) .
association was somewhat , but not much , weaker than after the additional presence of hkl .
in contrast to ifn - i , hkl alone did not stimulate pol ii binding ( figure  5c ) .
this result indicates ( 1 ) that the histone acetylation caused by nf-b is not an absolute requirement for pol ii binding and ( 2 ) that there is a mechanistic difference between isgf3 and nf-b in their mode of activating the nos2 promoter .
ifn and stat - dependent recruitment of pol ii predicts that binding of tfiid and its tbp subunit displays the same requirement .
figures 5d and 5e indeed show that both pol ii and tbp binding was completely abrogated when stat1 , stat2 , or irf9 macrophages were infected with l. monocytogenes .
pol ii , once stably bound to the initiation site , must be phosphorylated at its ctd to associate with proteins required for promoter clearance , capping of the mrna , and elongation ( chapman et  al . , 2008 ; hirose and ohkuma , 2007 ) .
serine 5 ( s5 ) of the ctd amino acid heptarepeat becomes phosphorylated first , followed by s2 , to proceed to productive elongation . with nf-b playing only a minor role in pol ii recruitment
, we wondered whether it might play a role in distinct steps of transcriptional initiation .
s5-phosphorylated pol ii was precipitated from the nos2 initiation site only after treatment with both hkl and ifn - i , but not after treatment with ifn - i alone ( figure  6a ) .
it is a multiprotein transcription factor containing the ctd s5 kinase cdk7 and a number of additional subunits including p62 ( egly , 2001 ) . as in the case of s5-phosphorylated pol ii
, cdk7 was associated with the nos2 initiation site after stimulation with hkl and ifn - i , but not after treatment with ifn - i alone ( figure  6b ) .
in contrast to ifn - i , hkl treatment alone produced as much cdk7 binding as the combined ifn - i - hkl treatment ( figure  6c ) .
the kinetics of cdk7 binding as induced by l. monocytogenes demonstrated association with the nos2 promoter at 2 hr postinfection ( figure  6d ) . at this time
, nf-b is associated with nos2 chromatin , but no or very little isgf3 is present ( figure  3c ) .
binding of cdk7 as well as that of tfiih p62 was abrogated by both nf-b p65 and ikk deficiency ( figures 6e6h ) .
together , these data confirm the hypothesis that a tfiih complex is recruited by nf-b , providing kinase activity for the pol ii ctd at s5 .
comparing the kinetics of nf-b and tfiih binding in the course of infection suggested that tfiih remains bound at the promoter even after dissociation of nf-b ( figures 3 , 6 g , and 6h ; figure  s2 ) .
we tested the possibility that nf-b , by depositing tfiih , primes the nos2 promoter for subsequent isgf3 activity , thus providing a  transcriptional memory  effect . to this end , macrophages were given a 2 hr pulse of hkl treatment , a period sufficient for cdk7 recruitment ( figure  6d ) .
the pulsed cells were left without further stimulation for various intervals , followed by a 4 hr treatment with either ifn- alone , hkl alone , or a combination of ifn- and hkl .
the data show that for at least 24 hr , the level achieved by ifn- treatment of pulsed cells exceeded the level achieved by ifn- treatment of unpulsed cells ( figure  6i ) .
this result is in agreement with the notion of a transcriptional memory or priming effect of nf-b - recruited cdk7 .
the results obtained by studying nos2 regulation raise the question why the nos2 gene requires both isgf3 and nf-b to achieve elongation competence .
they also predict that genes induced by either ifn - i or the nf-b pathway alone should demonstrate promoter binding of both cdk7 and pol ii after single treatment with ifn - i or hkl .
this assumption was tested by analyzing pol ii and cdk7 binding to the promoters of the ifn - i - induced mx2 gene or of the nfkbia gene , which is activated by nf-b for the production of ib to establish a feedback inhibition loop .
the data summarized in figures 7a7d show that ifn- treatment for 2 or 4 hr caused association of both pol ii and cdk7 with the mx2 but not the nfkbia promoter .
conversely , treatment with hkl to activate the nf-b pathway increased association of pol ii and cdk7 with the nfkbia but not the mx2 promoter . both stimuli provided together
consistent with this , transcriptional priming by the nf-b pathway was not observed with either the mx2 or the nfkbia gene ( figures 7e and 7f ) .
the data are consistent with our notion that the establishment of elongation competence by cooperative signals is a gene - specific attribute and a major contribution to the regulation of the nos2 promoter by the transcription factors nf-b and isgf3 .
no production is a hallmark of innate immune responses , but its influence on infected cells or organisms varies . for some pathogens ,
by contrast , l. monocytogenes stimulates macrophages to synthesize large quantities of no , but appears to be relatively insensitive to its toxic effects under our experimental conditions ( zwaferink et  al . , 2008 ) .
the main effect of no is to promote the death of listeria - infected macrophages .
our studies of nos2 regulation were prompted by the findings of several labs that the gene expression signature of cells infected with pathogens , or exposed to their pathogen - associated molecular patterns ( pamps ) , results to a significant extent from cooperative signaling by pattern recognition and ifn - i receptors ( doyle et  al .
by using the nos2 gene as a well - studied example , we show that the need for cooperation between the isgf3 complex and nf-b arises from the inability of the former to provide ctd kinase activity and the lack of pol ii recruitment by the latter .
the prevalent mode of rendering a gene competent for transcription is to assemble a tfiid - tfiib - pol ii complex prior to the association with tfiih ( roeder , 1996 ) .
the combined isgf3 and nf-b activity at the nos2 promoter results in an unconventional transcription initiation complex assembly where tfiih binds the promoter first to provide kinase activity for the subsequent recruitment of pol ii .
three lines of evidence led us to this conclusion : ( 1 ) the kinetics of transcription factor and cdk7 binding during l. monocytogenes infection show that tfiih - cdk7 recruitment occurs before pol ii binding , ( 2 ) ifn - i alone is able to bring about the recruitment of pol ii and hkl alone are able to stimulate cdk7 binding , and ( 3 ) cdk7 binding is abrogated in absence of the nf-b pathway and tbp - pol ii binding is abrogated in absence of isgf3 .
cdk7 binding trails that of nf-b by about 1 hr , suggesting that a complex forms at the promoter that is not preassembled and may require intermediate steps and partner proteins .
similarly , pol ii binding occurs roughly 1 hr after the observed increase in stat1 association .
cdk7 remains associated with nos2 chromatin once nf-b p65 leaves the promoter ( best seen in figure  s2 ) , suggesting that the function of nf-b is to load the promoter with cdk7-tfiih , but not to maintain this association once it has been established . to our knowledge
this is the first time this mode of initiation complex assembly is shown for a gene in the context of the cellular genome and as a result of isgf3-nf-b interaction , although several recent studies are in line with our findings ( spilianakis et  al . , 2003 ) .
the most compelling evidence that tfiih - cdk7 recruitment by nf-b may be more widely used was provided in studies on the activation of the hiv ltr in response to tnf ( kim et  al . , 2006 ) .
contrasting the situation with nos2 , an initiation complex including a hyperphosphorylated rna pol ii was preassembled at the ltr , but , similar to our findings with nos2 , elongation competence required tnf and nf-b to attract tfiih - cdk7 .
the authors propose that nf-b both associates with tfiih and stimulates release of the inhibitory cdk8 from the mediator complex .
genes induced by lps differ concerning the rate - limiting regulatory step for the onset of transcription , consisting either in the release of an elongation block to a paused polymerase or the pol ii recruitment step ( adelman et  al . , 2009 ) .
whether and how nf-b - mediated tfiih recruitment contributes in both situations is not known .
therefore it will be of interest to determine to what extent this mechanism contributes to the large impact of the nf-b pathway on infection - related gene expression and in how far nos2 represents a paradigm valid for the many genes synergistically induced by stats and nf-b .
the mechanism of pol ii recruitment to the many genes regulated by nf-b in absence of isgf3 requires further investigation and , conversely , the mode of tfiih recruitment to isgf3 target genes in the absence of nf-b remains to be clarified .
this may generally be determined by cooperative transcription factors bound to their target promoters and/or by differences in the preexisting chromatin structure and composition .
our studies allow some conclusions about the mechanism of isgf3 action beyond the functional division of labor with nf-b . at the nos2 promoter ,
isgf3 stimulated binding of rna pol ii without requiring stat1 phosphorylation at s727 or the helper function of irf1 .
this differs from the stat1 dimer , which is transcriptionally more active with its transactivating domain phosphorylated and requires irf1 to induce the expression of nos2 and other genes in response to ifn- ( kamijo et  al . , 1994
data from our lab addressing ifn- induction of the gbp2 promoter showed that the stat1 dimer alone can not recruit rna pol ii ( ramsauer et  al .
this allows speculation that the stat2 transactivating domain may generally supersede the requirement for stat1 s727 phosphorylation and the ancillary activity of irf1 . in accordance with our findings about acetylation of the nos2 promoter , stat2-dependent transcriptional initiation via mediator and tfiid subunits
correlates with the ability of the stat2 tad to contact the hats gcn5 and pcaf ( lau et  al .
histone acetylation is an important regulatory step for both nf-b and stat target genes ( chen and greene , 2004 ; ramsauer et  al . , 2007 ) .
nf-b as well as isgf3-dependent acetylation of nos2 promoter chromatin was restricted to the nucleosomes adjacent to their binding sites .
this resembles virus - induced histone acetylation at the ifn- promoter or the promoter of the ifn - i - induced ifi-56k gene that was similarly restricted to a region around the transcription factor binding sites and the transcription start ( parekh and maniatis , 1999 ) .
reviewing our findings and corroborating studies in the perspective of l. monocytogenes infection or pathogen infection in general raises the question why some , but not all , isgs are coupled to the nf-b pathway .
ifn - i synthesis during infection occurs in response to nucleic acid pamps in the cytoplasm , when endosomal tlrs are stimulated , or when tlrs resident at the plasma membrane travel to late endosomes in the process of pathogen uptake ( kagan et  al . ,
2008 ) . with the notable exception of the cytoplasmic dna receptor ( stetson and medzhitov , 2006 ) ,
all prrs stimulating ifn - i synthesis will also stimulate the nf-b pathway , thus providing both signals necessary for nos2 induction .
vice versa , some prrs capable of activating nf-b are not normally coupled to ifn - i synthesis . examples of these are tlr2 , tlr5 , and the nlr family receptors nod1 and nod2 , which have been associated with ifn - i synthesis only in a limited number of cell types or under specific circumstances ( barbalat et  al . , 2009 ; pandey et  al . ,
furthermore , a large number of stress or inflammatory signals , most notably those emanating from the tnf receptor family , provide nf-b activity without concomitant ifn - i production and signaling ( dempsey et  al . , 2003 ) .
we hypothesize that such receptors and their signals provide a tfiih - dependent transcriptional memory effect for nos2 expression , independently of pathogen uptake . vigorous inos expression and no production
are limited , however , to situations where a pathogen is engulfed and processed by host cells , and when pamps appear in the late endosome and cytoplasm .
this mechanism is consistent with our results in figure  6 showing that the hkl - stimulated nf-b pathway can provide the nos2 promoter with transcriptional memory for a subsequent treatment with ifn - i .
it ensures that large amounts of no are made only when its antipathogen activity is needed inside cells .
continuing along these lines , the reason why classical isgs do not require this prime - and - trigger mechanism may be that their products are less harmful and cells can afford to prepare for pathogen entry without running the risk of inflicting damage upon themselves ( zwaferink et  al .
although our study provides a mechanism for signal integration and a potential paradigm for cooperativity between the stat and nf-b pathways during infection , further experiments must reveal the biological impact of stat - nf-b convergence .
recombinant ifn- was purchased from biomedica ( nova scotia , canada ) and added to culture medium to a final concentration of 250 u / ml . the inhibitors trichostatin a ( tsa ) ( wako biochemicals , osaka , japan ) , sp600125 for c - jun kinase inhibition ( sigma - aldrich , st louis , mo ) , sb203580 for p38mapk inhibition ( sigma - aldrich ) , and u0126 for mek inhibition ( calbiochem , nottingham , uk ) were used in a final concentration of 150 nm , 25  nm , 4 nm , and 10 nm , respectively .
the listeria monocytogenes strain lo28 was cultured in brain heart infusion broth overnight at 37c .
infection of cells at moi 10 was performed as described ( stockinger et  al . , 2002 ) .
heat - killed listeria ( hkl ) were generated by incubation of an overnight culture of lo28 in a waterbath at 70c for 20 min .
animal experiments were discussed and approved by the university of veterinary medicine , vienna , institutional ethics committee and carried out in accordance with protocols approved by the austrian law ( gz 680 205/67-brgt/2003 ) .
mice ( wt c57bl/6 , ifnar1 [ muller et  al . , 1994 ] , stat1 [ durbin et  al . , 1996 ] , stat1s727a [ varinou et  al . , 2003 ] , stat2 [ park et  al . , 2000 ] , irf1 [ reis et  al . , 1994 ] , irf3 [ sato et  al . , 2000 ]
, irf7 [ honda et  al . , 2005 ] , irf9 [ harada et  al . , 1996 ] , and ikbkb and rela [ greten et  al . , 2007 ] ) were sacrificed for bone marrow between 7 and 10 weeks of age .
poly i : c - mediated deletion of ikk and nf-b p65 in bone marrow cells was performed as described ( greten et  al . , 2007 ) .
bone marrow - derived macrophages ( bmdm ) were obtained by culture of bone marrow in l - cell - derived colony - stimulating factor 1 as described previously ( baccarini et  al . , 1985 ) .
rna preparation was performed with nucleospin rna ii kit purchased from macherey - nagel ( dren , germany ) according to the manufacturer 's protocol .
quantitative real - time pcr was performed on mastercycler ep realplex s , purchased from eppendorf ( vienna , austria ) .
primer for inos mrna expression and qrt - pcr were described previously ( stockinger et  al . , 2004 ) .
primer for mx2 and ib mrna expression were as follows : mx2 fwd 5-ccagttcctctcagtcccaagatt-3 ; mx2 rev 5-tactggatgatcaagggaacgtgg-3 ; ib fwd 5-gcaatttctggctggtggg-3 ; ib rev
5-gatccgccaggtgaaggg-3. chromatin immunoprecipitation ( chips ) were performed according to the protocol described in nissen and yamamoto ( 2000 ) .
antibodies used were described recently ( anti - stat1c [ kovarik et  al . , 1998 ] , anti - stat2 [ park et  al . , 2000 ] ) , purchased from santa cruz ( santa cruz , ca ) and used at a 1:20 dilution ( anti - nf-b p65 , anti - nf-b p50 , anti - rna pol ii , anti - cdk7 , and anti - p62-tfiih ) purchased from bethyl ( montogomery , tx ) and used in a dilution of 1:100 ( anti - ps5 ctd pol ii ) , purchased from abcam ( cambridgeshire , uk ) and used in a dilution of 1:100 ( anti - histone 3 and anti - tbp ) , or purchased from upstate and used in a dilution of 1:100 ( anti - acetyl histone 4 ) .
chip data were normalized to input and , in case of histone acetylation , further normalized to total h3 and to the untreated sample to correct for histone eviction . in the re - chip experiments
, the immunecomplexes were eluted by adding 10 mm dtt and incubation for 30 min at 37c .
primers used for pcr and q - pcr of the nos2 promoter were as follows : inos dis fwd 5-ccaactattgaggccacacac-3 ; inos dis rev 5-gcttccaataaagcattcaca-3 ; inos prox fwd 5-gtcccagttttgaagtgactacg-3 ; inos prox rev 5-gttgtgaccctggcagcag-3 ; mx2 prox fwd 5-acccagccaaggccccctta-3 ; mx2 prox rev 5-gcagctgccagggctcagac ; ib prox fwd 3-ggaccccaaaccaaaatcg-5 ; ib prox rev 3-tcaggcgcggggaatttcc-5. macrophages were infected with an overnight culture of l. monocytogenes for 8 hr ( moi10 ) or treated 4 hr with ifn-. rna was extracted with trizol and qiagen rneasy kit according to the manufacturers ' protocol .
cdna syntheses and array - hybridizations were performed according to the manufacturers ' protocol ( amersham - biosciences ; ge healthcare ) .
 summarytranscriptional regulation of the nos2 gene encoding inducible nitric oxide synthase ( inos ) requires type i interferon ( ifn - i ) signaling and additional signals emanating from pattern recognition receptors . 
 here we showed sequential and cooperative contributions of the transcription factors isgf3 ( a complex containing stat1 , stat2 , and irf9 subunits ) and nf-b to the transcriptional induction of the nos2 gene in macrophages infected with the intracellular bacterial pathogen listeria monocytogenes . 
 nf-b preceded isgf3 at the nos2 promoter and generated a transcriptional memory effect by depositing basal transcription factor tfiih with the associated cdk7 kinase for serine 5 phosphorylation of the rna polymerase ii ( pol ii ) carboxyterminal domain ( ctd ) . 
 subsequent to tfiih deposition by nf-b , isgf3 attracted the pol ii enzyme and phosphorylation at ctd s5 occurred . 
 thus , stats and nf-b cooperate through pol ii promoter recruitment and the phosphorylation of its ctd , respectively , as a prerequisite for productive elongation of inos mrna . 
Introduction
Results
Discussion
Experimental Procedures