Mol. Hum. Reprod. Advance Access originally published online on January 18, 2008
Molecular Human Reproduction 2008 14(1):53-59; doi:10.1093/molehr/gam086
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Prostaglandin H synthase-2 gene regulation in the amnion at labour: histone acetylation and nuclear factor kappa B binding to the promoter in vivo
1 Mothers and Babies Research Centre, Hunter Medical Research Institute, Newcastle, Australia 2 College of Life Sciences, University of Dundee, Dundee DD15EH, UK 3Division of Obstetrics and Gynaecology, University of Newcastle, Australia 4John Hunter Hospital, Locked Bag 1, Hunter Region Mail Centre, Newcastle, NSW 2310, Australia
5 Correspondence address. Tel: +61-2-4921-4383; Fax: +61-2-4921-4394; E-mail: tamas.zakar{at}newcastle.edu.au
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Abstract |
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Increased prostaglandin H synthase-2 (PGHS-2) expression in the amnion is critical for the production of prostaglandins that induce labour. The aim of the present investigation was to determine whether PGHS-2 gene activity is controlled by NF
B transcription factors in term amnion in vivo as suggested by in vitro findings. Amnion membranes were collected after elective Caesarean section (n = 14) or spontaneous labour (n = 12) at term, and histone acetylation and transcription factor binding to the PGHS-2 and IB
promoters were determined in fresh tissues by chromatin immunoprecipitation. High level of histone-3 and -4 acetylation was detected in the proximal 1000 bp region of the PGHS-2 promoter indicating permissive chromatin structure in an area that contains two consensus NFB binding sites and other transcription factor binding motifs. The TATA-box was occupied by TATA-binding protein (TBP) demonstrating that the PGHS-2 gene was transcriptionally active before and after labour. NFB (p65 and p50) binding to the consensus sites, however, was detected only before, but not after, labour. Moreover, NFB factor binding before labour was unrelated to TBP binding to the PGHS-2 TATA-box in the same tissues. Further, p65 binding to the NFB-responsive IB promoter increased at labour and correlated strongly with TBP binding to the TATA-box of this gene. We conclude that the proximal 1000 bp region is involved in PGHS-2 promoter regulation in term amnion. The NFB system is activated at labour and stimulates the IB gene, but the NFB factors do not drive PGHS-2 transcription using consensus promoter sites in normal term amnion in vivo.
Key words:
amnion/NF-B/parturition/PGHS-2/labour
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Introduction |
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Prostaglandin (PG) E2 and F2
produced by the gestational tissues (amnion, chorion and decidua) play important roles in human parturition by stimulating myometrial contractility, cervical softening and membrane rupture (Gibb, 1998). PGE2 and PGF2 synthesis is dependent on the prostaglandin endoperoxide H synthase isoenzymes PGHS-1 and -2 (PTGS1 and PTGS2), which catalyse the committing and limiting step of the PG-biosynthetic pathway. In the amnion, PGHS-1 expression decreases during pregnancy (Johnson et al., 2006), whereas PGHS-2 mRNA, protein and activity levels increase towards term and during labour (Teixeira et al., 1994; Mijovic et al., 1999; Slater et al., 1999; Sadovsky et al., 2000). This increase is critical to the production of PGs that stimulate labour and delivery. We have shown recently that the gestational age-dependent increase of amniotic PGHS-2 mRNA expression is due predominantly to an increase of PGHS-2 gene activity (Johnson et al., 2006). It is unclear that, however, what mechanisms control PGHS-2 gene transcription in the amnion as pregnancy advances to term.
In cell cultures derived from amnion, the PGHS-2 gene is induced by a variety of agonists including pro-inflammatory cytokines, lipopolysacharide, corticosteroids, growth factors and by hypotonic or mechanical stretch (Lundgren et al., 1997; Zakar and Hertelendy, 2001; Mohan et al., 2007). The human PGHS-2 promoter contains numerous transcription factor binding motifs that can mediate the actions of these inducers. The regulatory motifs cluster in the upstream 2 kb region of the promoter (Kosaka et al., 1994). Two consensus binding sites for the NFB transcription factors are also found here, at 213–222 and 438–447 bp upstream of the transcriptional start site. These sequences may be particularly important in PGHS-2 gene regulation, because the NFB transcription factors can mediate the up-regulation of PGHS-2 expression by pro-inflammatory factors, thereby providing a mechanistic link between intrauterine infection, inflammation and PG production that can trigger term and preterm labour (Lindstrom and Bennett, 2005). Indeed, studies with cultured amnion cells and cell-free binding assays showed that NFB family proteins, acting through the consensus sites, can mediate the stimulation of PGHS-2 promoter activity by the pro-inflammatory cytokines IL-1 (Allport et al., 2001; Yan et al., 2002a,b) and TNF (Ackerman et al., 2005) and by stretch (Mohan et al., 2007).
The purpose of the present investigation was to determine whether the NFB system is involved in PGHS-2 promoter regulation in the amnion in vivo as suggested by the in vitro cell culture studies. To achieve this, we have determined transcription factor binding to the promoter in freshly delivered amnion tissues, where in vivo conditions are preserved, using chromatin immunoprecipitation (ChIP), which is a technique that detects DNA-protein interactions in native chromatin (Orlando et al., 1997). Specifically, we have measured the level of histone acetylation at the proximal 2.6 kb region of the PGHS-2 promoter to define whether chromatin structure is permissive of transcription factor binding at the NFB binding sites. We have also determined the binding of p65 (RelA) and p50, the two principal NFB factors in amnion (Lee et al., 2003), to the promoter regions where the NFB binding sites are located. NFB factor binding was correlated subsequently with the occupancy of the PGHS-2 TATA-box by the TATA-binding protein (TBP), a key component of the transcriptional preinitiation complex, to assess the functional significance of regulatory transcription factor binding. Furthermore, we have measured p65, p50 and TBP binding to the promoter of IB, a well characterized NFB-regulated gene (Le Bail et al., 1993; Sun et al., 1993), to explore gene-specific regulation by NFB. Histone acetylation at the PGHS-2 promoter and transcription factor binding to the PGHS-2 and IB promoters were compared in tissues collected before and after term labour to determine labour-associated changes.
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ChIP-certified antibodies with established specificity were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA [anti-TFIID(TBP): SI-1, Cat. No. sc-273; anti-NF
B p65: H-286, Cat. No. sc-7151; anti-NFB p50: H-119, Cat. No. sc-7178] and Upstate Biotechnology, Charlottesville, VA, USA (anti-acetyl-Histone H3: Cat. No. 06-599; anti-acetyl-histone H4: Cat. No. 06-598). PCR primers were supplied by Invitrogen, Carlsbad, CA, USA. Protein A-agarose was from Santa Cruz Biotechnology. Glass beads (425–600 microns), protease inhibitors, sonicated salmon sperm DNA and the remaining chemicals were bought from Sigma (St Louis, MO, USA).
Patients
Placentas delivered at term by elective Caesarean section (TNL, n = 14) and spontaneous labour (TL, n = 12) were obtained from uncomplicated pregnancies within 30 min of delivery at the John Hunter Hospital, Newcastle, NSW, Australia. Women treated with non-steroidal anti-inflammatory drugs, or with a history of infection, chorioamnionitis or asthma, or undergoing induction were excluded. Informed consent was obtained from all participants as approved by the Hunter Area Research Ethics Committee and the University of Newcastle Human Research Ethics Committee.
Tissue extraction
Amnion membranes were isolated as described (Johnson et al., 2002). One gram tissue was fixed in phosphate-buffered saline (PBS) containing 1% formaldehyde for 20 min at room temperature. The tissues were washed in cold PBS containing 125 mM glycine and homogenized in 20 ml of swelling buffer (125 mM glycine, 2 mM EDTA, 0.5 mM EGTA, 10 mM Tris(HCl), pH8.0, 10 mM DTT, 0.1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin, 0.1 µM aprotinin) using a Polytron homogeniser on maximal setting with five 30 s bursts interrupted by 30 s cooling periods. Triton X-100 (0.5% v/v) and PMSF (1 mM) were added, and the samples were homogenized with a Potter type glass-teflon homogeniser in ice. The homogenate was pressed through a stainless steel mesh (380 µm pore size) and centrifuged at 1000g for 15 min at 4°C. The pellet was suspended in 2 ml ice-cold Buffer-1 (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES pH 6.5, 10 mM DTT, 0.1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin-A, 0.1 µM Aprotinin) for 10 min, centrifuged, resuspended in 1 ml of cold Buffer-2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES pH6.5, 10 mM DTT, 0.1 mM PMSF 1 µM leupeptin, 1 µM pepstatin-A, 0.1 µM aprotinin), and centrifuged and the pellet was stored overnight at 4°C.
Chromatin immunoprecipitation
To each pellet, 400 µl of cold Lysis Buffer (1% SDS w/v, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1), 16 µl of 1x protease inhibitor cocktail (Roche Molecular Biochemicals, Castle Hill, VIC, Australia) and 100 µl volume of glass beads were added, and the mixture was sonicated (5 x 30 s sonication with 30 s cooling periods in a salt/ice mixture) using a Branson Sonifier 250 (Branson Ultrasonic Corporation, Danbury, CT, USA) with a 1/8' micro tip at 40 W. The sonicate was cleared at 10 000g for 10 min, and 50 µl aliquots were diluted with 450 µl of Buffer-3 (1.12% Triton X-100, 0.11% deoxycholate (w/v), 1 mM EDTA, 0.56 mM EGTA, 170 mM NaCl, 10 mM Tris pH8.1, 1 mM PMSF). Each aliquot was pre-cleared with 40 µl of 50% (v/v) protein A-agarose and 2 µg of salmon sperm DNA at 4°C for 2 h and centrifuged. The supernatants were incubated with antibodies at 4°C for 18 h. Antibody amounts per reaction mixture were optimized in preliminary experiments and were 3 µg for the TBP, 5 µg for p65 and acetyl-histone-3 and -4 and 1 µg for p50. Immune complexes were captured by adding 40 µl of 50% protein A-agarose containing 2 µg of salmon sperm DNA for 1.5 h at 4°C. The agarose was sedimented and washed sequentially with 1 ml of TSE-I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris pH8.1), 1 ml of TSE-II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris pH8.1) and 1 ml of Buffer-4 (0.25M LiCl, 1% NP-40 v/v, 1% deoxycholate, 1 mM EDTA and 10 mM Tris pH8.1), with 10 min incubations on ice. The beads were washed a further three times with 1 ml TE Buffer (10 mM Tris pH8.0, 1 mM EDTA). The immune-complexes were eluted by three washes of elution solution (1 x 150 µl and 2 x 100 µl of 1% SDS, 0.1 M NaHCO3) for 10 min at 65°C each. Cross-links were reversed by incubating the pooled supernatants at 65°C overnight. The recovered DNA was purified using the Promega Wizard SV Gel and PCR Purification kit and quantitated using the Promega DNA Quantitation System (Promega Corporation, Annandale, NSW, Australia).
Real-time PCR
All real-time PCR reactions used 50 to 100 pg of DNA as template. Amplifications were performed using an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Scoresby, VIC, Australia) with SYBR® green detection. Each reaction mixture contained 12.5 µl of 2x SYBR® green PCR master mix (purchased from Applied Biosystems), primers (listed in Table I), template and water to 25 µl. Optimal primer concentrations, determined in preliminary experiments, were 200 nM for the PGHS-2 TATA binding site (R1), R4 and the PGHS-2 control site. Primer concentrations of 400 nM were used for all other sites. Reaction mixtures were assembled in 96-well PCR plates. No-template controls were included to detect primer interactions, and melt-curve analyses were performed to monitor the homogeneity of amplification products. The predicted size of the amplification products was verified by agarose gel electrophoresis.
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Data analysis
The abundance of promoter sequences in DNA immunoprecipitated with transcription factor antibodies (TBP, p65 and p50) was determined relative to an internal reference sequence, using the
CT values (Livak and Schmittgen, 2001). The reference sequence was a single copy DNA sequence residing in a different chromosome (i.e. in a different DNA molecule) and having no binding site for the transcription factors examined. The level of this reference sequence served as a measure of the non-specific background of DNA bearing the target sequence, since both sequences were single copy and the efficiency of PCR amplification in optimized real-time PCR systems was uniformly 2 (Livak and Schmittgen, 2001). We have used an intronic sequence of the PGDH gene for this purpose, since the corresponding PCR system has been thoroughly optimized (Johnson et al., 2004). The relative abundance values represented target sequence abundance relative to background. Immunoprecipitations without antibody were also performed with each tissue extract and were used in the statistical calculations as negative controls. Control immunoprecipitation experiments were done with rabbit IgG (5 µg/reaction) to ascertain that IgG did not cause non-specific immunoprecipitation under the conditions employed. The results of these control experiments are presented in Table II. In ChIP analyses with acetylated histone antibodies, CT values were generated using threshold cycle numbers obtained in PCR reactions where equal amounts (50 pg) of immunoprecipitated and non-immunoprecipitated DNA were used as template. The relative abundance values generated here represented the enrichment of target sequences due to immunoprecipitation.
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The relative abundance values were not normally distributed; therefore, non-parametric statistical methods were employed. These included the sign test paired according to individual tissues to determine transcription factor binding in the presence and absence of antibody and the non-parametric one-way ANOVA followed by multiple comparison test (Sidak) as appropriate. The Mann–Whitney U-test was used for two-sample comparisons. Correlation analyses were performed on relative abundance values after subtracting the no-antibody control values using the Spearman test with P-values corrected for multiple comparisons (Sidak). P < 0.05 was considered significant. The STATA software package (College Station, TX, USA) was used for statistical calculations.
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Histone acetylation
Acetylation of histones opens up chromatin structure and renders chromatin DNA accessible to transcription factors (Eberharter and Becker, 2002). We have assessed DNA accessibility at the PGHS-2 promoter by performing ChIP with antibodies against acetylated histones-3 and -4 (acH3 and acH4, respectively) and measuring the abundance of eight promoter sequence regions in the immunoprecipitated DNA using real-time PCR. These regions were distributed along the
2600 bp upstream promoter section of the PGHS-2 gene as illustrated by Fig. 1C. ChIP results for acH3 and acH4 (Fig. 1A and B, respectively) showed that acetylation of H3 and H4 was markedly higher in the proximal 1 kb region of the promoter than further upstream, or downstream in the control region. The highly acetylated region includes the two NFB binding motifs implicated in PGHS-2 gene regulation in transfected amnion cells (Allport et al., 2001). H4 acetylation was significantly higher after labour than before (P = 0.003, Mann–Whitney test). No significant labour-associated change was detected in the levels of acH3.
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P65, p50 and TBP binding to the PGHS-2 promoter
Two NF
B transcription factors, p65 and p50, bind to a consensus NFB-binding oligonucleotide in human amnion extracts in vitro (Lee et al., 2003). We determined the binding of these factors to the PGHS-2 promoter in vivo by ChIP. We used antibodies against p65 and p50 and measured the abundance of the two NFB-binding regions of the PGHS-2 promoter in immunoprecipitated DNA by real-time PCR (Site 1 and Site 2, which include the proximal and distal NFB-biding regions, respectively, as listed in Table I). TBP binding to the TATA-box region of the PGHS-2 promoter was also determined to assess whether the transcriptional start site is occupied by the preinitiation complex, which contains TBP. Significant TBP binding to the TATA-box region was detected at term before and after labour (Table III), indicating that the PGHS-2 gene was transcriptionally active. Labour had no significant effect on TBP binding to the TATA-box (ANOVA). Further, we have detected significant binding of p65 to Site 1, but not to Site 2, before labour (TNL group). Binding of p50 was significant to both Site 1 and Site 2 in these tissues. After labour (TL group), however, neither p65 nor p50 bound to the NFB sites (Table III). These results raised the possibility that p65 and p50 might be involved in PGHS-2 gene activation by interacting with Sites 1 and 2 before, but not after, term labour. To examine this possibility, we have correlated the binding of TBP, p65 and p50 to their respective binding sites in individuals in the TNL group. The correlation matrix is presented in Table IV, with significance values adjusted to allow for multiple comparisons. No significant correlation was detected between the binding of the transcription factors in individual tissues. The lack of correlations suggested that the binding of TBP and NFB factors to their consensus sites was unrelated and did not represent common complex(es) where functional interactions could occur.
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P65, p50 and TBP binding to the I
B promoterThe I
B gene promoter contains several NFB response elements and is positively regulated by NFB factors (Le Bail et al., 1993; Ito et al., 1994). The IB protein is expressed in the amnion, therefore binding of TBP, p65 and p50 to the IB promoter is expected to occur and this should indicate the activity of the amniotic NFB system. Table V shows the binding of TBP, p65 and p50 to the IB promoter in the amnion before and after labour as determined by ChIP. In this ChIP assay, Region 1 included the TATA-box region and two proximal NFB binding sites, while Region 2 included the second site and a third upstream NFB binding site (see Table I for the positions of the PCR primers). Significant binding of TBP and p65 to Region 1 was detected both before and after term labour, but there was no labour-associated difference between the medians (ANOVA). Binding of p50 to Region 1 did not reach statistical significance either in the TNL or in the TL group (although binding had approached significance after labour). Binding of p65 to Region 2 was not detectable in the TNL group, but after labour, p65 binding to Region 2 became highly significant. No p50 binding was detected to Region 2 in both patient groups. Further, in the TNL group, TBP and p65 binding to Region 1 did not correlate in individuals (P = 0.45, Spearman test). In the TL group of individuals, Table VI shows the matrix of correlations between TBP and p65 binding to Regions 1 and 2. Significance values have been adjusted to allow for multiple comparisons. The correlation between TBP binding to Region 1 and p65 binding to Region 2 was highly significant, but there was no correlation between TBP and p65 binding to Region 1 and between p65 binding to Regions 1 and 2. These correlations were in agreement with the possibility that TBP bound to the TATA box and p65 bound to the upstream NFB site (Region 2) formed a complex occupying the IB promoter after labour.
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Discussion |
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ChIP is a powerful technique for detecting DNA-protein binding in native chromatin (Orlando et al., 1997; Aparicio et al., 2005). ChIP is used widely to study the dynamics of chromatin structure, histone modifications and transcriptional complex formation in cultured cells in response to agonists. When adapted for use with fresh tissues, ChIP can detect DNA-protein interactions in vivo, making it possible to study chromatin structure and transcription factor binding to specific genes in different physiological states. We have utilized this potential and determined the histone acetylation status and transcription factor binding at the PGHS-2 promoter in amnion tissues collected before and after term labour. Critical features of our ChIP procedure were the use of quantitative real-time PCR instead of the traditional end-point PCR (Aparicio et al., 2005) and the measurement of DNA amount recovered after immunoprecipitation, which ensured equal DNA inputs and uniform amplification efficiencies in the subsequent PCR reactions. In addition, an internal reference was introduced to correct for sample variation, and data from control and immunoprecipitated samples from the same chromatin preparations were paired for statistical analysis to reduce patient-to-patient and between-assay variations. The analysis of histone acetylation along the
2.6 kb upstream promoter region revealed significantly elevated acetyl histone-3 and acetyl histone-4 levels in the proximal 1 kb section. This indicated that chromatin structure was permissive in an area where several transcription factor binding sites are located including NFB, NF-IL6 (C/EBPβ), SP1 sites and a CRE/E-box sequence (Kosaka et al., 1994). These motifs have been suggested to participate in PGHS-2 gene regulation in vitro in cultured amnion cells treated with agonists, inhibitors, subjected to stretch and transfected with PGHS-2 promoter-reporter constructs (Allport et al., 2000; Ackerman et al., 2005; Lee et al., 2005; Lindstrom and Bennett, 2005; Mohan et al., 2007). The NFB motifs and the cognate transcription factors were prime candidates for a regulatory role in vivo. Two principal NFB transcription factors, p65 and p50, are expressed in the amnion (Yan et al., 2002a,b) and bind to consensus NFB-binding oligonucleotides as shown by EMSA (Lappas et al., 2002; Lappas et al., 2003; Lee et al., 2003). IL-1, which activates the NFB system and stimulates PGHS-2 expression in amnion cells (Mitchell et al., 1993; Allport et al., 2001; Yan et al., 2002a,b), is present in human amniotic fluid (Romero et al., 1990, 1992; Tsunoda et al., 1990) and amnion tissue (Keelan et al., 1999) in increasing concentrations at labour. The persistent activation of the amniotic NFB system during labour has also been reported (Allport et al., 2001). Therefore, it was reasonable to expect that PGHS-2 gene activity was controlled in vivo at term by the NFB system through the binding of p65 and p50 to the consensus NFB binding sites in the promoter. ChIP analysis has indeed detected p65 binding to the proximal NFB site and p50 binding to both sites in the term not in labour group (Table III). Moreover, TBP binding to the TATA-region was significant, which was consistent with the transcriptional activation of the PGHS-2 gene in vivo (Johnson et al., 2002). TBP binding to the TATA-region remained significant following labour; however, p50 and p65 binding became undetectable. This was unexpected, because it suggested that PGHS-2 transcription was not driven by the NFB system after labour despite the fact that labour represents a strong pro-inflammatory stimulus (Cox et al., 1993; Allport et al., 2001; Lee et al., 2003) activating NFB. Moreover, the binding of TBP, p65 and p50 to their respective binding sites was not correlated in individuals in the TNL group. This suggested that these transcription factors did not bind to the PGHS-2 promoter as components of a complex. Collectively, our ChIP analysis results did not support the possibility that PGHS-2 gene transcription in term amnion was controlled in vivo by p65 and p50 interacting with the two consensus NFB-binding sites in the promoter.
IB is an inhibitory member of the NFB family of transcriptional regulators. IB expression is stimulated by p65/p50, and the protein acts as a negative feedback regulator of NFB activity (Sun et al., 1993). The IB gene promoter contains several NFB binding sites that mediate the stimulatory effect of the NFB factors on IB transcription (Le Bail et al., 1993; Ito et al., 1994). These properties indicated that NFB transcription factor binding to the IB promoter would show the activation of the NFB system independently of the effect of NFB on the PGHS-2 gene. ChIP analysis detected TBP binding to the IB TATA-region both before and after labour indicating that the IB gene was transcriptionally active. RelA (p65) binding was also significant to the proximal IB promoter region (Region 1, containing two functional NFB binding sites) in both the TNL and TL groups, but the lack of correlation with TBP binding in either group argued against the possibility that this interaction participated in IB transcription control. The binding of p65 to the distal promoter region (Region 2, containing an additional upstream functional NFB binding site), however, became highly significant after labour and correlated strongly with TBP binding to the TATA site. This was in agreement with the reported inflammatory activation of the NFB system during labour (Allport et al., 2001) and showed that the IB gene responded to this activation involving p65 binding to the upstream NFB site. The PGHS-2 gene, on the other hand, was refractory to this labour-associated NFB activation, at least in terms of increased p65 and p50 binding to the consensus binding sites. Restrictive chromatin structure was unlikely to limit access to these sites, since H3 acetylation was unchanged and H4 acetylation levels increased in the proximal PGHS-2 promoter region with labour (Fig. 1).
Thus, using a ChIP protocol modified for processing fresh tissues, we have gained insight, for the first time, into interactions between transcription factors and gene promoters in the amnion in vivo. It is to be noted, however, that ChIP detects DNA-protein binding at tissue collection providing a ‘snapshot’ of bound proteins, and conclusions on the functioning of the proteins are inferred on the basis of correlations between the binding of basic and regulatory transcription factors. Still, our results raise important questions. For example, if it is assumed that the function of promoter-bound p65 is to facilitate the recruitment of basic transcription factors and cofactors, why was p65 binding uncoordinated with TBP binding in all but one setting examined (binding to the distal NFB site of the IB gene after labour, Table VI)? The answer probably lies in the range of post-translational modifications that influence p65 function [reviewed by N. D. Perkins (2006)]. Permissive chromatin may allow the binding of p65 to promoters possessing binding sites, but post-translational activation (e.g. by phosphorylation and acetylation) is needed to perform the transactivator functions including the recruitment of basic transcription factors. Post-translational modification(s) can also influence p65 binding to promoters in a gene selective fashion (Perkins, 2006), potentially explaining why p65 binds to the IB promoter but not to the PGHS-2 promoter after labour. Women with chorioamnionitis were not involved in this investigation, therefore, the pathological activation of NFB did not occur. Hence, our results do not exclude the possibility that the NFB factors play a role in PGHS-2 gene stimulation in cases of intrauterine inflammation, e.g. by activating promoter-bound NFB proteins. Clearly, more studies are needed to explore the involvement of post-translational modifications in NFB factor binding and function in the fetal membranes. Furthermore, ChIP with fresh gestational tissues will provide valuable data on the in vivo control of PGHS-2 gene transcription by other transcription factors in normal pregnancies and in cases of the preterm stimulation of PGHS-2 expression.
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Funding |
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National Health & Medical Research Council of Australia (Project Grant 252431); John Hunter Hospital Chariable Trust; The University of Newcatle, Australia.
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References |
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Submitted on June 12, 2007; resubmitted on November 10, 2007; accepted on December 4, 2007.
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