Transcription regulation from a TATA and INR-less promoter: spatial segregation of promoter function
2006; Springer Nature; Volume: 25; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7600966
ISSN1460-2075
AutoresAnuja A. George, Manish Kumar Sharma, Badri Nath Singh, Naresh Sahoo, Kanury V. S. Rao,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle26 January 2006free access Transcription regulation from a TATA and INR-less promoter: spatial segregation of promoter function Anuja A George Anuja A George Search for more papers by this author Manish Sharma Manish Sharma Search for more papers by this author Badri N Singh Badri N Singh Search for more papers by this author Naresh C Sahoo Naresh C Sahoo Search for more papers by this author Kanury VS Rao Corresponding Author Kanury VS Rao Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Anuja A George Anuja A George Search for more papers by this author Manish Sharma Manish Sharma Search for more papers by this author Badri N Singh Badri N Singh Search for more papers by this author Naresh C Sahoo Naresh C Sahoo Search for more papers by this author Kanury VS Rao Corresponding Author Kanury VS Rao Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India Search for more papers by this author Author Information Anuja A George, Manish Sharma, Badri N Singh, Naresh C Sahoo and Kanury VS Rao 1 1Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India *Corresponding author. Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. Tel.: +91 11 2617 6680; Fax: +91 11 267 5114; E-mail: [email protected] The EMBO Journal (2006)25:811-821https://doi.org/10.1038/sj.emboj.7600966 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The mode of regulation of class II genes that lack the known core promoter elements is presently unclear. Here, we studied one such example, the murine CD80 gene. An unusual mechanism was revealed wherein the pre-initiation complex (PIC) first assembled on an upstream, NF-κB enhancer element. Notably, this assembly occurred independent of contributions from the core promoter domain, and resulted in a PIC that was competent for transcription initiation. Positioning was subsequently achieved by exploiting the intrinsic architecture of the promoter, by virtue of which the tethered PIC was spatially juxtaposed with the transcription initiation site. Bridging interactions then ensued, through protein–protein contacts, which then enabled the elongation phase of CD80 transcription. Introduction The control of gene expression represents a combinatorial process involving a multiplicity of events that include chromatin recognition and reconfiguration, covalent modification of histones, recruitment of cofactors and the basal trans-cription components, and the assembly of an elongation-competent transcription complex (Jones and Kadonaga, 2000; Lemon and Tjian, 2000). Although transcriptional activation mechanisms for several eucaryotic genes have been deciphered to varying degrees of detail, all of these represent genes that contain well-defined core promoter elements (Smale and Kadonaga, 2003). In contrast, while genes that lack any of the conventional core promoter elements constitute a significant proportion of the eucaryotic genome, little is known about how they are regulated. Here, we examined mechanisms regulating transcriptional activation of the gene coding for the murine CD80 protein, a member of the B7 family of costimulatory molecules. CD80 is primarily expressed on the surface of professional antigen-presenting cells of the mammalian immune system, and plays a critical role in the activation of T lymphocytes (Carreno and Collins, 2002). An intriguing aspect of the CD80 gene is that it does not possess any of the canonical promoter elements that have been described for class II genes so far (Smale and Kadonaga, 2003; Lim et al, 2004). It lacks the TATA, the Initiator (Inr), the downstream promoter (DPE), the TFIIB recognition (BRE) elements, and the recently identified motif ten element (MTE). Further, the 5′-proximal sequence is also devoid of consensus sequences for Sp1 binding, a characteristic of both Inr-dependent promoters and those associated with CpG islands (Supplementary Figure 1). Finally, both the transcription start site and its proximal regions are shown to be completely histone-free, with an isolated nucleosome that was centered at −619 bp relative to the transcription start site. A novel mechanism of gene activation was revealed wherein assembly and positioning of the pre-initiation complex (PIC) took place in two discrete and spatially segregated steps. A functional PIC competent for transcription initiation was first assembled in an NF-κB-dependent manner, at a distal site that was encapsulated by the upstream nucleosome. Activation-induced remodeling of the nucleosome regulated this assembly. The intrinsic architecture of the promoter, supported by cofactor binding, then provided for a superstructure wherein the PIC was brought to within spatial proximity of transcription start site. This juxtapositioning of PIC with the transcription start site enabled its appropriate positioning through a bridging effect, mediated by the Sp1 that was constitutively bound to the core promoter domain. Thus, cooperativity between discontiguous DNA sequence elements, operating within a three-dimensional structural context, can provide for a transcriptional regulatory mechanism that circumvents the need for a conventional core promoter element. Results The murine CD80 promoter and the induction of its gene product Stimulation of the murine plasmacytoma, J558L cells with hydrogen peroxide (H2O2) for a brief (15 min) period led to an increase in CD80 mRNA levels between 1 and 2 h poststimulation, and in a manner that was devoid of de novo protein synthesis. Transcription was initiated from the originally identified (Selvakumar et al, 1993; Borriello et al, 1994) transcription initiation site (TS1) and in vitro transcription assays established that the promoter sequence extending up to −957 nt from TS1 was sufficient to support expression of the CD80 gene. These cumulative results are shown in Supplementary Figure 1. We next examined the CD80 promoter sequence (extending up to −955 nt) for the presence of nucleosomes. Initial Southern blot hybridization analysis on MNase-digested DNA, using the probes depicted in Figure 1A, yielded a positive signal only with the probe Pr4 (Supplementary Figure 2), although all of the remaining probes were equally capable of hybridizing with the CD80 promoter. We then performed ligation-mediated PCR (LM-PCR) experiments on MNase-digested, mononucleosomal DNA preparations. Using nested primer sets internal to each of the probe sequences shown in Figure 1A, we again obtained amplification in both 3′- and 5′-directions only when primer sets from within the Pr4 regions were employed (Figure 1B). These results indicated the presence of a solitary nucleosome that was positioned between −693 and −545 nt with reference to TS1. This was further confirmed by mononucleosome chromatin immunoprecipitation (ChIP) assays using antibodies specific for H3 histone (Supplementary Figure 2). A similar analysis on the B lymphoma cell line, A20, and on splenocytes derived from BALB/c mice gave identical results, confirming that the nucleosomal positioning described here was not artefactual to J558L cells (Supplementary Figure 2). Finally, in vivo DNase I footprinting analysis of this nucleosome yielded the typical 10 bp ladder (Figure 1C, lanes 3 and 4), establishing that this single nucleosome was both translationally and rotationally positioned (illustrated in Figure 1D). Figure 1.MNase and DNase analysis-based positioning of a solitary nucleosome. (A) The probes used for Southern blot analysis and the start site (arrow) are depicted in a line diagram (not to scale). (B) LM-PCR-based positioning of the nucleosome. Lane 1 represents the amplified product of Primer A, and lane 2 of Primer B as depicted in (D). The lengths of LM-PCR products are indicated on right. The number in parentheses corresponds to the lengths minus that of the 25 bp linker. M is pBR322MspI-digested marker. Lanes 3–6 are products of sequencing for accurate positioning. (C) In vivo DNase I footprinting of the positioned nucleosome. Lanes 1, 2 represent naked DNA and lanes 3, 4 represent nuclei digested with DNase I. M is pBR322MspI-digested marker. Arrows indicate laddering in lanes 3 and 4. (D) Schematic representation of mouse CD80 proximal promoter region. A and B represent primer positions used for LM-PCR mapping. The shaded oval represents the positioned nucleosome. (E) Time kinetics of histone acetylation in response to H2O2 stimulation, as determined by chromatin immunoprecipitation (ChIP). Antibodies specific for the various acetylated derivatives of the lysine residues are indicated and the specificity of immunoprecipitation was ensured in parallel experiments where the primary antibody was omitted (NA). (F) Time-dependent recruitment of the indicated nucleosome modifiers, following H2O2 stimulation of the cells. Results are representative of four separate experiments. Download figure Download PowerPoint Stimulation of cells with H2O2 induces histone acetylation and nucleosome remodeling Stimulation also led to histone acetylation, which was first detectable by 15 min. Acetylation occurred at both H3 and H4 histones, with a temporal increase in the spectrum of lysine residues that were acetylated (Figure 1E). Both Lys14 of H3 and Lys18 of H4 are known substrates for PCAF/GCN5, implying that these early acetylation events are probably PCAF dependent (Grant et al, 1999; Roth et al, 2001). This is consistent with our findings that, in addition to HDAC2 and HDAC6, PCAF was also constitutively associated with the promoter region (Figure 1F). Whereas the association of HDAC2 remained stable, stimulation of cells led to an immediate dissociation of HDAC6 (Figure 1F). Thus, we postulate that the H2O2-induced dissociation of HDAC6 leads to a shift in steady-state equilibrium from HDAC to PCAF-associated HAT activity, and the consequent activation of histone acetylation events. That treatment of cells with the HDAC inhibitor, TSA, yields a similar profile of histone acetylation (Supplementary Figure 3) supports such a possibility. The subsequent expansion of the spectrum of lysine residues acetylated is probably due to the recruitment of CBP that is first detected at 30 min of poststimulation, to then saturate by 1.5 h (Figure 1F). On the other hand, the transience of the acetylation at Lys 8 and 23 of H3 could be a consequence of the HDAC5 engagement observed at 3 h (Figure 1F). Acetylation also led to recruitment of BRG1, the ATPase subunit component of SWI/SNF remodeling complexes. This was initiated by 15 min poststimulation, with saturation being obtained by 30 min (Figure 1F). Since ChIP assays on mononucleosome preparations revealed that BRG1 was recruited on the nucleosome (Supplementary Figure 3), we examined if there was any reconfiguration of the nucleosome as a consequence. While nucleosome sliding was not detected (not shown), increased accessibility of the AccI restriction site indicated in Figure 2A was, however, observed. Stimulation of cells with H2O2 led to a substantial increase in the accessibility of this site by 30 min poststimulation, which then remained constant up to 4 h (Figure 2B). Thus, stimulation of J558L cells with H2O2 results in rotational, but not translational, repositioning of the nucleosome. Figure 2.Stimulation with H2O2 induces remodeling of the nucleosome. (A) Schematic representation of the restriction enzyme sites analyzed in (B) corresponding to the positioned nucleosome. (B) Restriction enzyme accessibility was performed on unstimulated and H2O2-stimulated J558L cells for the indicated time points. Arrows to the right denote the fragments resulting from in vitro and in vivo digestion, respectively. M is pBR322MspI-digested marker. (C) Time course for the recruitment of indicated factors as analyzed by ChIP in unstimulated and H2O2-stimulated J558L cells. The specificity of immunoprecipitation was ensured in parallel experiments where the primary antibody was omitted (NA). The corresponding time kinetics of CD80 mRNA in unstimulated (0.0) and H2O2-stimulated cells is represented in the lowest panel. The results are representative of four separate experiments. (D) Restriction enzyme accessibility was performed on J558L cells transfected with BRG1 and BRM targeting-siRNA that were either unstimulated (lane1), or stimulated with H2O2 for 1 h(lane2). A parallel set of cells was transfected with nonsilencing siRNA (GFP-specific), prior to stimulation with H2O2 (lane 3). The corresponding products are indicated. Results are from one of three experiments. (E) J558L cells were either transfected with siRNA targeted to BRG1 and BRM or to GFP (Mock). After 24 h, they were stimulated with H2O2 for 1 h and ChIP was performed with the indicated antibodies. Download figure Download PowerPoint Increased accessibility of the AccI site at 30 min poststimulation was also observed for BALB/c mouse splenic B cells stimulated with H2O2. In contrast, this site was already accessible in unstimulated A20 cells—a cell line that constitutively expresses high levels of CD80—with no further modification upon stimulation (Supplementary Figure 3). Collectively, these findings point to the relevance of nucleosome remodeling in defining the transcriptional status of the CD80 gene. Nucleosome remodeling facilitates formation of the PIC Components of the PIC, RNA polymerase II (Pol II), TFIIB, and TBP were also all recruited by 30 min of stimulation of cells with H2O2 (Figure 2C). This time point correlates with that of the nucleosome remodeling event (see Figure 2B), suggesting a causal relationship between these two events. Significantly, soon after its recruitment, Pol II was phosphorylated in the C-terminal domain (CTD) of its largest subunit at Ser5 (Figure 2C), indicative of its acquisition of an initiation-competent state. Phosphorylation at Ser2 of the CTD, a marker for entry of Pol II into the elongation phase (Prelich, 2002), was detected by 2 h (Figure 2C)—consistent with the time course of appearance of the CD80 transcript. We also detected the constitutive association of HMGI(Y), Sp1, and C/EBPβ with the promoter (Figure 2C), and three distinct HMG-binding sites located between −87 ando −72 nt, −380 and −363 nt, and −542 and −536 nt could be identified (Supplementary Figure 4). While HMGI(Y) and Sp1 associations were stable, C/EBPβ dissociated by 1.5 h after stimulation. Significantly, C/EBPβ was found to be coassociated with Cdk8, implying its presence in a nonactivated form wherein it functions as a repressor of gene expression (Figure 2C) (Mo et al, 2004). Finally, our ChIP experiments revealed that NF-κB complexes were also recruited at 30 min of stimulation, and this association then remained stable for the remainder of the experiment (Figure 2C). Importantly, inhibition of NF-κB recruitment with specific inhibitors (described below) also prevented the dissociation of C/EBP and Cdk8 (not shown). Although both nucleosome remodeling and recruitment of NF-κB, along with components of the PIC, occurred simultaneously (Figure 2B and C), we established that the recruitment events were in fact dependent upon the former process. First, inhibition of NF-κB activation with either capsaicin (Singh et al, 1996) or the NEMO peptide (May et al, 2000) yielded no significant effect on H2O2-induced nucleosome remodeling (not shown), suggesting that NF-κB does not mediate nucleosome remodeling. We then attempted to inhibit nucleosome remodeling by using the approach of siRNA. To avoid possible complications due to redundancy, we employed a combination of siRNAs that targeted both the BRG1 and BRM genes. Silencing of BRG1/BRM expression led to a marked suppression of H2O2-induced remodeling at the nucleosome (Figure 2D). Further, association of both NF-κB and the PIC components was also inhibited in BRG1/BRM-silenced cells, but not in those transfected with nonsilencing siRNA (Figure 2E). Thus, nucleosome remodeling constitutes an obligatory prerequisite for the recruitment of NF-κB, in addition to assembly of the PIC. A role for NF-κB in CD80 regulation An analysis of the promoter sequence for the presence of NF-κB target sites revealed only a cognate half-site for p50 binding (5′-GGGAA-3′), located between −545 and −541 nt relative to TS1. Interestingly, this sequence was present towards the 3′ end of the nucleosome; and 60 nt downstream of the AccI restriction site that displayed increased accessibility upon stimulation of cells with H2O2 (Figure 2B). Electrophoretic mobility shift assays (EMSA) using a double-stranded probe spanning the positions from −551 to −530 yielded retarded mobility for this probe when incubated with nuclear extracts from peroxide-stimulated, but not unstimulated, cells (Figure 3A). Further, binding of the native probe with the extract from stimulated cells could be competitively inhibited with an excess of an unlabeled probe bearing the consensus sequence for NF-κB recognition (Figure 3A). Finally, binding was also substantially inhibited in the presence of antibodies specific for either p50 or p65, and completely inhibited with a combination of both (Figure 3A). Collectively, the results in Figure 3A identify the sequence between −545 and −541 nt as the locus for recruitment of the p50-p65 NF-κB heterodimer, in stimulated cells. Figure 3.NF-κB is critical for recruitment of the pre-initiation complex. (A) EMSA was performed with an oligonucleotide spanning the NF-κB half-site and nuclear lysate obtained from unstimulated J558L cells (lane 1) and H2O2-stimulated cells for 2 h (lanes 2–7). Specificity of shifted protein–DNA complex (lane 2) was assessed through competition with a 100-fold excess of unlabeled oligonucleotide representing the NF-κB consensus sequence (lane 3), and by the absence of binding to a mutant of the oligonucleotide used in lane 2 containing the GG → CC mutation (lane 7). Also shown are the effects of inclusion of antibodies specific for p50 (lane 4), p65 (lane 5), and a mixture of both anti-p50 and anti-p65 antibodies (lane 6). (B) Plasmids containing the native CD80 promoter, or its mutated derivatives, cloned upstream of EGFP (see text) were transfected into J558L cells and examined for EGFP expression by flow cytometry. Profiles obtained in unstimulated cells transfected with either the native (thin line), the NF-κB mutant promoter (thick line), and mock-transfected cells (dotted line) are given in panel ‘a’. Panels ‘b’ and ‘c’ show the extent of EGFP induction (thick line) obtained in cells transfected with native (b) or mutant (c) promoter at 4 h after stimulation with H2O2, along with the corresponding profile in unstimulated cells (thin line). Panel ‘d’ compares EGFP induction in unstimulated cells transfected with the native promoter alone (thin line), along with the p65 expression plasmid (thick line). The subpopulation of cells lacking EGFP expression in the latter group likely represents an untransfected subset. (C) J558L cells were pretreated with capsaicin or NEMO prior to stimulation with H2O2. After 1.5 h, ChIP analysis was performed with the indicated antibodies, using CD80 promoter-specific primers. Parallel experiments established that both capsaicin and the NEMO peptide also inhibit H2O2-dependent CD80 mRNA induction by >85% (data not shown). Download figure Download PowerPoint We next cloned the CD80 promoter fragment between −955 and +372 nt into the EGFP vector such that EGFP expression was now under its control. An additional construct was also generated with a mutation within the cognate NF-κB half-site. These constructs were separately transfected into J558L cells, and EGFP expression analyzed by flow cytometry. Low levels of EGFP expression were detected in unstimulated cells transfected with either construct (Figure 3B, panel a). Stimulation with H2O2 led to a significant increase in EGFP expression in cells transfected with the plasmid encoding the native (Figure 3B, panel b), but not the mutant (Figure 3B, panel c), promoter. These results, therefore, identify a central role for the nucleosome-localized NF-κB binding half-site in regulating H2O2-dependent CD80 expression. Importantly, cotransfection of J558L cells with an NF-κB p65-expressing plasmid along with the above native CD80 promoter-containing EGFP-1 vector resulted in constitutive expression of high levels of EGFP (Figure 3B, panel d). Thus in addition to it being obligatory, the recruitment of NF-κB is alone sufficient to activate the CD80 promoter. To investigate the regulatory role of NF-κB, J558L cells were stimulated with peroxide either in the presence or absence of the inhibitors of NF-κB, and then subjected to ChIP assays using antibodies against p65, CBP, Pol II, TBP, or TFIIB. In addition to inhibition of NF-κB recruitment, the inclusion of NF-κB inhibitors also led to a marked reduction in recruitment of CBP, Pol II, TFIIB, and TBP; with the NEMO peptide showing the more pronounced inhibitory effect (Figure 3C). Thus, NF-κB recruitment appears to be critical for assembly of the PIC. NF-κB-dependent PIC assembly does not require the core promoter domain Since such interactions have been previously described (Schmitz et al, 1995; Vanden Berghe et al, 1999), we next ascertained whether CBP, Pol II, TFIIB, and TBP also associate with p65 in J558L cells. As expected, stimulation of cells with H2O2 led to an increase in nuclear accumulation of phosphorylated p65 (Figure 4A). Phosphorylation of p65 has been reported to enhance DNA binding, and the transactivation activity of p65 containing NF-κB dimers (Naumann and Scheidereit, 1994). Significantly, immunoprecipitation of p65 from peroxide-stimulated cells also resulted in the co-precipitation of CBP, Pol II, TFIIB, and TBP (Figure 4A), supporting that NF-κB can actively provide an interacting surface that contributes towards at least stabilizing PIC assembly/recruitment in the context of CD80 expression. Furthermore, ChIP assays using mononucleosome preparations verified that it was the nucleosome-localized p50-p65 NF-κB heterodimer that was involved in engagement of components of the PIC (Figure 4B). Figure 4.NF-κB nucleates formation of a functional PIC. (A) Nuclear lysates from unstimulated cells (U), cells stimulated with H2O2 for 1.5 h (S) were immunoprecipitated with antibodies specific for p65. Co-immunoprecipitated proteins were detected by Western blot using the antibodies indicated on the right. IgH was used as the loading control, and rabbit anti-mouse IgG was used as the mock-immunoprecipitation control (CL). (B) Nuclei were isolated from formaldehyde-fixed unstimulated and H2O2-stimulated (2 h) J558L cells. Mononucleosome-sized chromatin was immunoprecipitated with antibodies indicated on the left. PCR was performed with primers within the region protected by the nucleosome. The efficiency of MNase digestion was verified by using primers immediately outside the nucleosome (data not shown). (C) The results of a pull-down experiment wherein nuclear extracts from unstimulated (U) and H2O2-stimulated (S) cells were incubated with a biotinylated-ds-DNA fragment representing the native promoter sequence (−753 to +154) are shown. In parallel, nuclear extracts from stimulated cells were also incubated with biotinylated probes that contained a mutation within the nucleosomal NF-κB half-site (M) and a deletion of the region between −120 and +154 nt (D). These probes were then precipitated with streptavidin–agarose, and the bound proteins identified by Western blot using indicated antibodies. Lane C indicates the control group where streptavidin–agarose beads alone were used. Results are from one of four experiments. (D) The deletion mutant (D) described in (C) above was cloned into an EGFP-1 vector (see text), and then transfected into J558L cells. After 36 h, transfected cells were then either left unstimulated (U), or stimulated with H2O2 for 1 h (S), followed by ChIP analysis using the indicated antibodies. Immunoprecipitated DNA was analyzed by PCR using promoter and EGFP-specific primers. Results are a representative of three separate experiments. Download figure Download PowerPoint To rule out the possibility that additional noncanonical-NF-κB-binding sites may also exist, we generated a biotinylated-ds DNA probe representing the segment between −753 and +154 nt relative to TS1. This probe was incubated with the nuclear extract from either stimulated or unstimulated cells and then precipitated with streptavidin–agarose prior to examination by Western blot analysis. Binding of the p65 subunit of NF-κB could clearly be detected when the probe was incubated with extracts from stimulated, but not from unstimulated cells (Figure 4C). Further, coassociation with TBP, TFIIB, and Pol II was also detected in this case (Figure 4C). Importantly, a mutation within the NF-κB cognate half-site completely abolished the ability of the probe to interact with activated NF-κB, as also with TBP, TFIIB, and Pol II (Figure 4C). Thus, the nucleosome-encapsulated domain contains the sole NF-κB-binding site that facilitates PIC assembly. Notably, a 3′ truncation of the above probe from −120 to +154 nt had no effect on the binding of activated p65-containing NF-κB. Further, this 3′-deletion also did not affect the coassociation with Pol II, TBP, and TFIIB (Figure 4C). These findings were additionally verified by in vivo ChIP experiments where the truncated promoter was cloned upstream of EGFP into an EGFP-1 vector, and then transfected into J558L cells. Stimulation of these cells with H2O2 led to assembly of the PIC on the transfected template even in the absence of the core promoter region (Figure 4D). Thus, the above results together suggest that NF-κB recruitment at its cognate half-site is alone sufficient to ensure formation of a stable PIC, and that contributions from the TS1-core promoter domain are not necessary for this process. A significant aspect of the data in Figure 4D is the fact that phopshorylation of CTD on Ser5, but not on Ser2, could also be detected. This suggests that nucleosomal recruitment of the p65-p50 NF-κB heterodimer is alone sufficient to nucleate formation of a mature PIC that is functionally capable of promoter clearance. DNA-encoded structural elements facilitate communication between the nucleosome and TS1 While a functional PIC was assembled at a distal, 5′-upstream, site, transcriptional activation would—nonetheless—require it to interact with TS1 and/or its proximal sequences. To probe whether bending of the intervening DNA sequence could potentially support this long-range effect, we performed T4 ligase-mediated DNA cyclization assays (Kahn and Crothers, 1992) with a 704 bp probe, representing the sequence between −667 and +36 nt. A significant amount of the monomeric circular DNA product was indeed generated in a time-dependent fashion. (Figure 5A). Notably, the presence of HMGI(Y) during the ligation reaction resulted in a concentration-dependent increase in the amount of the closed circular DNA product formed (Figure 5B). Thus, the intervening sequence between the nucleosome and TS1 possess an intrinsic bending ability, which is further stabilized through HMGI(Y) binding. This bending, in turn, is likely to bring the nucleosome to within spatial proximity of the transcription start site (TS1). Figure 5.Stabilization of the intrinsic bending ability of the CD80 promoter. (A) Time-dependent ligation of the region from −667 to +36 of CD80 promoter in the absence of HMGI(Y). Upper panel shows ligation of the CD80-derived segment, whereas the lower panel shows ligation of a control DNA taken from the cDNA region of the murine LAB gene. Reaction products were resolved on an agarose gel (1.2%) followed by staining with ethidium bromide (0.5 μg/ml). Linear DNA (L) molecules run faster than the closed circular products (CC). (B) The results of the ligation reaction that was performed in the presence of increasing concentrations of HMGI(Y) with a constant reaction time of 20 min. (C) Schematic representation depicting Sau3AI and HindIII cut sites. Sau3AI has multiple cut sites within the CD80 promoter but one of the fragments spanning −770 to +165 relative to the start site defines the region of interest. HindIII cuts once within the promoter at −217 bp. PCR primers spanning −427 to +53 bp are represented by arrows. (D) 3C assay-based ligation products detected by PCR. Lane 1–3 are unstimulated, lanes 4–7 are stimulated with H2O2 (2 h), and lane 5 is pretreated with the NEMO peptide prior to stimulation. Lanes 1 and 7 are not subjected to either restriction digestion or ligation. Lanes 2 and 6 are digested with Sau3AI and HindIII only. Lanes 3–5 were first restriction digested followed by ligation. M is the 1 kb Promega ladder. (E) The time kinetics for loop configuration as identified by 3C assay on J558L cells stimulated with H2O2 for the indicated time points. Control sample with no ligase is marked as NL. Download figure Download PowerPoint To further confirm the spatial colocalization of the nucleosome and TS1 in situ, we adapted the Chromosome Conformation Capture assay, a technique that permits the identification of DNA segments that are spatially juxtaposed (Dekker et al, 2002). Using the combination of restriction enzymes and the p
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