Department of Biology, University of Konstanz, D-78457 Konstanz, Germany
Correspondence to: H.-g. Hu; E-mail: hong-gang.hu{at}uni-konstanz.de
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Abstract |
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Keywords: CD21/CR2, chromatin immunoprecipitation, DEK, promoter
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Introduction |
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In fact, DEK is an abundant (>106 copies per HeLa cell nucleus) and ubiquitous chromatin protein. The human DEK protein consists of 375 amino acids with a central DNA-binding domain of the SAP/SAF type and several stretches rich in acidic residues. It preferentially binds to cruciform or superhelical DNA and shows little, if any, sequence specificity. A remarkable feature of DEK is its ability to induce positive superhelical turns into SV40 minichromosomes and closed circular DNA (review: 5). It is thus likely that DEK functions as an architectural protein in chromatin. Indeed, over-expressed DEK has been detected in a complex with the co-repressor Daxx and an associated histone deacetylase (6) and with other transcription factors such as AP-2 (7) indicating that it may modulate the structure of chromatin around active genes.
To better understand its in vivo function on chromatin, we decided to investigate the distribution of DEK around the developmentally regulated promoter of the gene encoding the human CD21/complement receptor 2 (CR2). This gene is primarily expressed in B lymphocytes and follicular dendritic cells as well as in subsets of thymocytes and epithelial cells. During B cell development, CD21 is present in early activated and mature B cells, but not in pro- and pre-B cells, and is absent in lymphocytes at the plasma cell stage. CD21 in a complex with CD19 and CD81 forms the receptor for activating fragments of the complement component C3 and amplifies the B cell response to antigens by several orders of magnitude. The CD21 receptor is also involved in negative regulation during the selection of autoreactive B cells and absence or de-regulated expression is linked to autoimmunity (review: 810).
The expression of the CD21/CR2 gene is regulated by signal transduction pathways that target proximal promoter elements (within 120 bp around the transcriptional start) as well as regulatory sequences in the first intron (1113). These sequence elements are highly conserved between mouse and man and serve as binding sites for a host of negatively and positively acting transcription factors including SP1, AP-1/AP-2, nuclear factor -
B, NFAT-4, YY1 and others (1418).
Furthermore, gene activity is controlled by the conformation of chromatin since both the promoter-proximal and the intronic region contain DNase-I-hypersensitive sites in CD21-expressing cells but not in non-expressing cells (13, 19); potent inhibitors of histone deacetylases stimulate gene expression in a cellular background where the CD21 gene is normally silent (20). In fact, the intronic regulatory region, termed complement receptor 2 silencer (CRS) (13), includes a binding site for the C promoter-binding factor 1 protein, a component of the Notch signaling pathway, that is known to attract a histone deacetylase and may therefore be responsible for the silencing of the CD21 gene in non-expressing cells (21).
Thus, the regulatory region of the CD21 gene constitutes an array of adjacent positive and negative sequence elements conferring cell type specificity and developmental regulation and is therefore well suited to investigate whether the DEK protein associates with particular elements, and, if yes, whether this is associated with CD21 expression.
To address this point, we compare B cell lines representing two developmental stages, namely Ramos, a line of mature B cells shown to express the CD21 gene (see below), and Nalm-6, a pre-B cell line which does not express CD21.
The procedure that we have chosen is the immunoprecipitation of cross-linked chromatin by DEK-specific antibodies [chromatin immunoprecipitation (ChIP)] and the identification of specific cross-linked DNA sequences by quantitative PCR. We present evidence showing that DEK occurs at many sites in the CD21 gene regulatory region with a clear preference for a promoter-proximal site in active genes.
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Methods |
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For 5-aza-2'-deoxycytidine (aza-dc) treatment, Nalm-6 cells were diluted to 3.5 x 105 ml1 and allowed to grow overnight. Freshly prepared aza-dc was added to a final concentration of 4 µM and the cells were allowed to grow for 24, 48 or 72 h.
Reverse transcriptionPCR for endogenous CD21 expression
Total RNA from Nalm-6 and Ramos cells were isolated using reagents and protocols of the RNeasy RNA isolation kit (Qiagen). The reverse transcription (RT) reaction was performed using 2 µg total RNA primed with oligo(dT) according to the protocol of ThermoScriptTM RTPCR System (Invitrogen). Reverse transcripts were used in the PCR reaction with the following primers: CD21, 5'-ATCACCTCCCCCTATTCTCA-3' and 5'-GTCTATCTTCCTTT TGCCATT-3' (corresponding to positions 900920 and 13211342 of CD21 cDNA sequence) (22) and GAPDH, 5-CATCATCTCT GCCCCCTCT-3' and 5'-GCCTGCTTCACCACCTTCT-3' (corresponding to positions 432450 and 851869 of GAPDH cDNA sequence) (23). The cycling protocol used was 90 s at 94°C, then 30 s at 94°C, 30 s at 55°C and 60 s at 72°C for 30 cycles and finally 7 min at 72°C. PCR products were analyzed by electrophoresis on a 1.5% agarose gel.
In vivo cross-linking and nucleoprotein preparation
Formaldehyde was added to the Nalm-6 and Ramos cells at a final concentration of 1% and incubated at 37°C for 2 min if not otherwise indicated (24). The reaction was stopped by the addition of glycine to a final concentration of 0.125 M. The cells were collected by centrifugation and washed three times with cold PBS. All centrifugation steps were carried out at 670 x g for 5 min at 4°C. The cells were re-suspended in hypotonic RSB buffer (10 mM TrisHCl, 3 mM MgCl2, pH 8.0). All buffers contained 10 mM sodium bisulfite (pH 8.0) as a protease inhibitor. After 10 min incubation with RSB buffer on ice, the cells were disrupted by Dounce homogenization. The nuclear material was collected by centrifugation and washed twice in RSB buffer and once in high-salt SNSB buffer (1M NaCl, 10 mM TrisHCl, 0.1% NP-40, 1 mM EDTA, pH 8.0) and then incubated on ice for 5 min. Finally, the nuclear material was re-suspended in low-salt NSB buffer (0.1 M NaCl, 10 mM TrisHCl, 0.1% NP-40, 1 mM EDTA, pH 8.0) and loaded onto a step gradient consisting of 1.3, 1.5 and 1.75 mg ml1 CsCl in gradient buffer (20 mM TrisHCl, 1 mM EDTA, 0.5% sarcosyl; pH 8.0). Nucleoprotein complexes were collected after ultracentrifugation (37 000 revolutions per minute, 24 h and 18°C) and dialyzed against TE (10 mM TrisHCl, 1 mM EDTA, pH 7.4) overnight at 4°C. Nucleoprotein was then sonicated by a total number of 100 pulses on ice. Nucleoprotein fragments <1 kb were obtained by digestion with 7 U micrococcal nuclease mg1 nucleoprotein in the presence of 3 mM CaCl2 for 15 min at 37°C. The reaction was stopped by adding EDTA to a final concentration of 20 mM. The nucleoprotein fragments were analyzed on a 1% agarose gel (25).
ChIP
Immunoprecipitations were performed in NET buffer (50 mM TrisHCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 0.5% NP-40). Nuclease-digested nucleoprotein was centrifuged for 10 min at 15 000 x g at 4°C. The supernatants from 1 mg of digested nucleoprotein were incubated with 10 µg of affinity-purified DEK antibodies, 6 µg of AP-2 antibodies (SantaCruz Biotechnology) from rabbit or 10 µg of control rabbit IgG overnight at 4°C on a rolling platform. Then 50 µl of 50% protein A-sepharose was added and further incubated for 4 h at 4°C. Immunocomplexes were washed eight times with RIPA (50 mM TrisHCl, pH 8.0; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS), three times with LiCl buffer (10 mM TrisHCl, pH 8.0; 250 mM LiCl; 0.5% NP-40; 0.5% sodium deoxycholate; 1 mM EDTA) and five times with TE buffer. Beads were transferred to new tubes after each change of buffer in order to reduce contamination of unspecific DNA sticking to the tube walls. The washed precipitates were divided for protein and DNA extraction.
For western blotting experiments, proteins were eluted with 2% SDS in H2O twice for 15 min at 37°C. Cross-links were reversed by incubation at 65°C for 4 h and proteins were extracted with methanol/chloroform (26). Input and supernatant were treated accordingly. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes and treated with monoclonal DEK antibodies from mouse.
For DNA extraction, nucleoproteins were eluted with 1% SDS in TE twice at 37°C for 15 min. Proteins were digested with 200 µg ml1 protease K overnight at 37°C. Cross-links were reversed by incubation at 65°C for 4 h and DNA was purified by the standard phenolchloroform extraction, ethanol precipitation and dissolved in 40 µl TE. One-twentieth of the sample was used for quantitative real-time PCR.
Quantitative real-time PCR
Real-time PCR was performed with the Light Cycler instrument (Roche Diagnostics) using a ready-to-use hot start reaction mix (FastStart DNA Master SYBR Green I, Roche Diagnostics). The mix contains Taq DNA polymerase and a fluorescent dye, SYBR Green I, for real-time detection of double-stranded DNA. Reactions were set up in 10 µl volumes including 0.5 mM of each primer. PCR reactions were performed at 45 cycles routinely, using the standard settings recommended by the manufacturer. Annealing temperatures of individual primer pairs are indicated in Table 1. Standard DNA samples (human genomic DNA) were serially diluted to 30 and 3 ng and 300, 30 and 3 pg. After PCR, the x-axis crossing point of each standard sample was plotted against the logarithm of concentration to produce a standard curve. Genomic equivalents of DNA samples were determined by extrapolation from the standard curve (25).
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Results |
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Distribution of DEK
One condition for the determination of DEK at and around the CD21 gene promoter is that the available antibodies react well with and precipitate DEK on cross-linked chromatin. To investigate this point, we compared DEK-specific antibodies with unspecific antibodies in immunoprecipitations. As shown in Fig. 3(A), DEK antibodies, but not control antibodies, recognized and precipitated DEK, covalently linked to chromatin, although a substantial fraction of cross-linked DEK remained in the supernatant. The immunoprecipitable fraction could not be increased using higher antibody concentrations (not shown), suggesting that DEK in the supernatant fraction of cross-linked chromatin is either not accessible to antibody or altered by formaldehyde treatment such that it is unable to react with the antibody. In either case, we have extracted and then analyzed the DNA in immunoprecipitated chromatin by quantitative PCR in a Light Cycler system.
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We first determined the amount of amplifiable DNA in the input, i.e. in the DNA prepared from cross-linked chromatin before immunoprecipitation. These values were used as reference in comparisons with the amount of amplifiable DNA in the immunoprecipitates. As a first result, we note that unspecific antibodies precipitated very little amplifiable DNA (IgG in Fig. 3D), while DEK-specific antibodies precipitated significant amounts of all CD21 gene sequences tested. For comparisons, we expressed the results as the ratio of amplifiable specific DNA in the precipitates relative to the specific DNA in the input (Fig. 3D).
The data for the Nalm-6 cells show that all investigated CD21 gene sequences appeared in immunoprecipitated chromatin in similar amounts, namely between 5 and 10% of the input CD21-specific DNA sequences. This indicates that DEK seems to be rather uniformly distributed along the length of the non-expressed CD21 gene (Fig. 3D, left panel).
We also detected all amplifiable CD21 gene sequences in the immunoprecipitated chromatin from Ramos cells but recovered 2- to 3-fold higher amounts of the promoter-proximal DNA than of DNA from the other parts of the gene (Fig. 3D, right panel). This was found in four independent experiments and indicates that more DEK assembles at a promoter site of an active CD21 gene.
As a control, we investigated whether transcription factor AP-2, known to be involved in CD21 gene regulation (11, 17, 29), was present on the promoter sequence. For this purpose, we used AP-2-specific antibodies for the precipitation of cross-linked chromatin and analyzed the precipitated DNA as in Fig. 3(D). Our results indicate that AP-2, just as DEK, accumulated at the CD21 promoter in Ramos but not in Nalm-6 cells (Fig. 3E). This is interesting as Campillos et al. (7) had shown that DEK and AP-2 functionally interact as DEK modulates the transcriptional activation of AP-2.
Effects of aza-dc
It has been determined that the cytosine residues in a promoter-proximal CpG island are extensively methylated in the inactive CD21 gene and that a treatment with aza-dc induces demethylation and the expression of the CD21 gene in pro-B cell lines, Nalm-16 and KM3 (27). If the accumulation of DEK at the CD21 gene promoter were linked to gene expression, we would expect that more DEK appeared at the promoter of Nalm-6 cells after aza-dc treatment. In Fig. 4(A), we confirm that CD21-specific mRNA appears in Nalm-6 at 4872 h after addition of aza-dc, although the level of mRNA did not reach the level of CD21-specific mRNA in Ramos cells.
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In conclusion, we showed that the chromatin protein DEK is more or less evenly distributed over the analyzed parts of the active and inactive CD21 gene and that 23 times more DEK assembles at promoter-proximal sites in lymphocytes that actively express the CD21 gene.
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Discussion |
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We show here that DEK can occur at many sites of the CD21 gene in lymphocytes regardless of whether the gene is actively transcribed or not. Clearly, however, DEK accumulates 2- to 3-fold at a promoter-proximal region in cells with active CD21 genes. Thus, DEK, like many other global chromatin-modifying activities, seems to be widely distributed on chromatin but can also be recruited to specific sites such as the promoters of expressed genes. The accumulation of DEK on the active CD21 promoter is accompanied by a binding of the transcription factor AP-2 known to be involved in CD21 gene regulation. This is most likely of physiological significance as DEK has been described as being able to modulate the transcriptional activity of AP-2 (7).
The binding of DEK occurs on a sequence which includes a CpG island. This CpG island is known to be highly methylated in pro- and pre-B cells, which do not express CD21, and to be un- or hypomethylated in mature CD21-expressing B cells (27). Decreasing the methylation status by aza-dc results in an expression of the CD21 gene in pro- and pre-B cells, including Nalm-6 (27), and this, as we show here, is accompanied by an enhanced binding of DEK. As a control, we searched for but failed to detect an accumulation of DEK on the promoter of the (silent) CD21 gene in HeLa cells. Conversely, however, we detected significant amounts of DEK on a genetic control region of the (active) TOP1 gene in HeLa as well as in proliferating Ramos and Nalm-6 cells (Hu et al., manuscript in preparation) excluding the possibility that Nalm-6 cells may be intrinsically unable to recruit DEK to gene promoters.
We speculate that DEK with its preference for superhelical and bent DNA may function to stabilize a higher order DNA structure as requirement for gene expression (review: 5). The precedent here is the better-characterized class of architectural chromatin proteins of the HMG-A family. Protein HMG-A1a has been shown to be an essential constituent of a complex of transcription factors (enhanceosome) at the human IFN-ß gene (31, 32) where it functions to bend the DNA. It is a task for the future to determine whether DEK may perform a related function at the CD21 promoter.
Finally, we like to point out that ChIP is a useful procedure for the investigation of the distribution of non-histone proteins such as DEK in the chromatin. However, it has its limitations. First, not all cross-linked chromatin was accessible or responsive to DEK-specific antibodies, either because DEK may be buried among other chromatin components or because it is so altered by formaldehyde that it no longer reacts with the antibody. We can therefore not exclude the possibility that it may be a specific subset of DEK that was detected and analyzed. Second, the screening of DNA-binding sites relied on PCR amplification of DNA in immunoprecipitated chromatin. It was impractical to use primer pairs that cover the entire genomic region under investigation. We have instead selected particular primer pairs that correspond to sequences of established or assumed regulatory function. Therefore, we could have missed additional gene regions that may or may not be enriched relative to the average immunoprecipitated chromatin.
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Acknowledgements |
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Abbreviations |
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aza-dc | 5-aza-2'-deoxycytidine |
CR2 | complement receptor 2 |
ChIP | chromatin immunoprecipitation |
CRS | complement receptor 2 silencer |
FBS | fetal bovine serum |
RT | reverse transcription |
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Notes |
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Received 2 September 2004, accepted 14 March 2005.
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References |
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