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Nucleic Acids Research 27:3881-3890 (1999)
© 1999 Oxford University Press

Article

Presetting of chromatin structure and transcription factor binding poise the human GADD45 gene for rapid transcriptional up-regulation

Dawn M. Graunke, Albert J. Fornace Jr1 and Russell O. Piepera

DNA Damage and Repair, Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Room C2058-7, Baton Rouge, LA 70808, USA and 1Division of Basic Science, National Cancer Institute, Bethesda, MD 20982, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GADD45 has been suggested to coordinate cell cycle regulation with the repair of DNA damage following ionizing radiation (IR). Although the GADD45 gene is transcriptionally up-regulated in response to IR, alterations in in vivo transcription factor (TF) binding or chromatin structure associated with up-regulation have not been defined. To understand how chromatin structure might influence TF binding and GADD45 up-regulation, key regulatory regions of the gene were identified by in vivo DNase I hypersensitivity (HS) analysis. Chromatin structure and in vivo TF binding in these regions were subsequently monitored in both non-irradiated and irradiated human ML-1 cells. In non-irradiated cells expressing basal levels of GADD45, the gene exhibited a highly organized chromatin structure with distinctly positioned nucleosomes. Also identified in non-irradiated cells were DNA–protein interactions at octamer binding motifs and a CCAAT box in the promoter and at consensus binding sites for AP-1 and p53 within intron 3. Upon irradiation and a subsequent 15-fold increase in GADD45 mRNA levels, neither the chromatin structure nor the pattern of TF binding in key regulatory regions was altered. These results suggest that the GADD45 gene is poised for up-regulation and can be rapidly induced independent of gross changes in chromatin structure or TF binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The GADD gene family consists of genes cloned on the basis of their up-regulation following DNA damage and/or growth arrest (1,2). The most widely characterized of these genes is GADD45. In human cells, GADD45 is up-regulated in response to growth arrest conditions or after exposure to ultraviolet radiation (UV), hydrogen peroxide, methyl methane­sulfonate (MMS) or ionizing radiation (IR) (1,3). The direct action of increased levels of GADD45 protein remains uncertain although increases in GADD45 protein levels are correlated with a block in S phase progression as well as a stimulation of excision repair (4–6). GADD45 has, therefore, been suggested to link the processes of cell cycle arrest with DNA repair. As such, an understanding of the mechanism by which GADD45 is up-regulated is important in understanding the cellular response to DNA damage.

Up-regulation of GADD45 occurs primarily at the transcriptional level and appears to follow one of two pathways. Under conditions of growth arrest or following exposure of cells to UV or MMS, GADD45 expression is up-regulated 5- to 10-fold (3). Because similar up-regulation was noted in transient gene expression systems in which the GADD45 promoter was linked to a reporter gene, GADD45 up-regulation in response to UV has been suggested to be promoter-dependent (3). Although it remains unclear which sequences in the GADD45 promoter control GADD45 up-regulation, a variety of consensus binding sites for transcription factors have been identified, including those for WT-1 and POU family members (3). Transcriptional up-regulation mediated by the GADD45 promoter therefore potentially involves the action of a number of transcription factors. While GADD45 up-regulation following UV exposure or stress is promoter-dependent, a second regulatory pathway is activated following exposure of cells to IR. This pathway is more complex than that noted following UV exposure in that it appears, at least in transient transfection systems, to require intron 3 sequence of the GADD45 gene in addition to promoter sequence (7,8). The intron 3 region of the GADD45 gene contains a consensus p53 binding site and may involve DNA–protein interactions in both the promoter and intron 3 regions and potentially interactions between complexes in both regions (8).

Although the identification of transcription factor binding sites in the GADD45 gene has proven useful in beginning to understand GADD45 regulation, an important and as yet uninvestigated factor in GADD45 gene regulation is chromatin structure. It is becoming increasingly appreciated that while information can be derived by studying TF/DNA interactions in vitro or in transient transfection systems, many interactions in vivo require transcription factors to gain access to and bind to recognition sequences. This accessibility is in turn dependent upon the chromatin organization of regulatory sequences. For many inducible genes (e.g. steroid receptor targets and the yeast PHO5 gene) up-regulation first requires the movement or ‘remodeling’ of nucleosomes (9,10). The changes in chromatin structure allow TF binding which in turn drives gene expression. For other inducible genes, regulatory regions are maintained in a conformation in which nucleosomes are arranged around transcription factor binding sites with TF free to bind at all times (11–13). In these so-called ‘preset’ genes, up-regulation is independent of chromatin remodeling and nucleosomal movement but rather is dependent upon the binding of newly synthesized TF or on the modification of prebound TF (11–13). With regard to the GADD45 gene, it is not known how chromatin structure relates to TF binding in the presumed regulatory regions of the gene nor is it known whether induction of expression involves changes in this chromatin structure. As the chromatin structure of regulatory regions shapes the series of events necessary for the induction of gene expression, we examined the chromatin structure of the GADD45 gene in vivo both prior to and following IR and correlated the chromatin structure with TF binding. The results of this study show that prior to up-regulation, the GADD45 gene is highly organized, with nucleo­somes positioned around but not within the promoter and intron 3 regions. These accessible regulatory regions of DNA are in turn occupied in non-irradiated cells by a variety of TFs. Surprisingly, upon irradiation and GADD45 up-regulation, neither the chromatin structure of the GADD45 gene nor the pattern of TF binding in the key regulatory regions was altered. These results suggest that the presetting of both chromatin structure and TF binding poise the GADD45 gene for rapid transcriptional up-regulation following DNA damage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and cell treatment
A human myeloid leukemia cell line (ML-1) was grown in RPMI-1640 medium (2 mM L-glutamine, penicillin/streptomycin and 10% bovine calf serum). For the irradiation studies, log phase ML-1 cells (1 x 106 cells, 50 ml) were exposed to 20 Gy of {gamma}-irradiation using a 137Cs source (5.7 Gy/min). Following irradiation, cells were collected by centrifugation and either harvested immediately or resuspended in fresh growth medium and incubated at 37°C for 4 h prior to analysis. ML-1 cells exposed to chemical agents were incubated in fresh medium containing the agent prior to exposure to 20 Gy of IR and then either harvested immediately or grown for an additional 4 h prior to harvesting.

DNA probes used in northern blot analysis and in DNase I/MNase hypersensitivity assays
A 1.4 kb GADD45 cDNA and a 900 bp glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA were used as probes for northern blot analysis. The 1.4 kb GADD45 cDNA probe was isolated from the pHUL145B2 plasmid by sequential digestions using the restriction enzymes KpnI and NotI. The final digestion products were size fractionated through a 0.8% low melting temperature agarose gel. The KpnI–NotI fragment of ~1.4 kb was excised from the gel and purified by centrifugation through glass wool (13 000 g, 30 s). The 900 bp GAPDH cDNA was isolated from a plasmid (provided by Mark R. Kelley, Indiana University) by PstI digestion and agarose gel purified as described above.

Two probes were used in the hypersensitivity assays. The 5'D probe (nt –816 to nt –232) recognizes the 5'-end of a 3.3 kb BamHI fragment of the GADD45 gene (nt –816 to nt +2455) (Fig. 1C) and was isolated by BamHI and BssHII digestion of a plasmid (pHg45) containing the entire GADD45 gene. The 584 bp BamHI–BssHII DNA fragment was gel purified as described above. The 3'D probe (nt +1721 to +2499) recognizes the 3'-end of the same 3.3 kb BamHI fragment of the GADD45 gene (Fig. 1C) and was isolated by restriction enzyme digestion of the pHg45 plasmid using BamHI and EcoRI. The resulting 778 bp fragment was gel purified as described above.



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  		Figure 1. The GADD45 gene is transcriptionally up-regulated in response to IR. (A) Northern blot analysis of GADD45 expression in ML-1 cells following 20 Gy of IR. (B) Northern blot analysis of the effect of ActD on GADD45 expression in non-irradiated and irradiated cells. (C) A schematic representation of the 3.3 kb BamHI fragment of the GADD45 gene. The white box represents the CpG island sequence, shaded boxes represent exons of the GADD45 gene and the arrow at +1 marks the transcription start site. Braces denote the regions of the GADD45 gene in which DNA–protein interactions were investigated. The location of DNA probes used in DHS and MNase HS analysis are noted.

 
DNA probes were uniformly radiolabeled using the random primer method (Prime-A-Gene; Promega) incorporating [{alpha}-32P]dCTP (sp. act. >1 x 109 c.p.m./µg). For use in DNase I and MNase hypersensitivity assays, either a 1 kb DNA ladder or a 100 bp DNA ladder were radiolabeled using [{gamma}-32P]ATP by the phosphate exchange reaction (Gibco Life Technologies). Following labeling and removal of unincorporated nucleotides by spin column chromatography, incorporation of 32P was quantified using a scintillation counter.

Analysis of GADD45 mRNA up-regulation in response to IR
RNA from 1 x 107 ML-1 cells (irradiated and non-irradiated) was isolated using a standard guanidinium isothiocyanate lysis procedure (14). RNA (15 µg) was fractionated through a 0.8% denaturing agarose gel and transferred to a nylon membrane by capillary action. The membrane was prehybridized in a solution of 50% formamide, 5x SSPE (1 M NaCl, 50 mM NaH2PO4, 5 mM EDTA, pH 7.7), 10% dextran sulfate, 1% SDS, 1x Denhardt’s solution, with 0.25 mg/ml sheared salmon sperm DNA for 3 h, after which a 32P-end-labeled 1.4 kb GADD45 cDNA probe was added to a final count of 2 x 106 c.p.m./ml. After hybridization (36 h, 42°C), the membrane was washed as previously described (15). The amount of probe hybridized to the membrane was quantified using a Betascope 603 analyzer (BetaGen). Following removal of the GADD45 probe, the membrane was rehybridized using the radiolabeled GAPDH cDNA probe (15 h, 42°C). Unbound probe was removed from the membrane by washing (6 min, 60°C) in a solution of 0.1% SSPE, 0.1% SDS in a modified Disk-Wisk washing system (Schleicher & Schuell). The amount of probe hybridized to membranes was quantified as described above. Following quantification, membranes were exposed to X-ray film (X-Omat AR-5; Kodak) for 16–20 h to generate auto­radio­graphic data.

The effect of actinomycin D and cycloheximide on up-regulation of GADD45 in response to IR
Control ML-1 cells were incubated with the RNA synthesis inhibitor actinomycin D (ActD, 5 µg/ml, 37°C, 250 min) as previously described (16) prior to RNA isolation. ML-1 cells treated with ActD and exposed to IR were incubated with ActD (10 min, 37°C), irradiated as described above, supplied with fresh medium containing ActD and incubated (4 h, 37°C) prior to isolation of RNA for northern blot analysis. ML-1 cells were treated with cycloheximide (CHX, 5 µg/ml) in a manner consistent with previously published work (17). Control ML-1 cells were incubated with CHX (16 h, 37°C) prior to RNA isolation. Cells treated with both CHX and IR were incubated overnight (12 h) in CHX, irradiated as described above, then supplied with fresh medium containing CHX and incubated (4 h, 37°C) prior to isolation of RNA for northern blot analysis. Northern blot analysis was performed as described above.

Isolation of nuclei for DNase I hypersensitivity assays
ML-1 cells (5 x 107 non-irradiated or irradiated) were collected by centrifugation, washed in ice-cold PBS and lysed in 600 µl lysis buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 2 min, 4°C). Nuclei were collected by centrifugation at 10 000 g for 30 s, washed in ice-cold RSB (10 mM Tris, pH 8.0, 10 mM NaCl, 3 mM MgCl2), resuspended in cold RSB and divided equally among six reactions.

Analysis of DNase I hypersensitivity within the GADD45 gene
Nuclei from ML-1 cells, the equivalent of 25 µg of DNA/sample, were incubated with DNase I (0, 2.5, 5, 10, 25 or 50 U, 10 min, 37°C) and then lysed (20 mM Tris, pH 8, 20 mM NaCl, 20 mM EDTA, 1% SDS, 600 µg/ml proteinase K) (18). Following protein digestion, RNase A (50 µg/ml) was added to the samples and incubated for a minimum of 1 h at 37°C. The DNase I-cleaved DNA was phenol:chloroform extracted, ethanol precipitated, resuspended and digested overnight with BamHI. For preparation of the ‘naked’ DNA samples used in these studies, protein-free DNA from ML-1 cells was incubated with DNase I (0.005, 0.01, 0.02 and 0.05 U, 5 min, 37°C). DNase I was inactivated by phenol:chloroform extraction and the DNA was ethanol precipitated, resuspended and cleaved with BamHI. For Southern blot analysis, DNA (20 µg) was size-fractionated through a 1.1% agarose gel (25 V, 14 h) and transferred to a nylon membrane by capillary action. Either the radiolabeled 5'D or 3'D probe (see Fig. 1C) was hybridized to the membrane-bound DNA as described above (>48 h, 42°C). Unbound probe was removed from the membranes as described above for the GAPDH probe. Autoradiographs were generated by exposing hybridized membranes to X-ray film for 16–20 h.

Ligation-mediated PCR (LMPCR) analysis of DNA–protein interactions
Non-irradiated and irradiated ML-1 cells were analyzed for in vivo DNA–protein interactions within the GADD45 gene by LMPCR. In vivo DMS-treated DNA and LMPCR-suitable DNA devoid of DNA–protein interactions was generated as previously described (19). LMPCR was performed using a modification of the method of Garrity et al. (20). Following primer extension and 18 rounds of PCR amplification using a gene-specific primer and the linker-specific primer, two additional rounds of amplification were performed using a third, nested, 32P-end-labeled primer so that amplification products could be analyzed by autoradiography following polyacrylamide gel electrophoresis. LMPCR primers used in the analysis of the GADD45 promoter and intron 3 regions were: linker primer LLP, 5'-GCGGTGACCCGGGAGATCTGAATTC-3'; GADD45 primers GAD4, 5'-GATCTGTGGTAGGTG­G­G­GGTC-3'; GAD5, 5'-GGAGGGTGGCTGCCTTTGTCC­G­ACT-3'; GAD6, 5'-GGAGGGTGGCTGCCTTTGTCCGACTAGAG-3'; GAD10, 5'-CGCCTCCCGCGTGGCTCCT-3'; GAD11, 5'-CCCTTTT­CCGCTCCTCTCAACCTGA-3'; GAD12, 5'-CCCTTTTCC­G­C­TCCTCTCAACCTGACTCC-3'.

Isolation of nuclei for MNase analysis
Non-irradiated ML-1 cells (5 x 107) were collected, washed once in ice-cold PBS and lysed in 600 µl ice-cold MNase digestion buffer (50 mM Tris, pH 7.4, 60 mM KCl, 3 mM CaCl2, 0.34 M sucrose) plus 0.5% Nonidet P-40 (2 min, 4°C). Nuclei were collected by centrifugation (10 000 g, 30 s), washed and resuspended in ice-cold MNase digestion buffer and divided equally among six reactions.

Analysis of MNase hypersensitivity within the GADD45 gene
Nuclei from non-irradiated ML-1 cells (the equivalent of 25 µg of DNA/sample) were incubated with MNase (0, 0.01, 0.02, 0.04, 0.05 or 0.1 U, 10 min, 37°C), lysed and incubated with RNase A (1 h, 37°C). Following RNase A incubation, the DNA was phenol:chloroform extracted, ethanol precipitated, resuspended and digested using BamHI (10 U/µg DNA, 12 h, 37°C). DNA purified from ML-1 cells was cleaved with MNase (0.01 or 0.02 U, 5 min, 37°C), phenol:chloroform extracted, ethanol precipitated, resuspended and cleaved using BamHI. Southern blot analysis was performed as previously described, using the 5'D probe.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IR-induced up-regulation of GADD45
To verify that GADD45 mRNA levels in ML-1 cells increase following exposure to IR, up-regulation of GADD45 mRNA levels in ML-1 cells was measured by northern blot analysis 4 h following a 20 Gy exposure to IR. In ML-1 cells, GADD45 mRNA levels increased 15.7 ± 1.5-fold over basal levels (n = 7) (representative results shown in Fig. 1A) following exposure to IR, consistent with previously published results (2,6,15). Previous studies have also demonstrated increased transcription of the GADD45 gene following exposure to IR (2). To verify this response, ML-1 cells were incubated with the RNA synthesis inhibitor ActD prior to irradiation. Control ML-1 cells displayed a 15-fold induction of GADD45 mRNA levels 4 h following irradiation, while cells pretreated with a concentration of ActD previously shown to block RNA synthesis in ML-1 cells (16) showed no IR-induced increase in GADD45 mRNA levels (representative results shown in Fig. 1B). To determine the role of protein synthesis in GADD45 up-regulation, ML-1 cells were incubated with the protein translation inhibitor CHX prior to irradiation. While control cells displayed a 15-fold induction of GADD45 mRNA levels following irradiation, cells preincubated with a concentration of CHX that blocked protein synthesis by 98% demonstrated a 6.9 ± 2.7-fold induction of GADD45 mRNA levels following irradiation (data not shown), consistent with previously published results (17). As a whole, these studies verify that GADD45 induction following IR occurs at least in part at the transcriptional level and that this IR-induced GADD45 up-regulation can occur in the absence of protein synthesis.

Localization of sites of DNase I hypersensitivity (DHS) within the GADD45 gene
TF interactions often occur at nucleosome-free, DNase I hypersensitive regions of DNA. In order to localize potential transcriptional control sites within the GADD45 gene in vivo and to determine whether or not these sites changed in number or location following IR, regions of DHS were identified within the GADD45 gene. DHS analysis was performed by isolating nuclei from either non-irradiated or irradiated ML-1 cells and exposing the nuclei to varying concentrations of DNase I. DNA was purified, cleaved with BamHI and subjected to Southern blot analysis using probes complementary to either the 5'- or the 3'-end of a 3.3 kb BamHI fragment of the GADD45 gene (Fig. 1B). As shown in Figure 2A, exposure of nuclei to increasing concentrations of DNase I increased the cleavage of genomic DNA. The results of Southern blot analysis using this DNA and a probe complementary to the 5'-end of the GADD45 gene are shown in Figure 2B. In DNA from non-irradiated nuclei not exposed to DNase I (lane 1), the probe hybridized to a 3.3 kb DNA fragment. In DNA from non-irradiated nuclei exposed to increasing concentrations of DNase I in vivo (lanes 2–5), DNase I cleavage occurred within the 3.3 kb GADD45 BamHI fragment, resulting in gradual disappearance of the fragment. Rather than being random, however, this DNA cleavage preferentially occurred in two regions of the 3.3 kb GADD45 BamHI fragment (upper and lower arrows, Fig. 2B). The locations of these DHS regions were confirmed by rehybridization of the membrane-bound DNA to a probe (3'D) complementary to the 3'-end of the GADD45 BamHI fragment. The 3'D probe hybridized to the 3.3 kb GADD45 BamHI DNA fragment in DNA from non-irradiated cells not exposed to DNase I (lane 1) and to lower molecular weight fragments generated by DNase I cleavage (upper and lower arrows, Fig. 2C). Given the resolution of the technique used, DHS could be localized to ~700–1000 bp and 2.3–2.5 kb from the 3'-end of the 3.3 kb fragment or ~800–1000 bp and 2.3–2.6 kb from the 5'-end of the GADD45 BamHI fragment, corresponding to two regions of DHS located 1400 bp apart (see Fig. 1C). Alignment of the two DHS sites with the GADD45 gene sequence revealed that the identified regions of preferential DNase I cleavage coincided with the promoter and intron 3 regions of the gene. These data suggest that even in the uninduced state, two regions of DHS exist within the GADD45 gene, one in the 5' promoter, the other in intron 3.



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  		Figure 2. DHS exists within the GADD45 gene in both non-irradiated and irradiated ML-1 cells. Nuclei from non-irradiated cells were incubated with increasing concentrations of DNase I. (A) A photograph of an ethidium bromide (EtBr) stained agarose gel following fractionation of DNA. Lanes 1–5, nuclei isolated from non-irradiated ML-1 cells; lanes 6–10, nuclei isolated from ML-1 cells 4 h after exposure to 20 Gy of IR. Lane L, 1 kb DNA ladder. (B) Autoradiograph of Southern blot analysis performed using the 5'D probe. Arrows to the right of the figure denote DNase I cleavage fragments. (C) Autoradiograph generated by stripping the membrane used in (B) and rehybridization using the 3'D probe. Results shown are representative of four independent experiments.

 
Having identified regions of DHS within the GADD45 gene prior to IR, it was next addressed whether these hypersensitive regions were altered following induction of gene expression by IR. Southern blot analyses using nuclei from irradiated ML-1 cells were performed in parallel with studies using nuclei from non-irradiated cells. In DNA samples that had not been exposed to DNase I, the 5' probe recognized the expected 3.3 kb DNA fragment (Fig. 2B, lane 6). In DNA samples exposed to increasing concentrations of DNase I in vivo, the 3.3 kb DNA fragment disappeared as DNase I concentrations increased (Fig. 2B, lanes 7–10). As in the studies using the nuclei of non-irradiated cells, two regions of DHS were identified in the GADD45 BamHI fragment (arrows, Fig. 2B) from irradiated ML-1 cells. The location of these sites was confirmed by rehybridization of the membrane-bound DNA to the 3'D probe, which revealed DNA fragments in the same size range as those identified in non-irradiated cells (arrows, Fig. 2C). The location and size of regions of DHS in irradiated ML-1 cells, as analyzed using either the 5'D or 3'D probe, were identical to the DNase I hypersensitive sites in non-irradiated cells (compare lanes 1–5 to lanes 6–10 in Fig. 2B and C). These data suggest that the regions of DHS in the promoter and intron 3 regions of the GADD45 gene present in non-irradiated cells do not change following up-regulation of GADD45 expression.

In order to confirm that regions of DHS identified by the studies represented in Figure 2 were a result of in vivo chromatin structure and were not merely due to sequence-specific DNase I cleavage, Southern blot analyses similar to those described above were performed with purified, non-chromatin-associated DNA isolated from ML-1 cells (referred to as naked DNA). Incubation of naked DNA in vitro with increasing concentrations of DNase I yielded DNA fragments digested to the same degree as the in vivo DNase I-treated samples (compare Fig. 3A to 2A). In Southern blot analyses of this DNA, the 5'D probe hybridized to a 3.3 kb GADD45 BamHI fragment in naked DNA not exposed to DNase I (Fig. 3B, lane 2). At higher concentrations, DNase I cleaved within the 3.3 kb fragment, resulting in its gradual disappearance (Fig. 3B, lanes 4–6). Unlike the results of in vivo DNase I cleavage, however, there were no sites of preferential DNase I cleavage within the 3.3 kb GADD45 BamHI fragment in naked DNA. These results suggest that preferential DNase I cleavage within the GADD45 gene in nuclei is not due to a sequence preference of DNase I, but rather is due to in vivo chromatin structure.



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  		Figure 3. DHS present in ML-1 nuclei is not present in naked ML-1 DNA. Purified DNA from ML-1 cells was incubated with increasing concentrations of DNase I. The following DNA samples were used: 1 kb DNA ladder (lane 1); BamHI-digested ML-1 DNA (lane 2); 32P-radiolabeled 1 kb DNA ladder (lane 3); DNase I-digested ML-1 DNA (lanes 4–6); 32P-radiolabeled 100 bp DNA ladder (lane 7). (A) Photograph of the EtBr stained gel prior to capillary transfer. (B) Autoradiograph of Southern blot analysis using the 5'D probe. The results shown are representative of three independent experiments.

 
LMPCR analysis of DNA–protein interactions within the GADD45 promoter
As regions of DHS are typically nucleosome free and contain DNA–protein interactions in vivo, the DHS regions of the GADD45 gene were analyzed for DNA–protein interactions in ML-1 cells. Specific DNA–protein interactions in the GADD45 promoter were examined by LMPCR amplification of DNA from ML-1 cells exposed to dimethyl sulfate (DMS). DMS methylates the N7 position of guanine and, to a lesser degree, the N3 position of adenine (21). This modification of DNA is blocked by sequence-specific DNA-binding proteins (e.g. TFs) but is not blocked by nucleosomes (22). DMS-treated DNA can be cleaved at sites of adduction by incubation with piperidine. For LMPCR, the 3'-ends of piperidine-cleaved DNA are ligated to a linker molecule. Following PCR amplification using a gene-specific primer and a linker-specific primer, PCR products are analyzed by polyacrylamide gel electrophoresis and autoradiography. This process results in the generation of a ladder of fragments, the 3'-end of each corresponding to a site of DMS adduction and subsequent cleavage. Using this method, sites of in vivo DNA–protein interactions appear as holes or ‘footprints’ in the LMPCR-generated guanine ladder. Analysis of DNA–protein inter­actions in the GADD45 gene were carried out in non-irradiated ML-1 cells, immediately following IR (identifying rapidly induced DNA–protein interactions) and 4 h following IR (persistent DNA–protein interactions would be noted). An LMPCR analysis of in vivo DNA–protein interactions in the transcribed strand of the GADD45 promoter is shown in Figure 4. LMPCR analysis using the plasmid pHg45, which contains the entire GADD45 gene sequence, yielded a sequence ladder consistent with the published GADD45 sequence (lanes 1 and 9). LMPCR analysis using ML-1 DNA which was purified prior to DMS exposure also yielded the expected sequence ladder (lane 2) and confirmed that the more complex genomic setting of the GADD45 promoter had little effect on DMS reactivity. Results from LMPCR analysis using as template DNA from cells prior to irradiation (B), from cells immediately following exposure to IR (IR-0) and from cells 4 h following irradiation (IR-4) are shown in lanes 3–8. The sequence ladder in lanes 3 and 4 differed slightly from that in lanes 1 and 2 in the disappearance of DNA fragments corresponding to cleavage at guanines –83 and –76 (open circles, Fig. 4). The guanine at nt –83 (lower circle), although not located within one of the consensus TF binding sites in the GADD45 promoter (bars to the left of Fig. 4), was most likely protected by the same DNA–protein interaction as the guanine at nt –76, an interaction at a consensus octamer binding motif. The sequence ladder in lanes 3 and 4 also differed from that in lanes 1 and 2 in the appearance of adenines (nt –96, –92, –53 and –39, marked with asterisks in Fig. 4) hypersensitive to DMS-induced damage and subsequent cleavage. Hyper-reactive adenines at nt –92 and –96 (lower asterisks) were located within an additional octamer binding motif. Adenines at nt –53 and –39 (upper asterisks), while not located within a consensus TF binding site, appeared hypersensitive to DMS-induced damage, most likely due to a DNA–protein interaction within the CCAAT box they flank. These data suggest the presence of three DNA–protein interactions in the GADD45 promoter of ML-1 cells prior to irradiation, two involving the POU family of transcription factors (possibly the ubiquitously expressed Oct-1 protein or the lymphoid cell-specific Oct-2 protein), the other most likely involving the CCAAT box-binding C/EBP protein. The LMPCR-generated sequence ladder from reactions using template DNA from irradiated cells (Fig. 4, lanes 5–8) was identical to the results from amplification of DNA from non-irradiated cells (lanes 3 and 4), suggesting that in the transcribed strand of the GADD45 promoter the DNA–protein interactions present during basal transcription do not change upon GADD45 up-regulation. Similar analyses of DNA from non-irradiated and irradiated ML-1 cells performed using primers specific for the non-transcribed strand of the GADD45 promoter revealed no DNA–protein interactions (data not shown), suggesting that the interactions noted in the GADD45 promoter were single-stranded in nature.



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  		Figure 4. Identification of DNA–protein interactions within the transcribed strand of the DNase I HS GADD45 promoter. LMPCR was performed using primers GAD10–12 and plasmid DNA (P, lanes 1 and 9), naked ML-1 DNA (N, lane 2), DNA from non-irradiated cells (B, lanes 3 and 4) and DNA from cells exposed to IR, either immediately following irradiation (IR-0, lanes 5 and 6) or 4 h after irradiation (IR-4, lanes 7 and 8). Putative TF binding sites are represented by bars to the left of the panel while guanines and adenines involved in DNA–protein interactions are marked with a circle or an asterisk, respectively. Results shown are representative of three independent experiments.

 
LMPCR analysis of DNA–protein interactions within intron 3 of the GADD45 gene
The results of LMPCR analysis of the transcribed strand of intron 3 are shown in Figure 5. LMPCR analysis using as template either a plasmid containing the entire GADD45 gene or purified ML-1 DNA yielded the expected sequence ladder (Fig. 5, lanes 1 and 2). The results of LMPCR analysis using DNA from non-irradiated ML-1 cells immediately post-irradiation (IR-0) and cells 4 h post-irradiation (IR-4) are shown in lanes 3–8. The sequence ladder generated using DNA from non-irradiated cells (lanes 3 and 4) differed slightly from that generated from plasmid DNA and purified DNA (lanes 1 and 2). Specifically, in DNA from non-irradiated cells the guanine at nt +1629 (top open circle, Fig. 5) was protected from DMS adduction and cleavage. This guanine occurs in a consensus AP-1 binding site (bar to the left of Fig. 5), suggesting a DNA–protein interaction at this site during basal expression of the GADD45 gene. Other guanines within intron 3 of the GADD45 gene were also involved in DNA–protein interactions. Two guanines located within a p53 consensus binding site (nt +1588 and +1578) were protected in DNA from non-irradiated cells (lanes 3 and 4) when compared to plasmid or ‘naked’ DNA (lanes 1 and 2, respectively). The p53 consensus binding site within the GADD45 intron 3 has been shown in vitro to be bound by p53 post-IR (23) and with regard to the present data suggests an interaction of the GADD45 intron 3 DNA with p53 prior to irradiation of ML-1 cells. No DNA–protein inter­actions at the consensus p53 binding site were identified on the non-transcribed strand of intron 3 (data not shown), which is consistent with the single-stranded nature of the p53 interaction (23). The sequence ladder generated using DNA from irradiated cells (lanes 5–8) was very similar to that derived from amplification of DNA from non-irradiated cells (lanes 3 and 4), i.e. there were DNA–protein interactions at both the AP-1 and p53 consensus binding sequences. These results suggest that DNA–protein interactions exist within the promoter and intron 3 of the GADD45 gene during basal transcription and that no new DNA–protein interactions are created following induction of GADD45 expression.



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  		Figure 5. Analysis of DNA–protein interactions within the transcribed strand of the DNase I HS intron 3 of the GADD45 gene. LMPCR was performed using primers GAD4–6 with DNA from the following sources: plasmid (P, lane 1); naked ML-1 DNA (N, lane 2); non-irradiated ML-1 cells (B, lanes 3 and 4); cells exposed to IR immediately following irradiation (IR-0, lanes 5 and 6) or 4 h after irradiation (IR-4, lanes 7 and 8). Putative TF binding sites are represented by bars to the left of the panel. Guanines involved in DNA–protein interactions are marked with a circle. Results are representative of three independent experiments.

 
Nucleosome-like positioning in the GADD45 gene
The results of DHS and LMPCR analyses suggest that the GADD45 gene assumes a chromatin structure that favors TF binding in two regulatory regions and that neither this chromatin structure nor TF binding in these regions changes following irradiation. To further define this chromatin structure, nucleosome positioning in the GADD45 gene was investigated using MNase. MNase is an endonuclease that preferentially cleaves DNA between nucleosomes in vivo. Regions of DNA in which nucleosomes are randomly positioned will be randomly digested in vivo with MNase while regions in which nucleosomes are positioned in the same location in all or most cells will display periodic cleavage.

Nuclei from non-irradiated ML-1 cells were isolated and incubated with varying concentrations of MNase. The DNA was purified, digested with BamHI and subjected to Southern blot analysis using the same 5'D probe as used in the DHS analysis. As shown in Figure 6A, increasing the in vivo exposure of nuclei from ML-1 cells to MNase resulted in increased cleavage of ML-1 DNA. Note that because MNase cleaves between nucleosomes in vivo, a nucleosomal ladder results from incubation of nuclei with increased concentrations of MNase (Fig. 6A, lanes 2–5). As MNase cleavage can display sequence specificity, naked ML-1 DNA exposed to varying concentrations of MNase in vitro and cleaved to the same degree as experimental groups was also included in this study (compare lanes 8 and 9 to lanes 4 and 5 in Fig. 6A). Figure 6B shows the results of Southern blot analysis using the 5'D probe. In DNA from nuclei not exposed to MNase, the 5'D probe hybridized to a 3.3 kb DNA fragment (lane 1). DNA isolated from nuclei exposed to increasing amounts of MNase was cleaved within the 3.3 kb GADD45 BamHI fragment. Rather than being random, however, this MNase cleavage of the 3.3 kb fragment preferentially occurred ~400, 560, 720, 820, 950, 2300 and 2500 bp from the 5'-end of the GADD45 BamHI fragment (range noted by the left bracket, Fig. 6B). Periodic cleavage was not noted in ML-1 DNA digested to an equal extent by in vitro MNase exposure (lanes 8 and 9). Alignment of the sites of MNase hypersensitivity with the GADD45 gene sequence suggested that MNase cleaved at the transcription start site, the boundaries of regions of DHS and at periodic intervals around regions of TF binding (Fig. 7A). The appearance of these periodically spaced cleavage sites in DNA from nuclei exposed to MNase suggests the positioning of nucleosome-like structures upstream of the TF-binding GADD45 promoter region and between the promoter and the TF-binding intron 3 region (Fig. 7A). The higher order chromatin structure of the GADD45 gene in cells expressing basal levels of GADD45, in combination with the lack of changes in TF binding and chromatin structure following irradiation, suggests that the GADD45 gene is organized as depicted in Figure 7B. Based on the data presented, the GADD45 gene appears to be poised for rapid up-regulation in the absence of gross changes in TF binding or chromatin structure.



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  		Figure 6. Analysis of local chromatin structure of the GADD45 gene. Nuclei from non-irradiated ML-1 cells were incubated with increasing amounts of MNase. (A) Photograph of the EtBr stained agarose gel containing the following DNA samples: non-radiolabeled 1 kb DNA ladder (lane 1); DNA isolated from nuclei exposed to increasing concentrations of MNase (lanes 2–5); 32P-radiolabeled 100 bp DNA ladder (lane 6); non-radiolabeled 100 bp DNA ladder (lane 7); ML-1 DNA incubated with MNase in vitro (lanes 8 and 9). (B) Southern blot analysis using the 5'D probe. The bracket denotes the region in which MNase HS sites were identified. The results shown are representative of two independent experiments.

 


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  		Figure 7. (A) Map of the MNase HS sites and proposed nucleosomes within the GADD45 gene. The position of MNase HS sites relative to the transcription start site and the regions of DNase I HS (stippled boxes) are denoted by downward arrows. Proposed nucleosome-like structures are represented by filled, numbered circles. (B) Model for the organization of the GADD45 gene in vivo. Basal expression of GADD45 is associated with two nucleosome-free regions of TF binding that are surrounded by at least five positioned nucleosome-like structures. No detectable changes in TF binding or nuclease HS are associated with induction following IR. Large arrowheads denote regions of DNase I HS, filled, numbered circles represent positioned nucleosome-like structures while the reverse shaded, non-numbered circles represent hypothetical positioned nucleosome-like structures. In vivo DNA–protein interactions are represented by the geometric shapes located within the regions of DNase I HS.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented in this study suggest that the rapid up-regulation of GADD45 expression following IR does not require synthesis of new transcription factors, changes in chromatin structure or novel IR-induced DNA–protein interactions. As such, the GADD45 gene appears to fall into the category of ‘preset’ genes whose up-regulation is independent of changes in chromatin structure. The data supporting these conclusions are internally consistent as well as being consistent with previous studies concerning GADD45 expression and inducible gene expression. Prior to irradiation, DNA–protein interactions in the GADD45 promoter exist at two consensus octamer binding motifs and at a CCAAT box sequence, consistent with previously published in vitro data (3). An interaction at the consensus AP-1 site was noted in the intron 3 region of the GADD45 gene during basal expression, consistent with both the presence of a consensus AP-1 site in this region, as well as with the presence of AP-1 interactions in regulatory regions of other IR-inducible genes prior to irradiation (23). A DNA–protein interaction at a consensus p53 binding site in the intron 3 region of the gene is also consistent with previously published in vitro data (6,7,23) and with the observation that non-irradiated ML-1 cells express wild-type p53 (7; data not shown). The results of the present study differ slightly from those of Chin et al. who, using DNase I as an in vivo ‘footprinting’ agent, suggested that the p53 interaction with the GADD45 intron 3 binding site only occurs 30 min following irradiation (23). The interaction identified in the present study, and in the aforementioned study, is relatively weak, making an assessment of when the interaction occurs and how it changes over time difficult. DMS and DNase I also have differing sensitivities to DNA–protein interactions and perhaps the constitutive p53 binding demonstrated using DMS was not detectable in the previous study using DNase I and the interaction detected in the previous study using DNase I was perhaps a conformational or other qualitative change in the p53 interaction. Nonetheless, both studies suggest an early and relatively constant interaction between p53 and intron 3 of the GADD45 gene. The regions of the GADD45 gene shown in the current study to interact with proteins in vivo coincided both with those regions previously suggested to be involved in GADD45 gene regulation (3,6,7) and with regions identified to be nucleosome free and DNase I HS. During basal expression, MNase cleaved the GADD45 gene at the outer boundaries of TF-binding regions, at the transcription start site or at nucleosome sized intervals. The pattern of MNase hypersensitivity could be a result of interaction of the GADD45 gene with non-nucleosomal proteins. This is unlikely, however, due to the periodicity of cleavage, the lack of detectable DNA–protein interactions (nucleosomes, as opposed to sequence-specific DNA-binding proteins, do not protect DNA from DMS-induced damage) and the lack of DHS within the region of proposed nucleosome-like structures. Rather, the periodic cleavage of the GADD45 gene at regular nucleosome sized intervals by MNase is consistent with the positioning of nucleo­somes. Such nucleosome-like positioning is in turn consistently found in CpG islands (24,25), one of which spans the TF-binding regions of the GADD45 gene. The lack of change in DNA–protein interactions in the GADD45 gene following IR-induced transcriptional up-regulation is consistent with the observation that GADD45 up-regulation can occur in the absence of protein synthesis, as well as with the lack of chromatin reorganization of the gene following IR.

One issue that complicates interpretation of the present study is the fact that DNA damage, including damage caused by IR, induces GADD45 expression. Therefore, the DNA-damaging techniques used to identify regions of in vivo DNA–protein interactions and regions of DNase I/MNase HS may themselves induce GADD45 expression. As such, the DNA–protein interactions and hypersensitive sites seen in non-irradiated cells may be artifacts of the processes used for their identification and may obscure IR-induced alterations. If this were true, however, the DNA damage-induced changes in DNA–protein interactions and chromatin structure would be immediate as exposures of cells to DNase I/MNase or DMS were for 10 and 2 min, respectively. Additionally, with regard to DMS, it should be noted that the large concentration of DMS (10 mM) used for in vivo footprinting analysis reduced, rather than induced, expression of GADD45 and completely blocked IR-induced GADD45 up-regulation in ML-1 cells (data not shown). As such, it appears that the large concentrations of DMS used for in vivo footprinting extensively methylated cellular components (DNA and proteins), ‘froze’ cells in their pre-exposure state and precluded cellular responses to DMS-induced DNA damage. Finally, and most importantly, it should be noted that the GADD45 gene contains a CpG island beginning upstream of the promoter region and ending at the third intron (26). DNA damage-based analysis of DNA–protein interactions and chromatin structure have demonstrated that all CpG islands examined to date contain TF-binding, nucleosome-free regions which coincide with the regulatory regions of genes (24,25). The identification of nucleosome-free, TF-binding regions in the CpG islands of genes not inducible by DNA damage as well as in the damage inducible GADD45 gene suggests that the TF-binding, nucleosome-free regions noted in the GADD45 gene are not likely an experimental artifact, but rather are present in non-irradiated as well as irradiated cells.

The present study has significant impact on how GADD45 up-regulation can be viewed. As is the case for most genes whose expression is transcriptionally up-regulated, many control mechanisms can be envisioned. IR may trigger changes in chromatin structure which could subsequently allow TF binding to regulatory regions previously not available for interaction. Alternatively, exposure to IR could increase the levels of, or modify, a number of different TF, any one of which could interact with the GADD45 regulatory regions and increase transcription. Finally IR could alter GADD45 expression in the absence of changes in TF binding and chromatin structure simply by altering prebound TF interactions. With regard to the first scenario, while changes in gene expression are commonly associated with changes in chromatin structure (9,27,28), the present studies provide little evidence for IR-induced changes in chromatin structure in the GADD45 gene. With regard to the second possibility, because GADD45 mRNA levels can increase following IR even in the absence of protein synthesis, it seems unlikely that new transcription factors relevant for GADD45 expression are synthesized in response to IR. IR could, however, induce post-translational modification of TF, which could result in increased TF binding and subsequent GADD45 up-regulation. IR-induced post-translational modification of p53 is a particularly appealing possibility as p53 is known to play a role in IR-induced GADD45 up-regulation and is known to be stabilized following IR by post-translational modification. The issue of increased levels of p53 binding to intron 3 of the GADD45 gene following IR is only partially addressed in the present study. Non-irradiated, logarithmically growing ML-1 cells express p53 and data from the present study show that p53 binds to the GADD45 intron 3 region even prior to irradiation. Additionally, p53 binding did not quantitatively change following IR, suggesting that GADD45 up-regulation was not likely a result of increased p53 binding. Rather, the presence of TF interactions in the GADD45 regulatory regions prior to IR is most consistent with the possibility that IR alters the interactions between prebound TF. In this regard it is worth noting that p53 has recently been shown to activate UV-induced GADD45 expression not by direct DNA interaction, but rather by interaction with bound TF and/or the transcriptional machinery itself (29). If p53 functioned similarly following IR, GADD45 up-regulation could be accomplished by IR-induced stabilization of p53 followed not by increased p53 DNA binding in the GADD45 exon 3 region, but rather by increased p53 interaction with prebound TF or the transcriptional machinery. Such up-regulation could be accomplished in the absence of protein synthesis and would not require changes in chromatin structure or TF binding, all of which would be consistent with the data presented in this study.

In summary, the present studies, by linking chromatin structure of the GADD45 gene with the DNA–protein interactions that contribute to GADD45 transcriptional up-regulation, suggest that the presetting of both chromatin structure and TF binding poise the GADD45 gene for rapid transcriptional up-regulation following DNA damage. Further understanding of GADD45 up-regulation will likely require a better understanding of how IR triggers changes in TF activation and/or coupling to the transcriptional machinery.


   ACKNOWLEDGEMENTS
 
This work was supported by a fellowship from the Arthur J. Schmidt Foundation (D.M.G.) and by NIH grant CA-55064 (R.O.P.).


   FOOTNOTES
 
a To whom correspondence should be addressed at: UCSF Cancer Center, 2340 Sutter Street, Room N261, Box 0128, San Francisco, CA 94115, USA. Tel: +1 415 502 7132; Fax: +1 415 502 3179; Email: rpieper{at}cc.ucsf.edu Back


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Received May 7, 1999. Revised and Accepted August 19, 1999.


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