©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Mouse Histone H2A.X Gene Promoter by the Transcription Factor E2F and CCAAT Binding Protein (*)

(Received for publication, May 2, 1995 )

Hirotaka Yagi Tomohisa Kato Toshi Nagata Toshiyuki Habu Masami Nozaki Aizo Matsushiro (1) Yoshitake Nishimune Takashi Morita (§)

From theDepartment of Molecular Embryology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan and the Department of Biotechnology, Faculty of Biological Science, Kinki University, 930 Nishimitani, Uchdiacho, Nakagun, Wakayama 649-64, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have molecularly cloned the genomic gene encoding the mouse histone variant H2A.X and characterized the promoter. The promoter region of the H2A.X gene was characterized by chloramphenicol acetyltransferase analysis using Balb/c 3T3 cells. Maximal promoter activity was found in the construct containing up to -282 base pairs H2A.X upstream region. Within this region, we found two sequences regulating the promoter activation: one was an E2F site and another was a CCAAT box. These sequences were also required for the DNA/protein binding activities. Thus, these activities corresponded to the promoter activities, implying that the promoter activity of H2A.X gene was controlled by both the transcription factor E2F and H1TF2 through the E2F and CCAAT element. The CCAAT box binding activity was constitutive when cell cycle was progressed by release from G1 arrest, but transiently transfected chloramphenicol acetyltransferase activity slightly increased when cells entered S phase. Similarly, the level of the smallest form of E2F (free E2F) became higher when cells reentered the cell cycle, indicating that the free E2F was one capable of inducing the promoter activation. Thus, the free E2F and CCAAT DNA binding activity correlated with regulation of the promoter activity.


INTRODUCTION

The nucleosome consists of DNA and histone octamer cores winding around DNA. One octamer core consists of eight molecules constituted by two molecules of respective core histones H2A, H2B, H3, and H4(1) . In the past, the histone octamer was thought to play a passive role in packaging DNA. However, now it is recognized that histones play crucial roles in DNA replication (2) and transcription(3) .

Four kinds of molecular variants belong to the H2A family. They are different in length and sequence. The histone H2A.1 and H2A.2 of mammalian cells are called major histones because they are primarily synthesized in concert with DNA replication during S phase of the cell cycle, while the other two, H2A.X and H2A.Z, are minor components and are synthesized constantly at low levels throughout the cell cycle(4, 5) . In vertebrate, the H2A.X protein represents 10% of the total H2A proteins, but the major H2As in lower eukaryotes contain the C-terminal amino acid sequence homologous to H2A.X(6, 7) . It seems likely that histone H2A.X functions as nucleosomes associated with active genes, such as the immunoglobulin kappa chain gene(8) .

In mammalian cells, the major histone proteins are synthesized in DNA replication-dependent manners, and the expressions are regulated at both transcriptional and post-transcriptional levels (reviewed in (9, 10, 11) ). Histone gene transcription occurs throughout the cell cycle, but in the case of some major histone genes their transcription levels increase severalfold at the onset of S phase. Transcriptional regulation during the cell cycle of those genes is mediated by several consensus elements. In the case of post-transcriptional regulation, those mRNA levels are regulated in the following two steps: alterations in 3`-processing efficiency of mRNA and in mRNA stability(12) . Those genes are transcribed into non-polyadenylated mRNAs. These mRNA contain a common feature characteristic of a 3`-stem-loop motif, which regulates their mRNA stability, thereby controlling their mRNA levels during the cell cycle. Thus, the replication-dependent protein synthesis is post-transcriptionally controlled by the mRNA levels.

The minor histone H2A.X protein is encoded by a single genomic gene without introns(13) . Our previous study (7) indicated that two mRNA species were transcribed from the H2A.X gene: one was in a non-polyadenylated form, and the expression increased in S phase by post-transcriptional regulation; the other was not processed at the 3`-processing site and was polyadenylated at 0.8 kb (^1)downstream of the processing site, and this mRNA was not expressed in a DNA replication-dependent manner. It is likely that the H2A.X protein is synthesized from those two forms of mRNAs in a partially cell cycle-dependent manner.

In this paper, we report characterization of the mouse histone H2A.X promoter and of interactions between proteins and promoter activating sequences. CAT reporter gene transfection assay indicates that there are two activating sequences within the promoter region: one element is an E2F binding site and the other is a CCAAT box. However, these two elements are not the same as those required for the human promoter, even though the promoter sequence between mouse and human is highly conserved. The studies of the protein-DNA interactions suggest that the factor binding to the CCAAT box is a previously characterized factor H1TF2(14) . There are various forms of E2Fs due to multiple proteins binding to the E2F element(15, 16) . Among these E2Fs, a certain form of E2F (free E2F) is capable of inducing the promoter activation (16, 17, 18, 19) . In the case of H2A.X promoter, the level of the active form of E2F increases when cells reenter cell cycle. The increase explains the partial cell cycle-dependent activation of H2A.X promoter activity measured by CAT analysis.


MATERIALS AND METHODS

Cell Culture

Balb/c 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum.

RNase Protection Assay

The pBluescript-containing 1.8-kb SacI-XhoI H2A.X promoter fragment (see Fig.1A) was linearized at -174 bp by SmaI, and a riboprobe was synthesized in vitro from the T7 promoter. To determine the transcription initiation site, the probe was hybridized with 10 µg of cellular RNA for overnight at 45 °C. The annealed RNA was then digested with RNase T1 and RNase A for 1 h at 30 °C. The resulting protected fragments were electrophoresed beside DNA ladders and the non-digested riboprobe.


Figure 1: Analysis of the H2A.X promoter. From the genomic mouse H2A.X clone, the 1.8-kb SacI-XhoI DNA fragment was subcloned into pBluescript plasmid (pSX2). The plasmid DNAs were used for restriction enzyme mapping, DNA sequencing, and determination of mRNA transcription site. A, a map of 5`-promoter region of the mouse H2A.X gene. The stippledbox indicates a 3`-non-coding sequence of GPT gene. The 5`-leader sequence of H2A.X mRNA is represented by the openbox, and the coding region is shown by the solidbox. B, DNA sequence of the 5`-portion of the mouse H2A.X gene. Potential control elements are indicated. The transcription initiation site (also see D) is indicated by a bentarrow over the sequence (position +1). The translation start ATG codon is in a box. The upstream boundaries of various promoter fragments used for CAT assay are indicated by bentarrows underneath the sequence (also see Fig.2A). C, a comparison of the mouse and human H2A.X promoter region. Each CCAAT box is indicated by a line with an arrowhead. TATA box is shown by a stippledbox. D, transcription initiation site of the mouse H2A.X gene. An antisense riboprobe including a region from the XhoI site in the coding region to the SmaI site at -174 bp was generated by T7 polymerase and used for this RNase protection experiment. On the basis of the expected size of the non-digested riboprobe, the mobility of the RNase probe was calculated to be 97.12% of the DNA ladder. The major RNase protected band was designated as the transcription start site (shown by an arrowhead).




Figure 2: Transcriptional activity of various upstream sequences of the mouse H2A.X gene. A, determination of the core promoter region of mouse H2A.X gene. DNAs of mouse H2A.X-CAT constructs containing a series of 5`-deleted promoter regions were transiently transfected into Balb/c 3T3 cells. The 5`-non-coding sequence of the transcript is represented by the openbox, and the CAT reporter gene is shown by the solidbox. Plasmids are described under ``Materials and Methods.'' CAT activities of the constructs are shown as the percentage of the activity elicited by the longest construct (pL). Standard deviations are also indicated. B, E2F element required for H2A.X promoter activity. The DNA sequence of the H2A.X promoter at nucleotide positions -282 to -243 is shown. This sequence contains two putative E2F sites. Mutated sequences in one or both E2F sequences are indicated by underlines. A construct truncating the two E2F sites (pA) is also shown. C, effect of the putative CCAAT elements at the -150- and -155-bp region on H2A.X promoter activation. The sequence of the H2A.X promoter at nucleotide positions -162 to -143 bp is shown together with the two CCAAT sequences. The mutations introduced into the putative elements are specified by underlines. The construct truncating these regions (pD) is also shown.



Preparation of CAT Constructs

All promoter fragments were constructed by PCR except for a CAT vector pL. Each of those PCR fragments contained an XhoI site at 5` terminus and a HindIII site at 3` terminus, both derived from the PCR primers. The fragments were digested with both XhoI and HindIII and then ligated to pSV1 vector, from which the SV promoter sequence had been removed by XhoI and HindIII digestion. The sequences of those PCR products were confirmed by DNA sequencing. The CAT vector pL was constructed as follows: 1) 1.8-kb H2A.X promoter containing plasmid DNA was digested with Eco43III and BssHII, and the 1.0-kb promoter-containing fragment was obtained; 2) pE2F CAT plasmid DNA was digested with XhoI and filled in by Klenow, followed by digestion with BssHII; 3) The H2A.X promoter fragment was ligated with the linearized CAT plasmid.

Transfection and CAT Assay

Balb/c 3T3 cells were transfected by a calcium phosphate protocol. For this, 8 µg of CAT expression plasmid DNA and 8 µg of beta-galactosidase plasmid DNA were used. For cell synchronization, lovastatin (a generous gift from Banyu-Merck Pharmaceutical Co.) was used to arrest cells in G1. Following transfection, cells were plated and cultured in fresh medium for 15 h. Then lovastatin (25 µM) was added to the cultures, and cells were incubated for another 32 h. At time zero, cells were re-fed with fresh medium containing mevalonic acid (2.0 mM) and harvested at indicated times; the cell cycle phases of particular cell populations were determined by flow cytometry analysis.

Preparation of Probes for Gel Shift Assays

DNA fragments containing E2F elements were generated by PCR. The DNA fragments contain an XhoI site and an EcoRI site derived from the PCR primers at their termini, respectively. These DNA fragments and pBluescript (Stratagene) were digested with both XhoI and EcoRI and ligated. After confirmation by DNA sequencing, E2F fragment-containing plasmids were digested with both XhoI and EcoRI. Then, DNA fragments containing E2F elements were isolated by acrylamide gel electrophoresis. In the case of other DNA fragments, plus and minus stranded oligo DNAs were separately synthesized by a model 381 A DNA synthesizer (Applied Biosystems) and later annealed. These probes were radiolabeled with [alpha-P]ATP by Klenow enzyme.

Analysis of DNA-Nuclear Factor Binding

Nuclear extracts were prepared from 3T3 cells according to Schreiber et al.(20) . Protein concentration of each nuclear extract was estimated according to Bradford(21) . For the standard assay of 16 µl of reaction mixture, each probe containing approximately 5,000-10,000 cpm was incubated with the nuclear extract in a buffer consisting of 80 mM KCl, 20 mM HEPES (pH 7.9), 0.3 mM EGTA, 12.5% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.05% Nonidet P-40 and then incubated for 30 min at 25 °C. When the CCAAT-binding factor was analyzed, MgCl(2) was also added to achieve a final concentration of 1.5 mM. The reaction products were electrophoresed on a 4.5% polyacrylamide gel (29:1 acrylamide/bisacrylamide) in 0.25 TBE.

Deoxycholate treatment was carried out as previously described(22) . The reaction mixture for the standard mobility shift assay was treated with 0.25% deoxycholate for 30 min. Then, Nonidet P-40 was added to the sample to achieve a final concentration of 0.8%, and then the reaction mixture was electrophoresed.


RESULTS

Characterization of the Mouse Histone H2A.X Promoter

To obtain a genomic DNA fragment containing the mouse histone H2A.X promoter, a mouse (129Sv) genomic DNA phage library (EMBL3) was screened. For this, a probe containing the 3` terminus of a previously described full-length mouse histone H2A.X cDNA clone (7) was used. The screening yielded a phage clone that contained the genomic H2A.X gene. A SacI-XhoI 1.8-kb fragment containing the extreme 5`-end of the histone H2A.X coding region was subcloned into pBluescript (Fig.1A). Sequence analysis of the fragment revealed that this fragment contained 5` part of histone H2A.X coding region. Furthermore, within the fragment, we found the 478 bp of 3`-non-coding region of the gene encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosamine-phosphotransferase (GPT) (23) upstream of the 5`-end of histone H2A.X gene. The polyadenylation site of GPT gene was at 1.1 kb upstream of the H2A.X transcription initiation site (Fig.1B). Southern blot analysis of mouse DNA also indicated that GPT and H2A.X genes were located in the same manner as were observed in the above genomic clone (data not shown). The non-coding region upstream of the 5`-coding region of H2A.X had several characteristic nucleotide motifs for various transcription factors. H2A.X promoter contained one consensus TATA box 30 bp upstream of the transcription initiation site and four CCAAT boxes as were seen in the other histone promoters but did not contain the Sp1 site. Also found were two tandem E2F sites located between -277 and -248 bp, one of which showed a complete match and the other showed a 7 out of 8 match to the consensus E2F site. Besides, one EGR-1 site and one PEA3 site were located just upstream of the TATA box (Fig.1B).

Recently, human H2A.X gene was isolated, and the proximal promoter sequence was determined(13) . The nucleotide identity between the human and mouse promoter regions was highly conserved (Fig.1C). In those sequences, the two CCAAT elements were conserved, but the other two CCAAT sequences were not. A middle CCAAT box in the human sequence was not conserved in the mouse sequence. In contrast, the most distal CCAAT box and two other consensus sites (EGR-1 and PEA3) in the mouse sequence were not conserved in the human sequence.

To map the H2A.X transcription start site, RNase protection assay was carried out. A radiolabeled antisense RNA probe, extended from the XhoI site in the coding region to the SmaI site at -174, was produced. By this method, the major transcription start site was mapped at 68 bp upstream of the translation initiation site ATG (Fig.1, B and D).

Identification of Mouse H2A.X Promoter Activity

To identify specific sequences required for promoter activity, a series of deleted promoter-CAT reporter gene plasmids were constructed. All of these plasmids contained a region of both the translation initiation site ATG and the following 2-bp derived from H2A.X gene. This region was connected with CAT gene in frame.

DNAs of these CAT constructs containing a series of deleted promoter sequences (Fig.2A) were transfected into Balb/c 3T3 cells. The CAT activity of the plasmid pL, which included the 1.3-kb sequence upstream of the H2A.X translation start site, was used as the standard 100% activity. The construct with a -282-bp fragment (pE2F) showed a maximal level of CAT activity. This construct included the TATA box, the two E2F sites, and the four CCAAT boxes. The construct pC, which included the TATA box, the three CCAAT boxes, and deletion of the two E2F sites, produced 52.5% relative activity. The construct without the two CCAAT boxes at -155 and -150 bp (pD) produced a basal level CAT activity (25.8%). These results demonstrated that the H2A.X core promoter was localized within the 5`-region of 282 bp upstream of the transcription start site and that the two E2F sites at -275 and -255 region and the two CCAAT boxes at -155 and -150 region were contained in the major core promoter region (Fig.1B).

To determine whether either one or both of the putative E2F-binding sites were responsible for the promoter activity, we introduced mutations singly or in combination into these two sites and then assayed their promoter activity (Fig.2B). When the upstream E2F site was mutated (pE2F-MW), the CAT activity was slightly reduced (Fig.2B). In contrast, the introduction of two mutations to the downstream E2F site (pE2F-WM) decreased the activity as low as that observed with the construct with mutations at both E2F sites (pE2F-MM) or the E2F-deleted construct (pA). Thus, the downstream E2F site was important for the promoter function, i.e. transcription of histone H2A.X gene.

We next examined which of the two putative CCAAT elements at -155 and -150 region could regulate the histone H2A.X promoter activity (Fig.2C). When two mutations were introduced into the -150 CCAAT box (pC-M3), the mutated construct produced only a basal level of CAT activity as low as that of the construct without the CCAAT sites (pD). Another construct with two mutations within the -155 CCAAT box (pC-M2) persistently showed high activity, about 80% CAT activity, as compared with the wild-type CCAAT construct (pC). Thus, the -150 CCAAT box was essential for the promoter activity of histone H2A.X gene.

Cell Cycle Regulation of Histone H2A.X Promoter Activity

To examine whether the histone H2A.X promoter could confer cell cycle-regulated expression, the histone H2A.X promoter-CAT gene plasmid was transfected into Balb/c 3T3 cells. Following exposure to the calcium phosphate precipitate, cells were arrested in early G1 phase in medium containing lovastatin (24) for 32 h. Following addition of fresh medium containing mevalonic acid, cells were released and reentered the cell cycle synchronously. The cell extracts were made at indicated time points, and the CAT activities were assayed. For normalization, the cell extracts were adjusted with beta-galactosidase activity directed by a cotransfected beta-galactosidase expression vector, pCH110, yielding stable activity throughout the cell cycle. The parallel Balb/c3T3 cultures of cells were stained with ethidium bromide, and cell DNA content in each culture was measured by flow cytometry to examine the cell cycle status. It was found that in cells transfected with pL (Fig.2A), CAT activity began to increase gradually 20 h after release, when the cells almost entered S phase (Fig.3). CAT activity directed by pF with the minimal promoter region showed a slight increase after the cells entered S phase (Fig.3). In contrast, the control plasmid with SV40 promoter showed a constant CAT activity, independent of the cell cycle (Fig.3).


Figure 3: Activity of CAT reporter genes in synchronized Balb/c 3T3 cells following transient transfection. Experiments were performed as described under ``Materials and Methods.'' Balb/c 3T3 cells were arrested at early G(1) phase by lovastatin. After release from the lovastatin block, at each time point, half of the cells were used for preparation of cell lysate and the other half were used for cell cycle analysis by the fluorescence-activated cell sorter. For each construct, CAT activities were standardized by beta-galactosidase activity and shown as the relative activities compared with 0 h after release. pL and pF contained H2A.X promoter sequences up to -1.3 kb and -52 bp, respectively. Control pSV2CAT contained SV 40 enhancer element.



Gel Retardation Assays for H2A.X Promoter Elements

To test whether the elements necessary for promoter activation could bind transcription factors, oligonucleotides containing the E2F or CCAAT elements were prepared (Table. I) and used for gel shift assays. As shown in Fig.4A, the oligonucleotide carrying the two E2F sites (E2F-WW, see Table1) revealed three protein-DNA complexes (bandsA, B, and C), whose pattern was similar to those of E2F-containing complexes shown by others and the fastest migrating complex (D). All of these complexes disappeared by the addition of more than 10-fold molar excess of the same oligonucleotide. To determine which E2F site was involved in the protein-DNA complex formation, an oligonucleotide with the two E2F sites each containing the mutations or with either one (see Table1) was used as a competitor. Competition mobility shift assay exhibited that the oligonucleotide containing the mutation within the upstream E2F site (E2F-MW) competed specifically for complex formation of the bands C and D but not for the bands A and B, suggesting the upstream E2F site was responsible for the complex formation of bands A and B. In contrast, the oligonucleotide containing the mutations either in the downstream E2F site (E2F-WM) or in both E2F sites (E2F-MM) did not affect the complex formation. These results suggested that the bands C and D were specifically associated with only one E2F site, the downstream E2F site, and that those binding activities were associated with the increase of the CAT activity.


Figure 4: Gel shift analysis of the E2F element in the H2A.X promoter. A, the labeled H2A.X promoter fragment containing the two E2F elements (E2F-WW, see Table1) was incubated with 2.5 µg of the nuclear extract from Balb/c 3T3 cells in the absence or presence of indicated unlabeled competitor oligonucleotides. The arrows show the specific gel shift bands. As shown in Table1, mutant competitors contain base substitutions in each or both E2F sites. B, competition of the H2A.X E2F with other promoters containing E2F elements derived from other genes. The competitor sequences derived from other genes are shown in Table1. The labeled E2F-WW probe was incubated with the Balb/c 3T3 nuclear extract in the absence or presence of 50- or 200-fold molar excess of each competitor derived from other genes. C, the same mixture of E2F-WW as in A was incubated in the presence of 0.25% deoxycholate and then treated with 0.8% Nonidet P-40 before subjected to electrophoresis.





To characterize the binding specificity of the H2A.X E2F, we labeled the E2F-WW fragment and carried out competition mobility shift assay with other known E2F sequences (Fig.4B). Five different sequences corresponding to E2F factor binding sites from the promoters of murine dihydrofolate reductase(25) , B-myb(26) , thymidine kinase(27) , human cdc2(28) , and cyclin D1 (29) genes were used (Table1). Among these, the E2F sequences from murine dihydrofolate reductase, B-myb, and thymidine kinase competed for the complex formation of band C but were only slightly competitive to the bands A, B, and the fastest migrating complex (D). Furthermore, the E2F sequences from human Cdc2 and cyclin D1 also exhibited competition activities for the band C complex formation at the same molar excess, though the activities were slightly weaker than those of the other E2F sequences. These results indicated that all of the E2F sequences examined were able to form the complex C and that the sequence heterogeneity among them appeared not to alter the affinity between the protein and the E2F elements.

The transcription factor E2F binds to the consensus sequence(30) . However, the gel shift entities also called ``E2F'' are present in various forms due to multiple proteins binding to the E2F factor(15) . Thus, different migrating forms were seen by gel shift analysis. Treatment with deoxycholate abolished some complex formation between proteins(31) . To investigate whether or not these observed gel shift bands were derived from free E2F or other E2F complexes, the sample containing the wild-type oligonucleotide carrying the two E2F sites was treated with deoxycholate and examined by gel shift assay. After deoxycholate treatment, only a single band was detected at the same position as that of the band C (Fig.4C), which implied that the band C would contain the smallest protein unit binding to the E2F consensus sequence called ``free'' E2F. These results also suggested that the bands A, B, and D were complexes formed between the free E2F and other proteins.

Next, we have investigated the CCAAT elements by gel shift assay. The gel shift assay using a 36-bp oligonucleotide containing the wild-type CCAAT element at position -150 region (C-WT) revealed one protein-DNA complex (shown by an arrowhead in Fig.5A), and this complex disappeared by adding 50-fold molar excess of the same oligonucleotide. We have made five competitors with mutations within regions containing the CCAAT site at position -150 and its flanking regions (Table1). These competitors, except for one with the mutations in the CCAAT sequence (C-M3), showed competitive activity (Fig.5A). C-M1 and C-M5 showed the activity comparable to that of the intact wild-type oligonucleotide, C-WT. C-M2 and C-M4 were also able to compete but somewhat weakly as compared with C-WT. Among the above five mutant oligonucleotides, only the oligonucleotide C-M3 had the mutations within the CCAAT sequence, and only this oligonucleotide (C-M3) showed no competitive activity. Thus, these findings suggested that the -150 CCAAT sequence was involved in protein binding. Furthermore, the results showed that the -150 CCAAT flanking sequences were also important, if not essential, for interacting with the factor (compare C-WT with either C-M2 or C-M4 in Fig.5A, also see Table1).


Figure 5: Gel shift analysis of the CCAAT element at -150 region. A, gel shift assay performed with C-WT and 0.7 µg of the nuclear extract in the absence or presence of 50-fold molar excess of competitors. Each competitor containing the CCAAT at -150 region with or without mutations is shown in Table1. The position of the gel shift band is indicated by an arrowhead. B, competition of the H2A.X CCAAT with other promoters containing CCAAT elements derived from other genes. The competitor sequences derived from other genes are shown in Table1. The P-labeled probe containing the H2A.X CCAAT element (C-WT) was incubated with the Balb/c 3T3 nuclear extract in the absence or presence of 50-, 200-, or 1000-fold molar excess of each competitor derived from other genes.



Conversely, when mutant oligonucleotides, C-M2 and C-M3, were labeled with [-P]ATP and used as probes, C-M2 showed a 30-35% intensity of the gel shift band as compared with WT. On the other hand, C-M3 showed a trace of band (data not shown). It should be pointed out that both probes did not show any other band in Balb/c 3T3 extract. Thus, these observations agreed with the CAT promoter activities, which indicated that the CCAAT box mutated in C-M3 oligonucleotide was essential for the activation of transcription (see Fig.2C).

There were multiple proteins capable of binding CCAAT boxes. These proteins showed some distinct sequence-specific binding activity. NF-Y bound to CCAAT boxes derived from various genes, such as Ealpha (a murine class II MHC) gene and human alpha-globin gene(32) . H1TF2 bound to histone H1 gene promoter that included a CCAAT motif(14) . At first, CP2 was found as a CCAAT binding factor(33) . But later, it turned out that CP2 binding motifs did not always contain the CCAAT sequence but occasionally included CCAAT motifs(34) . We have tested whether the above CCAAT motifs, Ealpha, alpha-Glob 30-mer, and the H1TF2-binding sequence, could compete with the H2A.X CCAAT for the binding protein. For this, oligonucleotides containing the above CCAAT motifs were made (Table1). Besides, a CP2 consensus element derived from the mouse alpha-globin gene that lacked a CCAAT motif (34) (Table1) was also tested. Fig. 5B shows that the H1TF2 strongly competes with the labeled H2A.X CCAAT fragment, that the Ealpha and alpha-Glob do very weakly, and that the CP2 cannot compete at all. This result suggested that the protein binding to the H2A.X CCAAT box was most likely the H1TF2 factor.

Protein/DNA Binding Pattern during Cell Cycle Progression

We have examined whether the activities of these DNA binding proteins were controlled in the cell cycle. In this experiment, Balb/c 3T3 cells were arrested at G1 phase by lovastatin and then released to proceed the cell cycle synchronously. The nuclear extracts were prepared from the cells at various time points after release.

Fig.6B shows that the CCAAT binding factor was constitutively present throughout the cell cycle. In contrast, the E2F bands showed cell cycle dependence; E2F band A decreased at the time point of the G(1)-S phase transition 8-12 h after release; on the other hand, the bands C and D increased as the cells in G1 phase progressed into S phase (Fig.6A). When the binding reaction mixture was treated with 0.25% deoxycholate, the amount of band C was constant throughout the cell cycle (Fig.6A). Since the band C corresponded to the free E2F that was able to induce the promoter activity(19) , the increase in free E2F may have contributed to the limited enhancement of the transcription during cell cycle progression (see Fig.3).


Figure 6: Cell cycle analysis of protein-DNA complexes. Balb/c 3T3 cells were arrested at G(1) phase by lovastatin and reentered the cell cycle synchronously after release as described in Fig.3. At various time points after release, nuclear extracts were prepared and assayed for H2A.X promoter elements, and the cell populations of each phase in cell cycle were monitored by fluorescence-activated cell sorter. The E2F element (E2F-WW) (A) or CCAAT element (C-WT) (B) was incubated with the same amount of nuclear extract. +DOC indicates the treatment of the binding reaction mixture with 0.25% deoxycholate.




DISCUSSION

Analysis of the DNA sequence upstream of the mouse H2A.X gene revealed that at about 1 kb upstream of the translation initiation site there was the GPT gene polyadenylation site and that these two genes, H2A.X and GPT, are transcribed in the same orientation. Downstream of the mouse H2A.X gene, porphobilinogen deaminase gene (35) was located as was seen in human and is supposedly transcribed in the opposite orientation of the H2A.X gene. (^2)These results indicate that the H2A.X gene is not one of the clustered histone genes, unlike the case with the major histone genes.

We have shown that the maximal promoter activity was localized within the region of 282 bp upstream of the transcription initiation site, that the proximal promoter of the mouse histone H2A.X gene contained two transcription activation sequences, E2F and CCAAT elements, and that those elements were able to form protein-DNA complexes as demonstrated by gel shift assays. The 5`-promoter regions of the vertebrate histone genes contain many transcriptional regulatory elements: several general promoter elements, e.g. the TATA box and CCAAT motif; elements found in a subfamily of histone genes; and elements that are found only in particular classes of histone genes. So far, involvement of the E2F element in transcriptional regulation of any histone gene has not been demonstrated. However, our results showed that the free E2F was closely associated with the promoter activation. Thus, it is likely that the E2F element is a transcriptional activation element for some histone genes containing the E2F element.

Recently, human H2A.X gene was isolated, and its promoter was also characterized(13) . Between the human and mouse genes, the proximal promoter sequence was highly conserved. Two conserved CCAAT elements existed, but their properties were different in promoter activity. In mouse, our CAT assay indicated that the distal CCAAT element was involved in the promoter activation and that the proximal one was not. However, the different result has been reported in the human promoter. This different observation may have resulted from the diversification of promoter function between human and mouse. Although our CAT assay did not show the effect of the proximal CCAAT element, the conservation suggests that its effect on the promoter activity might be observed by a different experimental method.

A protein-DNA complex was observed by gel retardation assay when the promoter activation region containing the two CCAAT motifs was tested. In the case of mouse H2A.X, only one CCAAT sequence in this region, which was conserved between the human and mouse H2A.X, was needed for the protein-DNA interaction. The necessity of this same CCAAT motif for promoter activation was also demonstrated by the CAT assay, as discussed above. Thus, it is likely that a factor binding to the CCAAT element is required for mouse H2A.X promoter activation. The competition analysis showed that the mutations in the CCAAT flanking sequences also affected DNA binding activity. These results were also consistent with those of the CAT reporter gene studies.

As mentioned above, the CCAAT sequence is essential for the protein to bind to DNA, but the flanking sequences also affect its binding activity. Double-stranded oligonucleotides containing various types of CCAAT elements competed for the H2A.X promoter binding factor to various degrees. Their competition activities were as follows: C-WT≅H1TF2>>>Ealpha>alpha-Glob. Since the H1TF2 fragment most strongly competed the H2A.X CCAAT box, it is likely that the factor H1TF2 binds to the H2A.X CCAAT sequence.

A DNA binding factor, H1TF2, has been characterized as a transcriptional activator as well as a cell cycle regulator in HeLa cells(14, 36) . It has been reported that its DNA binding activity increased 2-5-fold in S phase as compared with that in G(1) phase. In contrast, our experiment showed that the H2A.X CCAAT box binding activity was not much altered during the cell cycle in Balb/c 3T3 cells and that the H2A.X CCAAT box apparently bound to H1TF2, as discussed above. Although the above report showed the pattern of complex formation in the cell cycle different from ours, we would like to consider that H1TF2 is the one binding to the CCAAT element in H2A.X promoter. As indicated by the transient CAT assay and gel retardation assay, the constitutive complex formation was correlated with the promoter activity. Thus, it seems likely that the H2A.X CCAAT binding factor activates the H2A.X transcription throughout cell cycle.

Gel retardation assay showed that the E2F factor bound to the E2F consensus element of H2A.X gene and that E2F could exist in free E2F or in complex with other partner proteins such as a family of the retinoblastoma susceptibility gene protein (pRB, p107, and p130) (17, 37, 38, 39, 40) . Bands A and B in our gel retardation experiments are likely to be formed between the free E2F and those pRB family proteins. Since pRB and p107 inhibited E2F-dependent gene expression (16, 17, 18, 19) , it is likely that only band C (free E2F) could activate the gene expression. Our results indicated that the amounts of E2F complexes A and B were remarkably reduced as cells entered S phase and that free E2F increased when cells re-entered the cell cycle. Thus, we consider that the promoter of histone HA.X was activated partially, when the cells entered the G(1)/S boundary, due to the increase in free E2F. Since the amount of band C after the treatment with deoxycholate was constant throughout the cell cycle, the other bands are likely formed between the free E2F and its partner proteins depending on the cell cycle. Though the free E2F are thought to have transcription-stimulating activity, we cannot rule out the possibility that the fastest migrating complex D, which emerged from the cells entering S phase, might have a positive role in H2A.X transcription.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) D43966[GenBank].

§
To whom correspondence should be addressed. Tel.: 81-6-879-8308; Fax: 81-6-875-3268.

^1
The abbreviations used are: kb, kilobase pair(s); bp, base pair(s); CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction.

^2
H. Yagi, T. Kato, T. Nagata, T. Habu, M. Nozaki, A. Matsushiro, Y. Nishimune, and T. Morita, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. A. Tanaka for helpful comments on the manuscript.


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