(Received for publication, May 2, 1995 )
From the
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.
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 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 ()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.
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.
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.
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).
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.
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 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
-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.
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 E (a murine class II MHC) gene
and human
-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, E
,
-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
-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 E
and
-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.
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-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 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.
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. ()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>>>E>
-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 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/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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) D43966[GenBank].