(Received for publication, October 18, 1994)
From the
Inosine-5`-monophosphate dehydrogenase (IMPDH) activity and mRNA levels are induced up to 15-fold upon mitogenic or antigenic stimulation of human peripheral blood T lymphocytes. This increase in IMPDH activity is required for cellular proliferation and has been associated with malignant transformation. We have cloned the human IMPDH type II gene and show that it contains 14 exons and is approximately 5.8 kilobases in length. Exons vary in size from 49 to 207 base pairs and introns from 73 to 1065 base pairs. The transcription start site was mapped to a position 50 nucleotides upstream of the translation initiation site. The 5`-flanking region consisting of 463 base pairs upstream of the translation initiation site confers induced transcription and differential regulation upon a chloramphenicol acetyltransferase reporter gene when transfected into Jurkat T cells and human peripheral blood T lymphocytes, respectively. DNase I footprinting analysis using Jurkat T cell nuclear extract identified four protected regions in the promoter which coincide with consensus transcription factor binding sites for the nuclear factors AP2, ATF, CREB, Egr-1, Nm23, and Sp1. These findings suggest that several of these nuclear factors may play a critical role in the regulation of IMPDH type II gene expression during T lymphocyte activation.
Inosine-5`-monophosphate dehydrogenase (IMPDH, ()EC
1.1.1.205) is positioned at the branch point of adenine and guanine
nucleotide biosynthesis from IMP and constitutes the rate-limiting
enzyme in the de novo synthesis of guanine nucleotides. The
enzyme catalyses the NAD-dependent oxidation of IMP to XMP and is
responsible for maintaining cellular guanine deoxy- and ribonucleotide
pools required for DNA and RNA biosynthesis, respectively. Enzyme
activity varies with the cell cycle, exhibiting maximal activity in S
phase(1) .
Total cellular IMPDH activity is accounted for by the expression of two distinct genes, IMPDH type I located on chromosome 7 and IMPDH type II located on chromosome 3(2, 3) . The human IMPDH type I and type II cDNAs have been isolated and found to contain open reading frames encoding 514 amino acid proteins of 56 kilodaltons(4, 5) . These enzymes are 84% identical at the amino acid level and demonstrate very similar kinetic parameters(4, 6, 7) .
The close correlation between elevated IMPDH activity and cell proliferation and the observation of high activity in neoplastic cells have linked IMPDH activity to malignant transformation(8, 9) . This association has led to the search for and development of several inhibitors with demonstrated antineoplastic and immunosuppressive potential(10, 11) . Such inhibitors of IMPDH activity have been demonstrated to inhibit cell proliferation and induce cellular differentiation as a consequence of the reduction of guanine nucleotide levels(12, 13, 14, 15, 16) .
Regulation of IMPDH activity during cellular growth and differentiation has been largely attributed to changes in the expression of the IMPDH type II gene. The type II 2.3-kb mRNA transcript is the predominant species in neoplastic cells and is selectively up-regulated in replicating cells(17, 18, 19) . Conversely, when neoplastic cells are induced to differentiate, the enhanced levels of the type II transcript and total cellular activity are down-regulated(13, 14, 15) . In contrast, the 3.5-kb type I transcript remains constitutively expressed during cell proliferation and induction of cell differentiation(4, 17, 18, 19) . The modulation of cellular IMPDH activity during cell growth and differentiation suggests a critically important role for the regulation of IMPDH type II gene expression in the progression of normal cell development. In order to assess the molecular mechanisms governing the expression of IMPDH type II in quiescent, replicating, and differentiating cells, we have cloned the IMPDH type II gene and characterized the gene and its 5`-flanking region.
Figure 1: Schema and partial restriction map of the human IMPDH type II gene. Restriction sites for SacI and EcoRI are shown. Exons are represented by open bars and intron and flanking sequences by solid lines.
Figure 2:
Analysis of the transcription initiation
site of the IMPDH type II gene. A, primer extension product
produced from Jurkat T cell poly(A) RNA. DNA sequence
was obtained from the 1536-bp EcoRI promoter fragment using
the identical oligonucleotide used for primer extension; lanes contain
primer alone; primer and yeast tRNA; primer, tRNA, and Jurkat T cell
poly(A
) RNA; and
X174 DNA/HaeIII
markers. The transcription initiation site and sequence surrounding the
site is indicated on the left. B, ribonuclease protection
assay of Jurkat, HL60, and Raji poly(A
) RNA hybridized
to the 466-bp
P-labeled RNA template extending 5` of the
translation initiation site. Lanes contain probe plus yeast tRNA; probe
plus poly(A
) RNA from Jurkat, HL60, and Raji cells,
respectively; and
X174 DNA/HinfI
markers.
Figure 3: Nucleotide sequence of the 466-bp EcoRI/NcoI fragment containing the 5`-flanking region of the IMPDH type II gene. The adenine from the ATG initiation codon is designated +1. The transcription start site is indicated by the arrow. Underlined sequences indicate the four regions protected from DNase I digestion in the presence of Jurkat T cell nuclear extract. Putative transcription factor binding sites are indicated above the sequence, and the sequence is italicized.
To evaluate the functional significance of the
putative promoter region in the regulation of IMPDH type II expression,
CAT reporter plasmids were constructed and transiently transfected into
exponentially growing Jurkat T cells. The constructs were derived from
pBSCAT and contained the 1536-bp genomic EcoRI fragment
(position -463 to +1073) and the 466-bp 5` EcoRI/NcoI fragment (position -463 to +3)
in 5`3` and 3`
5` orientations. Fig. 4demonstrates
that all constructs containing the IMPDH type II 5` region have
promoter activity. The 1536-bp construct exhibited 200-fold higher
activity than did the pBSCAT vector alone. The pBS466(5`
3`)CAT
construct manifested 70-fold and the pBS466(3`
5`)CAT construct
10-fold increased activity over the vector alone, demonstrating that
the 463-bp DNA fragment immediately upstream of the human IMPDH type II
gene's initiation codon functions as a promoter upon transfection
into Jurkat T cells, with substantially less activity in the reverse
orientation.
Figure 4:
Functional analysis of the 5` region of
the IMPDH type II gene. A, the 1536-bp EcoRI fragment
extending 463 bp 5` of the translation initiation site and 1073 bp 3`
into exon 4 was subcloned into the reporter construct pBSCAT in the
5`3` orientation. The 5` 466-bp EcoRI/NcoI
fragment was excised and subcloned into pBSCAT in both orientations.
The translation initiation site is indicated by the arrow. B,
Jurkat T cells were transiently transfected with the above constructs
and with the plasmid p
Ac-lacZ and assayed 48 h posttransfection
for CAT and
-galactosidase activities, respectively.
Chloramphenicol acetyltransferase activity was corrected for extract
protein concentration. To normalize for differences in transfection
efficiencies between experiments, CAT values were standardized to
-galactosidase values. Values represent the mean of two
independent experiments performed in duplicate. The bars indicate the SD.
Figure 5:
Transcriptional regulation of the IMPDH
type II promoter construct in resting and activated peripheral blood T
lymphocytes. Peripheral blood T lymphocytes were prestimulated as
described under ``Materials and Methods'' and transiently
transfected with the constructs indicated. Cells were maintained in
medium in the absence of stimulation or treated with 10 ng/ml PMA and
125 ng/ml ionomycin and continued in culture for 48 h. Cells extracts
were assayed for CAT activity. Stimulated T lymphocytes exhibited a
2.4-fold higher [H]thymidine incorporation than
nonstimulated cells. Values are the mean of a single experiment
performed in duplicate and represent data obtained from three
independent experiments.
, control; &cjs2113;, PMA +
ionomycin.
Figure 6: DNase I footprint analysis of the 5`- flanking region of the IMPDH type II gene. A 466-bp DNA fragment containing the IMPDH type II 5`-flanking region was labeled at the 3` end on the coding strand and subjected to DNase I treatment in the absence of nuclear extract (0), or in the presence of 120 µg or 240 µg of Jurkat T cell nuclear extract. The position of four regions protected in the presence of nuclear extract (A-D) are indicated on the right. The location of the transcription start site is indicated by the arrow.
The association of increased IMPDH enzymatic activity with cellular proliferation and transformation has been known for 20 years, originating in observations on rat hepatoma cells (8) and resulting in an intensive search for IMPDH inhibitors as potential antineoplastic agents. Such inhibitors have been demonstrated to result in inhibition of cell growth and the induction of cellular differentiation, in conjunction with inhibition of DNA synthesis directly attributable to the depletion of guanine nucleotides(12, 13, 14, 15, 16) . In addition, several inhibitors have been found to be useful as immunosuppressive agents and to inhibit the activation of T lymphocytes in vitro(44, 45) . These observations underscore the potential importance of this enzymatic activity in modulating normal cell growth. The recent identification of two separate genes encoding IMPDH activity (4, 5) and the distinct association of increases in type II IMPDH mRNA with neoplastic transformation in several cell types (18, 19) have made it feasible to search for the molecular basis for the regulation of expression of the type II gene at different stages of cell development. In order to determine the structural basis for IMPDH gene regulation, we have cloned and characterized the type II IMPDH gene and its 5`-flanking sequence.
Type II IMPDH is a relatively small gene of approximately 5.8 kb consisting of 14 exons varying in size from 49 to 207 bp. The transcription initiation site, as determined by both primer extension analysis and RNase protection, occurs 50 bp upstream of the ATG and 9 bp 5` to the 5` terminus of the published cDNA(5) . The 5`-untranslated region of the cDNA is highly (70%) GC-rich. The 463-bp 5`-flanking region confers strong promoter activity on a CAT reporter gene when transfected into Jurkat T cells and peripheral blood T lymphocytes. When T lymphocytes are stimulated with the pharmacological agents PMA and ionomycin, promoter activity was increased by about 6-fold; in contrast, promoter activity was unaffected when Jurkat T cells were stimulated under the same conditions (data not shown).
Recent studies by our group have shown that activation of peripheral blood T lymphocytes with the mitogens PMA and ionomycin results in a 10- and 15-fold induction of IMPDH type II mRNA expression and total cellular enzymatic activity, respectively(40) . It has been suggested that the growth-regulated increase in IMPDH expression is due to a posttranscriptional nuclear processing event(46) . However, our data suggest that a major transcriptional component is responsible for the up-regulation of IMPDH type II gene expression in activated peripheral blood T lymphocytes. Although these data are not conclusive, they strongly suggest that the 463-bp upstream region contains at least a portion of the regulatory elements necessary for the proliferation-related expression of the gene. While we have not ruled out a direct effect of PMA and ionomycin on gene expression that is independent of proliferation, the lack of an effect of these agents on promoter-mediated CAT expression in Jurkat T cells makes this explanation less likely. It remains possible that other regions of the gene outside of the promoter region contribute to IMPDH type II up-regulation. Indeed, the 2-3-fold higher level of CAT activity found with the 1536-bp construct containing a portion of the proximal coding region of the gene in addition to the 463-bp 5`-flanking region suggest the existence of enhancer activity in this region.
Several
potentially important regulatory sites in the 463-bp 5`-flanking region
of the gene are protected from DNase I digestion in the presence of
Jurkat T cell nuclear extract and suggest functional relevance for
IMPDH type II expression. The presence of a consensus binding site
(CGCCCCCGC) for the transcription factor Egr-1(37, 38) (synonymous with Krox-24, NGFI-A, Zif268, and TIS-8) (47) at bp -163 is particularly notable. This site has
been shown to bind a family of zinc finger proteins that are immediate
early response genes important in growth regulation. Egr-1 expression
is rapidly and transiently induced by nerve growth factor in PC12 cells (48) and by serum in fibroblasts(37, 39) . Of
particular relevance is the observation that Egr-1 is induced during
the G/G
transition in the cell cycle after
mitogenic stimulation of T lymphocytes, as well as during G
as a separate event mediated by interleukin-2 (49) .
Exposure of T lymphocytes to an Egr-1 antisense oligonucleotide blocked
lymphocyte activation, strongly suggesting that Egr-1 is essential for
the expression of downstream genes required for the T lymphocyte
proliferative response. In previous studies, we have employed a
peripheral blood T lymphocyte model system to examine IMPDH expression
as a function of T cell activation(40) . We observed the
induction of IMPDH type II mRNA within 6 h after stimulation of T
lymphocytes with PMA and ionomycin, as well as with phytohemagglutinin
and allogeneic mononuclear cells, although maximum induction was
observed at 24 h. In addition, IMPDH type II mRNA levels increased in
response to PMA alone and to calcium ionophore alone, although the
combination of PMA and ionomycin was significantly more potent than
either agent alone. Similar results were obtained for Egr-1 expression;
the combination of phorbol ester and calcium ionophore lead to higher
expression than either agent alone(49) . Although the evidence
implicating Egr-1 in IMPDH type II expression is circumstantial at
present, the presence of a protected region (C) corresponding to the
Egr-1 binding site in the IMPDH type II promoter and the requirement
for increased IMPDH gene expression for T lymphocyte activation do
suggest that this site could be of considerable functional importance.
It should also be noted, however, that the putative Egr-1 site overlaps
with sites for the transcription factor AP2 (CCGCCCCCGC) (29) and Sp1 (GCTCCGCCCC)(30, 32) . These
overlapping sites offer the potential for more complex regulation based
on transcription factor interactions. It has been observed, for
example, that Egr-1 can act as a repressor of Sp1 activity at the
coincident binding site in the adenosine deaminase gene
promoter(50) .
A second region of interest in the IMPDH type
II promoter is the DNase I protected region D that occurs in close
proximity to the recognition sequence for the nuclear purine-binding
transcription factor Nm23 (GGGTGGG)(36) . Nm23, also known as
PuF, was previously shown to bind to a nuclease hypersensitive element
of the human c-myc P1 promoter and directly regulate c-myc transcription(51) . Nm23 is a nucleoside diphosphate
kinase enzyme, the phosphorylation status of which appears to vary as a
function of the metastatic potential of some cell types(52) .
The transcriptional regulatory function of this protein has been shown
to be independent of its enzymatic activity(53) . Recent
studies of Nm23 expression in peripheral blood lymphocytes have
demonstrated a strong correlation between Nm23 expression and
proliferative activity, with levels of Nm23 protein being significantly
higher in activated peripheral blood lymphocytes and in malignant
proliferating lymphoid cells than in resting leukocytes(54) .
The increase in the level of Nm23 occurred as a relatively late event,
suggesting a potential role for this protein in the late G and early S phases of the cell cycle. Similarly, increased IMPDH
expression has been demonstrated to be a requirement for continued
cellular proliferation(12, 16) . The finding that the
murine homologue of Nm23-H2 serves as a differentiation inhibiting
factor in mouse myeloid leukemia cells further supports a role for Nm23
in cell proliferation(55) . Whether or not Egr-1 and Nm23 are
integral to IMPDH type II expression will be determined by specific
mutagenesis experiments. Delineation of their respective roles should
provide further insight into the cascade of molecular events required
for the ultimate synthesis of guanine nucleotides necessary for the
initiation of DNA replication.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L39210[GenBank].