(Received for publication, September 6, 1995)
From the
A 42-kilobase pair region of rat DNA containing the
Ca/calmodulin-dependent protein kinase IV (CaM kinase
IV) gene has been cloned and characterized. The gene consists of 12
exons and 11 introns and is predicted to encode both
and
forms of CaM kinase IV as well as the testis-specific
calmodulin-binding protein calspermin. The promoter utilized to
generate the
-kinase isoform is located in intron 1, whereas the
promoter utilized to produce the calspermin transcript is contained in
intron 10. The calspermin promoter region which extends from -200
to +321 relative to the calspermin transcription initiation site
that contains two cyclic AMP response elements (CRE) at -70 and
-50 and has been shown previously to be inactive in NIH3T3 cells
(Sun, Z., Sassone-Corsi, P., and Means, A. R.(1995) Mol. Cell.
Biol. 15, 561-571) was ligated to the lacZ reporter
gene and used to generate transgenic mice. The promoter was expressed
exclusively in postmeiotic testis where
-galactosidase was found
predominantly in elongating spermatids. The cell and developmental
specificity of transgene expression was very similar to the pattern
shown by the endogenous gene. Although the transgene promoter was
silent in somatic tissues,
-galactosidase expression could be
restored in primary cultures of skin fibroblasts by introduction of
vectors encoding CREM
and CaM kinase IV.
Ca/calmodulin-dependent protein kinase IV (CaM
kinase IV, (
)also known as CaM kinase Gr) is a monomeric
multifunctional enzyme expressed in a tissue-restricted manner. It is
particularly abundant in T-lymphocytes, granule cells of the cerebellum
and meiotic male germ cells (Ohmstede et al., 1991; Means et al., 1991; Hanissian et al., 1993; Jones et
al., 1991; Miyano et al., 1992). Although CaM kinase IV
is the product of a unique gene in both mouse and human (Sikela et
al., 1989, 1990), two isoforms of the enzyme are present in the
cerebellum with relative M
of about 62,000 (
)
and 64,000 (
) (Ohmstede et al., 1989). The cDNA of the
smaller
isoform, which has a calculated M
of
53,159, has been expressed in Escherichia coli and baculovirus
systems, and many properties of the enzyme have been determined
(Cruzalegui and Means, 1993; Okuno and Fujisawa, 1993; Mosialos et
al., 1994; Kitani et al., 1994; Enslen et al.,
1994; Tokumitsu et al., 1994; Selbert et al., 1995).
The relationship between the
and
isoforms is controversial.
It has been suggested that
is a post-translational modification
of
(Mosialos et al., 1994). However, McDonald et
al.(1993) provided evidence that
could not be converted to
by dephosphorylation and argued for an independent gene product.
The latter contention was strengthened by Sakagami and Kondo(1993) who
obtained a cDNA from a rat brain library thought to encode the larger
isoform. The calculated M
was 55,705, and it
differed from the
isoform only by a unique 28-amino acid
N-terminal extension. Whereas the
mRNA was distributed
differently from
in parasagital sections of the brain, the
properties of CaM kinase IV
have yet to be reported. The
form of CaM kinase IV requires phosphorylation on a threonine residue
in the ``activation loop'' (Thr
) by a CaM
kinase IV kinase for full activity (Selbert et al., 1995).
This phosphorylation stimulates subsequent autophosphorylation on
several serine residues, resulting in a significant amount of
Ca
/calmodulin-independent activity (McDonald et
al., 1993; Selbert et al., 1995).
The C-terminal 169
amino acids of CaM kinase IV comprise a separate protein called
calspermin that is expressed exclusively in postmeiotic male germ cells
(Ono et al., 1989; Means et al., 1991). This is the
most abundant calmodulin-binding protein in spermatozoa but is of
unknown function. In heterologous cell lines, the transcript of
calspermin can be initiated from a promoter within an intron of the CaM
kinase IV gene (Means et al., 1991; Jones et al.,
1991; Ohmstede et al. 1991; Sun et al., 1995) and,
with the exception of the first 130 nucleotides, is identical to the
corresponding region of the CaM kinase IV transcript (Means et
al., 1991). Transcription from this promoter in NIH3T3 cells
requires two cyclic AMP response elements (CRE) at -70 and
-50 bp relative to the transcription start site (Sun et
al., 1995). These CREs bind the transcription factors CREB or
CREM and either factor markedly stimulates activity of the basal
promoter defined by Sun et al.(1995) as the genomic DNA
fragment extending from nucleotide -80 to nucleotide +361.
However, the addition of 120 bp to the 5` end of the basal promoter
markedly inhibits transcription in NIH3T3 cells. Some activity could be
restored by cotransfection of CREM
(or CREB) together with CaM
kinase IV (or protein kinase A). We have suggested that the -200
to +361 fragment might be sufficient to produce germ cell-specific
transcription.
We have cloned the entire CaM kinase IV gene which
spans greater than 42 kbp. The organization of this gene suggests that
CaM kinase IV and -
and calspermin are all produced by
alternative transcriptional initiation. The longest transcript would
produce CaM kinase IV
. The CaM kinase IV
promoter is located
within the first intron, whereas the promoter and first exon of
calspermin are located in intron 10. We also show the -200 to
+321 region of the calspermin gene is sufficient to target
expression of a reporter gene in transgenic mice. Expression of the
transgene is restricted to postmeiotic male germ cells and shows the
correct developmental pattern during the initiation of spermatogenesis.
However, transgene expression can be induced in primary cultures of
skin fibroblasts by transfection of expression vectors encoding
CREM
and CaM kinase IV.
Figure 1:
Schematic
representation of the organization of the rat CaM kinase IV gene. A, schematic representation of the organization of exons I and
II as well as intron A. The CaM kinase IV initiation codon is
nucleotide +1 which is in exon II. The CaM kinase IV
initiation codon is at nucleotide -524 which is in exon I. Both
isoforms of the kinase share exons II-XII illustrated in B. B, organization of the CaM kinase IV gene. Exons
are indicated by Roman numerals, and introns are indicated by capital letters. The dotted line between intron J and
exon XI represents the location of the testis-specific exon involved in
generation of the calspermin transcript. The arrows indicate
the location of EcoRI restriction sites that were used to
clone and order the genomic fragments of DNA. C, calspermin
gene structure. Exons are indicated by boxes, and the black box is the testis-specific calspermin exon (Ts). +1 is the calspermin transcription
initiation site. The numbers indicate the positions of intron
and exon junctions. Two CRE-like motifs are located at -50 and
-70 relative to the transcriptional initiation site. D,
structure of the construct used to generate transgenic mice. The
-200 to +321 region of the calspermin promoter was linked to
a lacZ reporter gene.
Figure 3:
The
nucleotide sequence of the 5`-flanking region of the rat CaM kinase IV
gene. The numbers are relative to the A of the ATG encoding
the initiation codon for CaM kinase IV which is +1. The numbers above the nucleotide sequences represent significant
regions of the 5`-flanking region: A-211, the
transcriptional start site of CaM kinase IV
(Means et
al., 1991); B+1 and B-69 the
translation initiation site and first nucleotide of the cloned cDNA for
CaM kinase IV
as described by Sakagami and Kondo (1993); -350, -460, -500, and -1060 are the 5` nucleotides of the promoter constructs used to generate
the data in Fig. 4. All of these DNA fragments ended at
-11. The vertical lines depict exon/intron and
intron/exon junctions.
Figure 4:
CaM kinase IV promoter activity as a
function of DNA length. HeLa cells were transfected with 10 µg of
DNA containing different portions of the CaM kinase IV
promoter
ligated to CAT (the sequence of the promoter regions used in each
construct is shown in Fig. 3). CAT activity is expressed as the
ratio of that obtained from the construct to be tested relative to pCAT
basic which is a promoterless CAT construct. The results shown are the
mean values of at least three independent experiments ±
S.E.
Figure 2: The sequences of intron-exon junctions of the CaM kinase IV and calspermin gene. The last column indicates the amino acid codons interrupted by each intron.
Figure 5:
-Galactosidase activity from various
tissues. Black bars indicate
-galactosidase activity from
tissues of postpubertal transgenic mice. White bars indicate
-galactosidase activity from tissues of nontransgenic age-matched
littermates. The experiment was repeated at least three times with
animals from each of the three transgenic mice. These data are the
results of a typical experiment.
Testes from postpubertal
transgenic mice were removed, briefly fixed, and then soaked in an
X-gal solution for 5 h. The transgenic testis stained an intense blue
as shown in Fig. 6A. A testis from an age-matched
nontransgenic littermate was treated in an identical way and is shown
in Fig. 6B. The control testis remained unstained and
thus contained little -galactosidase activity.
Immunohistochemistry was performed to determine which cell types
expressed
-galactosidase in postpubertal testis as shown in Fig. 6. The immunoreactive
-galactosidase protein was
stained black as described under ``Experimental Procedures,''
and the tissue sections were counterstained with hemotoxylin.
-Galactosidase was localized primarily in the central portion of
the seminiferous tubules in testis from transgenic mice as indicated by
the arrow (in Fig. 6C). Fig. 6E is a higher magnification of one representative tubule.
-Galactosidase is present predominantly where elongating
spermatids are located. No
-galactosidase expression was detected
in testis from nontransgenic mice (Fig. 4D). These
results reveal that
-galactosidase is expressed from the
calspermin promoter specifically in postmeiotic germ cells during the
late stages of spermiogenesis.
Figure 6:
Localization of the -galactosidase
transgene product in testis. A, a testis of a postpubertal
transgenic mouse was removed and stained in a solution containing X-gal
for 5 h. The blue color indicates the presence of
-galactosidase which catalyzed hydrolysis of X-gal into an
insoluble blue product. B, a testis from a nontransgenic
age-matched littermate treated in an identical manner to the testis
described in A. C and D, immunohistochemical localization of
-galactosidase protein in seminiferous tubules. Immunoreactive
-galactosidase is stained black. The tissue sections are
counterstained with hemotoxylin. C, section of testis from a
postpubertal transgenic mouse.
-Galactosidase is primarily located
in the central portion of the seminiferous tubules as indicated by the arrow. D, section of testis from a nontransgenic
age-matched littermate. This section was treated identically to the
section in Fig. 3C. Note the complete absence of
-galactosidase reaction product. E, a higher magnification of one
seminiferous tubule from the testis of a transgenic mouse (the
magnification is
200).
-Galactosidase is restricted to the
central region of the tubule that contains postmeiotic elongating
spermatids.
Expression of the transgene was also
examined as a function of postnatal development. The first cycle of
spermatogenesis begins at birth and is completed at about day 34
(Russell, 1990). Spermatogonia constitute the predominant germ cell
population in 6-8 day mice. Leptotene and zygotene spermatocytes
appear at day 10, pachytene spermatocytes at day 14, round spermatids
at day 19-20, and elongating spermatids at day 23-25
(Bellve et al., 1995). -Galactosidase activity of testis
from transgenic mice of various postnatal ages was measured as shown in Fig. 7. There was no significant change in
-galactosidase
activity before day 19. The first obvious increase of
-galactosidase activity occurred between days 22 and 24,
correlating with the initial appearance of elongating spermatids.
-Galactosidase activity continued to increase until day 35 when
the first round of spermatogenesis is completed. Similar changes of
-galactosidase activity during testis development were observed in
the other two lines of transgenic mice (data not shown). Collectively
the observations suggest that expression of the lacZ transgene
is primarily restricted to postmeiotic germ cells.
Figure 7:
-Galactosidase activity from testes
of transgenic mice of different postnatal ages. Testes from 7-, 14-,
19-, 22-, 24-, 30-, and 35-day-old transgenic mice were removed and the
-galactosidase activity was measured as described under
``Experimental Procedures.'' The experiment was repeated at
least three times on animals from each of the three transgenic lines.
The data shown are the results of a typical experiment and represent
the mean values of triplicate samples from each of three mice ±
S.E.
Figure 8:
Restoration of -galactosidase
activity in skin fibroblasts of transgenic mice. The cells were
prepared from 2-day-old transgenic mice and were either transfected
with expression vectors as indicated or treated with forskolin. The
fibroblasts were stained with a solution containing X-gal 4 days after
transfection. The data presented represent the total number of blue
cells present within 10 random microscopic fields. A pCMV-CAT construct
was cotransfected to control for transfection efficiency. All cells
transfected with CREM
and CaM kinase IV were forced to express
-galactosidase. The experiment was repeated three times with cells
prepared from each of the three transgenic lines. The data shown are
representative of all experiments.
The rat CaM kinase IV gene spans at least 42 kbp of DNA and contains 12 exons. With the exception of the second (362 bp) and last (456 bp) exons, the other nine average only 82 bp in length. This complicated structural organization, if the norm, might explain why other genes encoding calmodulin-dependent enzymes from multicellular organisms have yet to be sequenced in their entirety. The location of the introns in the coding region seems to disrupt at random, and without other CaM kinase gene structures to compare, little else can be concluded.
The unusual aspect of the CaM kinase gene organization is
that alternative promoters exist in the first and penultimate introns.
The intron 1 promoter presumably directs transcription of the smaller
or form of CaM kinase IV which is the enzyme on which most of the
biochemical characterization has been performed. We show that a
fragment extending about 150 bp from the transcriptional start site at
-211 (Fig. 3) can function as a promoter in HeLa cells.
This 150-bp fragment contains GC-rich regions typical for those present
in a variety of genes but does not contain a TATA box. In the only
other published study evaluating regulation of the expression of a CaM
kinase gene, Olson et al.(1995) show the importance of a TATA
box at -162 of the CaM kinase II
gene. Extending the 150-bp
promoter fragment of the CaM kinase IV gene 100 bp or more at the 5`
end severely reduces transcription in HeLa cells. Since CaM kinase IV
expression is generally limited to thymic lymphocytes,
cerebellum, and testicular germ cells undergoing meiosis, perhaps the
silencing due to addition of additional 5` sequences reflects such cell
specificity. Alternatively, it is possible that expression of CaM
kinase IV requires regulation, and HeLa cells do not contain such
putative regulatory factors. For example, a preliminary report
suggested that expression of the enzyme in an embryonic stem
cell-derived neuronal culture required thyroid hormone and the thyroid
hormone receptor
(Larson et al., 1995). The availability
of the 5`-flanking region of the CaM kinase IV gene will allow a
mechanistic approach to this problem.
The organization of the CaM
kinase IV gene also allows a plausible explanation for how the
and
isoforms of the enzyme are generated. Sakagami and
Kondo(1993) reported the isolation of a rat brain cDNA that encoded a
different form of CaM kinase IV than that cloned by Means et
al.(1991). The only differences were the presence of a 92-bp
extension of unique sequence at the 5` end and the absence of 151 bp of
5`-nontranslated region present in the clone obtained by Means et
al.(1991). The 92-bp extension was predicted to change the start
site of translation resulting in an additional 28 N-terminal amino
acids not present in the CaM kinase IV
. The first 8 amino acids
would be encoded by the last 24 bp of the unique sequence, whereas the
remaining 20 amino acids would be encoded by nucleotides present as
part of the 5`-nontranslated sequence of the
cDNA. Whereas
Sakagami and Kondo(1993) did not formally show the presence of a
protein containing the extra 28 amino acids, they did utilize in
situ hybridization to reveal that the unique segments of the two
cDNAs differently hybridized to anatomical structures present in
parasagital sections of rat brain. As shown in Fig. 3, the
flanking region of the CaM kinase IV gene contains all the unique
nucleotides present in both
and
CaM kinase cDNA clones. The
92 bp unique to
are separated from the initial common segment,
also suggested to be present by Bland(1993), by an intervening 464 bp.
We have interpreted this intervening segment to represent an intron due
to the presence of donor and acceptor splicing sites. The fact that the
first 151 bp of the
cDNA constitute the last segment of this
intron implied that additional sequences in the intron might function
as a promoter. Preliminary evidence supporting this possibility is
shown in Fig. 4. Whereas our data cannot prove the existence of
two CaM kinase IV isoforms derived from one gene, they do support the
contention of Sakagami and Kondo(1993) that
and
are the
products of distinct mRNAs.
What our results have proven is that the
10th intron of the CaM kinase IV gene contains the testis-specific
promoter responsible for the production of the calspermin protein. The
-200 to +321 region of the calspermin promoter fragment was
sufficient to target expression of -galactosidase to postmeiotic
germ cells in a cell- and developmentally specific manner that mimics
expression of the endogenous gene judged from in situ hybridization analyses (Means et al., 1991). Sun et
al.(1995) had shown that the -200 to +361 region of the
calspermin gene contained at least three elements that influenced
expression. First the -80 to +361 fragment could be
expressed in a variety of cultured cells. One required element included
the two CREs at -70 and -50. The second element was the
111-bp intron sequence separating the 3` end of the testis-specific
calspermin exon from the 5` end of exon XI that is common to both CaM
kinase and calspermin (Fig. 1C). This intron was
subsequently shown to function in an orientation-dependent but
distance-independent manner and was required for stimulation of
transcription by CREM
(Sun and Means, 1995). The third element was
contained in the DNA between -200 and -80, since the
addition of this 120 bp largely inhibited transcriptional activity in
NIH3T3 cells. We postulated that these negative elements could
contribute to the inhibition of calspermin gene expression in somatic
tissues in vivo. The fact that the transgene driven by the
-200 to +321 is not expressed in any somatic tissues
examined in the present studies supports this suggestion (Fig. 5).
Although the -200 to +361 calspermin
promoter had very little activity when acutely transfected into NIH3T3
cells, some activity could be restored by cotransfection of expression
vectors encoding CREB or CREM together with a protein kinase
capable of specifically phosphorylating the transcription factors on
Ser
or Ser
, respectively (Sun et
al., 1995). This modification is required for the transactivation
function (deGroot et al., 1993). Since NIH3T3 cells contain
CREB, which is found at low levels in most cells, we questioned whether
an important factor in the expression of the calspermin promoter might
be the concentration of CRE-binding protein. Indeed the pattern of
expression of CREM
is very similar to that of calspermin and the
calspermin promoter transgene. The mRNA for the transcriptional
activating form of the CREM gene is first produced in very late meiotic
cells (Foulkes et al., 1992), but the protein does not appear
until later and accumulates to very high levels in cells undergoing the
late stages of differentiation during spermiogenesis (Delmas et
al., 1993). Therefore high concentrations of CREM
are present
in the same cells that transcribe the endogenous calspermin gene (Means et al., 1991) or the transgene driven by the -200 to
+321 portion of the calspermin promoter (Fig. 6). We
reasoned that the transgene would have integrated in a random fashion
and conducted preliminary experiments, suggesting that the site of
integration was different in all three transgenic lines (data not
shown). Thus, if our hypothesis was correct, transgenic expression
should be restored in somatic cells by simply increasing the cellular
content of CREM
. The data in Fig. 8show, indeed, that the
transgene can be expressed in primary skin fibroblasts but only when
the cells are transfected with expression vectors for CREM
and CaM
kinase IV. Although the data shown are from a single transgenic line,
similar results were obtained with cells derived from all three lines
(data not shown). We would argue that one critical factor responsible
for the cell-specific expression of the calspermin gene is the
availability of high levels of CREM
in those cells at the
developmentally correct time.
Clearly, the abundance of CREM
cannot be the only factor required for calspermin gene expression, or
its presence would be sufficient to activate the endogenous gene in
somatic cells which is not the case ( Fig. 8and Sun et
al., 1995). This has been aptly shown for the testis-specific
promoter of the angiotensin converting enzyme gene which also contains
a requisite CRE that binds CREM
(Goraya et al., 1995).
Although this 85-bp regulatory region is sufficient to faithfully
target testis-specific expression in transgenic mice (Howard et
al., 1993), it is not active in heterologous cells even when
cotransfected with CREM
. As shown by Goraya et al. (1995), mutation of the cryptic TATA box (TCTTATT) to a consensus
TATAATT results in a promoter that can be expressed in heterologous
cells and is responsive to CREM
. Thus a second important factor
for germ cell-specific expression could be the nature of the basal
promoter elements. A third likely contributing factor could be a
reorientation or modification of the gene that occurs as chromatin is
remodeled following meiosis. One of the mechanisms determining the
accessibility of promoters to transcription factors is DNA methylation.
DNA methylation is thought to play a role in the regulation of
tissue-specific gene expression (Cedar, 1988). Methylation inhibits
gene expression by interfering with transcription factor binding to
DNA. Studies on the DNA methylation patterns of testis-specific genes
reveal a very good correlation between undermethylation and gene
expression. The testis-specific histone H2B (Choi and Chae, 1991), TP1
(Trasler et al., 1990), and PGK2 (Ariel et al., 1991)
genes are undermethylated in testis, but more methylated in somatic
tissues where they are not expressed. It would be interesting to study
the methylation patterns of the calspermin transgene and the endogenous
calspermin gene and evaluate the accessibility of CREM
to the CRE
motifs in the promoter regions of both genes.
Since CRE motifs are
conserved within promoters of many postmeiotic germ cell specific genes
(Howard et al., 1993; Delmas et al., 1992), it is
tempting to speculate that they also are exposed during meiosis by
chromatin rearrangement and/or demethylation and in this state also
respond to the high levels of CREM. Were this true, then the
rate-limiting signal could be the stimulus that results in the
production of the CREM
transcript from the CREM gene primary
transcript. That this event is controlled by follicle-stimulating
hormone (Foulkes et al., 1993) might provide one explanation
for the requirement of this hormone for complete spermatogenesis (Means et al., 1976).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X91964[GenBank].