(Received for publication, April 11, 1995; and in revised form, June 23, 1995)
From the
Calspermin is a high affinity Ca/calmodulin
binding protein that is found only in postmeiotic male germ cells. Our
previous studies have shown that the calspermin transcript is produced
by utilization of a testis-specific promoter located within an intron
of the calmodulin kinase IV gene. This promoter contains two cAMP
response element-like motifs that bind the testis-specific
transcription factor CREM
. This interaction is required for
transcriptional activation. Here we describe a novel regulatory
element, the 111-base pair first intron of the calspermin gene, which
is also required for enhancement of transcription by CREM
via the
cAMP response element motifs. Deletion or inversion of this intron
results in loss of CREM
-mediated stimulation of transcription.
However, CREM
stimulates calspermin promoter activity when the
intron is moved upstream of the promoter but only when inserted in the
proper orientation. Footprint, linker scanning, and deletion analyses
were used to identify regulatory elements in the intron. We suggest
that the intron functions as an orientation-dependent but
position-independent regulatory element to activate the calspermin
promoter by facilitating the stimulatory effect of CREM
on
transcription.
Calspermin is a high affinity Ca/calmodulin
binding protein that is present exclusively in testicular germ
cells(1, 2, 3) . In situ hybridization experiments have shown that the calspermin
transcript first appears in primary spermatocytes undergoing the final
stages of meiosis and increases in abundance throughout the postmeiotic
differentiation process known as spermiogenesis(2) . During
postnatal development of the rat testis, calspermin mRNA is first
detected at day 24 and reaches the high levels characteristic of the
adult at about day 35. Therefore, expression of the calspermin gene is
both tissue-specific and developmentally regulated. The calspermin
transcript is derived from a gene encoding the
Ca
/calmodulin-dependent protein kinase IV (CaM kinase
IV)(
)(2, 4, 5) . Calspermin
represents the carboxyl-terminal 169 amino acids of CaM kinase IV and
lacks the entire kinase catalytic domain. Primer extension assays have
mapped the calspermin transcription initiation site within an intron of
the CaM kinase IV gene, which is at least 35 kilobases downstream of
the kinase transcription initiation site. (
)Our previous
studies showed that the calspermin transcript is produced by
utilization of a testis-specific promoter within the same intron of the
CaM kinase IV gene that contains the initiation site of the calspermin
transcript(6) .
Multiple regulatory elements contribute to
the testis-specific expression of the calspermin gene. Results from
analysis of transgenic mice demonstrate that the -200 to
+321 region of the calspermin promoter is sufficient to direct
expression of a -galactosidase reporter gene specifically to
testis of postpubertal transgenic mice.
We suggested that
this region contained at least three regulatory elements. First, there
are negative regulatory elements between nucleotides -200 and
-80 that may contribute to silencing the calspermin gene in
somatic cells. This is based on the finding that whereas a transgene
containing the -80 to +361 region of the gene is
transcriptionally active in NIH3T3 cells, the -200 to +361
region is inactive(6) . Second, there are two CRE-like motifs
at -70 and -50 that bind the testis-specific transcription
factor CREM
. Interaction of CREM
with the CRE sites is
required for maximal transcription from the calspermin promoter in
NIH3T3 cells(6) . Ectopic expression of CREM
is sufficient
to restore activity to an otherwise inactive calspermin promoter in
NIH3T3 cells and to a calspermin promoter-LacZ chimeric transgene in
skin fibroblasts prepared from transgenic mice, which normally express
the transgene only in testis (6) .
From these data,
we suggested that CREM
is the transcription factor primarily
responsible for activation of the calspermin gene in testis. Third, we
postulated that the 111-bp intron from +130 to +241 might
also serve a regulatory role since maximal promoter activity was
obtained with constructs that included this region(6) . In this
paper, we show that this element is absolutely required for CREM
to activate transcription from the calspermin promoter.
We
demonstrate that CREM stimulates transcription from a wild-type
calspermin promoter in NIH3T3 cells but not from the promoter when the
intron is deleted or inverted. By placing the intron in different
positions and different orientations, we show that the intron serves as
a regulatory element in an orientation-dependent but
position-independent manner. A region within the intron is protected
from DNase I digestion by nuclear extract from NIH3T3 cells. 5`, 3`,
and internal deletions of the intron sequence were made to identify
regulatory elements. Based on these data we conclude that the entire
111-bp intron is required to facilitate the effect of CREM
. We
suggest that multiple factors must bind to the intronic sequence to
enable CREM
interacting with the CREs to regulate transcription of
the calspermin gene.
Transfection of DNA, CAT assays, preparation of NIH3T3 cell nuclear extract and DNase I footprinting were performed as described previously (6) .
Figure 1: Schematic representation of the calspermin gene. Exons are indicated by rectangles. +1 is the initiation site of the calspermin transcript, and the numbers indicate the positions of intron-exon junctions relative to the transcription initiation site.
Figure 2:
Analysis of the intron by deletion
mutagenesis. The structure of the parental plasmid is shown at the top. The 5`- and 3`-terminal nucleotides of each promoter
construct are shown in the left side of the figure. CAT
activity obtained from transfection with (whitebars)
or without expression vectors encoding CREM and CaM kinase IV (blackbars) is shown. CAT activity is calculated as
the ratio of the activity obtained for the construct to be tested to
that obtained for pCAT basic, which is a promoterless CAT construct.
The results are the mean values of at least three independent
experiments ± S.E.
Previous studies have shown that CREM or
CREB binding to the two CRE motifs greatly stimulates activity of the
wild-type calspermin promoter but not of a promoter in which both CREs
were either mutated or deleted (6) . Since the CREs and the
intron appear to be regulatory elements of the calspermin promoter, we
studied the relationship between these elements. The constructs in Fig. 2were cotransfected into NIH3T3 cells with or without
expression vectors for CREM
and a constitutively active fragment
of CaM kinase IV(6) . The blackbars indicate
the amount of CAT activity generated following transfection of the
promoter-CAT constructs alone, and the whitebars indicate the amount of CAT activity resulting from cotransfection
with expression vectors for CREM
and CaM kinase IV. When the
intron was intact, CREM
stimulated promoter activity (Fig. 2, constructs -80/+361, -80/+321,
-80/+300, and -80/+242). By deleting part of the
intron, CREM
stimulation of promoter activity was abolished ( Fig. 2constructs -80/+200, -80/+180, and
-80/+50). Deletion (-80/+50), inversion of the
intron, internal deletion of the entire intron or of the central 50
nucleotides of the intron (Fig. 2, last two constructs)
eliminated CREM
stimulation of promoter activity. These results
suggested that the intron was required for CREM
activation of
promoter activity.
Figure 3:
Position and orientation effects of the
intron. The intron was placed in different positions and orientations
relative to the calspermin promoter. The intron is denoted by a
rectangle containing an arrow, which indicates the orientation
of the intron. The structure of each promoter construct is shown in the left side of the figure. CAT activity obtained from
transfection with (whitebars) or without expression
vectors encoding CREM and CaM kinase IV (blackbars) is shown. CAT activity is calculated as the ratio
of the activity obtained for the construct to be tested to that
obtained for pCAT basic, which is a promoterless CAT construct. The
results are the mean values of at least three independent experiments
± S.E.
The constructs in Fig. 3were
also cotransfected into NIH3T3 cells with expression vectors for
CREM and CaM kinase IV. In Fig. 3, the blackbars indicate the amount of CAT activity generated
following transfection of the promoter-CAT constructs alone, and the whitebars indicate the amount of CAT activity
resulting from cotransfection with expression vectors for CREM
and
CaM kinase IV. Constructs II and III served as positive and negative
controls, respectively. CREM
stimulated CAT activity from the
-80/+361 promoter, which contained a properly positioned and
oriented intron (Fig. 3, construct III), but not from
the -80/+50 promoter, which contained no intron (II). Placing an intron upstream of the -80/+50
promoter in the plus orientation rescued the stimulatory effect of
CREM
(Fig. 3, construct IV). Switching this
upstream intron to the minus orientation resulted in a complete loss of
the stimulatory effect (Fig. 3, construct V),
suggesting an orientation-dependent requirement for the intron to
facilitate the stimulatory effect of CREM
. CREM
-mediated
enhancement of transcription was somewhat reduced when two copies
rather than one copy of the intron were placed upstream of the
-80/+50 promoter (Fig. 3, construct VI). An
additional copy of the intron placed in either orientation upstream of
the -80/+361 promoter containing an intron at the proper
position and in the plus orientation had no effect on CREM
stimulation of transcription (Fig. 3, constructs VII and VIII). If the intron in the -80/+361
promoter was in the correct position but in the minus orientation,
CREM
did not stimulate promoter activity even when one or two
copies of the intron were positioned upstream of the promoter in the
plus orientation (constructs IX and X). Therefore,
one copy of the intron in the proper position was dominant and either
stimulated or inhibited the ability of CREM
to enhance activity
from the calspermin promoter depending on its orientation.
Figure 4: DNase I footprint analysis of the intron. The probe sequence spanned -200 to +361. LanesA and B, bovine serum albumin controls. LaneC, NIH3T3 cell nuclear extract. LaneD, a competition assay with 20 ng of cold oligonucleotide representing sequence from +170 to +195. LaneE, a competition assay with 20 ng of cold random sequence oligonucleotide.
Figure 5:
Analysis of the intron by linker scanning
mutagenesis. The sequences between +143 and +213 were
subjected to linker scan analysis. The constructs contain a -80
to +361 fragment of the calspermin promoter, except the sequences
in rectangles (linker cassettes) are replaced by a restriction enzyme
site indicated on the top of rectangles. These
constructs were cotransfected with or without expression vectors for
CREM and CaM kinase IV. CAT activity obtained from transfection
with (whitebars) or without the expression vectors
for CREM
and CaM kinase IV (blackbars) is
shown. CAT activity is calculated as the ratio of the activity obtained
for the construct to be tested to that obtained for pCAT basic. The
results are the mean values of at least three independent
experiments.
Figure 6:
Deletion analysis of the intron. A series
of deletions was made from the 5` end of the intron as shown in the left side of the figure. The 5`-terminal nucleotides of each
deletion are indicated. CAT activity obtained from transfection with (whitebars) or without expression vectors encoding
CREM and CaM kinase IV (blackbars) is shown.
CAT activity is calculated as the ratio of the activity obtained for
the construct to be tested to that obtained for pCATbasic. The results
are the mean values of at least three independent experiments +
S.E.
The calspermin transcript is derived from a gene encoding the
CaM kinase IV gene in postmeiotic male germ
cells(2, 4, 5) . Our earlier studies have
shown that the calspermin transcript is produced by utilization of a
promoter located within an intron of the CaM kinase IV
gene(6) . About 15 CAT constructs were made to
analyze promoter activity from different DNA fragments of the
calspermin promoter and, in each case, the intron was required for
maximal promoter activity(6) . This result has been repeated in
several cell lines including NIH3T3, HeLa, and NS20 cells. The
-200 to +361 fragment of the calspermin promoter, which
contains the 111-bp intron accurately initiates transcription in
vitro in testis nuclear extracts. The same DNA fragment targets a
LacZ reporter gene specifically to postmeiotic male germ cells of
transgenic mice.
Therefore, it seems likely that the intron
is important for the function of the calspermin promoter in vitro as well as in vivo, although the latter has not been
assessed experimentally. We demonstrate here that the intron is
essential for stimulation of transcription from the calspermin promoter
by the testis-specific transcription factor CREM
.
Introns have been shown to regulate the expression of many genes (7, 8, 9, 10, 11, 12, 13) . When a 200-bp DNA fragment contained in the first intron of the human DRA gene was linked to the 5` or 3` end of a CAT reporter gene driven by the thymidine kinase promoter, it stimulated thymidine kinase promoter activity. The effect was independent of position, orientation, or distance(14) , which are the characteristics of the classically defined enhancer. This enhancer is actively involved in the regulation of DRA gene transcription, since several DNase I hypersensitive sites were mapped to the element, and high level expression of the DRA gene depends on its presence. Similarly, transcription of several other genes has been shown to be influenced by a regulatory element within an intron including those for the immunoglobulin heavy chain gene(15) , the renin gene(16) , and the type IV collagen gene(17) .
The
transcriptional activation function of CREM is dependent on the
presence of the entire 111-bp intron of the calspermin gene. Our
previous studies have identified two CRE-like motifs at -70 and
-50, which bind the testis-specific transcription factor
CREM
and stimulate transcription from the calspermin
promoter(6) . We demonstrate here that both the CRE motifs and
the intron are required for the stimulation by CREM
, since
CREM
no longer stimulates transcription from the calspermin
promoter in which the intron has been deleted or placed in reverse
orientation. When the intron is moved upstream of the promoter and
correctly oriented, CREM
stimulates promoter activity. However,
CREM
loses its effect when the orientation of the upstream intron
sequence is reversed. In addition, 5` deletions of even 20 nucleotides
resulted in the loss of activity as did 3` or internal deletions of the
intron in the original or upstream (data not shown) position.
Therefore, the effect of the intron on the function of CREM
is
position-independent but orientation-dependent.
There are at least
four theoretical ways that the intron could influence the stimulatory
effect of CREM. First, the most obvious possibility is that the
intron is necessary for stability of the primary transcript. Second,
the intron could also contain CRE-like elements. Third, the intron
could be required for the physical binding of CREM
to the upstream
CRE motifs. Fourth, there could be interactions between CREM
binding at the CRE motifs and intron binding proteins that stabilize
the transcription complex or are otherwise required for CREM
function. Since the intron is position-independent but
orientation-dependent, we do not favor the stability theory. Inversion
of the intron while carefully maintaining the integrity of the splicing
junctions or deletion of the central 50 bp of the intron both result in
the loss of activity. On the other hand, the 111-bp intron sequence can
be moved upstream of the CREs and maintains its regulatory role in this
position. We also excluded the second possibility that the intron
contains CRE-like elements, because no region in the intron is
protected by purified CREM
in a footprint analysis, whereas both
CRE-like motifs are protected(6) . The effect of the intron is
orientation-dependent. The CRE is an orientation-independent response
element, because CRE is an inverted repeat sequence element. In
addition, CREM
does not stimulate transcription from the intron
sequence when it is linked to an SV40 promoter (data not shown). We can
excluded the third possibility, because our previous studies
demonstrate that CREM
physically binds to the CRE motifs of the
calspermin promoter in the absence of the intron. The CRE motifs are
protected from DNase I digestion by purified CREM
or CREM
in
testis nuclear extract, even though the DNA probe (from -500 to
+50) contained no intron(6) . Bandshift assays indicate
that purified CREM
or the CREM
present in testis nuclear
extract binds to double-stranded oligonucleotides containing the CREs
but not the intron(6) . Collectively these results indicate
that CREM
binds to the CRE motifs, but in the absence of the
intron it does not stimulate transcription from the calspermin
promoter. Our results support the fourth possibility that there are
interactions between CREM
and the intron binding proteins. Since
the interactions require both CREs and the intron, mutation of either
CREs or the intron should have the same negative effect on the
calspermin promoter. In support of this idea, the levels of CAT
activity produced from the calspermin promoter with an internal
deletion of the intron is the same as that from the calspermin promoter
with both CREs mutated or deleted.
Interactions between
transcription factors are the basis for transcriptional
regulation(18) . Interactions between CRE binding factors and
other transcription factors have been postulated. Imai et al. (19) revealed that optimal transcription of the
phosphoenolpyruvate carboxykinase gene in response to glucocorticoids
required not only a glucocorticoid response element but also a CRE in
the promoter region. Since a monoclonal antibody to the glucocorticoid
receptor coprecipitated CREB, a protein-protein interaction between
glucocorticoid receptor and CREB was proposed to account for the role
of the CRE in the response of the phosphoenolpyruvate carboxykinase
gene to glucocorticoids. Unfortunately, this type of interaction does
not explain the calspermin promoter. While a region in the intron that
is protected from DNase I digestion in nuclear extract corresponds in
sequence to a glucocorticoid response element-like element, activity of
the calspermin promoter was not stimulated in response to
glucocorticoids, and the intron failed to bind glucocorticoid receptor
(data not shown). In another study, Delegeane et al. (20) identified an enhancer in the promoter region of the human
glycoprotein hormone -subunit gene. This enhancer had no
independent activity but, in combination with the CRE found upstream of
the enhancer, it significantly increased the tissue-specific activity
of both the
-subunit promoter and a heterologeous promoter. In
this case, the interactions between the CRE binding proteins and other
transcription factors facilitated tissue specific expression of the
gene.
Whereas we cannot experimentally prove the reason for the
orientation dependence of the intron, it might be explained in the
following way. Interaction of the intron with the calspermin promoter
likely involves several DNA-binding proteins. Productive interactions
between a series of DNA-binding proteins that recognize elements in the
5`-flanking region of the calspermin promoter, such as the CRE motifs,
and another series that binds the intron may require the correct
spatial and directional orientation of these proteins. Reversal of the
intron containing sequences crucial for these interactions might lead
to disruption of the normal spatial geometry of the transcription
complex. In other words, when the intron is in the plus orientation,
the interactions between the intron and the promoter elements
facilitate formation or stabilization of the transcription initiation
complex. When the intron is in the minus orientation, the proteins
bound to the intron may prevent the formation of the transcription
initiation complex or at least render it sufficiently unstable to
support transcription. Several CAT constructs that contain an inverted
intron in its natural position have no transcriptional activity (Fig. 3). In these cases, the inverted intron is placed
downstream of the transcription initiation site, thus the proteins
bound to the inverted intron may not only prevent formation of the
transcription initiation complex but also may physically block
progression of RNA polymerase II. This may be why two additional plus
orientation introns upstream of promoter containing the inverted intron
do not rescue the promoter activity. Our studies suggest that one of
the factors that binds to the 5`-flanking region of the promoter and
interacts with the intron binding proteins is CREM.
Three lines
of evidence demonstrate a requirement for CREM in the regulation
of testis-specific expression of the calspermin gene. 1) CREM
restores activity to a normally inactive calspermin promoter in NIH3T3
cells(6) . 2) Anti-CREM
antibody inhibits in vitro transcription activity in testis nuclear extract from the
calspermin promoter. 3) CREM
restores transcription from a
calspermin promoter-LacZ chimeric transgene in skin fibroblasts
prepared from transgenic mice.
The CREM
transcript is
generated exclusively in testis from an alternative splicing
event(21) , and CREM
protein is accumulated to high levels
in postmeiotic germ cells where the calspermin gene is
activated(2, 22) . Based on these results, we
suggested that appearance of abundant CREM
in postmeiotic germ
cells results in activation of the calspermin gene. The results
reported here suggest that the intron may contribute to the regulation
of testis-specific expression of the calspermin gene. We demonstrate
that the intron serves as a position-independent but
orientation-dependent regulatory element that may function to
facilitate the stimulatory function of CREM
.