From the Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have isolated and characterized a cDNA encoding a transcription activating factor for the mouse selenocysteine tRNA (tRNAsec) gene from mouse mammary gland. The full-length cDNA, designated m-Staf, has a 1878-base pair open reading frame encoding 626 amino acids. The predicted amino acid sequence of m-Staf is highly homologous to that of Staf, another selenocysteine tRNA gene transcription activating factor of Xenopus laevis. Like Staf, m-Staf contains seven tandemly repeated zinc fingers and four repeated motifs. Gel shift assays indicated that the recombinant m-Staf specifically bound to the activator element region in the mouse tRNAsec gene. Transient co-transfection experiments in Drosophila Schneider cells, which lack endogenous Staf-like binding activity, showed that m-Staf increased the mouse tRNAsec gene transcription about 15-fold, whereas it stimulated Pol II-dependent thymidine kinase promoter only 2-fold. Northern blot analysis detected the presence of a 3.4-kilobase pair m-Staf transcript, which was widely but differentially expressed in various murine tissues. The binding activity of m-Staf in mouse mammary gland was undetectable during virgin and postlactating periods but increased markedly in parallel with the increase of tRNAsec transcript during the periods of pregnancy and lactation, when the gland undergoes growth and development. These results indicate that m-Staf is a transcriptional activator of the mouse tRNAsec gene and that its binding activity in the mammary gland undergoes developmental alterations.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Selenium has been established as a nutritional requirement for the essential trace elements and shown to be indispensable for the biosynthesis of selenoproteins (1). These selenoproteins include type I thyroxine 5'-deiodinase in thyroid hormone metabolism (2), those of the glutathione peroxidase family in an antioxidative system (3, 4), and several other newly found ones, such as selenoprotein P, selenoprotein W, and thioredoxin reductase (5-8). Both type I thyroxine 5'-deiodinase and glutathione peroxidase play an important function in metabolic activities of mammary cells during lactation (9-12).
Selenium is incorporated into selenoproteins in the form of selenocysteine, and its incorporation is directed by a specific UGA codon, which normally functions as a stop codon in both procaryotes and eucaryotes (13). The selenocysteine tRNA (tRNAsec)1 serves as a donor of selenocysteine to nascent selenoproteins in response to the specific UGA selenocysteine codons (13, 14). This reaction also requires a selenocysteine insertion sequence, a cis-acting mRNA element, and a specialized elongation factor, the SELB protein (15, 16). It has been shown that transfection of plasmids expressing tRNAsec into human 293 cells, an embryonic kidney cell line, increases the level of 5'-deiodinase activity (17), suggesting that the amount of tRNAsec is critical for the regulation of selenoprotein biosynthesis.
Extensive analysis of the tRNAsec gene promoter has been reported for Xenopus laevis (18-21). Like other tRNA genes, this gene is transcribed by RNA polymerase III (Pol III) (22). Usually, transcription of tRNA genes requires two promoter elements, named A box and B box, situated inside the coding region. However, the tRNAsec gene is atypical in that its transcription is not dependent on the A box, which is naturally debilitated in this gene, but requires the B box and three other upstream elements, such as an activator element (AE) located in a distal sequence element, a proximal sequence element, and a TATA motif (18-21). These elements are well conserved among murine and bovine species as well as in X. laevis (23). The factors binding to the proximal sequence element, the TATA motif, and the B box have been well characterized, but the ones to the AE have not (14-17). Recently, a cDNA encoding a DNA-binding protein to the AE has been cloned from X. laevis and named Staf (selenocysteine tRNA gene transcription activating factor) (24). The structure of Staf is characterized by the presence of seven tandemly repeated zinc fingers that facilitate its DNA binding activity (24). Staf has been shown to transactivate tRNAsec gene transcription in a X. laevis oocyte system (24).
We have been studying the regulatory mechanisms involved in developmental and tissue-specific gene expression in the mouse mammary gland (25). During the course of our study to clone and characterize cDNA for mammary transcription factors, we have cloned a cDNA encoding a protein containing zinc finger motifs and named it m-Staf. In this report, we describe the molecular cloning and functional characterization of m-Staf and present evidence indicating that it is a mouse counterpart of Staf, which activates transcription of the mammalian tRNAsec gene. We also present data suggesting that it is involved in regulating mouse tRNAsec gene expression during the development of mouse mammary gland.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA Cloning-- A mouse mammary gland cDNA library (Stratagene) was screened using a 32P-labeled probe corresponding to the DNA binding domain of mouse YY1 cDNA (26). Among several clones obtained, one clone, named 1BA, was sequenced and analyzed further. The 5' and 3' regions of the gene were obtained from a mouse spleen marathon-ready cDNA (CLONTECH) by the method of 5'- and 3'-RACE (rapid amplification of cDNA ends) (27). Using a set of primers corresponding to the 5' or 3' non-coding regions, a full-length cDNA was amplified from mouse mammary gland poly(A)+ RNA and ligated to a TA cloning vector (Invitrogen). The resultant plasmid was named pCRStaf. The sequence was verified by sequencing three independent clones.
Plasmid Constructions--
Plasmids pCRfull, pCR5, pCR
3,
and pCR
53 were prepared in the following way. A part of m-Staf
cDNA (position 54-440) was amplified using primers
5'-GCGGCCGCAAGCTTCTCAGGGAATGACAGAATTTCC-3' and
5'-TCTAACTGAACGGCCTGAAGTGAGCTC-3' and then digested with
HindIII and SacI. The resultant fragment was
ligated into a HindIII-SacI-digested pCRStaf to
form pCRfull. The 5'-linker element having the initial methionine codon
was made by hybridization of oligonucleotides 5'-AGCTTCTCAGGGAATGACAGAATTTCCTGGAGGAGGAATGGAGCT-3' and
5'-CATTCCTCCTCCAGGAAATTCTGTCATTCCCTGAGA-3'. The resultant fragment was
ligated into the HindIII-SacI-digested pCRStaf to
form pCR
5. For construction of pCR
3 and pCR
53, pCRfull and
pCR
5 were digested with SacII and EcoRI to
remove a portion of the cDNA (positions 1696-1941) and ligated
with the 3'-linker element having the termination codon. The 3'-linker
element was made by hybridization of oligonucleotides
5'-GTAGTAAGGCCGGTAG-3' and
5'-AATTCTACCGGCCTTACTACGC-3'.
Preparation of Recombinant Proteins--
Recombinant m-Staf
proteins were expressed by using the His-patch ThioFusion System
(Invitrogen). The expression constructs pThio-full and pThio-53 were
made by using the pThioHis vector. Transformed bacteria were grown as
described previously (24). The cell suspension was then subjected to
three cycles of sonication-freezing and thawing followed by
centrifugation. The supernatants from pThio-full and pThio-
53 were
named rStaf-full and rStaf-
53, respectively.
Northern Blot Analysis-- Total RNA from various tissues was prepared by the CsCl precipitation method (29) or by the acid phenol extraction method (Ambion). For detection of m-Staf mRNA, total RNAs were electrophoresed on formaldehyde-agarose gels, blotted onto a Hybond-N+ (Amersham Pharmacia Biotech), and hybridized with a 32P-labeled probe bearing the +1/+252 region of m-Staf cDNA. For detection of tRNAsec, total RNAs were separated by electrophoresis on a 6% polyacrylamide-8 M urea gel (Novex), transferred to a Nylon+ membrane (Novex) by electroblotting, and hybridized with a 5'-end 32P-labeled oligonucleotide (5'-GCACCCCAGACCACTGAGGATCATCCGGGC-3') specific for the mouse tRNAsec.
Preparation of Nuclear Extracts and Gel Shift Assays-- Aged-matched virgin (3 months old) and pregnant (9-11 days of gestation) C3H/HeN female mice were obtained from the Animal Center of the National Institutes of Health. Animal care and study protocol were in full compliance with the National Institutes of Health guidelines.
Nuclear extracts were prepared from thoracic mammary glands according to the method described previously (25). Nuclear extracts or recombinant proteins were mixed with 3 µg of poly(dI-dC) (Sigma) in 17 µl of reaction buffer containing 14 mM Hepes, pH 7.9, 12% glycerol, 90 mM NaCl, 2.5 mM MgCl2, and 1 mM dithiothreitol, and incubated for 15 min on ice. Gel shift assays were performed using a double-stranded oligonucleotide corresponding to theTransfections and CAT Assays--
Drosophila SL2
cells were grown at 22 °C in Schneider's medium supplemented with
10% heat-inactivated fetal calf serum. For transfection, 3.5 × 106 cells were plated on each of 3.5-cm-diameter dishes.
Five µg of CAT construct, 5 µg of expression plasmid, and 2 µg of
ADH-gal were co-transfected by the calcium phosphate method (30).
After 48 h, cells were lysed by freezing and thawing for CAT
assays (30). Transfection efficiency was normalized to
-galactosidase activity (31).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular Cloning of m-Staf-- The DNA binding domain of mouse YY1-cDNA encoding transcription factor bearing four C2-H2 type zinc fingers (26) was used for screening a mouse mammary gland cDNA library at low stringency. One positive clone, 1BA, was further analyzed. The complete nucleotide sequence of this clone (Fig. 1) indicated that it contained a 1878-nucleotide open reading frame (positions 64-1941), which potentially encoded a 67.5-kDa polypeptide consisting of 626 amino acid residues. The predicted amino acid sequence revealed that this protein contained four repeated motifs between residues 39 and 135 and a zinc finger domain of the C2-H2 type tandemly repeated seven times between residues 220 and 428.
|
|
Tissue Distribution of m-Staf mRNA-- Total RNA prepared from various tissues of female mice was subjected to Northern blot analysis using m-Staf cDNA as a probe (Fig. 3). A single band of transcript (3.4 kb) was detected in all tissues examined. The level of the transcript relative to that of GAPDH mRNA was highest in the lung and lowest in the liver. These results indicate that the m-Staf gene is widely but differentially expressed in mouse tissues.
|
Recombinant m-Staf Binds to the AE Region--
We produced two
recombinant m-Staf proteins, rStaf-full, corresponding to amino acid
residues 1-626, and rStaf-53, corresponding to residues 118-542
(Fig. 4A). Their binding
activities to the AE region of the tRNAsec gene were
examined by gel shift assays using a double-stranded DNA probe
corresponding to the
233/
198 region of the mouse
tRNAsec gene promoter, which includes the AE region
(
222/
208). Table I shows the
sequences of mouse (MW) and X. laevis wild type competitors and four mouse mutant competitors (MM0, MM1, MM2, and MM3).
|
|
Recombinant m-Staf Functions as a Transcriptional Activator on
Mouse tRNAsec Promoter--
We next examined whether
m-Staf functions as a transcriptional activator of the mouse Pol
III-dependent tRNAsec gene. For transient
co-transfection experiments, Drosophila SL2 cells, which do
not have endogenous Staf-like binding activity (24), were used. The
expression vectors ADH-pEXPfull, ADH-pEXP53, and ADH-pEXP
3
contained either a wild type m-Staf gene or its truncated forms under
the control of the Drosophila alcohol dehydrogenase promoter. ADH-0, which lacks the m-Staf insert, served as a control. These plasmids were co-transfected with a CAT reporter plasmid linked
to either a wild type or a mutated element bearing the
235/+7 region
of the mouse tRNAsec gene promoter (Table I and Fig.
5A).
|
m-Staf Binding Activity in Mouse Mammary Gland at Various Reproductive Stages-- Cloning of the m-Staf cDNA and detection of its transcript in mouse mammary gland prompted us to examine the activity of m-Staf in the gland. Gel shift assays using nuclear extracts from mammary glands of pregnant mice revealed the presence of two retarded bands (Fig. 6A, lane 1). Competition experiments indicated that the DNA binding activity of the upper band had the same sequence specificity as that of the recombinant m-Staf (Fig. 6A, lanes 2-13, and Fig. 4B) whereas the lower band showed no sequence-specific DNA binding activity and thus was considered to be nonspecific. In addition, the migration rate of the upper band was found to be similar to that formed by the recombinant m-Staf (data not shown). These data indicate the presence of endogenous m-Staf in the mammary glands.
|
The Level of tRNAsec in Mouse Mammary Gland at Various Reproductive Stages-- Because m-Staf was shown to be a positive transcriptional regulator of the mouse tRNAsec gene, it was of interest to compare the levels of tRNAsec transcript and m-Staf binding activities in mouse mammary gland at various reproductive stages. As shown in Fig. 7, a mouse tRNAsec transcript having 87 bp was detected as a single band. Its level in the mammary gland increased substantially during pregnancy and lactation (Fig. 7, lanes 2 and 3), whereas it remained at relatively low levels during virgin (lane 1) and postlactating (lane 4) periods.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we cloned a cDNA encoding a zinc finger protein from mouse mammary gland. The predicted amino acid sequence of this clone indicates that it contains the seven tandemly repeated C2-H2-type zinc fingers and the four repeated motifs, which are highly homologous with Staf from X. laevis. Accordingly, we have designated this cDNA m-Staf. However, we found several structural differences between m-Staf and Staf. First, Staf has additional amino acid residues at the amino terminus and contains the third methionine residue at the position corresponding to the translation start site of m-Staf. Second, m-Staf contains an additional 54 amino acid residues downstream of the zinc finger domain. The nucleotide sequence of the inserted portion is 5'-GTCAACA- - - -CAG-3', which is identical to the consensus sequence of the intron donor and acceptor site (33), suggesting that this portion might originally have been an intron, which has evolved into an exon. The sequence is no longer spliced out because RT-PCR analysis demonstrated that mouse mammary gland does not contain any Staf gene products that lack the 54 amino acid residues (data not shown). In addition, these amino acid residues are also present in the same downstream region of human zinc finger protein ZNF143, suggesting that they are well conserved in mammalian species.
We obtained several lines of evidence indicating that m-Staf is a
transcriptional activator of the mouse tRNAsec gene. Like
Staf, the recombinant m-Staf, rStaf-full, was shown to bind to the
15-base pair AE region of the mouse tRNAsec gene in a
sequence-specific manner. Competition assays using various mutated
sequences indicate that the two sequences CCA (222/
220) and TGC
(
216/
214) in the AE region are important for the binding. The
truncated form, rStaf-
53, which has only amino acid residues
118-542, was also found to bind to the AE region, indicating that this
portion of m-Staf is sufficient to facilitate the DNA binding activity.
Because it contains an entire zinc finger domain (Fig. 4A),
our results are consistent with the previous findings that the zinc
finger domain of Staf is important for its DNA binding activity (24).
However, we do not know whether all of the seven zinc fingers are
necessary for DNA binding activity. It was reported that three of the
six C2-H2 zinc fingers in the Gfi-1 transcription factor were
sufficient for DNA binding (34).
Transfection experiments showed that expression vectors for both m-Staf
(ADH-pEXPfull) and one truncated form of m-Staf (ADH-pEXP3) could
induce the mouse tRNAsec promoter CAT activity, whereas the
other truncated form, ADH-pEXP
53, was ineffective. These results
suggested that amino acid residues 1-117 of m-Staf contain some region
necessary for transcriptional activation. This portion includes three
of the four repeated motifs, which are well conserved between Staf and
m-Staf and thus may have functional importance. Based on these
findings, m-Staf is considered to have two major functional regions,
one for transcriptional activation and the other for DNA binding, which
are characterized by the presence of the repeated motifs and the zinc
finger domain, respectively. These features of m-Staf are common to
many transcription factors (35).
We found that m-Staf stimulated transcription of tRNAsec Pol III-dependent promoter but had only minimal effect on AE-linked thymidine kinase Pol II-dependent promoter in transfection experiments using Drosophila cells. These cells were used because they do not contain any detectable level of Staf-like binding activities. In contrast to our present findings, it was reported that transfection of Staf in Drosophila cells was not effective in stimulating transcription of the X. laevis tRNAsec Pol III, although Staf markedly activated the AE-containing Pol II promoter (24). The transcriptional activity of Staf could only be demonstrated by using a X. laevis oocyte system into which Staf mRNA was microinjected prior to introduction of the tRNAsec-CAT reporter gene. The observed difference in the transcriptional activity of the two Stafs transfected in Drosophila cells could be explained by the aforementioned structural difference of the two proteins or by the source of tRNAsec Pol III promoter used in reporter plasmids, i.e. mouse versus X. laevis. These differences could influence the functional properties of Stafs in forming the active transcriptional complex.
Our studies of m-Staf in mouse mammary gland indicated that m-Staf binding activities changes as a function of reproductive stage. The binding activity of m-Staf in the mammary gland was undetectable at virgin and postlactating stages, when the gland is developmentally dormant. However, the binding activity increased markedly during the periods of pregnancy and lactation, when the mammary gland undergoes extensive growth and differentiation (36). Thus, the change in the m-Staf binding activity in the gland appears to be correlated to the developmental status of the mammary tissue. Because the development of the mammary gland is stimulated by various steroid and polypeptide hormones (36), the binding activity of m-Staf in the gland also may be hormonally regulated. In addition, we found that the level of tRNAsec in the mammary gland increased during pregnancy and lactation largely in parallel with that of m-Staf binding activity. These findings are consistent with the view that m-Staf is involved in the regulation of tRNAsec gene transcription. It is noted, however, that both virgin and postlactating mammary glands had no detectable m-Staf binding activity but showed low levels of tRNAsec transcript. It is possible that the basal level of tRNAsec gene transcription in developmentally dormant glands is maintained by other transcription factors acting on the basal promoter elements (18, 21).
The parallel increase in tRNAsec and m-Staf binding activity in the mammary gland during pregnancy and lactation is noteworthy because the production of at least two selenoproteins, glutathione peroxidase (9) and type I thyroxine 5'-deiodinase (10-12), is found to increase in the gland during these periods. Type I thyroxine 5'-deiodinase is the best characterized selenoprotein in the mammary gland; it catalyzes monodeiodination of the prohormone thyroxine (T4) to form a more active hormone, 3,5,3'-tri-iodothyronine (T3). Its activity in the mammary gland increases during lactation (10) and correlates well with lactational intensity, as judged by litter size (11). These observations are consistent with the view that the deiodinase plays a key role in maintaining lactogenesis by catalyzing the production of T3 (12). Although the mechanisms of induction of type I thyroxine 5'-deiodinase during lactation have not been elucidated, our present findings raise the possibility that m-Staf plays a role in the biosynthesis of type I thyroxine 5'-deiodinase, as well as other selenoproteins, by regulating expression of the tRNAsec gene in lactating mammary glands.
Recently, it was reported that Staf could activate not only the X. laevis tRNAsec gene but many small nuclear RNA and small nuclear RNA-type genes transcribed by RNA polymerase II or Pol III in a X. laevis oocyte system (37). Moreover, some of these genes, such as the U2 gene, were found to contain binding sites for both Staf and Oct factor in their distal sequence element regions (37, 38), in which the two transcription factors interact to activate transcription. Because the distal sequence element region of the mouse tRNAsec gene also contains a consensus octamer binding site (23), it is of interest to examine whether such an interaction is important for the regulation of murine tRNAsec gene promoter transcription. In addition, the question of whether m-Staf can also activate transcription of small nuclear RNA genes in mammalian tissues, including the mammary gland, remains to be investigated.
Previously, it was reported that human ZNF76 (39) was a human homologue of Staf (24, 37), although its biological function was not identified. The sequence of ZNF76 showed 85.1% identity (172 of 202 residues) with Staf in the zinc finger region, but its entire sequence had only 58.0% homology with Staf (298 of 514 residues). We found that m-Staf and human ZNF143, another human zinc finger protein (32), share 97.1% homology in their amino acid sequences. In view of our findings that m-Staf functions as a transcription activator of the mouse tRNAsec gene, ZNF143 may be the human homologue of m-Staf and have similar functions for the regulation of tRNAsec gene transcription in mammalian systems. Moreover, because the ZNF143 gene was mapped to chromosomal regions implicated in developmental and malignant disorders (32), it is of interest to examine the possible involvement of m-Staf in these disease states in the mouse model.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Deborah M. Hinton, Karen Usdin, and Michael D. Davis for their discussion and critical reading of our manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AF011758.
Present address: Dept. of Medicine III, Osaka University Medical
School, 2-2 Yamada-oka, Suita-City, Osaka 565, Japan.
§ Present address: Molecular Biology Dept., Nippon Shinyaku Co., Ltd., 3-14-1 Sakura, Tukuba-City, Ibaraki 305, Japan.
¶ To whom correspondence should be addressed: LMCB/NIDDK/NIH, Bldg. 8, Rm. 309, Bethesda, MD 20892. Tel.: 301-496-1404; Fax: 301-402-0053.
1 The abbreviations used are: tRNAsec, selenocysteine tRNA; Pol III, RNA polymerase III; Pol II, RNA polymerase II; AE, activator element; Staf, selenocysteine tRNA gene transcription activating factor; CAT, chloramphenicol acetyltransferase; TBE, 90 mM Tris base, 90 mM boric acid, 0.5 mM EDTA, pH 8.3; MW, mouse wild type competitor; MM, mouse mutated competitor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|