Mechanisms of MARCKS Gene Activation during XenopusDevelopment*

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a high affinity cellular substrate for protein kinase C. The MARCKS gene is under multiple modes of transcriptional control, including cytokine- and transformation-dependent, cell-specific, and developmental regulation. This study evaluated the transcriptional control of MARCKS gene expression during early development of Xenopus laevis. Xenopus MARCKS was highly conserved with its mammalian and avian homologues; its mRNA and protein were abundant in the maternal pool and increased after the mid-blastula transition (MBT). The Xenopus MARCKS gene was similar to those of other species, except that a second intron interrupted the 5′- untranslated region. By transiently transfecting XTC-2 cells and microinjecting Xenopus embryos with reporter gene constructs containing serial deletions of 5′-flanking MARCKS sequences, we identified a 124-base pair minimal promoter that was critical for promoter activity. Developmental gel shift assays revealed that a CBF/NF-Y/CP-1-like factor and an Sp1-like factor bound to this region in a manner correlating with the onset ofXenopus MARCKS transcription at MBT. Mutations in the promoter that abolished binding of these two factors also completely inhibited transcriptional activation of the MARCKS gene at MBT. The binding sites for these two factors are highly conserved in the human and mouse MARCKS promoters, suggesting that these elements might also regulate MARCKS transcription in other species. These studies not only increase our knowledge of the transcriptional regulation of the MARCKS genes but also have implications for the mechanisms responsible for zygotic activation of the Xenopus genome at MBT.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) 1 is a prominent, high affinity cellular substrate for protein kinase C (PKC) (1,2). The MARCKS protein is dramatically phosphorylated by PKC in many cell types in response to phorbol esters, diacylglycerols, neurotransmitters, drugs, and various growth factors, implying that it is involved in the signaling pathways linking PKC activation to specific cellular responses. MARCKS also plays an essential role in the early development of vertebrates (3), and its gene transcription is developmentally regulated (4). In the mouse, MARCKS mRNA and protein are first detected in the nascent neural folds early on embryonic day 8 (E8) and then become highly expressed throughout the developing central nervous system (4). Consistent with this expression pattern, MARCKS gene "knock-out" mice exhibited lethal defects in neurulation, cerebral hemisphere fusion, cortical and retinal lamination, and other central nervous system developmental processes (3,5).
The MARCKS gene is also regulated by other factors. It has been noted that MARCKS expression is severely decreased in cells transformed with a variety of oncogenes (6 -9). For example, in v-src-transformed murine fibroblasts, MARCKS transcription is down-regulated by 68% compared with untransformed cells (8); inhibiting the protein-tyrosine kinase activity of v-src with herbimycin A restores MARCKS mRNA levels to normal, suggesting that the reduced MARCKS mRNA levels are a direct effect of v-src activity. MARCKS transcription can also be dramatically stimulated by tumor necrosis factor ␣ or bacterial lipopolysaccharide in macrophages and neutrophils (10,11). In fact, it has been estimated that MARCKS constitutes 90% of all new protein synthesis in these cells in response to tumor necrosis factor ␣ or lipopolysaccharide (10). The promoter elements responsible for oncogene and cytokine regulation of the MARCKS gene, however, have not been determined.
Previous reports from this laboratory have described the cloning and mapping of the human and mouse MARCKS genes (11,12). Transient transfection experiments indicated that a sequence of about 240-bp of human promoter is sufficient for high level expression of the growth hormone reporter gene in fibroblasts (11). Recently, we used this 240-bp fragment to drive the expression of a MARCKS-lacZ fusion gene in transgenic mice. 2 This fragment was able to direct correct temporal and tissue-specific expression of the MARCKS-lacZ fusion protein during mouse embryogenesis, suggesting that this fragment contains most, if not all, important elements required for developmental regulation of MARCKS gene transcription in mice.
However, specifically what cis-and trans-regulatory elements are involved in MARCKS gene regulation has not been described. It is conceivable that by serial deletion/mutation analysis of the promoter in transgenic mice, it might be possible to identify regulatory elements that are important for developmental control of the MARCKS gene. However, such experiments would be expensive and time-consuming. Alternatively, Xenopus embryos provide a more convenient system in which "transient transgenic" experiments can be relatively easily performed, mainly because large numbers of embryos can be easily obtained and manipulated. Microinjection of DNA constructs into Xenopus embryos has proven to be an effective approach in the study of developmental regulation, not only of many amphibian genes but also of some mammalian genes (13)(14)(15)(16)(17)(18)(19)(20)(21).
The current studies were designed to identify potential cisacting elements and trans-acting transcription factors that regulate transcription of the Xenopus MARCKS gene. We cloned the cDNA and genomic sequences encoding Xenopus MARCKS and examined the mechanisms of its transcriptional control.
Our results indicate that a CBF/NF-Y-like factor and an Sp1like factor bind to the Xenopus MARCKS promoter and that mutations of their binding sites that prevent transcription factor binding also inhibit activation of the MARCKS gene at the midblastula transition (MBT). The regulatory expression patterns of these two factors suggest that they might play a general role in selectively activating the Xenopus genome at MBT.

In Vitro Phosphorylation
Enzymatically defolliculated oocytes were homogenized in 10 l/ oocyte of a buffer consisting of 20 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, 1 mM dithiothreitol, and 0.6% (v/v) Triton X-100. After incubating on ice for 30 min with occasional mixing, the homogenates were centrifuged at 12,000 ϫ g for 15 min at 4°C. Because MARCKS is heat-stable, we then boiled the supernatant for 10 min and recentrifuged. The supernatants were subjected to phosphorylation by using protein kinase M (PKM), the active catalytic fragment of PKC, as described previously (22,23). Briefly, to 54 l of the supernatant was added 1 ⁄10 volume of Mg 2ϩ /ATP/PKM for final concentrations of 20 mM MgCl 2 , 75 mM [␥-32 P]ATP (approximately 4 mCi/nmol; ICN, Irvine, CA), and a 1:480 dilution of the purified PKM. The reaction was incubated at 30°C for 10 min with shaking and stopped with 10 l of 5 ϫ SDS sample buffer (5% (w/v) SDS, 0.05 M EDTA, 1.25 M sucrose, 0.42 M dithiothreitol, 0.006% (w/v) pyronin Y). The reaction products were separated on one-or two-dimensional SDS-polyacrylamide gel electrophoresis followed by autoradiography.

Cloning of a Xenopus MARCKS cDNA and Genomic Clones
Degenerate Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-Two thousand oocytes were defolliculated in 2% (w/v) collagenase (type II; Worthington) in 1 ϫ MBS (88 mM NaCl, 1 mM KCl, 0.41 mM CaCl 2 , 0.33 mM Ca(NO 3 ) 2 , 0.82 mM MgSO 4 , 2.4 mM NaHCO 3 , 10 mM HEPES, 100 g/ml gentamicin, 100 units/ml penicillin, 100 g/ml streptomycin, pH 7.4), pulverized with a mortar and pestle under liquid N 2 , and homogenized with a Polytron homogenizer for 30 s at 30,000 rpm in 20 ml of phenol (prewarmed to 60°C) plus 20 ml of a buffer consisting of 100 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% (w/v) SDS. Standard phenol/chloroform extractions and oligo(dT)-cellulose chromatography were then performed to purify oocyte mRNA. PCR primers were chosen based on the conserved amino-terminal sequences of mammalian MARCKS proteins (2). The forward primer was (5Ј to 3Ј) AT-GGGTGCCCAGTTCTCCAAGA, and the reverse primer was (5Ј to 3Ј) TGGCCITTITCCTGICCATTIGCITT, where I stands for inosine. One g of mRNA was reverse transcribed using MuLV reverse transcriptase and 50 pmol of the reverse primer as described (24). Following the addition of the forward primer and Taq DNA polymerase, the DNA was amplified for 35 cycles (1 min at 94°C, 2 min at 42°C, and 3 min at 72°C). A single PCR fragment of 108 bp was subcloned into the PCR 1000 plasmid (Invitrogen, San Diego, CA) and sequenced using an automated sequencer.
cDNA Cloning-Sequence analysis showed significant homology between the PCR fragment and mammalian MARCKS cDNAs. Therefore, this fragment was used as a probe to screen two gt10 cDNA libraries made from stage 11 and stage 17 Xenopus embryos (generously provided by Dr. Douglas Melton, Harvard University, Cambridge, MA) under high stringency conditions. Positive clones were purified according to standard procedures (25). The EcoRI cDNA inserts from positive clones were subcloned into pBluescribe (Stratagene, La Jolla, CA) and characterized by restriction mapping. Restriction fragments were subcloned and sequenced by the dideoxy chain termination method (26) using Sequenase 2.0 (Amersham Life Science, Inc.). Sequences were analyzed with the University of Wisconsin Genetics Computer Group (GCG) sequence analysis package.
Cloning of Genomic Sequences-Cloning was performed essentially as described above, except that a Xenopus genomic FixII library (Stratagene) was used. Several positive clones were obtained, two of which were analyzed by restriction enzyme mapping and Southern blotting. A 2.6-kilobase pair AccI fragment was found in both clones that hybridized strongly to the cDNA probe. Therefore, this fragment was subjected to sequencing as described above.

Plasmid Construction and Site-directed Mutagenesis
For construction of the 1047-bp promoter-chloramphenicol acetyltransferase (CAT) fusion gene, an SspI fragment extending from nucleotide Ϫ344 to Ϫ99 of the Xenopus MARCKS 5Ј-flanking region (where ϩ1 represents the initiation codon) was subcloned into a promoterless pCAT-Basic vector (Promega, Madison, WI), which had been cut with AccI and treated with Klenow to fill in the 5Ј-overhang. For construction of other CAT reporter genes, different lengths of the Xenopus MARCKS promoter were amplified by PCR, using Pfu DNA polymerase (Stratagene) under standard conditions (25). The 5Ј-primers were designed to contain a HindIII site, and the 3Ј-primers were designed to contain a XbaI site. The PCR products were then digested with HindIII and XbaI and subcloned between the HindIII and XbaI sites of the pCAT-Basic vector. In all cases, the PCR-derived fragments were sequenced to confirm sequence fidelity.
Site-directed mutagenesis was performed using the Altered Sites II in vitro mutagenesis system (Promega). Mutated clones were analyzed by restriction mapping and by sequencing to ensure they were otherwise identical to the wild type sequence except for the indicated mutations. The oligonucleotide sequences used for PCR and mutagenesis are available upon request.
Low/high salt embryo extracts were prepared by a modification of the method of Brewer et al. (21). Pools of 10 embryos at each developmental stage were dejellied in 2% (w/v) cysteine-HCl (pH 8.0), washed in 0.1 ϫ MBS and then snap-frozen in liquid N 2 and stored at Ϫ70°C for no more than 1 week. Frozen embryos were homogenized by repeated pipetting in 100 l (10 l/embryo) of homogenization buffer (20 mM HEPES, pH 7.9, 2 mM MgCl 2 , 2 g/ml leupeptin, 2 g/ml pepstatin, 2 g/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM ␤-glycerophosphate). The lysed mixture was centrifuged at 13,000 ϫ g for 5 min at 4°C. 50 l of the clear liquid fraction located underneath the fat cake was carefully removed without disturbing the fat cake and was used as the low salt extract. The remaining 50 l of the supernatant fraction containing the fat cake was discarded. The pellet was reextracted with 50 l of the original homogenization buffer plus 300 mM KCl and centrifuged at 13,000 ϫ g for 10 min at 4°C. The resulting supernatant was diluted with 100 l of homogenization buffer (without KCl) to adjust the final salt concentration to 100 mM KCl and was used as the high salt extract. The low and high salt extracts were used immediately without freezing.
Cell Culture, Transfections, CAT Assays, and Luciferase Assays XTC-2 cells (a generous gift from Dr. David J. Shapiro, University of Illinois, Urbana, IL) were maintained and transfected according to Smith and Tata (30). Briefly, these cells were maintained at room temperature in 70% (v/v) Leibovitz's L-15 medium (Life Technologies, Inc.), 10% (v/v) heat-treated fetal calf serum, 100 mM HEPES, 100 g/ml gentamicin, 50 units/ml penicillin, 50 g/ml streptomycin, pH 7.4. The medium was switched to 70% (v/v) Dulbecco's modified Eagle's medium/F12 (Sigma), 10% heat-inactivated fetal calf serum, 100 g/ml gentamicin, 50 units/ml penicillin, 50 g/ml streptomycin, pH 7.4, 3-7 days before transfection. One day before transfection, approximately 10 6 cells were plated out on a 100-mm culture dish, and the medium was changed to fresh medium 3 h prior to transfection. DNA-CaPO 4 co-precipitation (25) was used to transfect 15 g of recombinant CAT constructs and 5 g of a pGL2 luciferase vector (Promega) in each plate. Twenty-four hours later, the cells were washed and fed with fresh medium. Cells from each plate were harvested in 100 l of 0.25 M Tris-Cl (pH 7.8)/5 mM EDTA 48 h after transfection and broken by four cycles of quick freezing in liquid N 2 and thawing at 37°C. The homogenates were centrifuged at 12,000 ϫ g for 10 min, and the supernatants were frozen in liquid N 2 and stored at Ϫ70°C or were assayed for CAT activity and luciferase activity immediately.
For CAT assays, 50 l of protein extract was incubated for 2 h at 37°C with 5 l of [ 14 C]chloramphenicol (0.025 mCi/ml; NEN Life Science Products) and 5 l of 4 mM acetyl-coenzyme A (Sigma) in a total volume of 120 l containing 0.25 M Tris (pH 7.8). The reaction products were extracted with 300 l of xylene and then back-extracted twice with 150 l of 0.25 M Tris (pH 7.8), after which 200-l aliquots of the xylene phase were subjected to liquid scintillation counting.
For luciferase assays, 20-l protein extracts were assayed using the Luciferase Assay Kit (Promega) and a LB 9501/16 luminometer (Berthold System Inc., Pittsburgh, PA) according to the manufacturers' instructions.

Microinjection of Embryos and CAT Assay
The procedures for maintenance of frogs, hormonal stimulation of egg laying, and in vitro fertilization have been described previously (13). One blastomere of two-cell stage embryos was injected with 10 nl of a solution containing 75 pg of the supercoiled DNA construct. The injections were performed in 1 ϫ MBS containing 5% (w/v) Ficoll, and the injected embryos were kept in the same solution for a period of 3-5 h at room temperature. After that, the incubation medium was changed to 0.1 ϫ MBS, and the embryos were allowed to develop until the desired stage. All embryos were staged according to Nieuwkoop and Faber (31). Groups of 10 -20 embryos were homogenized in a buffer containing 0.25 M Tris (pH 7.8), 5 mM EDTA (20 l/embryo) by repeated pipetting. The homogenates were centrifuged at 12,000 ϫ g for 10 min at 4°C, and five embryo-equivalents of supernatant were assayed for CAT activity as described above.

Gel Shift and DNase I Protection Analysis
For gel shift analysis, plasmid restriction fragments or synthetic double-stranded oligonucleotides (Life Technologies, Inc.; Table I) were labeled by filling in the 5Ј-overhangs with [␣-32 P]dCTP and purified by polyacrylamide gel electrophoresis, using standard methods (32). Binding reactions were carried out with the appropriate amount of protein and 2 ϫ 10 4 cpm probe in a total volume of 20 l containing 20 mM HEPES (pH 7.9), 2% (v/v) glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol, and 10 g of poly(dI-dC). One g of the XTC-2 nuclear extract, three embryo equivalents of embryo nuclear extract, or one embryo equivalent of low/high salt extract was used in each binding reaction. The mixtures were incubated at room temperature for 45 min and then separated on 4% polyacrylamide gels containing 0.5 ϫ TBE buffer (0.0045 M Tris borate, 0.001 M EDTA). For competition analysis, 0.1 g of unlabeled probe was added to the reaction mixture together with the other components. For supershift analysis, antibodies were incubated with the protein extracts for 20 min before the other components of the mixture were added. The antihuman NF-YA or anti-human NF-YB IgG were purchased from Rockland (Gibertsville, PA); 2 g of IgG was used in each reaction. The anti-Sp1 antiserum was kindly provided by Dr. Jonathan M. Horowitz (Duke University, Durham, NC) (33); 1 l of antiserum was used in each reaction. The anti-FRGY1 and anti-FRGY2 antisera were kindly provided by Dr. Alan Wolffe (NICHD, National Institutes of Health, Bethesda, Maryland (34)); 2 l of antiserum was used in each reaction.
DNase I footprinting was performed following a protocol described previously (35). The DNA probe was derived from the chimeric CAT construct containing the 245-bp (Ϫ344/Ϫ99) MARCKS promoter cloned between HindIII and XbaI sites. Ten g of this construct was digested with XbaI and labeled with [␣-32 P]dCTP. The labeled DNA was ethanol-precipitated and digested with HindIII to release the 245-bp promoter insert, which was then purified by polyacrylamide gel electrophoresis. 10 5 cpm of the purified probe was incubated with 50 g of XTC-2 nuclear extract in a final volume of 50 l containing 5 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 5% (v/v) glycerol, and 2 g of poly(dI-dC). After incubation at room temperature for 20 min, 5 l of 1 g/ml DNase I (Promega) in 25 mM MgCl 2 , 25 mM CaCl 2 was added to the reaction for exactly 1 min. The reaction was stopped by adding 90 l of stop solution (0.2 M NaCl, 0.03 M EDTA, 1% (w/v) SDS, 100 g/ml calf thymus DNA). The sample was extracted with phenol/chloroform and precipitated with ethanol. The DNA was separated on a 6% sequencing gel and subjected to autoradiography.

MARCKS, but Not MRP, Is Detected in Xenopus Oocytes-To
determine whether a MARCKS-like protein exists in Xenopus, heat-stable protein extracts were prepared from Xenopus oocytes and subjected to in vitro phosphorylation using PKM, the active catalytic fragment of PKC, in the presence of [␥-32 P]ATP. When the reaction products were separated by SDS-polyacrylamide gel electrophoresis, a single major phosphorylated protein of M r ϳ70,000 was detected (Fig. 1A). Two pieces of evidence suggested that this band corresponded to Xenopus MARCKS. First, this 70-kDa phosphoprotein displayed the acidic pI and characteristic migration pattern of MARCKS on two-dimensional gel electrophoresis ( Fig. 1B; Ref. 36). Second, it could be specifically immunoprecipitated by antiserum 94 (Fig. 1C), an antibody raised against a synthetic peptide corresponding to the highly conserved amino-terminal sequences of known MARCKS proteins (37).
An M r ϳ45,000, pI ϳ5.0 homologue of MARCKS, the MARCKS-related protein or MRP (38), is expressed at levels approximately as great as MARCKS in mouse embryos (3). Interestingly, no significant phosphoprotein of that approximate M r and/or pI was detected in the phosphorylation experiment ( Fig. 1, A and B), suggesting that MRP, if it is present in Xenopus oocytes, is expressed at very low concentrations compared with MARCKS. This suggested that Xenopus might represent a useful system in which to study the role of MARCKS in early development, since there would not be functional redundancy due to A, phosphorylation of a Xenopus oocyte heat-stable extract with PKM identified a major heat-stable PKC substrate of M r 70,000 (arrow); however, no major substrates at about M r 45,000 were found. B, the phosphorylation reaction shown in A was subjected to two-dimensional electrophoresis followed by autoradiography. The arrow indicates the major PKC substrate with apparent M r of 70,000 and pI of about 4.5. This protein has the characteristic two-dimensional electrophoretic mobility and morphology of MARCKS from other species. Again, note the absence of a major phosphoprotein corresponding to mammalian MRP, which has an apparent M r of 45,000 and pI of 5. C, the phosphorylation reaction shown in A was subjected to immunoprecipitation with antiserum 94, an antibody against an amino-terminal peptide common to all known MARCKS proteins (I), or with preimmune serum (PI); the arrow indicates presumed Xenopus MARCKS. high level expression of a known MARCKS homologue.
cDNA Cloning and Developmental Expression of Xenopus MARCKS-Two degenerate oligonucleotide primers based on conserved sequences within the amino terminus of MARCKS from other animal species were used to amplify a 108-bp fragment from Xenopus oocyte mRNA by RT-PCR, as described under "Experimental Procedures." Sequence analysis of this fragment revealed that it shared significant amino acid sequence homology with other MARCKS proteins (2). Therefore, it was used as a probe to screen stage 10 and stage 17 Xenopus embryo cDNA libraries. Several overlapping clones were isolated, two of which contained the entire open reading frame. One of the cDNA clones, XM17-9-2, contained 70 nucleotides of 5Ј-untranslated region (UTR), followed by 861 nucleotides of open reading frame, and 1286 nucleotides of 3Ј-UTR, ending in two AATAAA polyadenylation signals and a poly(A) tail (Gen-Bank TM accession number AF017299). Sequencing of Xenopus MARCKS genomic clones has shown that the intron splice site seen in MARCKS proteins from other species is identical to that in Xenopus (see below).
An alignment of the Xenopus MARCKS amino acid sequence with those of other species showed that Xenopus MARCKS is more closely related to chicken MARCKS than to the protein from various mammalian species, with an overall sequence identity of 69% (Fig. 2). This alignment confirms that the three consensus sequences previously described (2) are also conserved in Xenopus (underlined sequences in Fig. 2): the aminoterminal myristoylation domain; the site of intron splicing, which is homologous to a sequence within the cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor II receptor (39); and the 25-amino acid phosphorylation site domain, which includes the three to four serines that can be phosphorylated by PKC (22,23). This domain is also involved in binding to calmodulin (40 -42) and actin (43), both in a PKC phosphorylation-dependent manner. The highly conserved nature of these three regions through evolution implies that they are important functional domains.
The cloned cDNA was transcribed, and the mRNA was expressed in a rabbit reticulocyte lysate translation system. The translated product migrated at M r ϳ70,000 on SDS-polyacrylamide gel electrophoresis, the same size as the major phospho-protein shown in Fig. 1, confirming that the cDNA clone encoded the full-length protein. The predicted size of the protein was only 29.3 kDa; therefore, Xenopus MARCKS, like its mammalian and avian homologues (2), migrates with an anomalously high apparent M r on SDS-polyacrylamide gel electrophoresis. Like MARCKS from other species, Xenopus MARCKS is also heat-stable (Fig. 1).
We determined the expression pattern of MARCKS mRNA at different stages of early Xenopus development by RT-PCR (Fig.  3), using a well characterized set of cDNAs (27). Xenopus MARCKS mRNA was abundant at the four-cell stage; this mRNA is from maternal stores, since zygotic transcription does not occur until approximately 6 h after fertilization at MBT (generally by stage 9). The decreased level of MARCKS mRNA observed at stage 9 (lane 2) probably reflects continued decay of the maternal mRNA, since accumulation of newly transcribed mRNA has not yet become significant (44). Thereafter, zygotic transcription of the MARCKS gene appeared to be turned on and continued throughout development (lanes 3-9).
MARCKS protein levels at different stages were determined by the PKM phosphorylation assay described in Fig. 1A. The protein was also expressed in the maternal pool, with levels gradually increasing with development after blastulation (data not shown).
Isolation of Xenopus MARCKS Genomic Clones-To obtain a genomic DNA fragment containing the Xenopus MARCKS promoter, a Xenopus genomic FixII library was screened with a 5Ј Xenopus MARCKS cDNA probe as described under "Experimental Procedures." Two positive clones were isolated and analyzed by restriction enzyme mapping. A 2.6-kilobase pair AccI fragment from both clones hybridized strongly to the 5Ј cDNA probe on Southern blot analysis. This fragment was partially sequenced to obtain sequences extending approximately 1.5 kilobase pairs upstream of the initiation codon (position ϩ1); this sequence has been deposited in GenBank TM (accession number AF017300). Comparison of the genomic and cDNA sequences revealed a gene organization consisting of three exons and two introns (Fig. 4A). The second intron, located within the cDNA coding region, is also found in an identical position in the other MARCKS genes identified so far (11,12); however, the first intron, located in the 5Ј-UTR, is only found in the Xenopus gene. As is true for its mammalian homologues, the Xenopus MARCKS gene also lacks a TATA box. An alignment of the 5Ј portion of the Xenopus gene with that of the human or mouse gene revealed a significantly conserved region of approximately 250 nucleotides, which contains the proximal promoter and part of the 5Ј-UTR (Fig. 4B). The highly conserved nature of this region implied that it might contain important regulatory element(s); indeed, this region of the human gene was able to drive high level expression of the growth hormone reporter gene in fibroblasts (11) and to drive appropriate developmental expression of a lacZ transgene in transgenic mice. 2 Localization of cis-Regulatory Elements-To identify specific sequences required for promoter activity, a series of deletion constructs was made by linking different regions of the Xenopus MARCKS promoter to a CAT reporter gene (Figs. 4B and 5A). These constructs were transiently transfected into XTC-2 cells, a Xenopus embryo-derived cell line that constitutively expresses the MARCKS gene (data not shown), and the result-ing CAT activity was assayed (Fig. 5B). A pGL2 luciferase vector was cotransfected to standardize transfection efficiency. The 1047-bp (Ϫ1088/Ϫ42) promoter construct produced high level CAT activity that was comparable with that of the SV40driven CAT vector. This construct also expressed in a manner similar to that of endogenous MARCKS gene expression in Xenopus embryos (see below). The next shorter construct, which contains the 246-bp conserved region described above (Ϫ344/Ϫ99), produced similar CAT activity, confirming the importance of this region for promoter activity in these cells. Further deletions revealed that as little as 124 bp (Ϫ325/Ϫ201) of the proximal promoter was still capable of driving high level CAT gene expression. However, deletion of another 60 bp, from nucleotides Ϫ325 to Ϫ266, dramatically decreased CAT activity by approximately 85%, suggesting that these 60 bp contain strong positive regulatory element(s) that are important for MARCKS promoter activity in XTC-2 cells.
Previous studies have revealed different requirements for regulatory sequences identified in cultured cells and in transgenic animals (45). Therefore, we also examined the sequence requirements for MARCKS expression in Xenopus embryos. In this series of experiments, the chimeric CAT constructs were microinjected into two-cell stage embryos, and CAT activities were assayed 36 h later, at the tailbud stage (Fig. 5C). In contrast to the transfection studies, the 246-bp promoter and the 125-bp promoter constructs exhibited moderately lower levels of CAT activity than the 1047-bp promoter construct (23 and 35% less, respectively) in embryos. However, in agreement with the transfection studies, deletion of the 60 bp between nucleotides Ϫ325 and Ϫ266 caused the most dramatic decrease of CAT activity (95%). These data indicate that these 60 bp contain critical positive regulatory element(s) for the Xenopus MARCKS gene and also suggest the presence of other relatively minor positive regulatory element(s) in upstream sequences.
Interaction between the Proximal MARCKS Promoter and XTC-2 Nuclear Extracts-To explore potential DNA-protein interactions in this region of the Xenopus MARCKS promoter, gel mobility shift experiments were performed (Fig. 6A). Using a probe comprising the 124-bp of promoter (Ϫ325/Ϫ201), a major retarded band was seen with XTC-2 nuclear extracts (lane 1). This band could be abolished by an excess of unlabeled probe but not by other unrelated probes (data not shown), suggesting that its formation was due to specific DNA-protein interactions. This 124-bp probe contains an AluI site (AGCT) between positions Ϫ289 and Ϫ286; neither of the two resulting subfragments was able to form the retarded band (data not shown), suggesting that the binding sequence spanned this AluI site. Based on this, a double-stranded oligonucleotide probe, gs-6 ( Fig. 4B; Table I), containing the AluI site was synthesized and used as a cold competitor in the binding reaction. The addition of unlabeled gs-6 to the reaction inhibited formation of the retarded band (lane 3). To further localize binding sequences within gs-6, two shorter probes, gs-7 and gs-10 (Table I), which together overlap and cover gs-6, were used as competitors. While gs-7 had no effect on formation of the complex (lane 2), gs-10 efficiently inhibited its formation (lane 4). We then radiolabeled gs-6 and gs-10 and tested their ability to bind proteins in XTC-2 nuclear extracts. As shown in lanes 5 and 6, both probes could form the complex; however, the binding of gs-10 appeared to be much weaker than that of gs-6. Therefore, it appeared that the core protein binding site was located within gs-10 and that its 5Ј-flanking sequences were also required for optimal binding. DNase I footprinting was then used to further characterize potential binding site(s) (Fig. 6B). Throughout the 246-bp (Ϫ344/Ϫ99) region, a single footprint was observed with XTC-2 The Xenopus (X) sequences are numbered at the right, with the ATG initiation codon (boxed) designated as position ϩ1 and the intron sequence (lowercase letters) not counted. The small numbers/arrows on the top indicate the 5Ј-and 3Ј-boundaries of different promoter fragments linked to CAT for promoter functional analysis (also see Fig. 2A). Two of the most important synthetic probes used in gel shift analysis, gs-1 and gs-6, are specified, with the corresponding sequences overlined; the boldface letters within them indicate nucleotides that were changed in mutagenesis experiments. Sequence alignment of the Xenopus and human MARCKS promoters were performed with the GCG Gap program. The asterisks indicate identical nucleotides. The dots in the human sequence indicate gaps inserted to optimize the alignment. The bent arrow at the underlined G indicates the 5Ј-most transcription initiation site; the other three potential initiation sites are also underlined.
nuclear extracts (lane 2) but not with bovine serum albumin (lane 1). The sequence of this footprint, 5Ј-CTGATTGGCT-3Ј, is included in probes gs-6 and gs-10 and in the 60-bp sequence that was found crucial for promoter activity. Therefore, the results of DNase I footprinting are consistent with those of the gel shift and deletion analyses. This sequence contains an inverted CCAAT box, which is the core binding site for a number of transcription factors (46). To confirm the role of this CCAAT box in the formation of the retarded band, we changed the CCAAT site in probe gs-6 to GGGGT and compared the binding abilities of gs-6 and its mutated version (mut-gs-6; Table I) by competition assays. As shown in Fig. 6C, while gs-6 fully competed for binding (lane 2), mut-gs-6 had no effect (lane 3). Therefore, this CCAAT site is essential for the formation of the major complex between XTC-2 nuclear factor(s) and this region of the proximal Xenopus MARCKS promoter.
Since the inverted CCAAT site and flanking sequences are highly conserved between the Xenopus and mammalian MARCKS promoters (8 of 10 nucleotides; Fig. 4B), we investigated whether this site in the mouse promoter also binds to a nuclear factor. As shown in Fig. 6D, the mouse counterpart of probe gs-6, mgs-6 (Table I), formed a complex with a protein in XTC-2 nuclear extracts that co-migrated with the one formed when gs-6 was used (lanes 1 and 2). When nuclear proteins from mouse brain were used, a similar complex was detected (lane 3). The formation of this complex was inhibited by an excess of unlabeled gs-6 (lane 4) or mgs-6 (lane 5) but not by mut-mgs-6 (lane 6), a probe that was otherwise identical to mgs-6 except that the CCAAT site was changed to GGGGT (Table I). Taken together, these results suggest that both the binding site and the nuclear factor are conserved between Xenopus and mammals.
A number of transcription factors have been found to bind to CCAAT boxes or related sequences, including C/EBP (47), NF-1 (48), and CBF (also known as NF-Y or CP-1; Refs. 46, 49, and 50). To examine the relationship between these previously identified factors and the one described here, competition experiments were performed using unlabeled oligonucleotide probes corresponding to the known recognition sequences for C/EBP, NF-1, or CBF/NF-Y (Table I). Among these competitors, only the CBF/NF-Y probe, but not the C/EBP or the NF-1 probe, efficiently competed for binding (Fig. 7A), suggesting that the CCAAT site in the MARCKS promoter might be specifically recognized by a CBF/NF-Y-like factor. To further con-firm this, a supershift analysis was performed using antibodies against NF-YA or NF-YB, two different subunits of the human CBF/NF-Y protein (Fig. 7B). These antisera were incubated with the mouse brain nuclear extract before the Xenopus probe gs-6 (lanes 1-3) or the mouse probe mgs-6 (lanes 4 -6) was added to the reaction mixture. Incubation with either antibody resulted in a supershifted band, although the one produced by anti-NF-YA was much stronger than the one produced by anti-NF-YB. When an authentic CBF/NF-Y probe (Table I) was used in this assay, the same supershift pattern was seen (lanes 7-9). As controls, antibodies against FRGY1 and FRGY2, two other CCAAT-binding proteins (34), did not result in supershifts (lanes 10 -12). Taken together, these results strongly suggest that the nuclear factor that recognizes the CCAAT site in the proximal MARCKS promoter is a CBF/NF-Y-like factor.
Developmental Regulation of the Interactions between the Proximal MARCKS Promoter and Xenopus Embryo Extracts-To compare binding activities in embryos with those in XTC-2 cultured cells and to determine whether there is developmental regulation of these binding activities, we used nuclear extracts prepared from Xenopus embryos at different stages. Three major complexes, designated C1, C2, and C3, were detected with the 124-bp (Ϫ325/Ϫ201) probe (Fig. 8, lanes  2-6). Among these complexes, C2 migrated to the same position as that formed with XTC-2 nuclear extracts (lane 1), while C1 and C3 were never seen with XTC-2 nuclear extracts. These complexes were not visible at stage 7 (lane 2) but were readily detectable at stage 9 (lane 3), and their binding activities gradually increased throughout later development. Such a pattern of complex formation correlates well with the transcription of the endogenous MARCKS gene, which begins after the midblastula transition at stage 8.
As a further test, we used differential salt extraction to address the question of whether and when these binding activities become associated with chromatin (Fig. 8, lanes 7-18). Embryos were homogenized in low salt buffer, and the resulting pellet was then extracted with a high salt buffer. Based on the generally accepted premise that low salt does not interfere with specific DNA-protein interactions, the low salt extract should contain most cytosolic proteins and the nuclear factors not associated with chromatin, while the high salt extract should contain chromatin-associated factors. As shown in Fig.  8 (lanes 14 -18), the expression pattern of the three complexes formed with the high salt embryo extracts was similar to that seen with nuclear extracts, i.e. they first became detectable at stage 9 and gradually increased thereafter. A different set of complexes was formed when the low salt extracts were used (lanes 7-13). The most distinct of these co-migrated with C2 and was subsequently confirmed to be identical to C2 (see below). Interestingly, this complex (C2) was detected in low salt extracts of embryos from every developmental stage, including the twocell stage, suggesting that its potential DNA-binding protein is present in the maternal pool but does not become localized to the nucleus or associated with chromatin until after MBT.
To characterize the identity of C1, C2, and C3, we first attempted to determine the DNA sequences involved in their formation. Synthetic double-stranded oligonucleotides spanning different portions of the 124-bp promoter region were used in competition assays (Fig. 9). The formation of C3 could be inhibited by almost every unlabeled probe used, including an unrelated probe containing random DNA sequences (lanes 1  and 2). Thus, C3 probably reflects nonspecific DNA-protein interactions, and it was not studied further. Complex C2 is probably identical to the retarded band detected with XTC-2 nuclear extracts because of their co-migration. This was confirmed by competition with unlabeled gs-6 and with several other CBF/NF-Y-specific probes, which all completely inhibited the formation of C2 (lanes 5 and 6; data not shown). Probe gs-6 and the CBF/NF-Y probes also inhibited formation of the distinct band from low salt extracts that co-migrated with C2 (data not shown), suggesting that this co-migrating band is identical to C2. These results strongly suggest that C2 represents a complex between CCAAT-containing sequences and a CBF/NF-Y-like factor and that this CBF/NF-Y-like factor is expressed in the maternal stores of early embryos but does not become chromatin-associated until the mid-blastula transition. A single region of protection (bracketed; Ϫ280/ Ϫ271) was detected, and its corresponding sequence is shown on the right. C, a competition assay revealed the importance of the CCAAT site in binding nuclear factors. Formation of the complex between the XTC-2 extract and probe gs-6 (lane 1) could be inhibited by an excess of cold gs-6 (lane 2) but not by mut-gs-6 (lane 3), a synthetic double-stranded oligonucleotide that is otherwise identical to gs-6 except that the CCAAT site was changed to GGGGT (Table I). D, formation of this complex occurs with mouse brain nuclear extracts. Similar complexes were found with either probe gs-6 or mgs-6 (the mouse counterpart of gs-6, Table I) and XTC-2 nuclear extracts (lanes 1 and 2). Using nuclear extracts from mouse brain (lanes 3-6), a similar complex was formed with probe mgs-6 (lane 3), which could be competed away by cold gs-6 (lane 4) or mgs-6 (lane 5), but not by mut-mgs-6 (a mutated version of mgs-6 in which the CCAAT site was changed to GGGGT (see Table I) (lane 6).
Among the oligonucleotide competitors tested, only one, gs-1 ( Fig. 4; Table I), could inhibit the formation of complex C1 (Fig.  9, lanes 3 and 4). When gs-1 itself was labeled and used as probe in the gel shift assay, it formed a complex that migrated to approximately the same position as C1 (lane 8). Therefore, gs-1 contained sequences required for the formation of complex C1. Within gs-1, there is a CCCCTCCCC motif that is a potential Sp1 binding site, with the T replaced by a G in a classical Sp1 site (51,52). In fact, the corresponding sequence of this motif in the human and mouse promoters is exactly CCCCGC-CCC. Given the considerable variation in Sp1 binding sequences (53), we suspected that C1 might represent a complex between the CCCCTCCCC motif and an Sp1-like factor. To test this theory, we used as competitors a cold gs-1 probe, a mutated gs-1 probe (mut-gs-1; Table I) in which the CCCCTCCCC motif was mutated to AAAATCCCC, and a probe containing a classical Sp1 binding site (Table I; Ref. 51). As shown in Fig. 10, both gs-1 (lane 2) and the classical Sp1 probe (lane 3) completely abolished complex formation, while mut-gs-1 (lane 4) did not affect complex formation. To confirm the presence of Sp1 in this complex, a supershift analysis was performed using an antiserum raised against Sp1 (33). Incubation of the nuclear extract with the Sp1 antiserum prior to the standard gel shift assay with probe gs-1 resulted in a supershifted complex (lane 5), whereas preincubation with preimmune serum had no effect (lane 6). Parallel supershift assays using the classical Sp1 probe produced a similar supershift pattern (lanes 7 and 8). Taken together, these results show that an Sp1-like factor interacts with the CCCCTCCCC motif in the proximal MARCKS promoter and that this interaction accounts for the the formation of complex C1.
Functional Analysis of the CCAAT Site and the Sp1 Site-To examine the potential functional significance of the CCAAT site and the Sp1 site in the proximal Xenopus MARCKS promoter, the mutations shown to abolish binding affinities ( Fig.  6C; Fig. 10) were each introduced into the 1047-bp Xenopus MARCKS promoter by site-directed mutagenesis; a double mutant in which both sites were changed was also constructed. These mutants, as well as constructs containing the wild type promoter, were injected into two-cell stage embryos, and the resulting CAT activities were measured (Fig. 11). The wild type 1047-bp promoter was able to direct CAT expression in a man-ner similar to that of the endogenous MARCKS gene expression, in that both genes were specifically activated at MBT (about late stage 8 or 9). Both the Sp1 site mutant and the CCAAT site mutant were able to activate CAT expression at MBT but at a much lower level than the wild type construct. Most strikingly, double mutation of both sites resulted in a total loss of reporter gene activation. This double mutant remained transcriptionally silent throughout further development (data not shown). These results suggest that each of these two sites is important for the normal developmental activation of the Xenopus MARCKS gene at the MBT and that they may work together in an additive manner. DISCUSSION These investigations into the transcriptional regulation of the Xenopus MARCKS gene revealed a promoter region of about 250 bp that was significantly conserved between Xenopus and other animal species. The corresponding region of the human MARCKS promoter was able to drive high level expression of a reporter gene in mouse fibroblasts (11) as well as the developmentally appropriate expression of a MARCKS-lacZ fusion gene in transgenic mice. 2 These data suggest that most of the developmentally important domains are contained in this region of the promoter in various animal species.
We attempted to identify important cis-regulatory elements in this region of the Xenopus MARCKS promoter by comparing the results of transient transfection assays in XTC-2 cells with those from microinjection of developing Xenopus embryos. It is now becoming clear that regulatory element(s) identified in cultured cells are often not a precise reflection of those used in intact animals (45). However, in both systems, the most dramatic decrease of promoter activity occurred when a 60-bp sequence (Ϫ325/Ϫ266) was deleted, suggesting that this 60-bp region contained one or more strong positive regulatory element(s) essential for MARCKS promoter activity.
To investigate potential DNA-protein interactions in this region of the Xenopus MARCKS promoter, we performed gel shift assays with a 124-bp probe corresponding to the minimal promoter from nucleotides Ϫ325 to Ϫ201. Parallel experiments were carried out using nuclear extracts from XTC-2 cells and from Xenopus embryos. Two specific complexes (designated C1 and C2) were detected in embryo nuclear extracts, and one of

5Ј-TCGACAGGAAAATCCCCGTG-3Ј
This paper these, C2, was also detected in XTC-2 nuclear extracts. The core binding sequence for C2 was found to be an inverted CCAAT box. A number of transcription factors bind to a similar core sequence, with the specificity of binding determined by flanking sequences (46). Three well known CCAAT-binding factors are C/EBP (47), NF-1 (48), and CBF/NF-Y/CP1 (46,49,50). By competition assays and supershift experiments using antibodies, we determined that the nuclear factor binding to the CCAAT site of the MARCKS promoter was a CBF/NF-Y/ CP1-like protein. Using similar approaches, the core sequence involved in the formation of complex C1 was found to be CCCCTCCCC, and its binding factor was found to be an Sp1like protein. Neither CBF/NF-Y nor Sp1 has been cloned in  lanes 8 -10), and c/ebp (lanes [11][12][13]. cbf, nf-1, and c/ebp contain the previously described binding sites for transcription factors NF-Y/CBF/Cp-1, NF-1, and C/EBP, respectively (Table I). 1, 0.1, and 0.01 g of cold oligonucleotides were used for each competitor as indicated by the triangles. B, antibody supershift assays. Preincubation of mouse brain nuclear extracts (1 g of protein) with antibodies to NF-YA (aYA) or NF-YB (aYB) prior to gel shift assay using probe gs-6 resulted in supershifted bands (lanes 1-3). Similar supershift patterns were observed when probe mgs-6 (lanes 4 -6) or cbf (lanes 7-9) was used. As controls, antibodies to FRGY1 and FRGY2, two other CCAATbinding factors, had no effect on complex formation (lanes 10 -12).

FIG. 8. Developmental regulation of complex formation between the proximal Xenopus MARCKS promoter and embryo extracts.
All binding reactions were performed with the 124-bp (Ϫ325/Ϫ201) promoter fragment as a probe. The extracts used are XTC-2 nuclear extract (lane 1; 1 g of protein); nuclear extracts from staged embryos (lanes 2-6; three embryo equivalents of extract/reaction); low salt extracts from staged embryos (lanes 7-13; one embryo equivalent of extract/reaction); and high salt extracts from staged embryos (lanes 14 -19; one embryo equivalent of extract/reaction). C1, C2, and C3 indicate the three major retarded bands. Note that C2 co-migrated with the retarded band seen with the XTC-2 cell extract. The embryo stages are indicated at the top of the autoradiographs.  Table I and Fig. 4B), and gs-6 (lane 6). Lanes 7 and 8, gel shift assays with probes gs-6 and gs-1, respectively, and a high salt extract from stage 32 embryos (one embryo equivalent/reaction).
Xenopus to our knowledge.
During Xenopus embryogenesis, transcription from the zygotic genome is not activated until approximately 6 h after fertilization, at a point that has been termed the mid-blastula transition (MBT) (44). The mechanism by which Xenopus embryos remain transcriptionally quiescent until MBT and then selectively activate certain genes is not fully understood. Recently, Almouzni and Wolffe (54) have shown that the transcriptional quiescence before MBT reflects not only general repressive effects due to an excess of histones but also a deficiency of transcriptional activators. According to this model, activation of transcription factors at MBT contributes at least in part to the onset of zygotic gene activation that occurs at MBT. However, what specific transcription factors activate which target genes at MBT is not known.
In this study, we showed that an Sp1-like factor and a CBF/NF-Y-like factor were first detected in nuclear or high salt extracts of the mid-blastula stage embryos, implying that these two factors might be among those contributing to the activation of the zygotic genome at this time. To address this possibility with regard to MARCKS expression, we mutated the Sp1 site and/or the NF-Y site in the 1-kilobase pair Xenopus MARCKS promoter and tested the effects of these mutations on the developmental expression of the MARCKS gene. These mutations were shown to abolish binding of the Sp1-and CBF/NF-Y-like factors to the MARCKS promoter. We found that each mutation significantly decreased activation of the MARCKS promoter at MBT. Most strikingly, nonbinding mutations at both sites resulted in a total loss of gene activation at MBT. These results suggest that the factors binding to these two sites play an essential role in the activation of MARCKS transcription at MBT and possibly that of other Xenopus genes that are selectively transcribed at this time.
Although the CBF/NF-Y binding activity was first detected in nuclear or high salt extracts of stage 10 embryo, it was already present in cytosolic or low salt extracts of embryos at much earlier stages, including unfertilized eggs. Therefore, it appears that this CBF/NF-Y/CP1-like factor is expressed in the maternal pool of the embryos but does not become localized to the nucleus and/or associated with chromatin until MBT. This expression pattern is similar to those of two other CCAATbinding activities described previously (21,55). In one case, a CCAAT box-binding transcription factor, termed CBTF, was found to be expressed in the maternal pool of Xenopus embryos but did not interact with the chromatin until late blastula stage (55). In the other case, a functional CCAAT element identified in the Xenopus GATA-2 promoter was found to bind a maternal factor that appeared to be localized to the cytoplasm until stage 10, at which point it became translocated to the nucleus (21). In neither case has the identity of the CCAAT binding factors been defined. The similar expression patterns of these two factors with the CBF/NF-Y-like factor described in this paper prompted us to examine similarities among the three factors. Our results, based on competition analysis and antibody supershift assays, suggest that the two previously described factors are identical to the one described here and that they are most likely to be the Xenopus homologue of the mammalian transcription factor CBF/NF-Y/Cp1. Alignment of the Xenopus MARCKS promoter with those from mouse and human genes indicated that both the Sp1 site and the NF-Y site are highly conserved among species, suggesting that the mammalian MARCKS genes are also regulated by Sp1 and NF-Y. Both Sp1 and NF-Y are expressed in many mammalian tissues including the brain (56) and thus seem to be reasonable candidates for transcriptional regulators of the MARCKS gene. Although these two factors by themselves are unlikely to account for development-, cytokine-, and oncogeneregulated expression patterns of mammalian MARCKS genes, they may act in concert with other sequence-specific transcription factors to confer these types of regulated expression. For example, studies have shown that both Sp1 and NF-Y can interact with upstream sequence-specific transcription factors to modulate transcription of target genes (33,(57)(58)(59). In one case, studies on the constitutive and interferon-␥-induced expression of the major histocompatibility complex class II DRA gene have shown that the presence of a distal X box and a proximal CCAAT box at a proper distance from each other is required for normal promoter function (58); this requirement is apparently mediated by a direct interaction between CBF and X box-binding proteins (59). Sp1, although a common transcription factor, can also interact with other factors to exert very specific regulatory effects (33,57). Ongoing studies in our laboratory are now trying to identify more cis-and trans-acting regulatory elements in the mammalian promoters and to study how these elements together produce constitutive as well as regulated expression of the MARCKS gene.
The primary structure and biochemical features of the Xenopus MARCKS protein bore striking similarities to those of MARCKS from other species, suggesting that these proteins might have similar functional properties. Recent gene "knockout" experiments from our laboratory indicated that MARCKS is required for normal development of the central nervous system and postnatal survival in mice (3). However, using these mice to study the function of MARCKS at the cellular and molecular levels has proved difficult, partly because to date, no defects have been detected in a variety of cell types derived from the MARCKS-deficient animals. It is noteworthy that MRP, another member of the MARCKS family, exists in mice at levels comparable with MARCKS (3). The high level expression of MRP in the same cells and tissues might, therefore, prevent the MARCKS-deficient mice and cells derived from them from exhibiting a more severe or informative phenotype. In this study, we found that although MARCKS was abundant in Xenopus oocytes and embryos, the Xenopus equivalent of MRP could not be detected. Thus, early Xenopus development provides a potentially interesting system in which to study the molecular functions of MARCKS in the absence of functional redundancy due to confounding levels of MRP. Although germline mutation is not yet available in Xenopus, alternative approaches such as the use of antisense oligodeoxynucleotides could be used to circumvent this limitation. Such experiments are currently under way in our laboratory.