Nucleic Acids Research

Hexammine cobalt(lIl) chloride (Co(NH3)63+) provokes a B-DNA -4 Z-DNA -+ P-DNA conformational transition in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC). The circular dichroism spectrum of '-DNA is characterized by a manyfold increase of positive ellipticity in the range of 300-225 nm and the complete absence of a negative peak. In order to ascertain the helical handedness of '-DNA, we used a recently developed enzyme immunoassay technique. This method consisted of treating the polynucleotides with Co(NH3)63+ to convert them to the Zor '-DNA forms and immobilizing these conformations on a microtiter plate. The plates were subsequently treated with a monoclonal anti-Z-DNA antibody Z22, alkaline phosphatase conjugated, affinity purified immunoglobulins, and the phosphatase substrate. The enzyme-substrate reaction was monitored by reading the absorbance at 405 nm with a microplate autoreader. The monoclonal anti-Z-DNA antibody had no reactivity to the B-DNA form, but bound strongly to both the Zand '-DNA forms, showing that Co(NH3)63+-induced '-DNA form of the polynucleotides exists in the left-handed Z-DNA conformation. INTRODQUCTION Multivalent ions like Co(NH3)63+ and Ru(NH3)63+ are excellent promoters of the transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) from their usual right-handed B-DNA form to the left-handed Z-DNA conformation at micromolar concentrations of these counterions (1-4). Increasing concentrations of Co(NH3)63+ are also known to provoke a supramolecular organization of the polynucleotides from the Z-DNA form to a '-DNA form (5-6). The '-DNA form is a believed to be a twisted, tightly packaged assembly of DNA. Several agents, including polylysine, spermine, ethylene glycol, and salt are known to convert native DNAs to the '-DNA form (7-9). The formation of this structural form of DNA is characterized by a large (10to 100-fold) increase in the intensity of the molar ellipticity of DNA in circular dichroism (CD) spectroscopic measurements. This property has presented a basic problem in interpreting the CD spectrum of '-DNA and assigning its helical handedness. This uncertainty has recently led to the conclusion that '-DNA is the end product in the © I R L Press Nucleic Acids Research Volume 17 Number 1


INTRODUCTION
The v-myb oncogene and its cellular homologue, c-myb, encode nuclear proteins which feature a highly conserved DNA-binding domain1~3. Expression of c-myb is highest in immature [4][5][6] haemopoietic cells and decreases during differentiation While the molecular mechanisms by which myb proteins function are poorly understood, their known properties are consistent with a role in regulating the expression of other genes at the level of transcription. For example, studies of temperature-sensitive mutants of a virus (E26) that carries a v-myb oncogene imply that 7 DNA binding is necessary for v-myb function7. Moreover, the DNAbinding region of the myb proteins shows structural homology with 8 a maize regulatory gene We show here that the c-myb gene product can function as a transcriptional trans-activator. This function was assayed in transient expression experiments by cotransfecting a plasmid containing c-myb-coding region and a plasmid containing a test promoter and enhancer. Co-transfection of a c-myb cDNA expression plasmid with reporter plasmids carrying the CAT gene resulted in a 10to 20-fold stimulation of CAT activity and CAT mRNA. Stimulation was independent of the promoter at which CAT transcripts initiated, but rather, was mediated by tandem repeats of the SV40 enhancer present in the reporter plasmids.

MATERIALS AND METHODS
Plasmid construction. The effector plasmid pact-c-myb was constructed using the plasmid pactCAT which contains the 1350 bp fragment of the 5' reguratory region of the chicken 0cytoplasmic actin gene linked to the CAT gene9' 10t The HpaI site downstream of the CAT coding region of the pactCAT was converted to the XbaI site by linker, and the 1460 bp NcoI-XbaI fragment containing the CAT gene was replaced by the 1990 bp NcoI-XbaI fragment containing the complete c-myb protein-coding region to generate the plasmid pact-c-myb. The 1990 bp NcoI-XbaI fragment containing the c-myb gene was prepared from the plasmid which was constructed by joining the two cDNA clones via their common SspI site at nucleotide 172111 and the HaeII site at nucleotide 2026 was converted to the XbaI site by linker. Because the restriction endonuclease NcoI recognizes sequences including the initiation codon (ATG) of both the chicken S-actin gene and the mouse c-myb gene, pact-c-myb, generated by ligation at this NcoI site, encodes the complete c-myb protein. The plasmid pacti was constructed by deletion of NcoI-XbaI fragment containing the c-myb sequence from pact-c-myb. The plasmid pMFcolCAT-SVE contains sequences of the mouse a2(I) collagen gene from approximately 2000 bp upstream of the start of transcription (+1) to 159 bp downstream of this site fused to the CAT gene10' 12. To generate the pMFcolCAT+1SVE, pMFcolCAT+2SVE, pMFcolCAT43SVE and pMFcolCAT+4SVE plasmids, 1, 2, 3 and 4 tandem repeats, respectively, of the SV40 enhancer were inserted at the BamHI site located about 100 bp downstream of the poly(A) addition signal for CAT mRNA. This SV40 enhancer DNA fragment contains sequences between +113 and +270 of the SV40 map and includes one complete copy of the 72 bp enhancer and a second copy which is almost complete10. The pA10cat2+1SVE, pA10cat2+2SVE, pA10cat2+3SVE, and pA10cat2+4SVE plasmids were also constructed by insertion of 1, 2, 3, and 4 tandem repeat, respectively, of the SV40 enhancer fragment into the BglII site 13 upstream of the SV40 promoter of pA10cat2 DNA transfection and CAT assay. African green monkey kidney cells (CV1), were plated at a density of 4 x 105 per 10 cm Falcon tissue culture plate. Mixturs of 6 jig of reporter plamid DNA, 6 pg of effector plasmid DNA and 2 pg of pRSV-$-gal plasmid DNA were transfected by Ca-PO4 precipitation method 16-20 h after plating cells. The plasmid pRSV-a-gal was used as an internal control for differences in transfection efficiency between 14 precipitates . Forty h after transfection, cells were collected by scraping with a rubber policeman in phosphate-buffered saline. Cells were lysed by freeze-thawing and sonication to prepare extracts, and 0-galactosidase activities were measured. CAT enzyme reactions were performed as described by Gorman et al.15 using 0.6 pCi [14C] chloramphenicol at 370C for 1 h. The amounts of cell extract used for the CAT assays were normalized with respect to 0-galactosidase activity. Acetylated reaction products were resolved by TLC analysis using Baker-flex silica gel IB-F TLC plates. The degree of conversion was determined by removing spots containing either unreacted 14C-chloramphenicol or acetylated forms and measuring the amount of radioactivity in a liquid scintillation counter.
Southern blot and RNA analyses. To compare the copy number of different transfected plasmid DNAs, nuclei were isolated to prepare DNAs 40 h after transfection, and at the same time 6galactosidase activities were measured to confirm that transfection efficiencies were similar. Ten pg of nuclear DNA was digested with BamHI and HindIII and analyzed by the method of Southern16. The hybridization probe was a nick-translated 1.6 kb BamHI-HindIII fragment containing the CAT gene. Forty h after transfection, RNA was isolated by guanidium thiocyanate method17. Primer extension analysis was carried out by using 40 pg of total RNAs as described previously18. A  oligonucleotide complementary to nucleotides 26-49 of the coding sequence of the CAT gene was used as a primer.

RESULTS
Tandem repeats of the SV40 enhancer are responsible for the c-myb-induced stimulation of CAT gene expression from the collagen promoter. In order to express the c-myb protein at high levels, we constructed a plasmid, pact-c-myb, in which a murine c-myb cDNA11 was linked to the chicken 0-actin promoter9 (Fig.1).
This was cotransfected into CV1 cells with various reporter plasmids, in which different promoters and/or enhancers were linked to the CAT gene. CAT activity was compared to that in cells cotransfected with the reporter plasmid plus pact-1, which lacks c-myb sequences (Fig.1). The transfections also included the plasmid pRSV-$-gal14, which carries the $-galactosidase gene linked to the Rous sarcoma virus (RSV) LTR promoter. This enabled us to control for differences in transfection efficiency by measuring 0-galactosidase activity.
Cotransfection of pMFcolCAT+4SVE, which contains the mouse a2(I) collagen promoter10' 12  Mixtures of the indicated reporter plasmid DNA, effector plasmid DNA (pacti (-) or pact-c-myb (+)) and pRSV-6-gal plasmid DNA were transfected, and CAT activity assayed. c. Levels of CAT RNA enhanced by c-myb. Mixtures of the indicated reporter plasmid DNA, effector plasmid DNA (pacti (-) or pact-c-myb (+)), and pRSV-$-gal plasmid DNA were transfected, and RNA prepared. Primer extension analysis was performed using a primer complementary to the CAT mRNA. Correctly initiated transcripts 35om the mouse a2(I) collagen promoter are indicated by arrow. P-labeled Hinf-I-digested pBR322 was used as a size marker. the level of CAT activity measured after 40 h (Fig. 2b). In contrast, when CAT expression was controlled by the chicken 6actin promoter/enhancer, CAT activity was elevated only 2-fold by co-transfection with pact-c-myb, while no effect of myb was observed when the CAT gene was linked to the RSV LTR (data not shown).
To identify the sequences in pMFcolCAT+4SVE which are 111 tyb ------- ----------r responsible for c-myb-induced transcription activation, we have examined the effects of c-myb in cotransfection experiments using a series of deletion mutations in the mouse a2(I) collagen promoter. None of these mutations prevented c-myb-induced transcription activation (data not shown). This observation lead us to speculate that the tandemly repeated SV40 enhancer might mediate the action of c-myb. To test this possibility, we constructed a series of plasmids (pMFcolCAT+1SVE, pMFcolCAT+2SVE, and pMFcolCAT+3SVE) containing fewer SV40 enhancer repeats (Fig. 2a). Results of cotransfection experiments using these plasmids are shown in Figure 2b. While one repeat of the SV40 enhancer was insufficient to obtain c-myb-induced CAT activation, increasing the number of repeats to two (pMFcolCAT+2SVE) resulted in lowlevel actvation (3-fold) of the CAT gene. Further activation (10-fold and 20-fold) was observed by increasing the repeat number to three and four (pMFcolCAT+3SVE and pMFcolCAT+4SVE, respectively). Thus the SV40 enhancer contributes incrementally to transcriptional activation of the hybrid gene by c-myb.
Analysis of CAT RNA and copy number of the reporter plasmid after transfection. To check whether the c-myb-induced transcription initiated at the legitimate cap sites of the mouse a2(I) collagen promoter, we performed primer extension analysis using a primer complementary to a segment of CAT mRNA. If transcription of CAT mRNA starts at the correct positions in the mouse a2(I) collagen promoter, the extended primer should run as a doublet of 246-247 nucleotides (162 nucleotides of a2(I) collagen mRNA plus 85 nucleotides of CAT mRNA)10. Figure 2c shows that the CAT transcripts are indeed generated from the authentic cap sites of the mouse a2(I) collagen gene, as judged by the sizes (246-247 nucleotides) of the extension products; as expected, the levels of these transcripts correlated with the respective CAT enzyme activities described above.
We also tested the posibility that c-my. was acting by increasing the copy number of the reporter plasmid pMFcolCAT+4SVE after transfection into CV1 cells. After cells were transfected with the mixture of pMFcolCAT+4SVE, and either pact-c-myb or pact-1 DNA, the DNA was extracted from isolated nuclei and used pMFcoICAT +4SVE mybr_ + 6.6-4. for Southern blotting analysis . In both cases, the intensities of 1.6 kb bands detected by a CAT probe were similar (Fig. 3), showing that c-myb did not affect the copy number of pMFcolCAT+4SVE plasmid DNA after transfection.
C-myb-induced trans-activation is independent of the promoter in a reporter plasmid. To confirm that tandem repeats of the SV40 enhancer are sufficient for c-myb-induced transcription activation and that the mouse c2(I) collagen promoter is not specifically required, we constructed another set of chimeric CAT genes (pA10cat2+1SVE, pA10cat2+2SVE, pA10cat2+3SVE, and pA10cat2+4SVE) using the pA10cat2 plasmid13, as shown in Figure 4a. In those constructs the mouse a2(I) collagen promoter was replaced by the SV40 early gene promoter.
When one or two repeats of the SV40 enhancer were linked to the SV40 promoter, the extent of activation by c-myb of the resulting hybrid genes was low (Fig. 4b). However, when number of repeats  and pRSV-a-gal plasmid were transfected into CV1 cells, and CAT activity assayed. c. Primer extension analysis of CAT RNA. Mixtures of the indicated reporter plasmid DNA, effector plasmid DNA (pacti (-) or pact-c-y~(4), and pRSV-~-gal plasmid DNA were transfected into CV1 cells, and primer extension experiments were performed. Correctly initiated transcripts from the SV4O promoter are indicated by arrow. 3P-labeled Hinf I-digested pBR322 was used as a size marker. of the SV4O enhancer element was increased to three or four,, the hybrid genes were activated about 10-fold by c-my (Fig. 4b). To confirm that the CAT enzyme activities were correlated with the level of correctly initiated RNA, primer extension analysis was again carried out. If CAT RNA starts at the correct site in the SV4O early promoter, the extended primer should be 141 nucleotides long (56 nucleotides of SV4O mRNA plus 85 nucleotides of CAT RNA). In cells cotransfected with pact-c-myband pA1OCAT2+4SVE, there was an approximately 10-fold increase in the intensity of the 141 nucleotide band, indicating that c-yb stimulated specific and correct initiation at the SV40 early gene promoter. Taken together, these results imply that the presence of tandem repeats of the SV40 enhancer is sufficient to mediate the c-myb-induced transcriptional activation. DISCUSSION Although the c-jun, c-fos and c-myc gene products can function as transcriptional activators19-25, similar activity of the c-myb gene product has not previously been described. Here we have demonstrated that the mouse c-myb gene product either functions as or induces an activator of transcription that interacts with sequences in the SV40 enhancer; such an interaction may be either direct or mediated by another DNAbinding protein, by analogy with the interaction between the fos and un proteins26-28, Recent reports indicate that the SV40 enhancer region consists of multiple sequence elements29, each of which binds specific trans-activators such as AP-130 or AP-231, and that the individual elements can act autonomously when 32 present in multiple copies . In this light, our results suggest that one or more of these elements may, when tandemly repeated, create a functional enhancer that mediates c-mybinduced transcriptional activation. It will therefore be important to identify these sequences in the SV40 enhancer, as it is likely that the transcriptional control regions of cellular "target" genes modulated by c-myb will contain similar sequences.
Recently we have found that the c-myb protein binds to two sites in the SV40 enhancer directly (Nakagoshi et al., submitted), revealing that the c-myb protein is the second example of a proto-oncogene encoding a sequence-specific enhancer-binding protein; the first being the c-i_un/AP-1 protein20. Although the mechanism of an enhancer function of the sequence recognized by trans-acting factors including c-myb protein only when tandemly repeated is not clear at present, the cooperativity in binding might be most likely possibilty.
Finally, it is worth noting that the DNA-binding region of myb protein has 3-fold tandem repeats of 51 or 52 amino acids11' 1, and does not display any of the specific structural features such as the "helix-turn-helix" or "zinc-finger" motifs found in other DNA-binding and/or transcriptional regulator proteins.
Moreover, although the c-myb protein has the "leucine zipper"33_ like structure (the first Leu residue is at nucleotide 1,183 in the ref. 11), it is not linked to the DNA binding domain. Thus, analyses of the DNA-binding and trans-activator functions of m protein may lead to the definition of a new structural class of transcriptional regulators.