The Cytomegalovirus Enhancer Induces an Immediate Response to the Myosin Light Chain 2 v Promoter during P 19 CL 6 Cell Differentiation

The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.


Introduction
P19CL6 cells derived from P19 embryonic carcinoma cells can efficiently differentiate into cardiac muscle cells in the presence of 1% dimethyl sulfoxide (DMSO) [1].Such a property is a good tool for the study of cardiac myocyte differentiation in vitro.Before transcriptional activation of the genes for cardiac contractile proteins during the differentiation of P19CL6 cells, the gene for the GATA-4 transcription factor becomes activated [2].We have also been reported that the GATA-4 gene is immediately transcribed upon addition of DMSO through binding of GATA-6 to the upstream enhancer GATA motif, which is conserved in mammals [3].Not only the GATA-4 but also the MEF2C and Tbx5 transcription factors play crucial roles in cardiac myocyte differentiation [4].
Furthermore, genetic analysis suggested that NKX2.5 and GATA-6 together with these three transcription factors are essential for cardiac development [5] [6].
As for contractile proteins, transcripts of myosin heavy chains (α-MHC and β-MHC) were detected in parallel in P19CL6 cells at a late stage after the start of cardiac differentiation [1].Similar to these cardiac MHC isoforms, cardiac myosin light chain 2 (MLC-2v) is also transcribed during the course of differentiation [7].When the 250 base pair (bp) promoter for the rat cardiac MLC-2v gene was connected upstream of the gene for green fluorescent protein (GFP) together with the enhancer portion of the cytomegalovirus (CMV) immediate early promoter, and the resulting reporter plasmid was stably introduced into P19CL6 cells, expression of GFP was limited to developing cardiac myocytes [8].Although this system could be advantageous to quantify cardiac differentiation, it has not been addressed as to why the virus enhancer responds to such a differentiation signal.Actually, it has been demonstrated that the SV40 enhancer acted on both heart muscle and non-muscle cells [9].
In this study, we analyzed the MLC-2v promoter driven by the CMV enhancer in more details.Although the enhancer-promoter construct responded to the DMSO signal, the response was immediately before the start of transcription of native MLC-2v.We will discuss the role(s) of GATA motifs in the CMV enhancer sequence from the viewpoint of GATA-4 transcription in P19CL6 cells [3].

Construction of an Expression Plasmid for GFP under the
Control of the MLC-2v Promoter.
The cloned DNA was sequenced by the dideoxy chain-termination method [13] with a primer (M13 forward or M13 reverse) and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).The oligonucleotides used for PCR and sequencing are listed in Table 1.The molecular biological techniques were performed by published methods [14].

Cell Culture
P19CL6 cells [3] were cultured in α-Eagle's minimal essential medium (GIBCO Table 1.Sequences of oligonucleotides used in this study.
Restriction enzyme sites are underlined with bold letters.

HindIII
CMVenh-R 5'-TTA AGC TTC AAA ACA AAC TCC CAT TGA CG-3' BRL) supplemented with 10% (v/v) fetal bovine serum (GIBCO BRL) and antibiotics (100 μg/mL streptomycin sulfate and 100 u/mL benzyl-penicillin) (Wako).An expression plasmid was introduced into the cells (10 5 cells per 6 cm diameter dish) by means of the calcium-phosphate method as described previously [15].Cells were split into 10 cm diameter dishes after 48 hrs incubation, and then grown in the presence of 1 mg/mL G418 (Nacalai).Among six resistant colonies, one (clone B4) that showed strong green fluorescence in the presence of 1% (v/v) DMSO was analyzed.
The B4 clone (10 4 cells) was seeded into a 6 cm diameter dish.Cells were grown in the medium plus 1% (v/v) DMSO.The medium was changed to fresh medium containing DMSO on the fourth day, and then every two days.The expression of GFP was monitored under a microscope (Olympus IX-70) equipped with an AQUACOSMOS U7501 (HAMAMATSU PHOTONICS).

Chemicals
Restriction enzymes were obtained from NEB and Toyobo.Agarose LO3 and a DNA ligation kit ver.2.0 were purchased from TaKaRa.A GENECLEAN® III KIT and oligonucleotides were provided by MP Biomedicals and Gene Design Inc., respectively.All other chemicals used were of the highest grade commercially available.

Expression of GFP in the Stable Transfectant Cells
We constructed an expression plasmid for GFP under the control of the CMV enhancer and MLC-2v promoter (Figure 1).This expression plasmid was stably introduced into P19CL6 cells and a G418-resistant clone carrying the reporter gene was isolated.The clone was cultured in the presence and absence of 1% DMSO, and green fluorescence derived from GFP was monitored under a microscope.As shown in Figure 2, weak fluorescence was detected on day 4 in the presence of DMSO.The fluorescence increased gradually and was much stronger on day 12 under the differentiation conditions.However, without DMSO only a background level of fluorescence was detected on day 12.Previous study also showed that very little but detectable amounts of GFP was expressed in undifferentiated cells [8].

Comparison of the Expression of Native MLC-2v and GFP
The expression pattern of GFP was compared with that of native MLC-2v.As shown in Figure 3 (middle panel), the start of transcription of the native MLC-2v was clearly retarded in contrast to the appearance of the transcript of GFP.The transcript of MLC-2v appeared abruptly on day 4, and its amount was maintained constantly.Since the rat and mouse MLC-2v prompter sequences are essentially the same (Figure 4(a)), it is suggested that the immediate transcription of the GFP reporter gene could not be ascribed to the MLC-2v prompter moiety but rather to the CMV enhancer.

Presence of GATA Motifs in the CMV Enhancer
Since the CMV enhancer started to operate immediately after DMSO addition (Figure 3), it seems likely that the potential master regulator(s) that directs P19CL6 cells toward cardiomyocytes may instantaneously bind to and activate the enhancer in the presence of DMSO.Our previous study demonstrated that the binding of pre-existing GATA-6 to an upstream GATA motif is essential for the immediate activation of the GATA-4 gene upon DMSO addition to P19CL6  cells [3].Consistent with this observation, the CMV enhancer has multiple GATA-6 binding motifs, 5'-GAT(A/T)-3' [16]; two GATA and one GATT sequence are present in the downstream half of the 406 bp enhancer sequence (Figure 4(a)).However, such a motif could not be found in the 262 bp MLC-2v promoter sequence (Figure 4(b)).Since GATA-6 could activate reporter genes through GAT(A/T) sequences [17] [18], GATA-6 may bind to the CMV enhance in response to a cardiac differentiation signal.

Discussion
P19CL6 cells are used as a model of differentiation of cardiac myoblasts into myocytes [1] [8].In this study, we found that the MLC-2v promoter harboring the CMV enhancer responded immediately in P19CL6 cells upon the addition of DMSO, which is an induction reagent for cardiac differentiation [1].However, the intrinsic MLC-2v promoter was activated rather later during the differentiation (Figure 3).Thus, the transcription of GFP under the control of the CMV enhancer is not a result of cardiac myocyte differentiation, but rather parallel to activation of the start of differentiation.It should be noted that such comparison between native and hybrid MLC-2v promoters focused on the CMV enhancer has not been carried out previously [8].
As for GATA-4 gene expression, binding of GATA-6 to the distal enhancer GATA motif was obligatory for transcriptional activation under the differentiation conditions for P19CL6 cells [3].Since GATA-4 and GATA-6 function in transcription through a GAT(A/T) motif [17] [18], it seems likely that the CMV enhancer with GATA motifs (Figure 4(b)) immediately responds to pre-existing GATA-6 in P19CL6 cells on DMSO treatment.Actually, the CMV enhancer induces efficient cell-type specific expression of genes such as those of atrial natriuretic factor and MLC-2v in cardiomyocyte cell line HL-1 [19], which expresses GATA-4 and GATA-6 [20].Consistent with this finding, the SV40 enhancer composed of the 72 bp tandem repeat [21] without GATA motifs (NCBI Reference Sequence NC_001669) abolished the cardiac specificity [9].
The gradual increases in the amounts of GFP transcripts shown in Figure 3 could be explained by the participation of the pre-existing GATA-6, and increased amounts of GATA-4 and GATA-5 arising through transcription and translation [3].It is also suggested that factors like GATA-4 that potentiate transcription during development are inherently capable of initiating chromatin opening through binding to an enhancer [22].Thus, the CMV enhancer with GATA motifs may become open and activated in an environment in which GATA factors are expressed although modulation and/or a trigger such as DNA methylation and protein modification could be needed by DMSO.
Although roles of GATA factors expressed in P19CL6 cells and GATA motifs in the CMV enhancer should be further examined in detail, the specific and in-

Figure 1 .
Figure 1.Construction of an expression plasmid for GFP with the CMV enhancer and MLC-2v promoter.The CMV enhancer moiety (CMVenh, 406 bp) derived from pEGFP-N1 and the MLC-2v promoter (MLC-2v pro , 262 bp) cloned from rat liver genomic DNA were inserted into the pEGFP-N1 vector from which the human cytomegalovirus (CMV) immediate early enhancer and promoter sequences [CMV(e + p)] corresponding to nucleotide residue numbers 1 -589 (GenBank Accession No. U55762) had been deleted.Typical restriction enzyme sites used in this study (AseI, BamHI, EcoRI, HindIII, NotI and SmaI) were indicated in the figure.MCS, multi-cloning site.

Figure 2 .
Figure 2. Expression of GFP in the stable transfectant clone derived from P19CL6 cells.The expression plasmid (pCBVenh/MLC-2v pro /EGFP) constructed as in Figure 1 was stably introduced into P19CL6 cells.The G-418 resistant clone was cultured in the presence [DMSO (+)] or absence [DMSO (-)] of 1% (v/v) DMSO.Fluorescence images on the indicated days after the start of the experiment are shown.Bar, 200 μm.

Figure 3 .
Figure 3. Detection of transcripts of GFP and native MLC-2v.Total RNA was prepared on the indicated days after DMSO addition, and then subjected to RT-PCR to detect transcripts of GFP and native MLC-2v.β-actin mRNA was used as a positive control.Amplified fragments were analyzed by 2 % (w/v) agarose gel-electrophoresis.