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. 1996;6(1):45-57.

Characterization of the nonmuscle myosin heavy chain IIB promoter: regulation by E2F

L Weir  1 , D Chen
Affiliations

Characterization of the nonmuscle myosin heavy chain IIB promoter: regulation by E2F

L Weir et al. Gene Expr. 1996.

Abstract

To identify DNA sequences important for the transcriptional regulation of the nonmuscle myosin heavy chain IIB (NMMHC-IIB) gene we isolated and sequenced genomic clones that contain the promoter of the gene for both human and mouse. In addition to considerable homology in the first (untranslated) exon (91%) we found 80% sequence identity in the 700 base pairs immediately upstream of the major start of transcription (+1) as well as significant homologies as far as 1500 base pairs upstream. The promoter region was characterized using luciferase reporter constructs transiently transfected into NIH3T3 cells. Consensus binding sites for several known transcription factors are present that are completely conserved between the mouse and human genes, including CRE/ATF, Sp1, CAAT, and the cell-cycle transcription factor E2F. Gel shift assays indicated that E2F can bind to its putative binding site in vitro. To test whether this site is functional we cotransfected NMMHC-IIB promoter constructs driving luciferase with a vector expressing E2F-1. The E2F-1 vector stimulated luciferase activity from an intact promoter whereas mutation of the site eliminates binding and diminishes transactivation. These data provide strong evidence that E2F or an E2F-related transcription factor is involved in the regulation of nonmuscle myosin expression.

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Figures

FIG. 1
FIG. 1
Restriction maps of λ phage clones containing the 5′ end of the NMMHC-IIB gene. (A) Restriction map of λ clone containing 17 kb of the 5′ region of the human NMMHC-IIB gene. The position of exon 1 is indicated. (B) Restriction map of X clones containing the 5′ region of the mouse NMMHC-IIB gene. The positions of exons 1 and 2 are indicated. Abbreviations used for restriction enzyme sites: A, Apa I; B, Bam HI; Bs, Bss HII ; Bt, Bst YI; R, Eco RI; H, Hind III; P, Pvu II; Sa, Sac I; S, Sma I; X, Xho I.
FIG. 2
FIG. 2
Conservation of sequence in the promoter region of the NMMHC-IIB gene between human and mouse. The nucle-otide sequences of the human and mouse NMMHC-IIB promoter regions were aligned for comparison. The position of several conserved putative binding sites for DNA binding proteins are indicated. Identical nucleotides are indicated by dashes and spaces indicate gaps inserted to optimize homology. Exon 1 is overlined and the major transcription start site is numbered as + 1. Note that only part of the determined sequences are shown: the sequence of the entire 1959-bp Eco RI/ Bam HI fragment from the human NMMHC-IIB gene (–1492 to + 467) and the sequence of the 1814-bp Eco RI/Bam HI fragment from the mouse NMMHC-IIB gene (–1383 to + 431) were submitted to Genbank (accession Nos. U34301 and U34302, respectively).
FIG. 3
FIG. 3
Sequence of exon 2 of mouse NMMHC-IIB gene with deduced amino acid sequence. The complete sequence of the Hind III/Eco RI frament containing the second exon is presented. Positions of exon/intron junctions are indicated by slash marks.
FIG. 4
FIG. 4
Mapping of transcriptional start sites by ribonuclease protection. RNA probes containing the 5′-flanking sequence of the NMMHC-IIB gene including the first exon and part of the first intron were hybridized to total RNA and treated with RNase A and Tl. Protected fragments were analyzed on urea-6% polyacrylamide gels. Probe I was 509 nucleotides in length including 432 nucleotides of NMMHC-IIB sequence and 77 nucleotides of vector sequence as indicated in (C). Probe II was 293 nucleotides in length including 216 nucleotides of NMMHC-IIB sequence and 77 nucleotides of the vector. (A) Lane 1: molecular weight markers ϕX174 × Hinf I; lane 2: probe I alone; lane 3: probe I hybridized to yeast tRNA without RNase digestion; lane 4: negative control-probe I hybridized to yeast tRNA with RNase; lane 5: probe I hybridized to 50 μg total RNA from human endothelial cells. The major protected band of 75 nucleotides corresponding to the transcriptional initiation point designated as nucleotide + 1 is indicated. A number of other protected bands are also detected. (B) Lane 1: molecular weight markers ϕX174 × Hinf I; lane 2: probe II hybridized to 50 μg total RNA from human endothelial cells; lane 3: probe II alone; lane 4: probe II hybridized to yeast tRNA without RNase digestion; lane 5: negative control-probe hybridized to yeast tRNA with RNase digestion. The major protected band of 75 nucleotides corresponding to the transcriptional initiation point designated as nucleotide + 1 is indicated. Note the prominant band at 197 nt corresponding to protection of the whole of probe II except for 19 nucleotides of the first intron and 77 nt of vector.
FIG. 5
FIG. 5
Promoter activity in the 5′ region of the human NMMHC-IIB gene. Constructs containing portions of the human NMMHC-IIB promoter driving expression of luciferase were transfected into NIH3T3 cells along with a plasmid expressing alkaline phosphatase to control for transfection efficiency. The minus numbers indicate the endpoint of each deletion with respect to the major start site of transcription. The graph shows the relative luciferase activity produced by each construct normalized to the alkaline phosphatase activity. For comparison each result is expressed as a proportion of that obtained by construct –305, which contains the smallest promoter insert that still gave maximal activity. Each data point is the mean of at least three experiments ± SE. The luciferase vector with no promoter (pGL-2Basic) was transfected as a control.
FIG. 6
FIG. 6
Transcriptional activation of the NMMHC-IIB promoter by E2F-1. In each transient tranfection experiment 2 μg of construct D4 or D18 was cotransfected with increasing amounts of the E2F-1 expression vector into NIH3T3 cells. Construct D4 contains 389 bp of the NMMHC-IIB promoter driving luciferase. D18 contains a 4 bp deletion within the putative E2F binding site. To control for transfection efficiency a plasmid expressing β-galactosidase driven by the CMV promoter was cotransfected and luciferase results were normalized. All luciferase values are expressed relative to that obtained for D4 with carrier DNA alone. Each data point is the mean of three experiments ± SE.
FIG. 7
FIG. 7
Gel shift assay: the putative E2F element from the NMMHC-IIB promoter binds to the E2F activity in HeLa cell extract. Gel mobility retardation assays with a HeLa cell extract were used to test whether the sequence in the NMMHC-IIB promoter is a functional binding site for E2F. Oligonucleotides (25 bp) containing the sequence in question from the NMMHC-IIB gene (NMB wt) and the E2F consensus previously described by Helin et al. (10) were used as probes. Unlabeled competitor oligonucleotide was included, where indicated, at a 100 times molar excess. Products were separated on a 4% polyacrylamide gel run in 0.25 X Trisborate-EDTA. The gel was dried and exposed to X-ray film overnight at –70°C. The experiment was performed three times with similar results.
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