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. 1998 Nov 24;95(24):14220-5.
doi: 10.1073/pnas.95.24.14220.

Expression of myogenin during embryogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site

F Spitz  1 , J Demignon, A Porteu, A Kahn, J P Concordet, D Daegelen, P Maire
Affiliations

Expression of myogenin during embryogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site

F Spitz et al. Proc Natl Acad Sci U S A. .

Abstract

Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a beta-galactosidase transgene during embryogenesis. We show here that mutation of one of the DNA motifs present in this region, the MEF3 motif, abolished correct expression of this beta-galactosidase transgene. We have found that the proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin.

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Figures

Figure 1
Figure 1
The MEF3 motif is crucial for myogenin promoter activation during embryogenesis. One mouse founder embryo carrying the nls-β-galactosidase reporter under the control of a wild-type myogenin −184 promoter (a) and one F1 transgenic mouse carrying the β-galactosidase reporter under the control of a mutated MEF3 myogenin −184 promoter (b) were sacrificed at E11.5. At E11.5 the control transgene (a) gives a β-galactosidase activity in the limbs and in the jaws muscles (arrow heads), which is not observed with the mutated promoter. The transgenic mouse harboring the −184mutMEF3 transgene (b) was one of those with a faint expression of the transgene in a small stripe of cells in the myotomes and an additional stripe of cells in the lateral part of the myotome between fore and hind limbs. The ectopic expression in the brain was not reproduced in other independent transgenic lines.
Figure 2
Figure 2
Six1 and Six4 are able to bind the myogenin and aldolase A MEF3 motifs. (a) GMSAs performed with recombinant full-size Six4 (from in vitro T7-synthetized Six4 mRNAs translated in a rabbit reticulocyte lysate) and Six1 proteins (recombinant proteins purified after production in bacteria, see Materials and Methods). Mock lysate (lanes 1 and 4), Six4 translation products (lanes 2 and 5), or Six1 recombinant proteins (lanes 3 and 6) were incubated with labeled MEF3 sites from aldolase A (pM) or myogenin genes. ∗ indicates a nonspecific lysate binding activity. (b) GMSAs were performed with aldolase A MEF3 site (pM) and with either skeletal muscle (sk.m) nuclear extracts (Upper) or recombinant Six4 protein [corresponding to amino acids 1–240 of the AREC protein encompassing the DNA-binding domain (dbd) defined in ref. 15]. In lanes 2–5, 30 ng of double-stranded MEF3 site mutated in different nucleotides was added as competitor (lane 1, no competitor). ∗ indicate the G residues whose methylation inhibits protein binding in a dimethyl sulfate (DMS) interference assay using muscle nuclear extracts (not shown). The mutations were in the bases defined by DMS interference on the MEF3 complex, and the mutated MEF3 sequence site is indicated with mutated bases in small letters. Note that mutation of the T (mut5) in the sequence TCAGG completely abolished the competition, thus showing that this nucleotide is absolutely required for the binding of Six protein to the MEF3 site.
Figure 3
Figure 3
Six1 and Six4 are the proteins that form the MEF3 muscle-specific binding activities. (a) GMSAs performed with adult nuclear extracts from different tissues on the MEF3 site of aldolase A. Five micrograms of nuclear extracts from heart, spleen, and skeletal muscles from the limb (sk.muscle) were incubated on ice with 0.3 ng of labeled double-stranded MEF3 site (pM). Six1 Ab or Six4 Ab were added subsequently thereafter, and the incubation mix then was kept on ice for 5 min. ∗ indicate nonspecific DNA-protein complexes (faintly competed by an excess of double-stranded MEF3 site). MEF3-binding activity comprises a ubiquitous complex (ubiq. MEF3) detected in each nuclear extract (including liver, not shown) and two muscle-specific complexes that did not form in the presence of anti-Six1 and anti-Six4 sera, respectively. Preimmune sera were not able to displace these complexes (not shown). In contrast, Abs against both Six1 and Six4 abolished the fast and slow migrating bands of the MEF3 DNA-protein complexes, respectively. (b) GMSAs performed with adult skeletal muscle (sk.muscle) nuclear extracts in the presence of the myogenin MEF3 site. Five micrograms of nuclear extracts were incubated on ice with 0.3 ng of labeled double-stranded myogenin MEF3 site. Increasing amounts (5, 15, or 50 ng) of myogenin MEF3 site was added in the reaction mix, as competitor. Abs against Six1 (Six1 Ab) or Six4 (Six4 Ab) were added subsequently, and the reaction mix then was kept on ice for 5 min. (c) GMSAs performed with protein extracts from embryonic trunks at E10.5 in the presence of the myogenin MEF3 site. Twenty micrograms of E10.5 protein extracts were incubated on ice with 0.3 ng of labeled double-stranded myogenin MEF3 site. Abs against Six1 (Six1 Ab) or Six4 (Six4 Ab) were added subsequently, and the reaction mix then was kept on ice for 5 min. ∗ indicates a nonspecific complex, which is not competed by an excess of MEF3 oligonucleotide and is not reproducibly displaced by Six1ab. ubiq., ubiquitous complex.
Figure 4
Figure 4
Expression of Six genes: analyses by Northern blot and Western blot. (a) One microgram of poly(A)+ mRNA from adult limb muscle (M) or 5 μg of poly(A)+ mRNA from adult liver (L) was detected by successive hybridization with specific cDNA probes complementary to Six1, Six4, or Six5. Size marker positions are indicated on the left in kb. Exposure times were for 6 hr (Six1), 24 hr (Six4 and Six5/L), and 5 days (Six5/M). (b) Western blot analyses were performed with 50 μg of nuclear extracts from adult liver (L) or adult skeletal muscle (M). Immunoreactive proteins were revealed with 1/100 dilution of purified antiserum against Six1 or 1/2,000 dilution of antiserum against Six4. The proteins detected with these antisera were not detected with preimmune antisera (not shown). The size of molecular mass standards is indicated in kDa.
Figure 5
Figure 5
Nuclear localization of Six1 and Six4 proteins. C2C12 cells (a and c, differentiated myotubes; b and d, proliferating myoblasts) were fixed on gelatin-coated slides with 4% formaldehyde at room temperature. Six1 and Six4 proteins were detected by 1:100 dilution of purified antiserum against Six1 (a and b) or 1:2,000 dilution of antiserum against Six4 (c and d). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) was applied at 1:200 dilution, and then the immunoperoxidase reaction was used to reveal cellular localization of Six proteins. No signal was observed with the corresponding preimmune sera (not shown).
Figure 6
Figure 6
Cis- and trans-activating properties of MEF3/Six complexes. (a) Activity of the herpes virus simplex tk minimum promoter with (6xMEF3-tk; filled bars) or without (tk-105; empty bars) six multimerized MEF3 motifs, in chicken primary myoblasts (MB) and myotubes after 4 days of differentiation (MT). (b) 6xMEF3-tk or tk-105 reporter constructs were transfected with an increasing amount of pCR3 expression vectors containing cDNA for Six1 or Six4 in C2C12 myoblasts. The amount of expression vector used is indicated in μg. Activity was measured 48 hr after transfection.
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