Wong MW et al. (1994), Activation of Xenopus MyoD transcription by mem...

XB-ART-20492

Dev Biol 1994 Dec 01;1662:683-95. doi: 10.1006/dbio.1994.1347.

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Activation of Xenopus MyoD transcription by members of the MEF2 protein family.

Wong MW , Pisegna M , Lu MF , Leibham D , Perry M .

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Members of the MEF2 family of DNA binding proteins interact with a set of AT-rich sequences commonly found in the promoters and enhancers of muscle-specific genes. We have shown that a MEF2 binding site precisely overlaps the TFIID binding site (TATA box) in the Xenopus MyoDa (XMyoDa) promoter and appears to play an important role in muscle-specific activity of this promoter. To further investigate the potential role of MEF2 in the regulation of XMyoDa transcription, we have analyzed the appearance of factors that interact with the XMyoDa TATA/MEF2 site during early amphibian development. Proteins that bind specifically to this site were present at low levels during early development and increased in abundance during gastrulation and neurulation. Two related cDNAs were isolated that encode proteins that recognize the XMyoDa TATA motif. Both proteins are highly homologous to each other, belong to the MADS (MCM1 agamous deficiens SRF) protein family, and are most highly related to the mammalian MEF2A gene products. Xenopus MEF2A (XMEF2A) transcripts accumulated preferentially in forming somites after the appearance of XMyoD transcripts. Ectopic expression of XMEF2A and other members of the MEF2 gene family activated transcription of a reporter gene controlled by the XMyoDa promoter. Transcriptional activation of the XMyoDa promoter required only the conserved DNA binding domain of XMEF2A and was independent of a domain necessary for activity when this factor was bound to multiple upstream sites. These results suggest that the XMyoDa promoter can be activated by binding of MEF2 to the XMyoDa TATA motif and indicate that MEF2-dependent transcriptional activation occurs by different mechanisms depending on the location of the MEF2 binding site. We suggest that XMEF2 expression in myogenic cells contributes to the activation and stabilization of XMyoDa transcription during muscle cell differentiation.

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Species referenced: Xenopus laevis
Genes referenced: cat.2 fos lgals4.2 mef2a mef2d myod1 srf taf6 tbp

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	FIG. 1. (A) Appearance of MEF2 binding activity during Xe1w1ru~ embryonic development. A probe containing the TATA box motif (TGCTATAAATAGAG) f1Â·om the XMyoDa promoter was used in an electrophoretic mobility shift assay. Extracts were made from oocytes and embryos at the indicated stages. The complex formed from sequence- specific binding to the probe is indicated by the arrow. (B) The binding specificity of the complex formed with extracts from embryos at stage 18 tested using a 100-fold excess of oligonucleotide competitors that distinguish between the binding of TBP and MEF2. (C) Sequences of the oligonucleotide competitors. An oligonucleotide (OCTA) containing a binding site for Oct-1 and related POU proteins was used to test the DNA binding specificity of the observed compiex.
	FIG. 2. Amino acid sequence and structure of Xenop-u,11, MEF2 proteins. (Top) Alignment of the predicted amino acid sequence of XMEF2A1- lab2 (XMEF2Al), XMEF2A2-1a (XMEF2A2), and SL-1. The complete sequence of XMEF2Al is shown on the top line. Amii1o acid differences in XMEF2A2 and SL-1, relative to XMEF2Al, are shown on the second and third lines, respectively. Gaps (.) were introduced to optimize the alignment. The MADS domain and the MEF2-spccific region are indicated. (Bottom) The structure of the XMEF2A proteins. MADS domain (black box); MEF2 conserved region {shaded box); alternatively spliced exons (hatched boxes).
	FIG. 3. (A) XMEF2Al and XMEF2A2 bind to the XMyoDa TAT A box. Synthetic transcripts from each eDNA were injected into frog oocytes. Extracts from injected oocytes were used in a band shift assay with the oligonucleotide probe containing theXMyoDa TATA box. The mobility of the complexes formed was compared with that obtained using an extract from embryos at stage 18 (stage 18). (B) The DNA binding specificity of XMEF2Al tested using oligonucleotide competitors as described in Fig. 1.
	FIG. 4. Accumulation of XMEF2A transcripts in developing embryos. XMEF2A gene expression was examined by whole-mount in situ hybridization using a digoxygenin-labeled riboprobe. All embryos are oriented with their anterior end to the left. (A) Dorsal view of a stage 14 embryo. XMEF2A transcripts are observed in the paraxial mesoderm but not in the notochord. (B) Lateral view of a stage 22 embryo. (C) Dorsal view of a stage 22 embryo. XMEF2A expression in Band Cis observed in differentiating somites at the anterior end and the more posterior presomitic mesoderm. (D) The two stage 32 embryos were hybridized with an antisense riboprobe. The embryo on the bottom was hybridized with the corresponding sense strand riboprobe and is oriented with its head to the right.
	FJG. 5. The MADS and MEF2 domains of XMEF2A are sufficient for DNA binding and transcriptional activation of the XMyoDa promoter. (A) Proteins encoded hy truncated forms of XMEF2Al were synthesized Â·in vitro using [85SJmethionine and displayed by SDS-polyacry1amide gel electrophoresis. (B) The binding of truncated XMEF2Al proteins to the XMyoDa TATA box assayed using an electrophoretic mobility shift assay. Specific complexes formed by binding of the truncated proteins to the oligonucleotide probe are indicated with arrows. (C) The ability of XMEF2Al proteins to activate reporter gene transcription was analyzed in COSl cells using promoters that contain single or multiph~ copies of a MEF2 binding site. The XMyoDa-CAT reporter plasmid contained sequences from -272 to +73 of the XMyoDa promoter and a single MEF2 binding site at the TATA box. The 4XMEF2-CAT plasmid contained four copies of the MEF2 binding site from the murine MCK enhancer upstream of the minimal XM11oDa promoter containing a mutation that allows binding of TFIID but not MEF2 (D+/M-) to the TATA box. Reporter gene expression was analyzed by CAT assay and normalized to the expression observed in lhe absence of XMEF2Al expression. The deduced transcriptional activation domain (TAD) is depicted by a hatched box.
	FIG. 6. Identification of a transcriptional activation domain using GAL4-XMEF2A 1 gene fusions. The full-length XMEF2Al and a series ofNterminal and C-terminal deletions were each fused in frame with the GAL4 DNA binding domain. Chimeric proteins were tested in COSl cells for their ability to activate transcription of a minimal c-fos promoter containing four copies of a GAL4 binding site. Reporter gene expression was normalized to expression in the absence of transactivator. Identical resu1ts were obtained using proliferating and differentiated muscle cells.
	FIG. 7. Transcriptional activation of the XMyoDa promoter by MEF2C and MEF2D. (A) The binding of MEF2 proteins to the XMy~ oDa TATA box was examined using in vitro translated proteins and the mobility shift assay. (B) The ability of MEF2 proteins to activate expression of the 4XMEF2-CAT and XMyoDa-CAT reporter genes was tested in COSt cells as described in Fig. 5. Reported values are norÂ· malized to reporter gene expression in the absence of a cotransfected transactivator.