Dominant Negative Murine Serum Response Factor: Alternative Splicing within the Activation Domain Inhibits Transactivation of Serum Response Factor Binding Targets

Abstract

Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRF⌬5 to SRF. Increased levels of SRF⌬5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRF⌬5 variant relative to wild-type SRF. SRF⌬5 forms DNA binding-competent homodimers and heterodimers. SRF⌬5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal ␣-actin, cardiac ␣-actin, smooth ␣-actin, SM22␣, and SRF promoter-luciferase reporter activities. Expression of SRF⌬5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal ␣-actin and myogenin mRNAs. SRF⌬5 repressed the serum-induced activity of the c-fos serum response element. SRF⌬5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRF⌬5, may play an important regulatory role in modulating SRF-dependent gene expression.Alternative splicing is a commonly used molecular strategy for creating diverse gene products from a single genetic locus in most eucaryotic cells. The modular organization of transcription factor genes, through exon-encoded structural domains, may be conducive for forming a variety of alternatively spliced isoforms that affect DNA binding avidity and specificity, transactivation, subcellular localization, responsiveness to signaling pathways, and developmental regulation (reviewed in reference 31). For example, alternative splicing within the DNA binding domain of Pax-6 (9) and Wilm's tumor-associated protein 1 (5, 21) alters their DNA binding specificities. Alternative splicing of exons encoding the transactivating domains in the paired family proteins Pax-3 (59), , and Pax-9 (39), the POU homeodomain family proteins Pit-1 (37), Oct-1 (8), Oct-2 (1), and Brn-3a (38), and the zinc finger transcription factor GATA-5 (32) results in isoforms possessing activation domains with different potencies. Splicing out of select exons from the activation domain as in AML1a (56), Oct-2 (29), and CREB family proteins CREB (reviewed in reference 11), Drosophila CREB/CREM (61), and CREM (12) produces isoforms with dominant negative activity. Similarly, several splice variants containing only the DNA binding domain act as dominant negative isoforms presumably by heterodimerizing w...

Regulation and characteristics of vascular smooth muscle cell phenotypic diversity

“…Relevant to this review is that concentration gradients and alternatively spliced isoforms of SRF have specific effects on SMC gene transcription and consequently may contribute to SMC diversity. 22 In addition, myocardin has a heterogeneous expression pattern in SMCs of different tissues, 23 which may contribute to variations in marker gene expression and consequently SMC function. Whereas the transcription factors that regulate SMC phenotype are more and more defined, the list of factors that can activate or inhibit them continues to expand.…”

The Smooth Muscle γ-Actin Gene Promoter Is a Molecular Target for the Mouse bagpipe Homologue, mNkx3-1, and Serum Response Factor

“…The MADS box domain of SRF has been demonstrated to convey gene regulatory activity through associations with other factors, as well as being the DNA binding domain of the molecule (42,43,55,58). The SRF pm1 molecule contains three amino acid substitutions (Arg to Leu at position 143, Lys to Ala at position 145, and Leu to Gly at position 146) that interrupt DNA binding but do not alter the ability of the protein to dimerize (59). Cotransfection of the SRF pm1 mutant blocked SRF-mediated transcriptional activation of ␣-skeletal and ␣-cardiac actin promoters (43,46).…”

Calcium/Calmodulin-dependent Protein Kinase Activates Serum Response Factor Transcription Activity by Its Dissociation from Histone Deacetylase, HDAC4

“…Several mechanisms have been shown to regulate the SRF transcription activity, including physical interaction of SRF with a number of positive and negative cofactors (6 -10), phosphorylation-dependent change in the DNA and/or protein binding ability of SRF (18), regulated nuclear translocation of SRF (19), and alternative splicing of SRF mRNA primary transcript (20,21). Recently, we have shown that an alternative spliced isoform of SRF is highly expressed in the failing hearts of both humans and animals, which act as a dominant negative isoform to repress SRF-dependent cardiac muscle gene expression (22).…”