Introduction Ms1 (also known as STARS and ABRA) has been shown to act as an early stress response gene in processes as different as hypertrophy in skeletal and cardiac muscle and growth of collateral blood vessels. It is important for cardiac development in zebrafish and is upregulated in mouse models for cardiac hypertrophy as well as in human failing hearts. Ms1 possesses actin binding sites at its C-terminus and is usually found in the cell bound to actin filaments in the cytosol or in sarcomeres. It is assumed that it activates SRF dependent gene expression in an indirect manner by shifting the equilibrium between cytosolic actin from G to F. This releases MRTFs which can then translocate to the nucleus to bind to SRF.
Methods We used NMR spectroscopy to determine the structure of the only folded domain of Ms1 and characterised its binding to F-actin and DNA using a combination of biochemical and biophysical assays. To identify a specific DNA sequence we used the SELEX approach in vitro in combination with EMSA assays. This was followed up by monitoring Ms1 subcellular localisation in neonatal rat cardiomyocytes using detection of endogenous protein with a new antibody or overexpression of wild type and mutated proteins.
Results We determined the NMR structure of the only folded domain of Ms1 comprising the second actin binding site called actin binding domain 2 (ABD2, residues 294–375), and found that it is similar to the winged helix-turn-helix fold adopted mainly by DNA binding domains of transcriptional factors. In vitro experiments show specific binding of this domain, in combination with a newly discovered AT-hook motif located N-terminally, to the sequence (A/C/G)AAA(C/A). NMR and fluorescence titration experiments confirm that this motif is indeed bound specifically by the recognition helix. In neonatal rat cardiomyocytes endogenous Ms1 is found in the nucleus in a spotted pattern, reminiscent of PML bodies. In adult rat cardiomyocytes Ms1 is exclusively found in the sarcomere. A nuclear localisation site (NLS) in the N-terminus of the protein is required for nuclear localisation as shown by the abolition of nuclear translocation of over expressed protein in which the NLS was mutated. A phosphorylation site immediately N-terminal of this NLS could provide regulatory control of nuclear transport.
Conclusions Our data suggest that Ms1 has the potential to act directly in the nucleus through specific interaction with DNA in development and potentially as a response to stress in adult tissues. Ms1 could act in parallel to the established MRTF/SRF pathway to regulate the expression of hypertrophic genes directly which would explain recent findings from studies in C1C12 cells where inhibition of SRF signalling did not lead to a change in myotube differentiation rate
- DNA binding
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