During neural development, members of MTG family of transcriptional repressors are induced by proneural basic helix-loop-helix (bHLH) transcription factors and in turn inhibit the activity of the bHLH proteins, forming a negative feedback loop that regulates the normal progression of neurogenesis. Three MTG genes, MTG8, MTG16 and MTGR1, are expressed in distinct patterns in the developing nervous system. Various bHLH proteins are also expressed in distinct patterns. We asked whether there is a functional relationship between specific MTG and bHLH proteins in developing chick spinal cord. First, we examined if each MTG gene is induced by specific bHLH proteins. Although expression of NEUROG2, ASCL1 and MTG genes overlapped, the boundaries of gene expression did not match. Ectopic expression analysis showed that MTGR1 and NEUROD4, which show similar expression patterns, are regulated differently by NEUROG2 and ASCL1. Thus, our results show that expression of MTG genes is not regulated by a single upstream bHLH protein, but represents an integration of the activity of multiple regulators. Next, we asked if each MTG protein inhibits specific bHLH proteins. Transcription assay showed that NEUROG2 and ASCL1 are inhibited by MTGR1 and MTG16, and less efficiently by MTG8. Deletion mapping of MTGR1 showed that MTGR1 binds NEUROG2 and ASCL1 using multiple interaction surfaces, and all conserved domains are required for its repressor activity. These results support the model that MTG proteins form a higher-order repressor complex and modulate transcriptional activity of bHLH proteins during neurogenesis.
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