DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix–loop–helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin–Cdk complexes results in reduction in protein–protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.
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机译：ETS结构域转录因子的三元复合因子（TCF）亚家族的DNA结合受到分子内和分子间相互作用的严格调控。含有螺旋-环-螺旋（HLH）的Id蛋白是TCF与DNA结合的反式负调控因子。在TCF，SAP-2 / Net / ERP中，顺式作用NID区（也包含HLH样基序）促进了DNA结合的分子内抑制。 NID还充当转录抑制域。在这里，我们研究了HLH基序在通过TCF蛋白SAP-1调节DNA结合和转录中的作用，以及Cdk介导的磷酸化如何影响Id蛋白对TCF的抑制活性。我们证明，SAP-1的NID区是一种自抑制基序，其作用是抑制DNA结合并还起转录抑制域的作用。该区域可以通过将Id蛋白与SAP-1融合而在功能上被替换，从而使Id部分起到抑制顺式结合DNA的作用。细胞周期蛋白-Cdk复合物对Ids的磷酸化作用会导致Ids和TCF之间的蛋白质-蛋白质相互作用减少，并减轻其DNA结合抑制活性。在揭示HLH基元调节TCF活性的独特机制中，我们的结果因此提供了对HLH基元在调节TCF功能中的作用以及反式Id HLH蛋白自身的抑制特性如何通过磷酸化调节的进一步了解。
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