The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 had shown that each DBD can bind a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed binding sites for both paired and homeo domains. The present work investigated the implication of both DBDs in pioneer action. We now show that the composite sequence is a higher affinity Pax7 binding site compared to either paired or homeo binding sites and that efficient binding to this site involves both DBDs. We also show that a Pax7 monomer binds composite sites and that methylation of cytosines within the binding site does not affect binding, which is consistent with pioneer action exerted at methylated DNA sites within nucleosomal heterochromatin. Finally, introduction of single amino acid mutations in either the paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.

中文翻译：

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Pax7先驱因子作用需要配对和同源DNA结合域

先锋转录因子Pax7包含两个DNA结合结构域（DBD），一对和一个同源结构域。关于Pax7和相关Pax3的先前工作表明，每个DBD都可以结合同源的DNA序列，从而定义了两个结合靶点，并且可能定义了作用方式。Pax7导致染色质开放的先驱者行动的基因组靶点富含复合DNA靶点，这些靶点包含成对和同源域的并列结合位点。目前的工作调查了两个DBD在先驱行动中的含义。现在我们显示，与配对或同源结合位点相比，该复合序列具有更高的亲和力Pax7结合位点，并且与该位点的有效结合涉及两个DBD。我们还表明，Pax7单体结合复合位点，并且在结合位点内的胞嘧啶甲基化不会影响结合，这与在核小体异染色质内的甲基化DNA位点施加的先驱作用一致。最后，在成对或同源结构域中引入单个氨基酸突变会削弱与同源DNA序列的结合，这表明两个DBD必须完整无缺，才能发挥先驱作用。相反，异染色质位点的低亲和力结合仅需要配对结构域。因此，Pax7在异染色质上的先驱作用需要独特的蛋白质：DNA相互作用，与其在可访问的增强子靶位点上更简单的DNA结合方式相比，该相互作用更为复杂。在成对或同源结构域中引入单个氨基酸突变会削弱与同源DNA序列的结合，这表明两个DBD必须完整无缺，才能发挥先驱作用。相反，异染色质位点的低亲和力结合仅需要配对结构域。因此，Pax7在异染色质上的先驱作用需要独特的蛋白质：DNA相互作用，与其在可访问的增强子靶位点上更简单的DNA结合方式相比，该相互作用更为复杂。在成对或同源结构域中引入单个氨基酸突变会削弱与同源DNA序列的结合，这表明两个DBD必须完整无缺，才能发挥先驱作用。相反，异染色质位点的低亲和力结合仅需要配对结构域。因此，Pax7在异染色质上的先驱作用需要独特的蛋白质：DNA相互作用，与其在可访问的增强子靶位点上更简单的DNA结合方式相比，该相互作用更为复杂。