G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1^−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.

中文翻译：

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蛋白质-DNA相互作用的邻近连接测定检测—血红素加氧酶-1与G-四链体之间是否存在联系？

G-四链体（G4）是由血红素稳定的堆叠核酸结构。在细胞中，它们影响DNA复制和基因转录。它们被几种解旋酶解开，但是修复复合物的组成及其血红素敏感性尚不清楚。我们发现，G-四链体的积累受血红素加氧酶-1（Hmox1）表达的影响，但以细胞类型特异性的方式：Hmox1 ^-/-小鼠的造血干细胞（HSC）上调了G4的表达解旋酶（例如Brip1，Pif1），并且对G-四链体的染色较弱，而Hmox1尽管解旋酶上调，但鼠缺陷型多能干细胞（iPSC）却具有更多的G-四链体，尤其是在暴露于外源血红素后。使用仅表达Hmox1的核或仅细胞质形式的iPSC，我们发现核定位可促进G4的去除。我们证明了邻近连接测定法（PLA）可以检测G-四链体与解旋酶以及HMOX1的细胞共定位，表明HMOX1在G4修饰中的潜在作用。但是，这种共定位并不意味着可以使用免疫沉淀测定法检测到直接相互作用。因此，我们得出的结论是，HMOX1影响G4的积累，而不是作为调节血红素可用性的蛋白质之一，而不是作为限速因子。