Approximately 1% of the human genome has the ability to fold into G-quadruplexes (G4s)—noncanonical strand-specific DNA structures forming at G-rich motifs. G4s regulate several key cellular processes (e.g., transcription) and have been hypothesized to participate in others (e.g., firing of replication origins). Moreover, G4s differ in their thermostability, and this may affect their function. Yet, G4s may also hinder replication, transcription, and translation and may increase genome instability and mutation rates. Therefore, depending on their genomic location, thermostability, and functionality, G4 loci might evolve under different selective pressures, which has never been investigated. Here we conducted the first genome-wide analysis of G4 distribution, thermostability, and selection. We found an overrepresentation, high thermostability, and purifying selection for G4s within genic components in which they are expected to be functional—promoters, CpG islands, and 5′ and 3′ UTRs. A similar pattern was observed for G4s within replication origins, enhancers, eQTLs, and TAD boundary regions, strongly suggesting their functionality. In contrast, G4s on the nontranscribed strand of exons were underrepresented, were unstable, and evolved neutrally. In general, G4s on the nontranscribed strand of genic components had lower density and were less stable than those on the transcribed strand, suggesting that the former are avoided at the RNA level. Across the genome, purifying selection was stronger at stable G4s. Our results suggest that purifying selection preserves the sequences of functional G4s, whereas nonfunctional G4s are too costly to be tolerated in the genome. Thus, G4s are emerging as fundamental, functional genomic elements.

中文翻译：

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选择和热稳定性表明 G-四链体是人类基因组的新功能元件

大约 1% 的人类基因组能够折叠成 G 四链体 (G4)，即在富含 G 的基序上形成的非规范链特异性 DNA 结构。G4 调节几个关键的细胞过程（例如转录），并被假设参与其他过程（例如复制起点的激发）。此外，G4 的热稳定性不同，这可能会影响它们的功能。然而，G4 也可能阻碍复制、转录和翻译，并可能增加基因组不稳定性和突变率。因此，根据其基因组位置、热稳定性和功能，G4 位点可能在不同的选择压力下进化，这一点从未被研究过。在这里，我们对 G4 分布、热稳定性和选择进行了首次全基因组分析。我们发现 G4 在基因组件（启动子、CpG 岛以及 5' 和 3' UTR）中具有高代表性、高热稳定性和纯化选择，预计它们在其中发挥功能。在复制起点、增强子、eQTL 和 TAD 边界区域内的 G4 中观察到类似的模式，强烈表明它们的功能。相比之下，外显子非转录链上的 G4 代表性不足，不稳定且中性进化。一般来说，基因成分的非转录链上的 G4 比转录链上的 G4 具有较低的密度和稳定性，这表明在 RNA 水平上应避免前者。在整个基因组中，稳定 G4 的纯化选择更强。我们的结果表明，纯化选择保留了功能性 G4 的序列，而非功能性 G4 的成本太高，无法在基因组中耐受。因此，G4 正在成为基本的功能基因组元件。