Human lysine demethylase KDM5A is a chromatin-modifying enzyme associated with transcriptional regulation, because of its ability to catalyze removal of methyl groups from methylated lysine 4 of histone H3 (H3K4me3). Amplification of KDM5A is observed in many cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, lung cancer, and gastric cancer. In this study, we employed alanine scanning mutagenesis to investigate substrate recognition of KDM5A and identify the H3 tail residues necessary for KDM5A-catalyzed demethylation. Our data show that the H3Q5 residue is critical for substrate recognition by KDM5A. Our data also reveal that the protein-protein interactions between KDM5A and the histone H3 tail extend beyond the amino acids proximal to the substrate mark. Specifically, demethylation activity assays show that deletion or mutation of residues at positions 14-18 on the H3 tail results in an 8-fold increase in the KMapp, compared to wild-type 18mer peptide, suggesting that this distal epitope is important in histone engagement. Finally, we demonstrate that post-translational modifications on this distal epitope can modulate KDM5A-dependent demethylation. Our findings provide insights into H3K4-specific recognition by KDM5A, as well as how chromatin context can regulate KDM5A activity and H3K4 methylation status.

中文翻译：

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组蛋白去甲基化酶KDM5A对组蛋白H3尾巴的扩展识别。

人赖氨酸脱甲基酶KDM5A是一种与转录调控相关的染色质修饰酶，因为它具有催化从组蛋白H3（H3K4me3）的甲基化赖氨酸4中去除甲基的能力。在许多癌症中观察到KDM5A的扩增，包括乳腺癌，前列腺癌，肝细胞癌，肺癌和胃癌。在这项研究中，我们采用了丙氨酸扫描诱变技术来研究KDM5A的底物识别，并确定KDM5A催化脱甲基所必需的H3尾部残基。我们的数据表明，H3Q5残基对于KDM5A识别底物至关重要。我们的数据还表明，KDM5A和组蛋白H3尾巴之间的蛋白质相互作用超出了底物标记附近的氨基酸范围。特别，脱甲基活性测定表明，与野生型18mer肽相比，H3尾部14-18位上残基的缺失或突变导致KMapp增加8倍，这表明该远端表位对组蛋白的结合很重要。最后，我们证明该远侧抗原决定簇的翻译后修饰可以调节KDM5A依赖性脱甲基。我们的发现提供了对KDM5A对H3K4特异性识别的了解，以及染色质如何调节KDM5A活性和H3K4甲基化状态。我们证明在此远端表位上的翻译后修饰可以调节KDM5A依赖性脱甲基。我们的发现提供了对KDM5A对H3K4特异性识别的了解，以及染色质如何调节KDM5A活性和H3K4甲基化状态。我们证明在此远端表位上的翻译后修饰可以调节KDM5A依赖性脱甲基。我们的发现提供了对KDM5A对H3K4特异性识别的了解，以及染色质如何调节KDM5A活性和H3K4甲基化状态。