Base editor scanning charts the DNMT3A activity landscape
Base editor scanning charts the DNMT3A activity landscape
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DOI：
10.1038/s41589-022-01167-4
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发表时间：
2022-10-20
影响因子：
14.8
通讯作者：
Liau, Brian B.
中科院分区：
文献类型：
--
作者：
Lue, Nicholas Z.;Garcia, Emma M.;Liau, Brian B.
DNA甲基化对于调节基因表达至关重要，需要DNA甲基转移酶DNMT3A等酶对其进行精确定位。这一过程的失调已知会导致异常发育和肿瘤发生，但DNMT3A如何通过其三个结构域进行整体调控仍然是研究的挑战。在这里，我们将碱基编辑与DNA甲基化报告结合起来，对细胞中的DNMT3A进行原位突变扫描。我们在整个蛋白质中发现了干扰功能的突变，包括在区域间界面上阻止变构激活的突变。出乎意料的是，我们还发现了PWWP结构域（一个组蛋白解读器）的突变，该突变在保持组蛋白识别和蛋白质稳定性的同时调节酶活性。这些影响源于PWWP结构域DNA亲和力的改变，我们发现这是细胞充分活性所必需的非规范功能。我们的研究结果强调了区域间串扰的机制，并展示了一种探索非必需染色质调节因子序列-活性关系的通用策略。
DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation ofthis process is known to cause aberrant development and oncogenesis, yet how DNMT3A is regulated holistically by its three domains remains challenging to study. Here, we integrate base editing with a DNA methylation reporter to perform in situ mutational scanning of DNMT3A in cells. We identify mutations throughout the protein that perturb function, including ones at an interdomain interface that block allosteric activation. Unexpectedly, we also find mutations in the PWWP domain, a histone reader, that modulate enzyme activity despite preserving histone recognition and protein stability. These effects arise from altered PWWP domain DNA affinity, which we show is a noncanonical function required for full activity in cells. Our findings highlight mechanisms of interdomain crosstalk and demonstrate a generalizable strategy to probe sequence-activity relationships of nonessential chromatin regulators.