DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation of this 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.

DNA 甲基化对于调节基因表达至关重要，需要通过 DNA 甲基转移酶 DNMT3A 等酶对其进行准确定位。众所周知，这一过程的失调会导致异常发育和肿瘤发生，但 DNMT3A 如何通过其三个域进行整体调控仍然具有挑战性。在这里，我们将碱基编辑与 DNA 甲基化报告基因相结合，对DNMT3A进行原位突变扫描在细胞中。我们确定了整个蛋白质中扰乱功能的突变，包括阻止变构激活的域间界面处的突变。出乎意料的是，我们还在组蛋白阅读器 PWWP 域中发现了突变，尽管保留了组蛋白识别和蛋白质稳定性，但它仍能调节酶活性。这些影响来自改变的 PWWP 域 DNA 亲和力，我们表明这是细胞中完全活动所需的非规范函数。我们的研究结果突出了域间串扰的机制，并展示了一种可推广的策略来探测非必需染色质调节剂的序列-活性关系。