Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
Biosci Rep. 2020 Feb 28;40(2). doi: 10.1042/BSR20191958.
During gene expression, histone acetylation by histone acetyltransferase (HAT) loosens the chromatin structure around the promoter to allow RNA polymerase II (Pol II) to initiate transcription, while de-acetylation by histone deacetylase (HDAC) tightens the structure in the transcribing region to repress false initiation. Histone acetylation is also regulated by intracellular pH (pHi) with global hypoacetylation observed at low pHi, and hyperacetylation, causing proliferation, observed at high pHi. However, the mechanism underlying the pHi-dependent regulation of gene expression remains elusive. Here, we have explored the role of the chromodomain (CD) of budding yeast Eaf3, a common subunit of both HAT and HDAC that is thought to recognize methylated lysine residues on histone H3. We found that Eaf3 CD interacts with histone H3 peptides methylated at Lys4 (H3K4me, a promoter epigenetic marker) and Lys36 (H3K36me, a coding region epigenetic marker), as well as with many dimethyl-lysine peptides and even arginine-asymmetrically dimethylated peptides, but not with unmethylated, phosphorylated or acetylated peptides. The Eaf3 CD structure revealed an unexpected histidine residue in the aromatic cage essential for binding H3K4me and H3K36me. pH titration experiments showed that protonation of the histidine residue around physiological pH controls the charge state of the aromatic cage to regulate binding to H3K4me and H3K36me. Histidine substitution and NMR experiments confirmed the correlation of histidine pKa with binding affinity. Collectively, our findings suggest that Eaf3 CD functions as a pHi sensor and a regulator of gene expression via its pHi-dependent interaction with methylated nucleosomes.
在基因表达过程中，组蛋白乙酰转移酶（HAT）通过组蛋白乙酰化作用使启动子周围的染色质结构变松，从而允许 RNA 聚合酶 II（Pol II）起始转录，而组蛋白去乙酰化酶（HDAC）通过组蛋白去乙酰化作用使转录区域的结构变紧，从而抑制错误起始。组蛋白乙酰化还受到细胞内 pH 值（pHi）的调节，在低 pHi 时观察到整体低乙酰化，在高 pHi 时观察到导致增殖的高乙酰化。然而，pHi 依赖性基因表达调控的机制仍不清楚。在这里，我们研究了芽殖酵母 Eaf3 的染色质结构域（CD）的作用，Eaf3 是 HAT 和 HDAC 的常见亚基，被认为可以识别组蛋白 H3 上的甲基化赖氨酸残基。我们发现 Eaf3 CD 与组蛋白 H3 肽的赖氨酸 4 位（H3K4me，启动子表观遗传标记）和赖氨酸 36 位（H3K36me，编码区表观遗传标记）发生相互作用，以及与许多二甲基赖氨酸肽甚至精氨酸不对称二甲基化肽相互作用，但与未甲基化、磷酸化或乙酰化肽不相互作用。Eaf3 CD 结构揭示了一个意想不到的组氨酸残基位于芳香盒中，对于结合 H3K4me 和 H3K36me 至关重要。pH 滴定实验表明，组氨酸残基在生理 pH 值附近的质子化控制芳香盒的电荷状态，从而调节与 H3K4me 和 H3K36me 的结合。组氨酸取代和 NMR 实验证实了组氨酸 pKa 与结合亲和力的相关性。总之，我们的研究结果表明，Eaf3 CD 通过其与甲基化核小体的 pHi 依赖性相互作用，作为 pH 值传感器和基因表达的调节剂发挥作用。
