Histone variants are distinguished by specific substitutions and motifs that might be subject to post-translational modifications (PTMs). Compared with the high conservation of H3 variants, the N- and C-terminal tails of H2A variants are more divergent and are potential substrates for a more complex array of PTMs, which have remained largely unexplored. We used mass spectrometry to inventory the PTMs of the two heterochromatin-enriched variants H2A.W.6 and H2A.W.7 of Arabidopsis, which harbor the C-terminal motif KSPK. This motif is also found in macroH2A variants in animals and confers specific properties to the nucleosome. We showed that H2A.W.6 is phosphorylated by the cell cycle-dependent kinase CDKA specifically at KSPK. In contrast, this modification is absent on H2A.W.7, which also harbors the SQ motif associated with the variant H2A.X. Phosphorylation of the SQ motif is critical for the DNA damage response but is suppressed in H2A.W.7 by phosphorylation of KSPK. To identify factors involved in this suppression mechanism, we performed a synthetic screen in fission yeast expressing a mimic of the Arabidopsis H2A.W.7. Among those factors was the BRCT-domain protein Mdb1. We showed that phosphorylation of KSPK prevents binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and as a result hampers response to DNA damage. Hence, cross-talks between motif-specific PTMs interfere with the vital functions of H2A variants. Such interference could be responsible for the mutual exclusion of specific motifs between distinct H2A variants. We conclude that sequence innovations in H2A variants have potentiated the acquisition of many specific PTMs with still unknown functions. These add a layer of complexity to the nucleosome properties and their impact in chromatin regulation.

中文翻译：

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H2A变体特异性修饰之间的串扰影响重要的细胞功能

组蛋白变体的特征在于可能发生翻译后修饰（PTM）的特定取代和基序。与高度保守的H3变体相比，H2A变体的N和C末端尾部更加分散，并且是更复杂的PTM阵列的潜在底物，而这些PTM很大程度上仍未开发。我们使用质谱法对拟南芥的两个富含异染色质的变体H2A.W.6和H2A.W.7的PTM进行了清单，它们包含C端基序KSPK。该基序也存在于动物的macroH2A变体中，并赋予了核小体特定的特性。我们表明，H2A.W.6被细胞周期依赖性激酶CDKA磷酸化，特别是在KSPK处。相比之下，H2A.W.7缺少此修饰，它也包含与变体H2A.X相关的SQ主题。SQ基序的磷酸化对于DNA损伤反应至关重要，但在H2A.W.7中却被KSPK磷酸化抑制。为了确定参与这种抑制机制的因素，我们在裂殖酵母中进行了拟南芥H2A.W.7模拟的合成筛选。这些因素中有BRCT结构域蛋白Mdb1。我们表明，KSPK的磷酸化可防止BRCT域蛋白Mdb1与磷酸化的SQ结合，结果阻碍了对DNA损伤的反应。因此，基序特异的PTM之间的串扰会干扰H2A变体的重要功能。这种干扰可能是导致不同的H2A变体之间特定基序相互排斥的原因。我们得出结论，H2A变体中的序列创新增强了许多功能未知的特定PTM的获得。