Department of Cell Biology & Plant Biochemistry, Centre for Biochemistry, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany.
Institute for Biochemistry I, Centre for Biochemistry, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany.
Nucleic Acids Res. 2022 May 20;50(9):5014-5028. doi: 10.1093/nar/gkac293.
The heterodimeric histone chaperone FACT, consisting of SSRP1 and SPT16, contributes to dynamic nucleosome rearrangements during various DNA-dependent processes including transcription. In search of post-translational modifications that may regulate the activity of FACT, SSRP1 and SPT16 were isolated from Arabidopsis cells and analysed by mass spectrometry. Four acetylated lysine residues could be mapped within the basic C-terminal region of SSRP1, while three phosphorylated serine/threonine residues were identified in the acidic C-terminal region of SPT16. Mutational analysis of the SSRP1 acetylation sites revealed only mild effects. However, phosphorylation of SPT16 that is catalysed by protein kinase CK2, modulates histone interactions. A non-phosphorylatable version of SPT16 displayed reduced histone binding and proved inactive in complementing the growth and developmental phenotypes of spt16 mutant plants. In plants expressing the non-phosphorylatable SPT16 version we detected at a subset of genes enrichment of histone H3 directly upstream of RNA polymerase II transcriptional start sites (TSSs) in a region that usually is nucleosome-depleted. This suggests that some genes require phosphorylation of the SPT16 acidic region for establishing the correct nucleosome occupancy at the TSS of active genes.
由 SSRP1 和 SPT16 组成的异二聚体组蛋白伴侣 FACT 有助于各种依赖于 DNA 的过程中的动态核小体重排，包括转录。为了寻找可能调节 FACT 活性的翻译后修饰，从拟南芥细胞中分离出 SSRP1 和 SPT16 ，并通过质谱进行分析。在 SSRP1 的碱性 C 末端区域内可以映射到四个乙酰化赖氨酸残基，而在 SPT16 的酸性 C 末端区域中鉴定到三个磷酸化丝氨酸/苏氨酸残基。对 SSRP1 乙酰化位点的突变分析显示只有轻微的影响。然而，由蛋白激酶 CK2 催化的 SPT16 的磷酸化调节了组蛋白相互作用。非磷酸化的 SPT16 版本显示出降低的组蛋白结合能力，并在补充 spt16 突变体植物的生长和发育表型方面证明是无效的。在表达非磷酸化 SPT16 版本的植物中，我们在 RNA 聚合酶 II 转录起始位点（TSS）上游的一组基因中检测到组蛋白 H3 的富集，该区域通常缺乏核小体。这表明，对于在活性基因的 TSS 处建立正确的核小体占据，一些基因需要 SPT16 酸性区域的磷酸化。
