Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA.
EMBO J. 2012 Aug 15;31(16):3494-505. doi: 10.1038/emboj.2012.188. Epub 2012 Jul 13.
Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.
Paf1 复合物（Paf1C）是一种转录延伸因子，其募集受 Spt5 和 CDK 激酶 Kin28 和 Bur1 的刺激，后者在丝氨酸 2、5 和 7 上磷酸化 Pol II C 端结构域（CTD）。Bur1 通过磷酸化 Spt5 中的 C 端重复序列（CTRs）促进 Paf1C 的募集，我们发现 Kin28 通过促进 Bur1 的募集来增强 Spt5 的磷酸化。然而，不清楚 Kin28 或 Bur1 对 CTD 的磷酸化是否也能刺激 Paf1C 的募集。我们发现 Paf1C 及其 Cdc73 亚基在体外与双磷酸化 CTD 重复序列（pCTD）和磷酸化 Spt5 CTRs（pCTRs）结合，并且消除这两种活性的 cdc73 突变会减少体内 Paf1C 的募集。Spt5 CTR 中的磷酸模拟（酸性）取代在其他野生型细胞中维持高水平的 Paf1C 募集，但在 Bur1 或 Kin28 失活时则不然。此外，在 Cdc73 中失活 pCTD/pCTR 相互作用域（PCID）会降低含有不可磷酸化 Spt5 CTR 的细胞中 Paf1C 依赖性组蛋白甲基化。这些结果确定了一种 Spt5 pCTR 独立的 Paf1C 募集途径，需要 Kin28、Bur1 和 Cdc73 PCID。我们提出 pCTD 重复序列和 Spt5 pCTRs 提供了独立的相互作用表面，共同确保高水平的 Paf1C 募集。
