Sheng Yushou Center of Cell Biology and Immunology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065.
Proc Natl Acad Sci U S A. 2021 Jul 6;118(27). doi: 10.1073/pnas.2106148118.
DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17, and ENL or AF9, is dysregulated in most cases of mixed-lineage leukemia (MLLr), and has been believed to regulate transcriptional elongation on the basis of its colocalization with RNA polymerase II (Pol II), the sharing of subunits (AF9 and ENL) between the DOT1L and super elongation complexes, and the distribution of H3K79 methylation on both promoters and transcribed regions of active genes. Here we show that DOT1L depletion in erythroleukemic cells reduces its global occupancy without affecting the traveling ratio or the elongation rate (assessed by 4sUDRB-seq) of Pol II, suggesting that DOT1L does not play a major role in elongation in these cells. In contrast, analyses of transcription initiation factor binding reveal that DOT1L and ENL depletions each result in reduced TATA binding protein (TBP) occupancies on thousands of genes. More importantly, DOT1L and ENL depletions concomitantly reduce TBP and Pol II occupancies on a significant fraction of direct (DOT1L-bound) target genes, indicating a role for the DOT1L complex in transcription initiation. Mechanistically, proteomic and biochemical studies suggest that the DOT1L complex may regulate transcriptional initiation by facilitating the recruitment or stabilization of transcription factor IID, likely in a monoubiquitinated H2B (H2Bub1)-enhanced manner. Additional studies show that DOT1L enhances H2Bub1 levels by limiting recruitment of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. These results advance our understanding of roles of the DOT1L complex in transcriptional regulation and have important implications for MLLr leukemias.
DOT1L 是人类细胞中唯一的 H3K79 甲基转移酶，也是酵母 Dot1 的同源物，通常与 AF10、AF17 和 ENL 或 AF9 形成复合物，在大多数混合谱系白血病（MLLr）病例中失调，并且据信基于其与 RNA 聚合酶 II（Pol II）的共定位、DOT1L 和超级延伸复合物之间的亚基（AF9 和 ENL）共享以及 H3K79 甲基化在启动子和活跃基因的转录区域的分布来调节转录延伸。在这里，我们表明在红细胞白血病细胞中耗尽 DOT1L 会降低其整体占有率，而不会影响 Pol II 的行进比或延伸率（通过 4sUDRB-seq 评估），这表明 DOT1L 在这些细胞的延伸中不起主要作用。相比之下，对转录起始因子结合的分析表明，DOT1L 和 ENL 的耗竭都会导致数千个基因的 TATA 结合蛋白（TBP）占有率降低。更重要的是，DOT1L 和 ENL 的耗竭都会同时降低直接（DOT1L 结合）靶基因的 TBP 和 Pol II 占有率，这表明 DOT1L 复合物在转录起始中起作用。从机制上讲，蛋白质组学和生化研究表明，DOT1L 复合物可能通过促进转录因子 IIID 的募集或稳定来调节转录起始，可能以单泛素化 H2B（H2Bub1）增强的方式。进一步的研究表明，DOT1L 通过限制 Spt-Ada-Gcn5-乙酰转移酶（SAGA）复合物的募集来增强 H2Bub1 水平。这些结果提高了我们对 DOT1L 复合物在转录调控中的作用的理解，并对 MLLr 白血病具有重要意义。
