School of Medicine, Koc University, Istanbul, 34450, Turkey.
Department of Molecular Biology and Genetics, Koc University, Istanbul, 34450, Turkey.
Epigenetics Chromatin. 2021 Jul 2;14(1):32. doi: 10.1186/s13072-021-00406-7.
The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown.
We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes.
Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.
组蛋白 H3 赖氨酸 79（H3K79）甲基转移酶 DOT1L 是体细胞重编程的关键染色质障碍。然而，DOT1L 如何保护细胞身份和体细胞特异性转录程序的机制尚不清楚。
我们采用基于邻近标记的蛋白质组学方法来鉴定 DOT1L 相互作用蛋白，并研究它们对重编程的影响。在 DOT1L 相互作用蛋白中，通过 RNA 干扰或 CRISPR/Cas9 抑制 AF10（MLLT10）可显著提高重编程效率。在体细胞和诱导多能干细胞（iPSC）中，较高阶的 H3K79 甲基化依赖于 AF10 的表达。在 AF10 敲除细胞中，重新表达野生型 AF10，但不是 DOT1L 结合缺陷突变体，可恢复整体 H3K79 甲基化并降低重编程效率。重编程过程中的转录组分析表明，AF10 的抑制导致成纤维细胞特异性基因下调，并加速多能性相关基因的激活。
我们的研究结果确立了 AF10 通过调节 H3K79 甲基化成为重编程的新障碍，从而揭示了体细胞中细胞身份得以维持的机制。
