MLL-AF9 lates transcriptional initiation in mixed lineage leukemic cells

MLL-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce mixed lineage leukemia (MLL) through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II (Pol II) in HEL, a human cell line without MLL1 arrangement (MLLr). MLL1 and AF9 only co-regulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and MLL-AF9 occupy promoters and distal intergenic regions, exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia (AML) cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely-believed elongation. CUT&Tag, PRO-Seq, and RNA-Seq

混合谱系白血病融合蛋白（MLL-fusion proteins，MLL-FPs）被认为通过异常刺激转录延伸以维持基因激活并诱发混合谱系白血病（mixed lineage leukemia，MLL），但其具体分子机制尚未完全阐明。本研究发现，在未发生MLL1重排（MLLr）的人类细胞系HEL中，MLL1及其主要融合伴侣之一AF9主要结合于启动子与远端基因间区域，二者的染色质结合模式与RNA聚合酶II（RNA polymerase II，Pol II）高度相似。尽管二者在数千个基因上存在共结合位点，但仅共同调控十余个基因；二者之间不存在相互作用，且各自的染色质结合也互不依赖。此外，HEL细胞中AF9缺失会降低全局TATA盒结合蛋白（TATA-box binding protein，TBP）的结合水平，仅在少量基因上减少细胞周期蛋白依赖性激酶9（cyclin-dependent kinase 9，CDK9）的结合，这提示AF9在CDK9招募中发挥辅助作用，并可能通过招募转录起始因子在转录起始过程中承担主要功能。值得注意的是，在MLL重排细胞中，MLL1与MLL-AF9的结合位点及染色质结合模式完全一致；在可诱导表达MLL-AF9的小鼠急性髓系白血病（acute myeloid leukemia，AML）细胞系iMA9中，MLL-AF9缺失会降低其核心靶基因上TBP与转录因子IIE（transcription factor IIE，TFIIE）的结合水平，表明MLL-AF9同样可调控转录起始。上述结果表明，与学界此前的认知相悖，MLL1与MLL-AF9在结合位点的位置与尺寸上并无差异；且MLL-AF9除了被广泛认为可调控转录延伸外，还可能参与转录起始调控。本研究采用的实验技术包括CUT&Tag、PRO-Seq与RNA-Seq。