Genome-wide maps of NUP98 fusion and MLL1 co-bound targets and their chromatin state in NUP98 fusion transformed cells.. Mus musculus

By performing biotin-mediated chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) for two different NUP98 fusions, we defined the genome-wide direct binding sites of NUP98-HOXA9 or NUP98-HOXD13. To test whether NUP98 fusions and Mll1 were recruited to the same region of Hox genes promoters, Mll1 ChIP-seq analysis was carried out in murine NUP98-HOXA9 transformed cells using an anti-Mll1n antibody. In agreement with our results showing that NUP98-HOXA9 interacts with MLL1 in NSL/MLL1 complex, Mll1 binding targets significantly overlap with that of NUP98-HOXA9 at promoter region and gene body region. Given that MOF and MLL1 work in concert to modify H4K16ac and H3K4me3 at promoters, we further test whether H3K4me3 and H4K16ac marks are associated with NUP98-HOXA9-bound targets in murine NUP98-HOXA9 transformed cells by anti-H3K4me3 and anti-H4K16ac ChIP-seq. Our data show that both H3K4me3 and H4K16ac mark presents on NUP98-HOXA9 binding targets at promoters, and this is in line with the coordination role in the activities between MOF-mediated H4K16 acetylation and MLL/SET-mediated H3K4 methylation. In summary, our results confirmed that NUP98-HOXA9 and Mll1 are recruited to the same Hox loci, and this recruitment is associated with activating epigenetic marks, supporting the notion of the association between NUP98 fusions and NSL/Mll1 complex, suggesting that the recruitment of NUP98 fusions to the Hox gene loci maybe through MLL1. Overall design: To define the binding targets of NUP98 fusion proteins, and the co-bound sites with MLL1. To determine the status of 2 different histone modifications on NUP98 fusion binding targets using NUP98-HOXA9 transfromed mouse bone marrow LSK cells.

本研究通过对两种不同NUP98融合蛋白开展生物素介导的染色质免疫共沉淀结合染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq），明确了NUP98-HOXA9与NUP98-HOXD13在全基因组范围内的直接结合位点。为验证NUP98融合蛋白与Mll1是否共同招募至同源框基因（Hox genes）启动子的同一区域，本研究使用抗Mll1n抗体，在经NUP98-HOXA9转化的小鼠细胞中开展了Mll1的ChIP-seq分析。本研究前期结果显示NUP98-HOXA9可与NSL/MLL1复合物中的MLL1发生相互作用，本次分析结果与之相符：Mll1的结合靶点在启动子区域与基因体区域均与NUP98-HOXA9的结合靶点存在显著重叠。鉴于MOF与MLL1可协同调控启动子区域的组蛋白H4赖氨酸16乙酰化（H4K16ac）与组蛋白H3赖氨酸4三甲基化（H3K4me3）修饰，本研究进一步使用抗H3K4me3与抗H4K16ac抗体，通过ChIP-seq检测经NUP98-HOXA9转化的小鼠细胞中，H3K4me3与H4K16ac标记是否与NUP98-HOXA9的结合靶点相关联。本研究数据显示，启动子区域的NUP98-HOXA9结合靶点同时带有H3K4me3与H4K16ac标记，这与MOF介导的H4K16乙酰化及MLL/SET复合物介导的H3K4甲基化之间的协同调控作用相符。综上，本研究结果证实NUP98-HOXA9与Mll1共同招募至同一Hox基因座，且该招募过程与激活型表观遗传标记相关，这一发现支持NUP98融合蛋白与NSL/Mll1复合物存在关联的观点，并提示NUP98融合蛋白可能通过Mll1被招募至Hox基因座。实验整体设计：1. 明确NUP98融合蛋白的结合靶点及其与MLL1的共同结合位点；2. 使用经NUP98-HOXA9转化的小鼠骨髓Lin⁻Sca-1⁺c-Kit⁺（LSK）细胞，检测NUP98融合蛋白结合靶点上两种不同组蛋白修饰的状态。