Promoter-bound METTL3 maintains myeloid leukemia by m6A-dependent regulation of protein translation

N6-methyladenosine (m6A) is an abundant internal RNA modification, in both coding and non-coding RNAs, catalyzed by the METTL3/METTL14 methyltransferase complex. We identified METTL3 as an essential gene for acute myeloid leukemia (AML) cell growth in two distinct genetic screens. Down-regulation of METTL3 results in cell cycle arrest, differentiation of leukemic cells and failure to establish leukemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start site (TSS) of 83 active genes. The vast majority of these genes have a CAATT-box motif at their TSS, are occupied by a specific set of transcription factors including NFY, WDR5, KLF9 and they harbor specific histone modifications (e.g. H3R2me2s). Promoter bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript and it enhances translation due to relief of ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for AML-leukemia. Together, these data define a new mechanism of gene regulation by METTL3 and identify this enzyme as a novel therapeutic target for AML. Overall design: This experiment contains data for 3 high-throughput sequencing experiments; ChIP seq, m6A RNA IP seq and Riboprofiling. ChIP seq of METTL3 and METTL14 was conducted with 5 biological replicates each, with IgG and Input controls in triplicate and H3K4me3 in duplicate. All biological replicates were run across 2 lanes on an Illumina HiSeq 1500 providing technical replicates and increased coverage. m6A RNA IP was run in duplicate for two METTL3 shRNA inducible knockdown cell lines (KD1 and KD2) and a scramble shRNA control at day 8 following doxycyclin induction. In addition, matching non-immuno-precipitated Input controls were also sequenced. All biological replicates were run across 2 lanes on an Illumina HiSeq 4000 providing technical replicates and increased coverage. Riboprofiling was performed on two METTL3 shRNA inducible knockdown (KD) cell lines and a scramble shRNA control at day 5 and day 8 following doxycyclin induction. Matching Input controls were sequenced for each condition for direct comparison. All samples were run across 6 lanes on an Illumina HiSeq 1500 as technical replicates and to provide increased coverage. Files are named with the following structure: [Experiment Type]_[Sample Name]_[Biological Replicate Num]_[Technical Replicate Num].[File Extension].

N6-甲基腺嘌呤（N6-methyladenosine，m6A）是一类广泛存在于编码RNA与非编码RNA内部的丰度较高的RNA修饰，由METTL3/METTL14甲基转移酶复合物催化生成。我们通过两项独立的遗传筛选，鉴定出METTL3是急性髓系白血病（AML）细胞增殖所必需的核心基因。下调METTL3的表达会引发细胞周期阻滞、白血病细胞分化，并导致免疫缺陷小鼠无法成功构建白血病模型。研究发现，METTL3可不依赖METTL14与染色质结合，并定位于83个活跃基因的转录起始位点（TSS）。其中绝大多数基因在其TSS区域携带CAATT盒基序，可被NFY、WDR5、KLF9等特定转录因子结合，同时伴随特定的组蛋白修饰（如H3R2me2s）。结合于启动子区域的METTL3可在对应mRNA的编码区诱导m6A修饰，并通过解除核糖体停滞增强翻译效率。我们证实，通过该机制受METTL3调控的基因对AML发生发展至关重要。综上，本研究揭示了METTL3介导基因调控的全新机制，并将该酶鉴定为AML的新型治疗靶点。

实验设计概述：本实验包含三类高通量测序实验数据，分别为染色质免疫共沉淀测序（ChIP-seq）、m6A RNA免疫沉淀测序（m6A RNA IP-seq）与核糖体谱测序（Riboprofiling）。
1. METTL3与METTL14的ChIP-seq实验各设置5个生物学重复，同时设置IgG对照、Input对照（各3次重复）以及H3K4me3对照（2次重复）。所有生物学重复均在Illumina HiSeq 1500的2个测序泳道上完成，以实现技术重复并提升测序覆盖度。
2. m6A RNA免疫沉淀测序针对2株METTL3可诱导短发夹RNA（shRNA）敲低细胞系（KD1与KD2）以及1株乱序shRNA对照细胞系，于多西环素诱导第8天进行检测，同时对匹配的非免疫沉淀Input对照进行测序。所有生物学重复均在Illumina HiSeq 4000的2个测序泳道上完成，以实现技术重复并提升测序覆盖度。
3. 核糖体谱测序针对2株METTL3可诱导shRNA敲低细胞系与1株乱序shRNA对照细胞系，分别于多西环素诱导第5天与第8天进行检测，同时为每个实验组设置匹配的Input对照用于直接比较。所有样本均在Illumina HiSeq 1500的6个测序泳道上完成测序，以实现技术重复并提升测序覆盖度。

样本文件命名规则如下：[实验类型]_[样本名称]_[生物学重复编号]_[技术重复编号].[文件扩展名]。