Defining TFAP4 transcriptional regulation in pre-leukemic Eμ-MYC pre-B lymphoma cells

Many lymphoid malignancies arise from deregulated c-MYC expression in cooperation with additional genetic lesions. While many of these cooperative genetic lesions have been discovered and their functions characterised, DNA sequence data of primary patient samples suggest that many more do exist. However, the nature of their contributions to c-MYC driven lymphomagenesis have not yet been investigated. We identified TFAP4 as a potent suppressor of c-MYC driven lymphoma development in a previous genome-wide CRISPR knockout screen in primary cells in vivo. CRISPR deletion of TFAP4 in Eµ-MYC transgenic haematopoietic stem and progenitor cells (HSPCs) and transplantation of these manipulated HSPCs into lethally irradiated animals significantly accelerated c-MYC-driven lymphoma development. Interestingly, TFAP4 deficient Eµ-MYC lymphomas all arose at the pre-B cell stage of B cell development. This observation prompted us to characterise the transcriptional profile of pre-B cells from pre-leukaemic mice transplanted with Eµ-MYC/Cas9 HSPCs that had been transduced with sgRNAs targeting TFAP4. This analysis revealed that TFAP4 deletion reduced expression of several master regulators of B cell differentiation, such as Spi1, SpiB and Pax5, which are direct target genes of both TFAP4 and MYC. We therefore conclude that loss of TFAP4 leads to a block in differentiation during early B cell development, thereby accelerating c-MYC-driven lymphoma development. Paired-end RNA-sequencing data was generated for 14 samples of primary mouse pre-leukemic Eµ-MYC/Cas9 B-cells. Of these samples, 6 are control and 8 are Tfap4 knock-out (KO). Differential expression analyses were performed, identifying differentially expressed genes between the KO and control groups. This analysis included an adjustment for samples with the same FL ID.多种淋巴系统恶性肿瘤由c-MYC表达失调引发，并与其他遗传损伤协同致癌。尽管目前已鉴定出诸多此类协同遗传损伤并阐明其功能机制，但原代患者样本的DNA序列数据表明，仍存在大量未被发现的协同遗传损伤。然而，此类损伤对c-MYC驱动的淋巴肿瘤发生的具体贡献方式尚未得到研究。我们此前在原代细胞的体内全基因组CRISPR敲除筛选中，鉴定出TFAP4是c-MYC驱动的淋巴瘤发生的强效抑制剂。在Eµ-MYC转基因造血干细胞与祖细胞（Haematopoietic Stem and Progenitor Cells，HSPCs）中敲除TFAP4，并将这些经过基因编辑的HSPCs移植至经致死剂量辐照的受体动物体内，可显著加速c-MYC驱动的淋巴瘤发生进程。值得注意的是，TFAP4缺陷型Eµ-MYC淋巴瘤均起源于B细胞发育阶段的前B细胞阶段。这一观察结果促使我们对移植了经靶向TFAP4的单向导RNA（single guide RNA，sgRNA）转导的Eµ-MYC/Cas9 HSPCs的白血病前期小鼠的前B细胞转录组特征进行解析。该分析显示，TFAP4敲除会降低多个B细胞分化主调控因子的表达水平，例如Spi1、SpiB与Pax5，这些因子均为TFAP4与MYC的直接靶基因。因此我们得出结论：TFAP4缺失会阻断B细胞早期发育过程中的分化进程，进而加速c-MYC驱动的淋巴瘤发生。本数据集针对14例原代小鼠白血病前期Eµ-MYC/Cas9 B细胞样本生成了双端RNA测序（Paired-end RNA-sequencing）数据。其中6例为对照组样本，8例为TFAP4敲除（Knockout，KO）组样本。研究人员开展了差异表达分析，鉴定出KO组与对照组之间的差异表达基因，该分析对具有相同FL ID的样本进行了校正。