Department of Microbiology & Immunology and the Centre for Human Immunology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; Division of Genetics and Development, Children's Health Research Institute, Lawson Research Institute, London, ON, Canada.
Department of Microbiology & Immunology and the Centre for Human Immunology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
Exp Hematol. 2019 May;73:50-63.e2. doi: 10.1016/j.exphem.2019.03.004. Epub 2019 Apr 12.
The most frequently occurring genetic abnormality in pediatric B-lymphocyte-lineage acute lymphoblastic leukemia is the t(12;21) chromosomal translocation that results in a ETV6-RUNX1 (also known as TEL-AML1) fusion gene. Expression of ETV6-RUNX1 induces a preleukemic condition leading to acquisition of secondary driver mutations, but the mechanism is poorly understood. SPI-B (encoded by SPIB) is an important transcriptional activator of B-cell development and differentiation. We hypothesized that SPIB is directly transcriptionally repressed by ETV6-RUNX1. Using chromatin immunoprecipitation, we identified a regulatory region in the first intron of SPIB that interacts with ETV6-RUNX1. Mutation of the RUNX1 binding site in SPIB intron 1 prevented transcriptional repression in transient transfection assays. Next, we sought to determine to what extent gene expression in REH cells can be altered by ectopic SPI-B expression. SPI-B expression was forced using CRISPR-mediated gene activation and also using a retroviral vector. Forced expression of SPI-B resulted in altered gene expression and, at high levels, impaired cell proliferation and induced apoptosis. Finally, we identified CARD11 and CDKN1A (encoding p21) as transcriptional targets of SPI-B involved in regulation of proliferation and apoptosis. Taken together, this study identifies SPIB as an important target of ETV6-RUNX1 in regulation of B-cell gene expression in t(12;21) leukemia.
在儿科 B 细胞谱系急性淋巴细胞白血病中最常发生的遗传异常是 t(12;21)染色体易位，导致 ETV6-RUNX1（也称为 TEL-AML1）融合基因的表达。ETV6-RUNX1 的表达诱导白血病前状态，导致获得继发性驱动突变，但机制尚不清楚。SPI-B（由 SPIB 编码）是 B 细胞发育和分化的重要转录激活因子。我们假设 SPIB 被 ETV6-RUNX1 直接转录抑制。通过染色质免疫沉淀，我们在 SPIB 的第一个内含子中鉴定了一个与 ETV6-RUNX1 相互作用的调控区域。SPIB 内含子 1 中 RUNX1 结合位点的突变阻止了瞬时转染实验中的转录抑制。接下来，我们试图确定 REH 细胞中的基因表达在多大程度上可以通过异位 SPI-B 表达来改变。使用 CRISPR 介导的基因激活和逆转录病毒载体强制表达 SPI-B。SPI-B 的表达被强制表达导致基因表达改变，在高表达水平下，抑制细胞增殖并诱导细胞凋亡。最后，我们确定 CARD11 和 CDKN1A（编码 p21）作为 SPI-B 调节增殖和凋亡的转录靶点。总之，这项研究确定 SPIB 是 ETV6-RUNX1 在调节 t(12;21)白血病中 B 细胞基因表达的重要靶点。
