PO.TB05.03 · 肿瘤生物学
B细胞急性淋巴细胞白血病中RAB5B GTP酶表达的表观遗传调控
Epigenetic regulation of the RAB5B GTPase expression in B-cell acute lymphoblastic leukemia
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
脑内Ras类似物（Rab）蛋白是人类恶性肿瘤中小GTP酶信号通路的重要组成部分。RAB5B是一种GTP酶，可促进细胞增殖，并在包括白血病在内的大量人类癌症中过表达。RAB5B转录在人类血液系统恶性肿瘤中的调控大多尚不明确。在此，我们分析RAB5B转录在人类B细胞急性淋巴细胞白血病（B-ALL）中的调控。利用全局染色质免疫沉淀结合下一代测序（ChIP-seq），显示IKAROS肿瘤抑制蛋白在多个B-ALL细胞系和原代B-ALL细胞中RAB5B基因启动子上的占据增加。IKAROS与RAB5B启动子的直接结合经定量染色质免疫沉淀（qChIP）证实。通过功能获得和功能缺失实验评估了IKAROS在B-ALL中对RAB5B转录调控的作用。荧光素酶报告基因检测显示IKAROS直接抑制RAB5B转录。通过逆转录病毒转导在B-ALL细胞中过表达IKAROS，导致IKAROS与RAB5B启动子的结合增加，RAB5B转录减少。这与组成型异染色质标志物H3K9me3的富集以及活性染色质标志物H3K4me3和H3K27ac的丢失相关。相反，shRNA介导的IKAROS敲低抑制了IKAROS与RAB5B启动子的结合，导致RAB5B表达增加，同时H3K4me3和H3K27ac活性染色质标志物富集增加，以及RAB5B启动子处H3K9me3标志物的丢失。携带一个IKAROS等位基因缺失的原代B-ALL细胞具有增高的RAB5B表达，并伴有H3K4me3和H3K27ac的强烈富集，以及RAB5B启动子处H3K9me3的缺失。总之，所呈现的数据表明IKAROS在B-ALL中作为RAB5B的直接转录抑制因子发挥作用，且IKAROS抑制RAB5B的机制涉及在RAB5B启动子处形成异染色质。研究结果揭示了IKAROS发挥其肿瘤抑制作用并调控白血病中转录和细胞增殖的新机制。本研究由NIH/NCI R01 CA278226基金资助。
查看英文原文 English abstract
Ras analog in brain (Rab) proteins are important parts of small GTPase signaling pathways in human malignancies. RAB5B is the GTPase, which promotes cellular proliferation and is overexpressed in a large number of human cancers, including leukemia. Regulation of RAB5B transcription in human hematological malignancies is mostly unknown. Here, we analyze regulation of RAB5B transcription in human B-cell acute lymphoblastic leukemia (B-ALL). The use of global chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq), showed increased occupancy of IKAROS tumor suppressor protein at the promoter of RAB5B gene in several B-ALL cell lines and primary B-ALL cells. IKAROS direct binding to promoter of RAB5B was confirmed by quantitative chromatin immunoprecipitation (qChIP). The role of IKAROS in transcriptional regulation of RAB5BB in B-ALL was assessed by gain-of-function and loss-of-function experiments. Luciferase reporter assay showed that IKAROS directly represses transcription of RAB5B. Overexpression of IKAROS in B-ALL cells via retroviral transduction resulted in increased IKAROS binding to the RAB5B promoter and decreased RAB5B transcription. This was associated with enrichment in constitutive heterochromatin marker, H3K9me3 and the loss of active chromatin markers, H3K4me3 and H3K27ac. In contrast, shRNA-mediated knockdown of IKAROS inhibited IKAROS binding to the RAB5B promoter and resulted in increased RAB5B expression, along with increased enrichment of H3K4me3 and H3K27ac active chromatin markers, and the loss of H3K9me3 marker at RAB5B promoter. Primary B-ALL cells that carry deletion of one IKAROS allele have increased expression of RAB5B, associated with strong enrichment of H3K4me3 and H3K27ac, as well as the absence of H3K9me3 at RAB5B promoter. In conclusion, presented data suggest that IKAROS functions as a direct transcriptional repressor of RAB5B in B-ALL and that the mechanism of RAB5B repression by IKAROS involves formation of heterochromatin at the RAB5B promoter. Results uncovered a novel mechanism through which IKAROS exerts its tumor suppressor effect and regulate transcription and cellular proliferation in leukemia. Supported by NIH/NCI R01 CA278226 grant.
利益披露 Disclosure
K. Dovat, None..
Y. Ding, None..
D. Bogush, None..
R. Husain, None..
R. Chingakham, None..
D. Desai, None..
S. Dovat, None.