Regulation of heterochromatin formation and tumor suppression in leukemia by IKAROS, HDAC1 and EZH2 [Molt4_CT]

The IKZF1 gene encodes IKAROS – a DNA binding protein that acts as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). IKAROS can act as a transcriptional repressor via chromatin remodeling, however the mechanisms through which Ikaros exerts its tumor suppressor function via heterochromatin in T-ALL are largely unknown. We studied human and mouse T-ALL using loss-of-function and Ikzf1 re-expression approach, along with Ikzf1-wildtype primary human and mouse T-ALL and thymocytes to establish the role of Ikaros and Ikaros-associated protein histone deacetylase 1 (HDAC1) in global regulation of facultative heterochromatin and transcriptional repression in T-ALL. Results identified novel Ikaros and HDAC1 functions in T-ALL: Ikaros and HDAC1 are essential for EZH2 histone methyltransferase activity, and formation of facultative heterochromatin; Recruitment of HDAC1 by Ikaros is critical for establishment of H3K27me3 and repression of active enhancers; and Ikaros-HDAC1 complexes promote formation and expansion of H3K27me3 large organized chromatin lysine domains (LOCKs) and broad genic repression domains (BGRDs) in T-ALL. Our results establish that Ikaros' tumor suppressor function in T-ALL occurs via activation of EZH2 and HDAC1 function, global regulation of the facultative heterochromatin landscape and silencing of active enhancers that regulate oncogene expression. Overall design: The IKZF1 gene was deleted in human T-ALL cells MOLT4 using the CRISPR-Cas system to create MOLT-4 IKZF1-null cells

IKZF1基因编码IKAROS蛋白——一种在T细胞急性淋巴细胞白血病（T-cell acute lymphoblastic leukemia, T-ALL）中发挥肿瘤抑制功能的DNA结合蛋白。IKAROS可通过染色质重塑发挥转录抑制作用，但目前关于Ikaros在T-ALL中通过异染色质发挥肿瘤抑制功能的具体机制仍不甚明确。本研究采用功能丧失实验与Ikzf1基因重表达策略，并结合野生型Ikzf1原代人源、鼠源T-ALL细胞及胸腺细胞，探究了Ikaros及其互作蛋白组蛋白去乙酰化酶1（histone deacetylase 1, HDAC1）在T-ALL兼性异染色质全局调控与转录抑制中的作用。

研究结果揭示了Ikaros与HDAC1在T-ALL中的全新功能：Ikaros与HDAC1对EZH2组蛋白甲基转移酶活性及兼性异染色质的形成至关重要；Ikaros介导的HDAC1招募对于建立H3K27me3修饰与抑制活性增强子的表达具有关键意义；Ikaros-HDAC1复合物可促进T-ALL中H3K27me3大型有序组蛋白赖氨酸结构域（LOCKs）与宽域基因抑制结构域（BGRDs）的形成与扩展。

本研究证实，Ikaros在T-ALL中的肿瘤抑制功能是通过激活EZH2与HDAC1的功能、全局调控兼性异染色质景观，以及沉默调控癌基因表达的活性增强子来实现的。

研究整体设计：本研究通过CRISPR-Cas系统在人源T-ALL细胞MOLT4中敲除IKZF1基因，构建了MOLT-4 IKZF1基因敲除细胞系。