Abstract

Background

The sensitivity of myelocytomatosis oncogene (MYC) amplified medulloblastoma to class I histone deacetylase (HDAC) inhibition has been shown previously; however, understanding the underlying molecular mechanism is crucial for selection of effective HDAC inhibitors for clinical use. The aim of this study was to investigate the direct molecular interaction of MYC and class I HDAC2, and the impact of class I HDAC inhibition on MYC function.

Methods

Co-immunoprecipitation and mass spectrometry were used to determine the co-localization of MYC and HDAC2. Chromatin immunoprecipitation (ChIP) sequencing and gene expression profiling were used to analyze the co-localization of MYC and HDAC2 on DNA and the impact on transcriptional activity in primary tumors and a MYC amplified cell line treated with the class I HDAC inhibitor entinostat. The effect on MYC was investigated by quantitative real-time PCR, western blot, and immunofluorescence.

Results

HDAC2 is a cofactor of MYC in MYC amplified medulloblastoma. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein, inducing a downregulation of MYC activated genes (MAGs) and upregulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and by distinct enhancer-box distribution.

Conclusions

Our data elucidate the molecular interaction of MYC and HDAC2 and support a model in which inhibition of class I HDACs directly targets MYC’s transactivating and transrepressing functions.

中文翻译：

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MYC 染色质结合的减少是 MYC 扩增的髓母细胞瘤中 HDAC 抑制的关键作用

摘要

背景

骨髓细胞瘤病癌基因 ( MYC ) 扩增的髓母细胞瘤对 I 类组蛋白去乙酰化酶 (HDAC) 抑制的敏感性先前已显示；然而，了解潜在的分子机制对于选择临床使用的有效 HDAC 抑制剂至关重要。本研究的目的是研究 MYC 和 I 类 HDAC2 的直接分子相互作用，以及 I 类 HDAC 抑制对 MYC 功能的影响。

方法

共免疫沉淀和质谱用于确定 MYC 和 HDAC2 的共定位。染色质免疫沉淀 (ChIP) 测序和基因表达谱用于分析 MYC 和 HDAC2 在 DNA 上的共定位以及对原发性肿瘤和用 I 类 HDAC 抑制剂恩替司他处理的MYC扩增细胞系转录活性的影响。通过定量实时 PCR、蛋白质印迹和免疫荧光研究了对 MYC 的影响。

结果

HDAC2 是MYC扩增的髓母细胞瘤中MYC的辅因子。MYC-HDAC2 复合物与定义 MYC 依赖性转录谱的基因结合。I 类 HDAC 抑制导致 MYC 蛋白的稳定和降低 DNA 结合，诱导 MYC 激活基因 (MAG) 的下调和 MYC 抑制基因 (MRG) 的上调。MAGs 和 MRGs 的特点是相反的生物学功能和不同的增强子盒分布。

结论

我们的数据阐明了 MYC 和 HDAC2 的分子相互作用，并支持了一个模型，其中 I 类 HDAC 的抑制直接针对 MYC 的反式激活和反式抑制功能。