Proteasome inhibition silences oncogenic transcripts in multiple myeloma through HDAC3-mediated DNA condensation and gene relocation [ChIP-seq]. Proteasome inhibition silences oncogenic transcripts in multiple myeloma through HDAC3-mediated DNA condensation and gene relocation [ChIP-seq]

This study provides a genome-wide map of changes in histone mark modifications and HDAC3 binding in response to protesome inhibition in the multiple myeloma cell line MM.1S. Chromatin immunoprecipitation assays were carried out to determine the genomic locations of histone modifications (H3K27ac, H3K4me1, H3K4me3) and histone deacetylase 3 (HDAC3) binding locations in multiple myeloma cells following proteasome inhibition with either lactacystin, bortezomib or carfilzomib. In addition, we report the effects of the overexpression of the E3-ubiquitin ligase Siah2 and the impact of HDAC3 knockdown on H3K27 acetylation levels in multiple myeloma cells treated with lactacystin. Our global ChIP-seq analysis of histone marks showed that enhancer and promoter marks (H3K4me1 and H3K4me3, respectively) present little response to proteasome inhibition, while the acetylation of histone H3K27 was significantly up- or down-regulated after three-hour treatment with proteasome inhibitors. Treatment of the cells with lactacystin, bortezomib or carfilzomib strongly increased HDAC3 recruitment at cell cycle and mitochondrial promoters, indicating that proteasome inhibition stabilized HDAC3 locally at the promoter of these genes to induce their repression. Furthermore, genome-wide ChIP-seq analysis of H3K27ac profiles showed that overexpression of Siah2 enhanced H3K27 acetylation levels at cell cycle and mitochondrial promoters. Overall design: Multiple myeloma cells MM.1S were treated for three hours with the synthetic proteasome inhibitor lactacystin or the FDA-approved clinical proteasome inhibitors bortezomib or carfilzomib and compared to DMSO control-treated cells in ChIP-sequencing experiments. We performed ChIP-seq for histone modifications (H3K27ac, H3K4me1, H3K4me3) and the histone deacetylase HDAC3. We also studied the changes in H3K27 acetylation triggered by HDAC3 knockdown or Siah2 overexpression in MM.1S cells.

本研究构建了多发性骨髓瘤细胞系MM.1S中，响应蛋白酶体抑制时组蛋白修饰变化及组蛋白去乙酰化酶3（HDAC3）结合变化的全基因组图谱。本研究通过染色质免疫沉淀（Chromatin immunoprecipitation, ChIP）实验，明确了多发性骨髓瘤细胞经乳胞素（lactacystin）、硼替佐米（bortezomib）或卡非佐米（carfilzomib）处理实现蛋白酶体抑制后，组蛋白修饰（H3K27ac、H3K4me1、H3K4me3）及HDAC3结合的基因组定位区域。此外，本研究还报道了E3泛素连接酶Siah2过表达的效应，以及HDAC3敲低对经乳胞素处理的多发性骨髓瘤细胞中H3K27乙酰化水平的影响。针对组蛋白标记的全基因组ChIP测序（ChIP-sequencing, ChIP-seq）分析显示，增强子标记与启动子标记（分别为H3K4me1与H3K4me3）几乎不对蛋白酶体抑制产生应答；而经蛋白酶体抑制剂处理3小时后，组蛋白H3K27的乙酰化水平出现显著上调或下调。使用乳胞素、硼替佐米或卡非佐米处理细胞后，HDAC3在细胞周期相关启动子及线粒体启动子区域的招募显著增强，表明蛋白酶体抑制可使HDAC3在这些基因的启动子区域局部稳定富集，从而诱导其转录抑制。进一步的全基因组H3K27ac谱ChIP-seq分析显示，Siah2过表达可提升细胞周期相关启动子及线粒体启动子区域的H3K27乙酰化水平。实验整体设计如下：将多发性骨髓瘤细胞系MM.1S经合成蛋白酶体抑制剂乳胞素，或美国食品药品监督管理局（FDA）批准的临床蛋白酶体抑制剂硼替佐米、卡非佐米处理3小时，以二甲基亚砜（DMSO）处理的细胞作为对照，开展ChIP-seq实验。本研究针对组蛋白修饰（H3K27ac、H3K4me1、H3K4me3）及组蛋白去乙酰化酶HDAC3进行了ChIP-seq检测。此外，我们还研究了MM.1S细胞中HDAC3敲低或Siah2过表达所引发的H3K27乙酰化水平变化。