Cancer Virology Program, UPMC Hillman Cancer Center, and Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Department of Biomedical Informatics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
mBio. 2021 Dec 21;12(6):e0343121. doi: 10.1128/mbio.03431-21.
Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is causally associated with numerous cancers. The mechanism of KSHV-induced oncogenesis remains unclear. By performing a CRISPR-Cas9 screening in a model of KSHV-induced cellular transformation of primary cells, we identified epigenetic regulators that were essential for KSHV-induced cellular transformation. Examination of TCGA data sets of the top 9 genes, including glutamate-rich WD repeat containing 1 (GRWD1), a WD40 family protein upregulated by KSHV, that had positive effects on cell proliferation and survival of KSHV-transformed cells (KMM) but not the matched primary cells (MM), uncovered the predictive values of their expressions for patient survival in numerous types of cancer. We revealed global epigenetic remodeling including H3K4me3 epigenetic active mark in KMM cells compared to MM cells. Knockdown of GRWD1 inhibited cell proliferation, cellular transformation, and tumor formation and caused downregulation of global H3K4me3 mark in KMM cells. GRWD1 interacted with WD repeat domain 5 (WDR5), the core protein of H3K4 methyltransferase complex, and several H3K4me3 methyltransferases, including myeloid leukemia 2 (MLL2). Knockdown of WDR5 and MLL2 phenocopied GRWD1 knockdown, caused global reduction of H3K4me3 mark, and altered the expression of similar sets of genes. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analyses further identified common and distinct cellular genes and pathways that were regulated by GRWD1, WDR5, and MLL2. These results indicate that KSHV hijacks the GRWD1-WDR5-MLL2 epigenetic complex to regulate H3K4me3 methylation of specific genes, which is essential for KSHV-induced cellular transformation. Our work has identified an epigenetic complex as a novel therapeutic target for KSHV-induced cancers. By performing a genome-wide CRISPR-Cas9 screening, we have identified cellular epigenetic regulators that are essential for KSHV-induced cellular transformation. Among them, GRWD1 regulates epigenetic active mark H3K4me3 by interacting with WDR5 and MLL2 and recruiting them to chromatin loci of specific genes in KSHV-transformed cells. Hence, KSHV hijacks the GRWD1-WDR5-MLL2 complex to remodel cellular epigenome and induce cellular transformation. Since the dysregulation of GRWD1 is associated with poor prognosis in several types of cancer, GRWD1 might also be a critical driver in other viral or nonviral cancers.
卡波氏肉瘤相关疱疹病毒（KSHV）感染与许多癌症的发生有因果关系。KSHV 诱导致癌的机制尚不清楚。通过在 KSHV 诱导的原代细胞转化模型中进行 CRISPR-Cas9 筛选，我们鉴定了对 KSHV 诱导的细胞转化至关重要的表观遗传调节剂。对 TCGA 数据集前 9 个基因（包括谷氨酸丰富的 WD 重复包含 1（GRWD1））的分析表明，这些基因上调是 KSHV 转化细胞（KMM）而不是匹配的原代细胞（MM）增殖和存活所必需的。KSHV 转化细胞（KMM），但不是匹配的原代细胞（MM）。我们揭示了包括 H3K4me3 在内的全局表观遗传重塑，与 MM 细胞相比，H3K4me3 是一种表观遗传活性标记。GRWD1 抑制细胞增殖、细胞转化和肿瘤形成，并导致 KMM 细胞中全局 H3K4me3 标记下调。GRWD1 与 WD 重复结构域 5（WDR5）相互作用，WDR5 是 H3K4 甲基转移酶复合物的核心蛋白，以及几种 H3K4me3 甲基转移酶，包括髓样白血病 2（MLL2）。WDR5 和 MLL2 的敲低模拟了 GRWD1 的敲低，导致全局 H3K4me3 标记减少，并改变了相似基因集的表达。转录组测序（RNA-seq）和染色质免疫沉淀测序（ChIP-seq）分析进一步鉴定了由 GRWD1、WDR5 和 MLL2 调节的共同和独特的细胞基因和途径。这些结果表明，KSHV 劫持了 GRWD1-WDR5-MLL2 表观遗传复合物，以调节特定基因的 H3K4me3 甲基化，这对于 KSHV 诱导的细胞转化至关重要。我们的工作确定了一种表观遗传复合物作为 KSHV 诱导癌症的新的治疗靶点。通过进行全基因组 CRISPR-Cas9 筛选，我们鉴定了对 KSHV 诱导的细胞转化至关重要的细胞表观遗传调节剂。其中，GRWD1 通过与 WDR5 和 MLL2 相互作用并将其招募到 KSHV 转化细胞特定基因的染色质位点来调节表观遗传活性标记 H3K4me3。因此，KSHV 劫持了 GRWD1-WDR5-MLL2 复合物来重塑细胞表观基因组并诱导细胞转化。由于 GRWD1 的失调与多种癌症的预后不良有关，因此 GRWD1 也可能是其他病毒或非病毒癌症的关键驱动因素。
