PML modulates epigenetic composition of chromatin to regulate expression of pro-metastatic genes in triple-negative breast cancer [RNA-seq]

The promyelocytic leukemia (PML) protein organizes nuclear aggregates known as PML nuclear bodies (PML-NBs), where many transcription factors and transcriptional regulators converge to be regulated. Specific associations of PML and PML-NBs with chromatin are described in different cell types, further implicating PML in transcriptional regulation. However, a complete understanding of the functional consequences of PML association to DNA in a cellular context where it regulates relevant phenotypes is still lacking.We examined the role of PML in chromatin association and transcription in triple-negative breast cancer (TNBC), a pathological condition where PML exerts important oncogenic functions. We find that PML associates discontinuously with large heterochromatic PML-associated domains (PADs) that contain gene-rich euchromatic sub-domains locally depleted of PML. PML promotes heterochromatic organization in PADs and expression of pro-metastatic genes embedded in these sub-domains. Importantly, this occurs outside PML-NBs, suggesting that nucleoplasmic PML exerts a cell type-relevant function of transcriptional regulation. PML also plays an indirect regulatory function in TBNC cells by promoting the expression of pro-metastatic genes outside PADs.Our findings demonstrate that PML is an important transcriptional regulator of metastasis and pro-oncogenic metagenes in TNBC cells, via distinct molecular activities that include indirect transcriptional regulation and direct epigenetic organization of heterochromatin domains that embed regions of localized transcriptional activity. RNA-sequencing (RNA-seq) in control and PML silenced MDA-MB-231 cells.

早幼粒细胞白血病蛋白（promyelocytic leukemia, PML）可组装形成被称为PML核体（PML nuclear bodies, PML-NBs）的核内聚集结构，众多转录因子与转录调控因子在此汇聚并受到调控。已有研究在多种细胞类型中报道了PML与PML-NBs同染色质的特异性结合，进一步证实PML参与转录调控过程。然而，在PML可调控相关表型的细胞环境中，人们对PML与DNA结合所产生的全部功能性后果仍缺乏完整认知。
本研究以三阴性乳腺癌（triple-negative breast cancer, TNBC）为模型——该疾病中PML发挥关键致癌功能——探究了PML在染色质结合与转录调控中的作用。研究发现，PML以不连续的方式结合大型异染色质相关PML结构域（heterochromatic PML-associated domains, PADs），这类结构域内部包含局部缺失PML的基因富集型常染色质亚结构域。PML可促进PADs内的异染色质组装，并调控嵌入该亚结构域中的促转移基因的表达。值得注意的是，这一调控过程并不依赖于PML核体，提示核质定位的PML发挥了与细胞类型特异性相关的转录调控功能。此外，PML还可通过调控PADs以外的促转移基因的表达，在三阴性乳腺癌细胞中发挥间接转录调控作用。
本研究结果证实，PML是三阴性乳腺癌细胞中转移相关与促癌基因集的重要转录调控因子，其通过两种不同的分子机制发挥功能：一是间接调控转录，二是对嵌入局部转录活性区域的异染色质结构域进行直接表观遗传调控。本研究采用了对照组与PML敲低的MDA-MB-231细胞的RNA测序（RNA-seq）数据。