RNA seq after RNA pol III manipulation by Brf1 and Maf1 knockdown, Maf1 overexpression and ML60218 treatment in ST2 cells before, and during differentiation into osteoblasts

Maf1, a key repressor of RNA polymerase III-mediated transcription, has been shown to promote mesoderm formation in vitro. Here, we show, for the first time, that Maf1 plays a critical role in the regulation of osteoblast differentiation. We also found that, in contrast to MAF1 overexpression, other perturbations that repress RNA pol III transcription, including Brf1 knockdown and the chemical inhibition of RNA pol III by ML-60218, inhibited osteoblast differentiation. RNA-seq was used to determine the basis for these opposing actions on osteoblast differentiation. RNA polymerase III-dependent transcription was manipulated in ST2 stromal cells by Brf1 or Maf1 knockdown, ML-60218 treatment and MAF1 overexpression. The ST2 cells were then tested for their capacity to differentiate into osteoblasts. RNA-seq was performed on day 0, before differentiation and day 4 after differentiation medium was added. The modalities used to perturb RNA pol III transcription resulted in distinct changes gene expression, indicating that this transcription process is highly sensitive and triggers diverse gene expression programs and phenotypic outcomes. Specifically, Maf1 induced genes in ST2 cells known to promote osteoblast differentiation. Furthermore, genes that are induced during osteoblast differentiation displayed codon bias. Comparative gene expression profiling analysis of RNA-seq data for ST2 cells before (d0), and during (d4) osteoblast differentiation. cells were manipulated by shBrf1 (=shBr) or SCRB control, shMaf1(=shMA) or SCRM control, pinducer20-MAF1 (=MAF1) or pInducer20-empty control (=pInd), 40uM ML-60218 treatment (=ML) or DMSO control. each condition has 3 replicates

Maf1是RNA聚合酶III（RNA polymerase III）介导转录的关键阻遏因子，既往研究证实其可在体外促进中胚层形成。本研究首次证实，Maf1在成骨细胞分化调控中发挥关键作用。同时我们发现，与MAF1过表达相反，其他抑制RNA聚合酶III转录的干预手段——包括敲低Brf1以及使用ML-60218对RNA聚合酶III进行化学抑制——均会抑制成骨细胞分化。为阐明上述对成骨细胞分化的相反调控作用机制，本研究采用RNA测序（RNA-seq）开展相关分析。在ST2基质细胞（ST2 stromal cells）中，我们通过敲低Brf1或Maf1、ML-60218处理以及过表达MAF1来干预RNA聚合酶III依赖的转录过程，随后检测这些细胞向成骨细胞分化的能力。分别于分化前第0天以及添加分化培养基后的第4天收集样本进行RNA测序。不同的RNA聚合酶III转录干预手段可引发显著的基因表达变化，表明该转录过程具有高度敏感性，并可触发多样化的基因表达程序与表型结局。具体而言，Maf1可在ST2基质细胞中诱导已知的成骨细胞分化促进基因的表达。此外，在成骨细胞分化过程中被诱导的基因表现出密码子偏好性。本研究对ST2细胞在成骨细胞分化前（d0）及分化过程中（d4）的RNA测序数据进行了比较基因表达谱分析，所涉及的细胞干预手段包括：shRNA靶向Brf1（shBrf1，简称shBr）或其对照SCRB、shRNA靶向Maf1（shMaf1，简称shMA）或其对照SCRM、pinducer20-MAF1过表达载体（MAF1）或空载体pInducer20（pInd）、40μM ML-60218处理（ML）或二甲基亚砜（DMSO）对照，每组设置3次生物学重复。