Pontin Functions as A Transcriptional Co-activator for Retinoic Acid-induced HOX Gene Expression [GRO-seq]

Pontin is a AAA+ ATPase protein that has functions in various biological contexts including gene transcription regulation, chromatin remodeling, DNA damage sensing and repair, as well as assembly of protein and ribonucleoprotein complexes. Pontin is known to regulate the transcription of several important signaling pathways, including Wnt signaling. However, its role in early embryonic signaling regulation remains unclear. Retinoic acid (RA) signaling plays a central role in vertebrate development. Using an in vivo biotin tagging technology, we mapped the genome-wide binding pattern of Pontin before and after RA-induced differentiation in the pluripotent embryo carcinoma cell line NTEAR-2. Biotin ChIP-seq revealed significant changes in genome-wide Pontin binding sites upon RA stimulation. We also identified a substantial amount of overlapping binding peaks between Pontin and RARα, especially on all of the HOX gene loci (A-D clusters). Pontin knockdown experiments showed that its chromatin binding at the HOX gene clusters is required for RA-induced HOX gene expression. Furthermore, we performed Global Run-On sequencing (GRO-seq) to map de novo transcripts genome-wide and found that Pontin knockdown significantly diminished nascent HOX gene transcripts, indicating that Pontin regulates HOX gene expression at the transcriptional level. Finally, proteomic analysis demonstrated that Pontin associates with chromatin organization/remodeling complexes and various other functional complexes. Altogether, we have demonstrated that Pontin is a critical transcriptional co-activator for RA-induced HOX gene activation. Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq)

Pontin是一种AAA+ ATP酶 (AAA+ ATPase)，在多种生物学过程中发挥功能，涵盖基因转录调控、染色质重塑、DNA损伤感知与修复，以及蛋白质与核糖核蛋白复合物的组装。已知Pontin可调控多条重要信号通路的转录，其中包括Wnt信号通路。然而，其在早期胚胎信号调控中的作用仍未明确。视黄酸 (Retinoic acid, RA) 信号通路在脊椎动物发育中发挥核心作用。本研究借助体内生物素标记技术，在多能性胚胎癌细胞系NTEAR-2中，绘制了RA诱导分化前后Pontin的全基因组结合图谱。生物素标记染色质免疫沉淀测序 (Biotin ChIP-seq) 结果显示，RA刺激后Pontin的全基因组结合位点发生显著变化。研究团队还鉴定出大量Pontin与视黄酸受体α (RARα) 的结合峰重叠区域，尤其在所有HOX基因位点 (A-D簇) 中富集明显。Pontin敲低实验表明，Pontin在HOX基因簇上的染色质结合是RA诱导HOX基因表达所必需的。此外，本研究通过全局运行-on测序 (Global Run-On sequencing, GRO-seq) 绘制了全基因组新生转录本图谱，发现Pontin敲低可显著减少新生HOX基因转录本的生成，提示Pontin在转录水平调控HOX基因的表达。最后，蛋白质组分析结果显示，Pontin可与染色质组织/重塑复合物及多种其他功能复合物发生相互作用。综上，本研究证实Pontin是RA诱导HOX基因激活的关键转录辅激活因子。全局运行-on (Global Run On, GRO) 检测联合高通量测序 (GRO-seq) 技术