Department of Biological Sciences, NMIMS Sunandan Divatia School of Science, NMIMS (deemed to-be) University, Mumbai, 400056, India.
Symbiosis Centre for Stem Cell Research (SCSCR), Symbiosis International University, Pune, 412115, India.
Cell Biol Int. 2020 Jun;44(6):1299-1311. doi: 10.1002/cbin.11325. Epub 2020 Mar 2.
Polycomb group (PcG) proteins are histone modifiers which are known to perform transcriptional repression and have been shown to be critical during murine embryonic development. PcGs are broadly characterized into polycomb repressive complex 1 (PRC1) and 2 and (PRC2). RING1B, core catalytic unit of PRC1 performs H2AK119 monoubiquitination leading to transcriptional repression. We used human embryonic stem cell (hESC) line to study the fate of pluripotent stem cells (PSCs) under inhibition of RING1B, as its role in human development is still to be completely explored. Embryoid bodies (EBs) were generated to differentiate hESCs using hanging drop method. PRT4165 (synthetic RING1B catalytic activity inhibitor) was added to undifferentiated and differentiated cells for 24 h. When we inhibited RING1B in undifferentiated cells, OCT4 levels remained unchanged indicating RING1B does not regulate pluripotency. The drug when added to differentiated cells led to decrease in the levels of RING1B, BMI1, and H2AK119ub1. Interestingly, we also report that the differentiated cells show an increased expression of neuroectodermal markers: SOX1 and PAX6 as well as expression of other neuroectodermal markers such as TUJ1, FOXG1, and NCAM. However, there was reduction in expression of endodermal (SOX17 and FOXA2) mesodermal marker BRACHYURY and TBX5 in treated EBs compared with control EBs. In summary, alteration of RING1B catalytic activity in hESCs during differentiation promotes neuroectodermal differentiation thus, we demonstrate critical role of RING1B in regulating neural differentiation. The strategy of inhibiting RING1B could be used to direct PSCs towards early neuronal fate.
多梳抑制复合物（PcG）蛋白是组蛋白修饰物，已知其具有转录抑制作用，并且在小鼠胚胎发育过程中至关重要。PcG 广泛分为多梳抑制复合物 1（PRC1）和 2（PRC2）。PRC1 的核心催化亚基 RING1B 执行 H2AK119 单泛素化，导致转录抑制。我们使用人胚胎干细胞（hESC）系研究 RING1B 抑制下人多能干细胞（PSCs）的命运，因为其在人类发育中的作用仍有待完全探索。使用悬滴法生成类胚体（EBs）以分化 hESC。将 PRT4165（合成 RING1B 催化活性抑制剂）添加到未分化和分化细胞中 24 小时。当我们在未分化细胞中抑制 RING1B 时，OCT4 水平保持不变，表明 RING1B 不调节多能性。当将该药物添加到分化细胞中时，RING1B、BMI1 和 H2AK119ub1 的水平下降。有趣的是，我们还报告说，分化细胞表现出神经外胚层标记物的表达增加：SOX1 和 PAX6 以及其他神经外胚层标记物的表达，如 TUJ1、FOXG1 和 NCAM。然而，与对照 EBs 相比，处理的 EBs 中内胚层（SOX17 和 FOXA2）标记物 BRACHYURY 和 TBX5 的表达减少。总之，在分化过程中 hESCs 中 RING1B 催化活性的改变促进了神经外胚层分化，因此，我们证明了 RING1B 在调节神经分化中的关键作用。抑制 RING1B 的策略可用于指导 PSCs 向早期神经元命运分化。
