H2AK119ub dynamics controls hair follicle stem cell quiescence [RNA-Seq 2]

The transition of stem cells from a quiescent state to an active state is a finely tuned process that requires the dismantling of the quiescence program and the establishment of a cell cycle-promoting transcriptional landscape. Whether epigenetic processes control stem cell states to promote the regeneration of adult tissues remains elusive. In this study, we show that a repressive histone modification, H2AK119ub, is dynamic between quiescent and active hair follicle stem cells (HFSCs) in the adult murine skin. Ablation of H2AK119ub in HFSCs leads to failure in maintaining quiescence leading to premature activation and an eventual exhaustion of HFSC pool. Transcriptional and chromatin studies revealed that H2AK119ub directly represses a proliferation promoting transcriptional program in the HFSCs to preserve quiescence. Lastly, we identify that the inhibitory FGF signaling produced by the hair follicle niche keratinocytes maintains H2AK119ub in quiescent HFSCs. Together, these findings reveal that a repressive histone mark, H2AK119ub, is under the dynamic regulation of inhibitory niche signaling to prevent the untimely establishment of an activated state to preserve SC function and longevity. Overall design: Control and PRC1 i2KO mice were induced starting at P50. 15 days after completion of induction treatment, the back skin was collected and the adipose layer from the dorsal side was scraped off using a surgical scalpel. The scraped skin sample was washed with PBS prior to incubation with 0.25% Trypsin/EDTA at 37°C for 1 hour on a rotation plate set at 60 r.p.m. After incubation, the epidermal cells, including the HFSCs, was scraped off from the trypsinized skin into the plate. 25 mLs of E-media was added to the cell suspension and was then sequentially strained through 100µm and 40µm filters. The cell pellet was washed twice with DPBS before proceeding with staining with cell surface markers. The cells were stained with 1:200 PerCP-Cy5.5-Sca1 (Thermo Fisher Scientific, 45-5981-82), 1:200 FITC-a6-integrin (eBioscience, 11-0493-81), 1:100 APC-EpCAM (Biolegend, 324207) and 1:20 Alexa700-CD34 (eBioscience, 56-0341-82) in staining buffer (HBSS + 2% Fetal Bovine Serum) for 30 minutes on ice and then washed twice with DPBS before cell sorting. HFSCs were sorted by gating on EpCAM (+), Sca1(-), a6-integrin(high) and CD34(+).

干细胞从静息状态向激活状态的转变是一项受到精密调控的过程，该过程需要解除静息程序，并建立促进细胞周期的转录调控图谱。表观遗传过程是否通过调控干细胞状态以促进成体组织再生，目前仍不明确。
本研究发现，在成年小鼠皮肤的静息与激活毛囊干细胞（hair follicle stem cells, HFSCs）之间，抑制性组蛋白修饰H2AK119单泛素化（H2AK119ub）处于动态变化之中。在HFSCs中敲除H2AK119ub会导致无法维持静息状态，进而引发提前激活，并最终耗尽HFSC池。转录组与染色质研究表明，H2AK119ub可直接抑制HFSCs中促进增殖的转录程序，从而维持其静息状态。最后，本研究证实，毛囊微环境角质形成细胞分泌的抑制性成纤维细胞生长因子（FGF）信号，可在静息HFSCs中维持H2AK119ub的水平。综上，本研究结果表明，抑制性组蛋白标记H2AK119ub受到微环境抑制性信号的动态调控，从而阻止激活状态的过早建立，以维持干细胞的功能与存活寿命。
实验整体设计：于出生后第50天（P50）起诱导对照组与PRC1诱导型敲除（PRC1 i2KO）小鼠模型。诱导处理结束15天后，收集背部皮肤，并用手术手术刀刮除背部的脂肪层。将刮取的皮肤样本用磷酸盐缓冲液（PBS）洗涤后，置于37℃、转速60转每分钟的摇床中，用0.25%胰蛋白酶-EDTA混合液孵育1小时。孵育结束后，将包括HFSCs在内的表皮细胞从经胰酶消化的皮肤样本上刮取至培养板中。向细胞悬液中加入25毫升E培养基，随后依次通过100μm与40μm滤膜过滤。收集细胞沉淀，并用DPBS洗涤两次，随后进行细胞表面标志物染色。将细胞置于染色缓冲液（汉克斯平衡盐溶液HBSS + 2%胎牛血清）中，于冰上分别用以下抗体孵育：1:200稀释的PerCP-Cy5.5-Sca1（赛默飞世尔科技，货号45-5981-82）、1:200稀释的FITC-α6整合素（eBioscience，货号11-0493-81）、1:100稀释的APC-EpCAM（Biolegend，货号324207）以及1:20稀释的Alexa700-CD34（eBioscience，货号56-0341-82），孵育时长30分钟。孵育结束后用DPBS洗涤两次，随后进行细胞分选。HFSCs通过以下门控策略分选：EpCAM阳性、Sca1阴性、α6整合素高表达以及CD34阳性。