The detailed molecular mechanisms regarding how

The detailed molecular mechanisms regarding how H1foo enhances the reprogramming efficiency in iPSC generation and why OSKH-iPSCs exhibit improved quality remain elusive. The higher-order chromatin structure is crucially dependent on architectural chromatin proteins, including the family of linker histone proteins. Although somatic tsa hdac contain numerous linker histone variants, only one, H1foo, is present in mouse oocytes (Tanaka et al., 2001). In the mouse egg, somatic linker histones in sperm-derived chromatin are rapidly replaced by H1foo after fertilization (Tanaka et al., 2001). In SCNT oocytes, the somatic linker histone H1c in the donor chromatin is also rapidly replaced by H1foo in mice (Gao et al., 2004; Teranishi et al., 2004). In Xenopus SCNT, oocyte-specific linker histone B4 loading to genome-wide somatic chromatin is required for successful reprogramming (Jullien et al., 2010, 2014; Miyamoto et al., 2007). In the early phase of the reprogramming process, global loss of histone H3 lysine 27 trimethylation (H3K27me3) occurs and epigenetic modification affects the status of heterochromatin (Hussein et al., 2014). In our study, H1foo reduced the heterochromatin area, which is consistent with previous reports that H1foo keeps chromatin looser than somatic H1 and other linker histones, and may support the generation of a more suitable chromatin state for reprogramming. These data suggest that dominant occupancy of oocyte-specific linker histone in donor chromatin may be required for successful reprogramming and might erase the parental epigenetic status. To determine whether innate H1foo would cooperatively induce reprogramming during iPSC generation by OSK, we examined H1foo expression during iPSC generation by OSK. We did not detect H1foo expression, which suggests that H1foo is not essential for OSK-dependent reprogramming. Therefore, we did not perform loss-of-function experiments such as H1foo knockdown by small interfering RNA. H1foo induced successful reprogramming for iPSC generation in a stringent assay, thus contributing to chimerism and germline transmission. Although in vivo experiments cannot be performed in humans, it is important to generate high-quality iPSCs without variation among different iPSC lines.
Experimental Procedures Details of all procedures are available in Supplemental Experimental Procedures.
Author Contributions
Acknowledgments Nanog-GFP-IRES-puro transgenic mice were kindly provided from Dr. Shinya Yamanaka. Several ESCs were kindly donated: R1 ESC from Dr. John C. Roder, B6J-23ˆ(UTR) ESC from Dr. Fumihiro Sugiyama, and SCNT-ESC (B6mt-1) from Riken BioResource Center. This study was supported in part by research grants from Grants-in-Aid for Scientific Research (JSPS KAKENHI grant numbers 24117716, 26670408, 15H01521, 15K14431), the Highway Program for Realization of Regenerative Medicine from Japan Science and Technology Agency, the Program for Intractable Diseases Research utilizing Disease-specific iPSCs from the Japan Agency For Medical Research and Development (AMED), Translational Research Network Program from AMED, and Keio University Medical Science Fund. H.O. is a Founding Scientist and a paid SAB of San Bio Co. Ltd.
Introduction Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by ectopic expression of defined transcription factors (OCT3/4, KLF4, SOX2, and c-MYC, hereafter referred to as OKSM) (Takahashi and Yamanaka, 2006). While the changes in gene expressions and epigenetic modifications during reprogramming have been well studied (Hussein et al., 2014; Koche et al., 2011; Koga et al., 2014; Mikkelsen et al., 2008; O\'Malley et al., 2013; Polo et al., 2012), the changes in activities of signaling pathways have not been extensively studied. The Wnt signaling pathway controls the pluripotency of embryonic stem cells (ESCs) (Sato et al., 2004). Wnt ligands inhibit GSK3 activity, resulting in β-catenin stabilization. Stabilized β-catenin then translocates into the nucleus and regulates gene expression. Mouse ESCs secrete Wnt ligands, and the autocrine Wnt activity is required for the maintenance of pluripotency (ten Berge et al., 2011). Mouse ESCs can even be maintained in the so-called 2i culture condition, the GSK3 inhibitor plus the MEK inhibitor (Ying et al., 2008). While Wnt/β-catenin signaling activates self-renewal of ESCs, it also plays a critical role in the initiation of differentiation (Murry and Keller, 2008), suggesting its divergent role in ESCs.