Heterochromatin loosening by the Oct4 linker region facilitates Klf4 binding and iPSC reprogramming
2019; Springer Nature; Volume: 39; Issue: 1 Linguagem: Inglês
10.15252/embj.201899165
ISSN1460-2075
AutoresKeshi Chen, Qi Long, Guangsuo Xing, Tianyu Wang, Yi Wu, Linpeng Li, Juntao Qi, Yanshuang Zhou, Bochao Ma, Hans R. Schöler, Jinfu Nie, Duanqing Pei, Xingguo Liu,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle1 October 2019free access Transparent process Heterochromatin loosening by the Oct4 linker region facilitates Klf4 binding and iPSC reprogramming Keshi Chen orcid.org/0000-0003-0402-4555 CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Qi Long orcid.org/0000-0002-7003-0104 CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Guangsuo Xing CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Institute of Physical Science and Information Technology, Anhui University, Hefei, China Search for more papers by this author Tianyu Wang orcid.org/0000-0003-4169-8268 CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Yi Wu CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Linpeng Li CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Juntao Qi CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Yanshuang Zhou CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Bochao Ma CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Hans R Schöler Department for Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany Search for more papers by this author Jinfu Nie CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Duanqing Pei Corresponding Author [email protected] orcid.org/0000-0002-5222-014X CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Xingguo Liu Corresponding Author [email protected] orcid.org/0000-0001-7060-8204 CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Keshi Chen orcid.org/0000-0003-0402-4555 CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Qi Long orcid.org/0000-0002-7003-0104 CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Guangsuo Xing CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Institute of Physical Science and Information Technology, Anhui University, Hefei, China Search for more papers by this author Tianyu Wang orcid.org/0000-0003-4169-8268 CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Yi Wu CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Linpeng Li CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Juntao Qi CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Yanshuang Zhou CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Bochao Ma CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Hans R Schöler Department for Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany Search for more papers by this author Jinfu Nie CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Duanqing Pei Corresponding Author [email protected] orcid.org/0000-0002-5222-014X CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Xingguo Liu Corresponding Author [email protected] orcid.org/0000-0001-7060-8204 CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China Search for more papers by this author Author Information Keshi Chen1,2, Qi Long1,2, Guangsuo Xing1,2,3, Tianyu Wang1,2, Yi Wu1,2, Linpeng Li1,2, Juntao Qi1,2, Yanshuang Zhou1,2, Bochao Ma1,2, Hans R Schöler4, Jinfu Nie1,2, Duanqing Pei *,1,2 and Xingguo Liu *,1,2 1CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China 2Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China 3Institute of Physical Science and Information Technology, Anhui University, Hefei, China 4Department for Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany *Corresponding author. Tel: +86 02032015201; E-mail: [email protected] *Corresponding author. Tel: +86 02032015225; E-mail: [email protected] EMBO J (2020)39:e99165https://doi.org/10.15252/embj.201899165 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The success of Yamanaka factor reprogramming of somatic cells into induced pluripotent stem cells suggests that some factor(s) must remodel the nuclei from a condensed state to a relaxed state. How factor-dependent chromatin opening occurs remains unclear. Using FRAP and ATAC-seq, we found that Oct4 acts as a pioneer factor that loosens heterochromatin and facilitates the binding of Klf4 and the expression of epithelial genes in early reprogramming, leading to enhanced mesenchymal-to-epithelial transition. A mutation in the Oct4 linker, L80A, which shows impaired interaction with the BAF complex component Brg1, is inactive in heterochromatin loosening. Oct4-L80A also blocks the binding of Klf4 and retards MET. Finally, vitamin C or Gadd45a could rescue the reprogramming deficiency of Oct4-L80A by enhancing chromatin opening and Klf4 binding. These studies reveal a cooperation between Oct4 and Klf4 at the chromatin level that facilitates MET at the cellular level and shed light into the research of multiple factors in cell fate determination. Synopsis Chromatin relaxation is required for generation of pluripotent stem cells, but how factor-dependent chromatin opening is instructed remains poorly understood. Here, epigenetic profiling reveals Oct4 as a pioneer factor that loosens heterochromatin and cooperates with Klf4 and other chromatin remodellers during reprogramming. The Oct4 linker site L80 is required for chromatin decondensation and recruitment of Brg1. Oct4 facilitates Klf4 binding and epithelial gene expression. Oct4 reduces repressive histone marks H3K9me3 and H3K27me3. Vitamin C or Gadd45a restore the reprogramming deficiency of Oct4-L80A. Introduction Pluripotent stem cells (PSCs) can be derived from the inner cell mass of blastocysts (Evans & Kaufman, 1981) or induced from somatic cells by defined factors (Takahashi & Yamanaka, 2006; Yu et al, 2007). Induced pluripotent stem cells (iPSCs), similar to embryonic stem cells (ESCs), have an open chromatin state integrating with epigenetic and genetic programs to coordinately regulate PSC self-renewal and differentiation (Aoto et al, 2006; Meshorer et al, 2006; Guenther et al, 2010; Fussner et al, 2011; Orkin & Hochedlinger, 2011). iPSCs have attracted great attention and investigation for their potential applications in regeneration medicine. The reprogramming process has been investigated extensively at the mechanistic level (Brambrink et al, 2008; Xu et al, 2009; Mah et al, 2011). DNA methylation has been shown to play a critical role in the reestablishment of pluripotency (Meissner et al, 2008; Doege et al, 2012). Various histone modifications have been investigated. H3K4me2 is shown to undergo rapid changes, whereas the repressive H3K27me3 modification remains unchanged during reprogramming, illustrating the diversity of regulatory events at the epigenetic level (Koche et al, 2011; Yang et al, 2011; Zhang et al, 2011; Rao et al, 2015). Analysis of genome-wide binding patterns of reprogramming factors has shown that c-Myc plays a unique role during reprogramming in allowing Sox2, Klf4, and Oct4 (SKO) to engage their targets (Sridharan et al, 2009). Genome-wide mapping of reprogramming factors at an early stage of reprogramming has confirmed that c-Myc facilitates SKO chromatin engagement and has shown that Oct4, Sox2, and Klf4 serve as pioneer factors at the enhancers of genes that play critical roles in the reprogramming process (Soufi et al, 2012). Another global analysis showed that the induction process elicits two transcriptional waves—the first wave is driven by c-Myc/Klf4 and the second wave by Oct4/Sox2/Klf4 (Polo et al, 2012). By employing ATAC-seq, the reprogramming process has been mapped into two prominent phases characterized by chromatin changes: one at the initial phase defined by a massive closing of somatic chromatin and the second, prior to pluripotency, where the pluripotency chromatin opened (Knaupp et al, 2017; Li et al, 2017). Consistent with these findings, quantitative proteomics has also revealed a two-step resetting of the proteome during the first 3 days and last 3 days of reprogramming (Hansson et al, 2012). Mesenchymal-to-epithelial transition (MET) was revealed to be a necessary step for the success of MEF reprogramming, and Klf4 is responsible for the activation of epithelial marker gene Cdh1 (Li et al, 2010). At the chromatin level, remodeling factors such as Brg1, Baf155, and INO80, which are partners of Oct4, have been shown to promote reprogramming (Singhal et al, 2010; Wang et al, 2014; King & Klose, 2017), whereas other modifying enzymes such as Dot1l or Mbd3 have been shown to inhibit reprogramming (Onder et al, 2012; Rais et al, 2013). Such large datasets have enriched our understanding of the reprogramming process at multiple mechanistic levels. Chromatin structure is unique in PSCs. During reprogramming, the ectopic factors open and maintain the global chromatin state to ensure high expression of pluripotent genes (Gaspar-Maia et al, 2011). However, the mechanisms underlying the changes of chromatin state remain incompletely understood. Previously, we demonstrated that heterochromatin is reduced and becomes hyperdynamic in the early phase of reprogramming (Chen et al, 2016b). Here, we show that heterochromatin is opened by Oct4, dependent on its L80 residue. L80A-Oct4 failed to generate iPSCs and could inhibit Klf4's binding and the expression of epithelial genes. In addition, vitamin C and Gadd45 proteins were identified as capable of restoring the reprogramming deficiency of SKO-L80A. Our results show that Yamanaka factors cooperate at the chromatin level to initiate MET at the cellular level. Results Oct4 is responsible for the early dynamic changes in heterochromatin via its linker domain As we previously demonstrated that heterochromatin is reduced and becomes hyperdynamic in the early phase of reprogramming (Chen et al, 2016b), we then asked which of the three Yamanaka factors Oct4/Sox2/Klf4 is responsible for the observed chromatin loosening during reprogramming. We labeled HP1a with mCherry and histone H1 with GFP (Fig EV1A–C). HP1a-mCherry allowed us to distinguish heterochromatin and euchromatin. By selecting region of interest within HP1a foci, we performed fluorescence recovery after photobleaching (FRAP) of heterochromatin H1 and found that Oct4, but not Sox2 or Klf4, induced significant increase in mobile fractions (MFs) of heterochromatin on day 3 of reprogramming (Figs 1A and EV1C). Furthermore, we observed a decrease in HP1a foci in mouse embryonic fibroblasts (MEFs) infected with Oct4, while no significant differences were observed in MEFs infected with Klf4 or Sox2 (Fig 1B). These results suggest that Oct4 is responsible for the heterochromatin loosening in early reprogramming. Click here to expand this figure. Figure EV1. The FRAP of H1 in regions of interest (related to Fig 1) The infection efficiency of virus in reprogramming. Scale bar: 100 μm. The image of pre-bleach in FRAP analysis of H1-GFP. Hp1a-mCherry was used to distinguish heterochromatin and euchromatin. The circles indicate regions of interest. The yellow triangle indicates heterochromatin, while the blue triangle indicates euchromatin. Scale bar: 5 μm. The images of pre-bleach, bleach, and recovering nuclei in FRAP analysis of H1-GFP. The red circles indicate regions of interest which were bleached. The relative FRAP curves of heterochromatin (Het) are shown divided into two parts—the mobile fraction (MF) and the immobile fraction (IF) after bleaching recovery. Scale bar: 5 μm. Western blot showing the expression of Oct4 and its mutant Oct4-L80A. Download figure Download PowerPoint Figure 1. Oct4 loosens chromatin during reprogramming, dependent on its linker domain (see also Figs EV1 and EV2) FRAP curves of cells infected with single Yamanaka factors Sox2, Klf4, Oct4, or Oct4-L80A. The mobile fractions (MF) at 120 s post-bleach is shown in the right. n ≥ 18 cells for each group. Immunofluorescence detection of HP1a foci in MEFs transfected with single Yamanaka factor Sox2, Klf4, Oct4, and Oct4-L80A. Quantitative analysis of the ratio of the HP1a foci area to the total nuclear area revealed by DAPI staining (dashed lines) is shown in the right. Scale bar: 5 μm. n ≥ 20 cells were analyzed for each group. Metagene plot of ATAC-seq signal in MEFs infected with Flag, SKO, Sox2, Klf4, Oct4, and Oct4-L80A. Venn diagram depicting overlap among genes with more accessible chromatin in MEFs infected with SKO, Sox2, Klf4, and Oct4, compared with Flag control. Venn diagram depicting overlap between genes with more accessible chromatin in MEFs infected with Oct4 and Oct4-L80A, compared with Flag control. Number of genes with more accessible chromatin in MEFs infected with SKO, Sox2, Klf4, Oct4, and Oct4-L80A, compared with Flag control. Selected genomic views of the ATAC-seq data are shown for the indicated gene loci. Gene Ontology (GO) analysis for the genes with more accessible chromatin in MEFs infected with SKO, Sox2, Klf4, Oct4, and Oct4-L80A, compared with Flag control. White boxes mean the indicated GO terms are not involved. Transcription factor motif analysis of genes with more accessible chromatin in MEFs infected with SKO, Sox2, Klf4, Oct4, and Oct4-L80A, compared with Flag control. The motifs for transcription factors are indicated on the right of the heatmap. White boxes mean the indicated motifs are not significantly detected. ImmunoFISH images showing the localizations of endogenous Oct4 locus and HP1a foci in MEFs infected with wild-type or Oct4-L80A. Scale bar: 5 μm. Summary of percentage of co-localizations at the Oct4 locus and HP1a foci is shown. n ≥ 72 cells for each group. Data information: In (A), data are presented as mean ± SEM; in (B), data are presented as mean ± SD. P-values were calculated using an unpaired two-tailed t-test. *P ≤ 0.05, ***P ≤ 0.001 Download figure Download PowerPoint To understand the details of heterochromatin loosening by Oct4 in reprogramming, we performed transposase-accessible chromatin sequencing (ATAC-seq) on MEFs infected with Flag control, SKO, or each Yamanaka factor individually for three days. Peaks of open chromatin and gene loci with open chromatin were identified in the ATAC-seq results and were highly consistent between independent experiments (R > 0.9) (Fig EV2A and B). Quantification of the AT
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