Changhao Fu

All Publications

• LncRNA Osilr9 coordinates promoter DNA demethylation and the intrachromosomal loop structure required for maintaining stem cell pluripotency. Molecular therapy : the journal of the American Society of Gene Therapy Zhu, Y., Yan, Z., Fu, C., Wen, X., Jia, L., Zhou, L., Du, Z., Wang, C., Wang, Y., Chen, J., Nie, Y., Wang, W., Cui, J., Wang, G., Hoffman, A. R., Hu, J., Li, W. 2022

  Abstract

  Nuclear reprogramming of somatic cells into a pluripotent status has the potential to create patient-specific pluripotent stem cells (iPSCs) for regenerative medicine. Currently, however, the epigenetic mechanisms underlying this pluripotent reprogramming are poorly understood. To delineate this epigenetic regulatory network, we utilized a chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to identify long noncoding RNAs (lncRNAs) embedded in the 3-dimensional intrachromosomal architecture of stem cell core factor genes. By combining CRIST-seq and RNA-seq, we identified Osilr9 (Oct4-Sox2 interacting lncRNA 9) as a pluripotency-associated lncRNA. Osilr9 expression was associated with the status of stem cell pluripotency in reprogramming. Using shRNA knockdown, we showed that this lncRNA was required for the optimal maintenance of stem cell pluripotency. Overexpression of Osilr9 induced robust activation of endogenous stem cell core factor genes in fibroblasts. Osilr9 participated in the formation of the intrachromosomal looping required for maintenance of pluripotency. After binding to the Oct4 promoter, Osilr9 recruited the DNA demethylase TET1, leading to promoter demethylation. These data demonstrate that Osilr9 is a critical chromatin epigenetic modulator that coordinates the promoter activity of core stem cell factor genes, highlighting the critical role of pluripotency-associated lncRNAs in stem cell pluripotency and reprogramming.

  View details for DOI 10.1016/j.ymthe.2022.12.010

  View details for PubMedID 36523163

• Author Correction: Chromatin lncRNA Platr10 controls stem cell pluripotency by coordinating an intrachromosomal regulatory network. Genome biology Du, Z., Wen, X., Wang, Y., Jia, L., Zhang, S., Liu, Y., Zhou, L., Li, H., Yang, W., Wang, C., Chen, J., Hao, Y., Salgado Figueroa, D., Chen, H., Li, D., Chen, N., Celik, I., Zhu, Y., Yan, Z., Fu, C., Liu, S., Jiao, B., Wang, Z., Zhang, H., Gulsoy, G., Luo, J., Qin, B., Gao, S., Kapranov, P., Esteban, M. A., Zhang, S., Li, W., Ay, F., Chen, R., Hoffman, A. R., Cui, J., Hu, J. 2021; 22 (1): 272

  View details for DOI 10.1186/s13059-021-02487-9

  View details for PubMedID 34544465

• Chromatin lncRNA Platr10 controls stem cell pluripotency by coordinating an intrachromosomal regulatory network. Genome biology Du, Z., Wen, X., Wang, Y., Jia, L., Zhang, S., Liu, Y., Zhou, L., Li, H., Yang, W., Wang, C., Chen, J., Hao, Y., Chen, H., Li, D., Chen, N., Celik, I., Zhu, Y., Yan, Z., Fu, C., Liu, S., Jiao, B., Wang, Z., Zhang, H., Gulsoy, G., Luo, J., Qin, B., Gao, S., Kapranov, P., Esteban, M. A., Zhang, S., Li, W., Ay, F., Chen, R., Hoffman, A. R., Cui, J., Hu, J. 2021; 22 (1): 233

  Abstract

  BACKGROUND: A specific 3-dimensional intrachromosomal architecture of core stem cell factor genes is required to reprogram a somatic cell into pluripotency. As little is known about the epigenetic readers that orchestrate this architectural remodeling, we used a novel chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to profile long noncoding RNAs (lncRNAs) in the Oct4 promoter.RESULTS: We identify Platr10 as an Oct4 - Sox2 binding lncRNA that is activated in somatic cell reprogramming. Platr10 is essential for the maintenance of pluripotency, and lack of this lncRNA causes stem cells to exit from pluripotency. In fibroblasts, ectopically expressed Platr10 functions in trans to activate core stem cell factor genes and enhance pluripotent reprogramming. Using RNA reverse transcription-associated trap sequencing (RAT-seq), we show that Platr10 interacts with multiple pluripotency-associated genes, including Oct4, Sox2, Klf4, and c-Myc, which have been extensively used to reprogram somatic cells. Mechanistically, we demonstrate that Platr10 helps orchestrate intrachromosomal promoter-enhancer looping and recruits TET1, the enzyme that actively induces DNA demethylation for the initiation of pluripotency. We further show that Platr10 contains an Oct4 binding element that interacts with the Oct4 promoter and a TET1-binding element that recruits TET1. Mutation of either of these two elements abolishes Platr10 activity.CONCLUSION: These data suggest that Platr10 functions as a novel chromatin RNA molecule to control pluripotency in trans by modulating chromatin architecture and regulating DNA methylation in the core stem cell factor network.

  View details for DOI 10.1186/s13059-021-02444-6

  View details for PubMedID 34412677

• Therapeutic Antitumor Efficacy of Cancer Stem Cell-Derived DRibble Vaccine on Colorectal Carcinoma INTERNATIONAL JOURNAL OF MEDICAL SCIENCES Fu, C., Tian, G., Duan, J., Liu, K., Zhang, C., Yan, W., Wang, Y. 2021; 18 (14): 3249-3260

  Abstract

  Dendritic cell (DC)-based immunotherapy has been a promising strategy for colon cancer therapy, but the efficacy of dendritic cell vaccines is in part limited by immunogenicity of loaded antigens. In this study, we aimed to identify a putative tumor antigen that can generate or enhance anti-tumor immune responses against colon cancer. CD44+ colon cancer stem cells (CCSCs) were isolated from mouse colorectal carcinoma CT-26 cell cultures and induced to form defective ribosomal products-containing autophagosome-rich blebs (DRibbles) by treatment with rapamycin, bortezomib, and ammonium chloride. DRibbles were characterized by western blot and transmission electron microscopy. DCs generated from the mice bone marrow monocytes were cocultured with DRibbles, then surface markers of DCs were analyzed by flow cytometry. Meanwhile, the efficacy of DRibble-DCs was examined in vivo. Our results showed that CCSC-derived DRibbles upregulated CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II on DCs and induced proliferation of mouse splenic lymphocytes and CD8+ T cells. In a model of colorectal carcinoma using BALB/c mice with robust tumor growth and mortality, DC vaccine pulsed with CCSC-derived DRibbles suppressed tumor growth and extended survival. A lactate dehydrogenase test indicated a strong cytolytic activity of cytotoxic T-cells derived from mice vaccinated with CCSC-derived DRibbles against CT-26 cells. Furthermore, flow cytometry analyses showed that the percentages of IFN-γ-producing CD8+ T-cells were increased in SD-DC group compare with the other groups. These findings provide a rationale for novel immunotherapeutic anti-tumor approaches based on DRibbles derived from colon cancer stem cells.

  View details for DOI 10.7150/ijms.61510

  View details for Web of Science ID 000683304200020

  View details for PubMedID 34400894

  View details for PubMedCentralID PMC8364449

• Efficacy of atovaquone on EpCAM+CD44+ HCT-116 human colon cancer stem cells under hypoxia. Experimental and therapeutic medicine Fu, C., Xiao, X., Xu, H., Lu, W., Wang, Y. 2020; 20 (6): 286

  Abstract

  Tumor hypoxia contributes to the development of resistance to chemotherapeutic drugs in several human cancer cell lines. Atovaquone, an anti-malaria drug approved by the US Food and Drug Administration, has recently demonstrated anti-cancer effects in vitro and in vivo in several cancer models. To assess the potential of atovaquone as an anti-cancer agent under hypoxia in colorectal carcinoma, EpCAM+CD44+ colon cancer stem cells were isolated from HCT-116 human colon cancer cells through magnetic-activated cell sorting. The efficacy of atovaquone on cytotoxicity, tumorsphere formation, apoptosis, invasion and cell-cycle progression under hypoxic conditions were evaluated. MTS assays indicated that atovaquone inhibited the proliferation of EpCAM+CD44+ HCT-116 cells with a half-maximal inhibitory concentration of 15 M. Atovaquone inhibited tumorsphere formation and cell proliferation by causing cell-cycle arrest in S-phase, which induced apoptosis of EpCAM+CD44+ HCT-116 cells, as detected by Annexin V-FITC/PI double staining assays, and caused mitochondrial membrane potential depolarization, as determined by a JC-1 staining assay. Reverse transcription-quantitative PCR demonstrated increased expression of Bax and downregulation of Bcl-2. Transwell invasion assays indicated that atovaquone inhibited the invasiveness of EpCAM+CD44+ HCT-116 cells under hypoxia, which was associated with upregulation of MMP-2 and -9 and increased expression of tissue inhibitor of MMPs (TIMP)-1. Taken together, atovaquone reduced the tumorsphere formation and invasion ability of EpCAM+CD44+ HCT-116 cells, at least in part by increasing the expression of TIMP-1 and downregulating the expression of MMP-2 and -9, as well as the cells' viability by inducing cell-cycle arrest in S-phase and induction of apoptosis via the Bcl-2/Bax pathway under hypoxic conditions. Further studies are warranted to explore the mechanisms of action of atovaquone as a promising anticancer agent in the treatment of colorectal carcinoma.

  View details for DOI 10.3892/etm.2020.9416

  View details for PubMedID 33209130

• Osblr8 orchestrates intrachromosomal loop structure required for maintaining stem cell pluripotency. International journal of biological sciences Zhu, Y., Yan, Z., Du, Z., Zhang, S., Fu, C., Meng, Y., Wen, X., Wang, Y., Hoffman, A. R., Hu, J. F., Cui, J., Li, W. 2020; 16 (11): 1861-1875

  Abstract

  Induced pluripotent stem cells (iPSCs), derived from reprogramming of somatic cells by a cocktail of transcription factors, have the capacity for unlimited self-renewal and the ability to differentiate into all of cell types present in the body. iPSCs may have therapeutic potential in regenerative medicine, replacing injured tissues or even whole organs. In this study, we examine epigenetic factors embedded in the specific 3-dimensional intrachromosomal architecture required for the activation of endogenous pluripotency genes. Using chromatin RNA in situ reverse transcription sequencing (CRIST-seq), we identified an Oct4-Sox2 binding long noncoding RNA, referred as to Osblr8, that is present in association with pluripotency status. Osblr8 was highly expressed in iPSCs and E14 embryonic stem cells, but it was silenced in fibroblasts. By using shRNA to knock down Osblr8, we found that this lncRNA was required for the maintenance of pluripotency. Overexpression of Osblr8 activated endogenous stem cell core factor genes. Mechanistically, Osblr8 participated in the formation of an intrachromosomal looping structure that is required to activate stem cell core factors during reprogramming. In summary, we have demonstrated that lncRNA Osblr8 is a chromatin architecture modulator of pluripotency-associated master gene promoters, highlighting its critical epigenetic role in reprogramming.

  View details for DOI 10.7150/ijbs.45112

  View details for PubMedID 32398955

  View details for PubMedCentralID PMC7211171

• Osblr8 orchestrates intrachromosomal loop structure required for maintaining stem cell pluripotency INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES Zhu, Y., Yan, Z., Du, Z., Zhang, S., Fu, C., Meng, Y., Wen, X., Wang, Y., Hoffman, A. R., Hu, J., Cui, J., Li, W. 2020; 16 (11): 1861–75

  View details for DOI 10.7150/ijbs.45112

  View details for Web of Science ID 000528063100009