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Lu, Falong

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Lu, Falong
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Now showing 1 - 6 of 6
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    Embryonic Development following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation
    (Elsevier BV, 2014) MATOBA, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A; Inoue, Azusa; Zhang, Yi
    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.
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    Tet3 and DNA Replication Mediate Demethylation of Both the Maternal and Paternal Genomes in Mouse Zygotes
    (Elsevier BV, 2014) Shen, Li; Inoue, Azusa; He, Jin; Liu, Yuting; Lu, Falong; Zhang, Yi
    With the exception of imprinted genes and certain repeats, DNA methylation is globally erased during preimplantation development. Recent studies have suggested that Tet3-mediated oxidation of 5-methylcytosine (5mC) and DNA replication-dependent dilution both contribute to global paternal DNA demethylation, but demethylation of the maternal genome occurs via replication. Here we present genome-scale DNA methylation maps for both the paternal and maternal genomes of Tet3-depleted and/or DNA replication-inhibited zygotes. In both genomes, we found that inhibition of DNA replication blocks DNA demethylation independently from Tet3 function and that Tet3 facilitates DNA demethylation largely by coupling with DNA replication. For both genomes, our data indicate that replication-dependent dilution is the major contributor to demethylation, but Tet3 plays an important role, particularly at certain loci. Our study thus defines the respective functions of Tet3 and DNA replication in paternal DNA demethylation and reveals an unexpected contribution of Tet3 to demethylation of the maternal genome. •Tet3 only partially mediates paternal DNA demethylation•DNA replication is the major contributor to paternal DNA demethylation•Tet3-dependent DNA demethylation also occurs on the maternal genome•Zygotic gene activation is independent of Tet3 activity Using genome-scale DNA methylation analyses of manually isolated paternal and maternal pronuclei, Zhang and colleagues show that zygotic demethylation of both genomes is mediated by Tet3 and DNA replication, with the latter as the major contributor.
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    Loss of HDAC-Mediated Repression and Gain of NF-?B Activation Underlie Cytokine Induction in ARID1A- and PIK3CA-Mutation-Driven Ovarian Cancer
    (Elsevier BV, 2016) Kim, Minchul; Lu, Falong; Zhang, Yi
    ARID1A is frequently mutated in ovarian clear cell carcinoma (OCCC) and often co-exists with activating mutations of PIK3CA. Although induction of pro-inflammatory cytokines has been observed in this cancer, the mechanism by which the two mutations synergistically activate cytokine genes remains elusive. Here, we established an in vitro model of OCCC by introducing ARID1A knockdown and mutant PIK3CA into a normal human ovarian epithelial cell line, resulting in cell transformation and cytokine gene induction. We demonstrate that loss of ARID1A impairs the recruitment of the Sin3A-HDAC complex, while the PIK3CA mutation releases RelA from IκB, leading to cytokine gene activation. We show that an NF-κB inhibitor partly attenuates the proliferation of OCCC and improves the efficacy of carboplatin both in cell culture and in a mouse model. Our study thus reveals the mechanistic link between ARID1A/PIK3CA mutations and cytokine gene induction in OCCC and suggests that NF-κB inhibition could be a potential therapeutic option.
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    Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development
    (Elsevier BV, 2016) Lu, Falong; Liu, Yuting; Inoue, Azusa; Suzuki, Tsukasa; Zhao, Kejie; Zhang, Yi
    How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos.
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    Histone Demethylase Expression Enhances Human Somatic Cell Nuclear Transfer Efficiency and Promotes Derivation of Pluripotent Stem Cells
    (Elsevier BV, 2015) Chung, Young Gie; MATOBA, Shogo; Liu, Yuting; Eum, Jin Hee; Lu, Falong; Jiang, Wei; Lee, Jeoung Eun; Sepilian, Vicken; Cha, Kwang Yul; Lee, Dong Ryul; Zhang, Yi
    The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits its potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing efficient derivation of SCNT-derived ESCs using adult Age-related Macular Degeneration (AMD) patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine.
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    Role of Tet proteins in enhancer activity and telomere elongation
    (Cold Spring Harbor Laboratory Press, 2014) Lu, Falong; Liu, Yuting; Jiang, Lan; Yamaguchi, Shinpei; Zhang, Yi
    DNA methylation at the C-5 position of cytosine (5mC) is one of the best-studied epigenetic modifications and plays important roles in diverse biological processes. Iterative oxidation of 5mC by the ten-eleven translocation (Tet) family of proteins generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are selectively recognized and excised by thymine DNA glycosylase (TDG), leading to DNA demethylation. Functional characterization of Tet proteins has been complicated by the redundancy between the three family members. Using CRISPR/Cas9 technology, we generated mouse embryonic stem cells (ESCs) deficient for all three Tet proteins (Tet triple knockout [TKO]). Whole-genome bisulfite sequencing (WGBS) analysis revealed that Tet-mediated DNA demethylation mainly occurs at distally located enhancers and fine-tunes the transcription of genes associated with these regions. Functional characterization of Tet TKO ESCs revealed a role for Tet proteins in regulating the two-cell embryo (2C)-like state under ESC culture conditions. In addition, Tet TKO ESCs exhibited increased telomere–sister chromatid exchange and elongated telomeres. Collectively, our study reveals a role for Tet proteins in not only DNA demethylation at enhancers but also regulating the 2C-like state and telomere homeostasis.