Early reactivation of clustered genes on the inactive X chromosome during somatic cell reprogramming

Aizawa, Shiho; Nishimura, Ken; Mondejar, Gonzalo Seminario; Kumar, Arun; Bui, Phuong Linh; Tran, Thi Hai Yen; Kuno, Akihiro; Muratani, Masafumi; Kobayashi, Shin; Nabekura, Tsukasa; Shibuya, Akira; Sugihara, Eiji; Sato, Takaaki; Fukuda, Aya; Hayashi, Yohei; Hisatake, Koji

doi:10.1016/j.stemcr.2021.11.008

Cited by 3 publications

(3 citation statements)

References 56 publications

(77 reference statements)

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“…Total RNA was isolated from cells using ISOGEN (Nippon Gene), and reverse transcription was performed using Superscript III First-Strand Synthesis System (Thermo Fisher Scientific) using the primer (5′-TGGCCACTTTGTCACACTAC-3′) corresponding to SeV NP gene. Quantitative PCR (qPCR) analyses were performed as described previously [ 73 ]. Primers are listed in Table S 2 .…”

Section: Methodsmentioning

confidence: 99%

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors

Kishimoto,

Nishimura,

Morishita

et al. 2024

J Biol Eng

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Background Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer. Results We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes. Conclusion SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.

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Section: Methodsmentioning

confidence: 99%

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors

Kishimoto,

Nishimura,

Morishita

et al. 2024

J Biol Eng

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“…The amplified cDNAs were inserted into SeVdp vector cDNA using an In-Fusion HD Cloning Kit (TaKaRa Bio, Shiga, Japan). To prepare plasmids or retroviral vectors expressing gRNA for CAPTURE method, gRNA sequences designed by using CRISPR direct ( ) were inserted to pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene, Watertown, MA, USA) or pMCs∆YY1-U6-Puro plasmid [ 55 ], respectively. pMCs∆YY1-U6-Puro was also used to create a retroviral vector expressing shRNA (MLV(U6-shRNA)) as described previously [ 55 ].…”

Section: Methodsmentioning

confidence: 99%

“…To prepare plasmids or retroviral vectors expressing gRNA for CAPTURE method, gRNA sequences designed by using CRISPR direct ( ) were inserted to pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene, Watertown, MA, USA) or pMCs∆YY1-U6-Puro plasmid [ 55 ], respectively. pMCs∆YY1-U6-Puro was also used to create a retroviral vector expressing shRNA (MLV(U6-shRNA)) as described previously [ 55 ]. Sequences of shRNA were designed using BLOCK-iT™ RNAi Designer (Thermo Fisher Scientific, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Locus-Specific Isolation of the Nanog Chromatin Identifies Regulators Relevant to Pluripotency of Mouse Embryonic Stem Cells and Reprogramming of Somatic Cells

Burramsetty

Nishimura

Kishimoto

et al. 2022

IJMS

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Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the Nanog promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells. Gene ontology analysis revealed that the identified proteins, including many RNA-binding proteins (RBPs), are enriched in RNA-related functions and gene expression. ChIP-qPCR experiments confirmed that BCLAF1, FUBP1, MSH6, PARK7, PSIP1, and THRAP3 occupy the Nanog promoter region in mESCs. Knockdown experiments of these factors show that they play varying roles in self-renewal, pluripotency gene expression, and differentiation of mESCs as well as in the reprogramming of somatic cells. Our results show the utility of unbiased identification of chromatin-associated proteins on a pluripotency gene in mESCs and reveal the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.

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Downregulation of Odd-Skipped Related 2, a Novel Regulator of Epithelial-Mesenchymal Transition, Enables Efficient Somatic Cell Reprogramming

et al. 2022

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Somatic cell reprogramming proceeds through a series of events to generate induced pluripotent stem cells (iPSCs). The early stage of reprogramming of mouse embryonic fibroblasts (MEFs) is characterized by rapid cell proliferation and morphological changes, which are accompanied by downregulation of mesenchyme-associated genes. However, the functional relevance of their downregulation to reprogramming remains poorly defined. In this study, we have screened transcriptional regulators that are downregulated immediately upon reprogramming, presumably through direct targeting by reprogramming factors. To test if these transcriptional regulators impact reprogramming when expressed continuously, we generated an expression vector that harbors human cytomegalovirus upstream open reading frame 2 (uORF2), which reduces translation to minimize the detrimental effect of an expressed protein. Screening of transcriptional regulators with this expression vector revealed that downregulation of odd-skipped related 2 (Osr2) is crucial for efficient reprogramming. Using a cell-based model for epithelial-mesenchymal transition (EMT), we show that Osr2 is a novel EMT regulator that acts through induction of TGF-β signaling. During reprogramming, Osr2 downregulation not only diminishes TGF-β signaling but also allows activation of Wnt signaling, thus promoting mesenchymal-epithelial transition (MET) toward acquisition of pluripotency. Our results illuminate the functional significance of Osr2 downregulation in erasing the mesenchymal phenotype at an early stage of somatic cell reprogramming.

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Early reactivation of clustered genes on the inactive X chromosome during somatic cell reprogramming

Cited by 3 publications

References 56 publications

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors

Locus-Specific Isolation of the Nanog Chromatin Identifies Regulators Relevant to Pluripotency of Mouse Embryonic Stem Cells and Reprogramming of Somatic Cells

Downregulation of Odd-Skipped Related 2, a Novel Regulator of Epithelial-Mesenchymal Transition, Enables Efficient Somatic Cell Reprogramming

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