A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells

Fang, Fang; Xia, Ninuo; Angulo, Benjamin; Carey, Joseph; Cady, Zackery; Durruthy-Durruthy, Jens; Bennett, Theo; Sebastiano, Vittorio; Pera, Renee A. Reijo

doi:10.1038/s41467-018-06908-5

Cited by 32 publications

(42 citation statements)

References 57 publications

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“…3C). The functional relevance of this isoform-specific recapping is still unclear, but expression isoform 2 of ZNF207 is enriched in differentiated cells, with isoform 3 being more prevalent in cells that retain stem cell-like characteristics [25].…”

Section: Resultsmentioning

confidence: 99%

mRNA 5′ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced

2019

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Until cytoplasmic recapping was discovered, decapping was thought to irreversibly destine an mRNA to degradation. Contradicting this idea, we readily observe mRNAs targeted by cytoplasmic capping in uncapped, yet stable forms. 5′ rapid amplification of cDNA ends (RACE) shows that nearly all uncapped ends correspond to capped analysis of gene expression tags and that the recapping of ZNF207 mRNA may be restricted to a single splice isoform. Here, a modified RACE approach detected uncapped 5′ RNA ends mapping to 46 mRNAs in cells expressing a dominant negative cytoplasmic capping enzyme and in normal cells. Eleven of 46 cloned mRNAs also contained splice isoform‐limiting sequences. Collectively, these data reinforce earlier work and suggest that alternative splicing may play a role in targeting transcripts for – and/or determining the position of – cytoplasmic capping.

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Section: Resultsmentioning

confidence: 99%

mRNA 5′ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced

2019

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“…Here, we describe TALE-mediated Isolation of Nuclear Chromatin (TINC), a novel epigenetic technique that allows isolation of a single genomic locus from mammalian cells for further molecular interrogation. TINC utilizes epitope tagged TALE proteins, which have previously been shown to possess the capacity to enrich a single RE from mammalian cells (Fang et al 2018). In order to minimize background contribution from cytoplasmic contaminants as well as from proteins specifically enriched by the TALE but without target locus-related functions (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…These include nucleic acid hybridization-based approaches (Ide and Dejardin 2015;Déjardin and Kingston 2009;Antão et al 2012;Kennedy-Darling et al 2014) and approaches that utilize DNA-binding proteins including LexA (Byrum et al 2012;Fujita and Fujii 2011), TetR (Pourfarzad et al 2013), Cas9 (Waldrip et al 2014;Fujita and Fujii 2013;X. Liu et al 2017;Gao et al 2018;Schmidtmann et al 2016;Tsui et al 2018;Qiu et al 2019) or TALE proteins (Fujita et al 2013;Byrum, Taverna, and Tackett 2013;Fang et al 2018). Although nucleic acid hybridization-based approaches pioneered the field, they require intensive probe-specific optimizations (Ide and Dejardin 2015;Déjardin and Kingston 2009;Antão et al 2012), which may account for why this approach has not yet been widely adopted nor translated to single copy elements in mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

“…Although nucleic acid hybridization-based approaches pioneered the field, they require intensive probe-specific optimizations (Ide and Dejardin 2015;Déjardin and Kingston 2009;Antão et al 2012), which may account for why this approach has not yet been widely adopted nor translated to single copy elements in mammalian cells. To target such challenging genomic regions in the mammalian genome, TALE (Fang et al 2018) and CRISPR/Cas9 (Waldrip et al 2014;Fujita and Fujii 2013;X. Liu et al 2017;Gao et al 2018;Schmidtmann et al 2016;Tsui et al 2018;Qiu et al 2019) approaches are starting to emerge as the systems of choice as, unlike TetR and LexA proteins, they do not rely on genetic engineering of binding sites into the target sequence.…”

Section: Introductionmentioning

confidence: 99%

“…TINC utilizes an epitope-tagged TALE in combination with a nuclei isolation and chromatin enrichment step based on Kustatscher et al, 2014 to further increase the signal-to-noise ratio. Even though the only other mammalian cell-validated TALEbased single locus isolation technique utilizes whole cell lysate (Fang et al 2018) we chose to include these two additional purification steps (nuclei and chromatin) to reduce cytoplasmic and free nuclear protein contaminant contribution. Using TINC, we interrogated the protein complex formed at the Nanog promoter in mouse ESCs.…”

Section: Introductionmentioning

confidence: 99%

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TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novelNanogregulators in mouse embryonic stem cells

Knaupp

Mohenska

Larcombe

et al. 2020

Preprint

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Cellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon crosslinking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors. Enrichment and other statistical analysesProtein classification analyses were performed with Panther ( Thomas et al. 2003), and all other ontology analyses were performed with Metascape (Zhou et al. 2019). Transcription factors were defined by collected annotation from Riken and fantom five mouse TF datasets (Carninci et al. 2005;Kanamori et al. 2004). All other epigenetic modifiers and respective metadata were obtained from the epifactors database (Medvedeva et al. 2015). The pluripotency network was obtained from ESCAPE (Embryonic Stem Cells Atlas of Pluripotency Evidence) (Xu et al. 2013(Xu et al. , 2014. Enrichment of TINC proteins within the pluripotency network was calculated with Fisher's exact test. RNA-seq data of reprogramming fibroblasts into iPSCs was obtained from Knaupp et al. 2017. Raw sequencing files were processed to raw counts as described in the original publication. The raw counts were then TMM normalized and converted to log2 CPM for all analyses. The standardized log2CPM values were centred to the mean expression for each gene. Pearson's correlation coefficients were calculated on standardized log2CPM values for all genes corresponding to proteins identified in all three TINC replicates. Protein-protein enrichment networks were visualized with Cytoscape v3.7.1 (Shannon et al. 2003). ComplexHeatmap was used for any visualization of heatmaps with default clustering parameters and all other analyses outputs were graphed using GGplot2 unless otherwise stated (Gu, Eils, and Schlesner 2016;Wickham 2016). A set seed of 123 was used in all analys...

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PTPN21‐CDS_long isoform inhibits the response of acute lymphoblastic leukemia cells to NK‐mediated lysis via the KIR/HLA‐I axis

Wang

Zhu

et al. 2020

J of Cellular Biochemistry

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Protein tyrosine phosphatase non-receptor type 21 (PTPN21) is a member of the non-receptor tyrosine phosphatase family. We have found that PTPN21 is mutated in relapsed Philadelphia chromosome-negative acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation. PTPN21 consists of three types of isoforms according to the length of the protein encoded. However, the roles of different isoforms in leukemic cells have not been elucidated. In the study, PTPN21 isoform constitution in five ALL cell lines were identified by transcriptome polymerase chain reaction combined with Sanger sequencing, and the relationship between PTPN21 isoforms and sensitivity to natural killer (NK) cells mediated killing in ALL cell lines were further assessed by knock-out of different isoforms of PTPN21 using CRISPR-Cas9 technique. Subsequently, we explored the functional mechanisms through RNA sequencing and confirmatory testing. The results showed that there was no significant change when all PTPN21 isoforms were knocked out in ALL cells, but the sensitivity of NALM6 cells with PTPN21-CDS long knock-out (NALM6-PTPN21 lk ) to NK-mediated killing was significantly increased. Whole transcriptome sequencing and further validation testing showed that human leukocyte antigen class I (HLA-I) molecules were significantly decreased, accompanied by a significantly downregulated expression of antigen presentingrelated chaperones in NALM6-PTPN21 lk cells. Our results uncovered a previously unknown mechanism that PTPN21-CDS long and CDS short isoforms may play opposite roles in NK-mediated killing in ALL cells, and showed that Abbreviations: ALL, acute lymphoblastic leukemia; HLA-I, human leukocyte antigens class I; NK cells, natural killer cells; PCR, polymerase chain reaction; PTPN21, protein tyrosine phosphatase non-receptor type 21.the endogenous PTPN21-CDS long isoform inhibited ALL cells to NK cellmediated lysis by regulating the KIR-HLA-I axis. K E Y W O R D Sacute lymphoblastic leukemia, human leukocyte antigens class I molecules, isoform, NK killing, protein tyrosine phosphatase non-receptor type 21

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A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells

Cited by 32 publications

References 57 publications

mRNA 5′ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced

mRNA 5′ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novelNanogregulators in mouse embryonic stem cells

PTPN21‐CDS_long isoform inhibits the response of acute lymphoblastic leukemia cells to NK‐mediated lysis via the KIR/HLA‐I axis

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A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells

Cited by 32 publications

References 57 publications

mRNA 5′ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced

mRNA 5′ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novelNanogregulators in mouse embryonic stem cells

PTPN21‐CDSlong isoform inhibits the response of acute lymphoblastic leukemia cells to NK‐mediated lysis via the KIR/HLA‐I axis

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PTPN21‐CDS_long isoform inhibits the response of acute lymphoblastic leukemia cells to NK‐mediated lysis via the KIR/HLA‐I axis