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TET1 interactome by SILAC IP-MS in mouse ESCs

ABSTRACT: TET1 maintains hypomethylation at bivalent promoters through its catalytic activity in embryonic stem cells (ESCs). However, whether and how TET1 exerts catalytic activity-independent functions in regulating bivalent genes is not well understood. Therefore, we mapped the TET1 interactome in mouse ESCs using a SILAC IP-MS proteomics approach.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Xin Huang

LAB HEAD: Jianlong Wang

PROVIDER: PXD033587 | Pride | 2022-06-10

REPOSITORIES: Pride

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			Action	DRS
	MS121398_Tet1_IP-MS_SILAC_bioR1-1.raw	Raw
	MS121398_Tet1_IP-MS_SILAC_bioR1-2.raw	Raw
	MS121398_Tet1_IP-MS_SILAC_bioR1-3.raw	Raw
	MS121398_Tet1_IP-MS_SILAC_bioR1-4.raw	Raw
	MS121398_Tet1_IP-MS_SILAC_bioR1-5.raw	Raw

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Publications

A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency.

Huang Xin X Bashkenova Nazym N Hong Yantao Y Lyu Cong C Guallar Diana D Hu Zhe Z Malik Vikas V Li Dan D Wang Hailin H Shen Xiaohua X Zhou Hongwei H Wang Jianlong J

Cell reports 20220601 10

TET1 maintains hypomethylation at bivalent promoters through its catalytic activity in embryonic stem cells (ESCs). However, TET1 catalytic activity-independent function in regulating bivalent genes is not well understood. Using a proteomics approach, we map the TET1 interactome in ESCs and identify PSPC1 as a TET1 partner. Genome-wide location analysis reveals that PSPC1 functionally associates with TET1 and Polycomb repressive complex-2 (PRC2). We establish that PSPC1 and TET1 repress, and the ...[more]

PMID: 35675764

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Amplicon bisulfite sequencing of LINE-1 elements in TET catalytic mutants and DPPA3 KO ESCs rescued by doxycylin induced overexpression

Project description:Tet1, Tet2 and Tet1/Tet2 catalytic mutants as well as DPPA3 KO ESCs harboring a doxycyclin inducible Dppa3 transgene were induced for several days to monitor LINE-1 methylation levels.

2020-10-23 | E-MTAB-9654 | biostudies-arrayexpress

OCT4 IP-MS in Naive and Primed Human ESCs

Project description:Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes.

2021-09-12 | PXD026556 | Pride

TET1 is a maintenance DNA demethylase that prevents methylation spreading in adult cells [RNA-seq]

Project description:We report that full length TET1 (TET1-FL) overexpression fails to induce global DNA demethylation in HEK293T cells. The preferential binding of TET1-FL to hypomethylated CpG islands (CGIs) through its CXXC domain leads to its inhibited 5-hydroxymethylcytosine (5hmC) production as methylation level increases. TET1-FL-induced 5hmC accumulates at CGI edges, while TET1 knockdown induces methylation spreading from methylated edges into hypomethylated CGIs. However, TET1 can regulate gene transcription independent of its dioxygenase catalytic function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically maintains the DNA hypomethylation state of CGIs in adult cells. Genome-wdie profiling of gene expression in HEK293T cells following overexpression of wild type or catalytically mutant TET1-FL or TET1-CD

2014-06-04 | E-GEOD-49833 | biostudies-arrayexpress

A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency [ChIRP-Seq]

Project description:The mammalian Ten-eleven translocation (TET) family proteins regulate the epigenome through catalytic activity (CA)-dependent and -independent functions. While catalytic activity of TETs has been intensely studied as DNA demethylation enzymes, little is known about their CA-independent function in early embryo development. Here we defined the CA-independent function of TET1 in naïve-to-formative pluripotent states transition. Using a proteomics method, we mapped the TET1 interactome and identified Paraspeckle component 1 (PSPC1) as a partner of TET1 for transcriptional repression at the bivalent genes in early development. Genome-wide location analysis revealed that PSPC1-bound regions largely overlap with TET1 and Polycomb repressive complex 2 (PRC2) subunits. Functional studies with genetic methods indicated that PSPC1 and TET1 repress, while lncRNA Neat1 activates the bivalent genes during their transcriptional activation. In embryonic stem cell (ESC) state, Neat1 tethers TET1, PSPC1, and PRC2 at promoters. During the ESCs to formative Epiblast like stem cells (EpiLCs) differentiation, PSPC1 and TET1 repress the PRC2 affinity to nascent mRNA transcripts of bivalent genes, while Neat1 facilitates PRC2 binding to those transcripts. Our study reveals a molecular mechanism by which proteins TET1 and PSPC1, and lncRNA Neat1 dynamically regulate gene transcription by modulating the PRC2 activity in pluripotent states transition.

2022-06-07 | GSE199265 | GEO

A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency [ChIP-seq]

2022-06-07 | GSE182442 | GEO

A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency [RNA-seq]

2022-06-07 | GSE182441 | GEO

A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency [H3 ChIP-Seq]

2022-06-07 | GSE199266 | GEO

Tet1 regulates embryonic stem cell differentiation programs and development independent of its catalytic activity.

Project description:In this study: (1) we characterized the Tet1 non-catalytic functions in the regulation of ESC gene expression programs by performing transcriptomic analysis of Tet1 wild type (WT), Tet1 catalytic mutant (Mut) and Tet1 knockout (KO) mouse ESCs by RNA-seq to identify differentially expressed genes. (2) We mapped the genome-wide occupancy of endogenously FLAG-tagged Tet1-WT and Tet1-Mut in ESCs by CUT&Tag using a specific antibody against FLAG. (3) We determined how the genome-wide occupancy of the epigenetic modifiers: Ezh2, Sin3a, Chd4 and the enrichment of histone marks: H3K27me3, H3K4me3 and H3K27ac, are affected in Tet1-KO ESCs versus Tet1-WT and Tet1-Mut by CUT&Tag or CUT&RUN. (4) We analyzed methylation levels and distribution in Tet1-WT, Tet1-Mut and Tet1-KO ESCs by WGBS, to confirm Tet1 non-catalytic targets with no differential methylation. (5) We explored whether Tet1 non-catalytic functions play a role in chromatin accessibility by performing ATAC-seq in Tet1-WT, Tet1-Mut and Tet1-KO ESCs.

2022-01-25 | GSE176389 | GEO

eIF4A3 interactome by IP-MS in mouse ESCs

Project description:RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNAi screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). We mapped the eIF4A3 interactomes in mouse ESCs, revealing that eIF4A3 is widely involved in the post-transcriptional regulation of gene expression. Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes cellular differentiation via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during cell cycle progression of ESCs. Our results reveal a previously unappreciated role of eIF4A3 and its associated EJC in the post-transcriptional cell cycle control in maintaining stem cell pluripotency.

2023-03-11 | PXD031362 | Pride

TET1 is a maintenance DNA demethylase that prevents methylation spreading in adult cells [cDNA microarray]

Project description:We report that full length TET1 (TET1-FL) overexpression fails to induce global DNA demethylation in HEK293T cells. The preferential binding of TET1-FL to hypomethylated CpG islands (CGIs) through its CXXC domain leads to its inhibited 5-hydroxymethylcytosine (5hmC) production as methylation level increases. TET1-FL-induced 5hmC accumulates at CGI edges, while TET1 knockdown induces methylation spreading from methylated edges into hypomethylated CGIs. However, TET1 can regulate gene transcription independent of its dioxygenase catalytic function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically maintains the DNA hypomethylation state of CGIs in adult cells. HEK293T cells were transfected with wild type or catalytically mutant TET1 expression plasmids (TET1-CD, mTET1-CD, TET1-FL and mTET1-FL), or subjected to shRNA-mediated TET1 knockdown. Total RNA samples were extracted and assayed on Affymetrix microarrays

2014-06-04 | E-GEOD-50016 | biostudies-arrayexpress

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