Dataset Information

A nuclear receptor facilitates differentiation of human PSCs into more mature hepatocytes

ABSTRACT: To start examining the mechanisms by which THRB mediated the epigenetic changes, we performed immunoprecipitation (IP) of THRB in THRB-FLAG HepG2 cells with anti-FLAG antibody, followed by mass-spectrometry to identify THRB-binding proteins.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Cell Culture

DISEASE(S): Liver Cancer

SUBMITTER: Haiting Ma

LAB HEAD: Rudolf Jaenisch

PROVIDER: PXD030665 | Pride | 2022-03-25

REPOSITORIES: Pride

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			Action	DRS
	210914_19_3haiting_IgG-Control.mgf	Mgf
	210914_19_3haiting_IgG-Control.mzxml	Mzxml
	210914_19_3haiting_IgG-Control.raw	Raw
	210914_19_3haiting_IgG-Control_peptides.pep.xml	Pepxml
	210914_19_3haiting_IgG-Control_peptides_1_1_0.mzid.gz	Mzid

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Publications

The nuclear receptor THRB facilitates differentiation of human PSCs into more mature hepatocytes.

Ma Haiting H de Zwaan Esmée E Guo Yang Eric YE Cejas Paloma P Thiru Prathapan P van de Bunt Martijn M Jeppesen Jacob F JF Syamala Sudeepa S Dall'Agnese Alessandra A Abraham Brian J BJ Fu Dongdong D Garrett-Engele Carrie C Lee Tong Ihn TI Long Henry W HW Griffith Linda G LG Young Richard A RA Jaenisch Rudolf R

Cell stem cell 20220421 5

To understand the mechanisms regulating the in vitro maturation of hPSC-derived hepatocytes, we developed a 3D differentiation system and compared gene regulatory elements in human primary hepatocytes with those in hPSC-hepatocytes that were differentiated in 2D or 3D conditions by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Regulome comparisons showed a reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and active enhancers without a reduced transcription of THRB. The addit ...[more]

PMID: 35452598

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Project description:Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ÂºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ÂºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300Âµl beads and 150Âµg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ÂºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).