Overlapping and distinct roles of LIN28 paralogs in reprogramming and stem cell metabolism
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE66838 | GEO | 2015/12/31
SECONDARY ACCESSION(S): PRJNA278068
REPOSITORIES: GEO
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Project description:The RNA binding protein paralogs LIN28A and LIN28B play critical roles in embryonic development, tumorigenesis, and pluripotency. Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by overexpression of OCT4 and SOX2 together with NANOG and LIN28A (OSNA), but the exact roles LIN28A/B play are still poorly understood. Here we show that LIN28B likewise promotes reprogramming, and that reactivation of both endogenous LIN28A and B loci are required for maximal reprogramming efficiency. The LIN28B locus has open chromatin and is activated early during reprogramming, whereas LIN28A is activated at later stages by OCT4, marking the transition to bona fide iPS cells. By proteomic and metabolomic analysis, we demonstrate that LIN28A chiefly regulates let-7 and protein translation, whereas LIN28B promotes embryonic metabolism by repressing oxidative phosphorylation and enhancing glycolysis. Thus, LIN28A and LIN28B play independent and cooperative roles in reprogramming and pluripotent stem cell metabolism. 8 samples in total were analyzed. 2 replicates for each phenotypes. Controls are the wild type (flox) without lin28a/b loss of function.
2015-12-31 | E-GEOD-66838 | biostudies-arrayexpress
Project description:Lin28a or Lin28b single knockout or double knockout MEF cells reprogrammed to iPS cells Lin28a or Lin28b single knockout or double knockout MEF cells reprogrammed to iPS cells
2016-05-24 | GSE67568 | GEO
Project description:Human LIN28A and B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ~3,000 mRNAs at ~9,500 sites located in the 3M-bM-^@M-^YUTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines, and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell-cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some, but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. To assess whether the transcripts identified by PAR-CLIP are regulated by LIN28B we analyzed the mRNA levels of LIN28B overexpressing and LIN28B-depleted cells using microarrays. Transcripts crosslinked to LIN28B were slightly downregulated upon LIN28B knockdown compared to LIN28B overexpression indicating that LIN28B stabilizes transcripts. The RBP LIN28B was depleted by siRNAs and the expression levels was compared to mock-transfected HEK293 cells The RBP LIN28B was depleted by siRNAs and the expression levels was compared to mock-transfected HEK293 cells
2013-02-26 | E-GEOD-44613 | biostudies-arrayexpress
Project description:Human LIN28A and B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ~3,000 mRNAs at ~9,500 sites located in the 3M-bM-^@M-^YUTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines, and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell-cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some, but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. To assess whether miRNAs are regulated by LIN28B we analyzed the miRNA levels of LIN28B overexpressing and LIN28B-depleted cells using small RNA cDNA library sequencing. The RBP LIN28B was depleted by siRNAs and the expression levels was compared to mock-transfected HEK293 cells.
2013-02-26 | E-GEOD-44568 | biostudies-arrayexpress
Project description:A single protein can be multifaceted depending on the cellular contexts and interacting molecules. LIN28A is an RNA-binding protein that governs developmental timing, cellular proliferation, differentiation, stem cell pluripotency, and metabolism. In addition to its best-known roles in microRNA biogenesis, diverse molecular roles have been recognized. In the nervous system, LIN28A is known to play critical roles in proliferation and differentiation of neural progenitor cells (NPC). We profiled the endogenous LIN28A-interacting proteins in NPC differentiated from human induced pluripotent stem (iPS) cells using immunoprecipitation and liquid chromatography-tandem mass spectrometry. We identified over 500 LIN28A-interacting proteins, including 156 RNA-independent interactors. Functions of these proteins span a wide range of gene regulatory processes. Our analysis opens multiple avenues for elaborating molecular roles and characteristics of LIN28A.
| MSV000084371 | MassIVE
Project description:Lin28A and its paralog Lin28B are RNA binding proteins that regulate gene expression by inhibition of the maturation of the Per/Pri Let-7 miRNAs family and also via Let-7 independent mechanism. To study the effect of Lin28A ectopic-expression on mouse embryonic development we used a transgenic mice strain that combine a Cre-Lox system and a Tet-On system (herein Lox-stop-Lox-TetOn-Lin28A mice) and enable spatial and temporal control of transgenic Lin28A over-expression. Here we show that global Lin28A over-expression (by crossing Lox-stop-Lox-TetOn-Lin28A males with VasaCre females) in the mouse embryo prevents embryonic lung development. To study the precise developmental stage affected by Lin28A expression we preformed RNA sequencing analysis for transgenic and control lungs from E12.5, E14.5, E16.5 and E18.5 embryos. This analysis revealed that the development of the lung was delayed but not completely abrogated by Lin28A expression. We further showed that the effect of Lin28A expression in the mesenchymal cells (by crossing Lox-stop-Lox-TetOn-Lin28A males with Wt1Cre females) of the lung leads to the same phenotype as global Lin28A expression while expression in the lung epithelial cells (by crossing the Lox-stop-Lox-TetOn-Lin28A males with Nkx2.1Cre females) result in a milder developmental phenotype.
2019-09-22 | GSE122919 | GEO
Project description:Human LIN28A and B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ~3,000 mRNAs at ~9,500 sites located in the 3’UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines, and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell-cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some, but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. To assess whether the transcripts identified by PAR-CLIP are regulated by LIN28B we analyzed the mRNA levels of LIN28B overexpressing and LIN28B-depleted cells using microarrays. Transcripts crosslinked to LIN28B were slightly downregulated upon LIN28B knockdown compared to LIN28B overexpression indicating that LIN28B stabilizes transcripts. The RBP LIN28B was depleted by siRNAs and the expression levels was compared to mock-transfected HEK293 cells
2013-02-26 | GSE44613 | GEO
Project description:Ten-eleven translocation (TET) family enzymes can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) in DNA and have been proposed as potential DNA demethylase candidates1. Evidences from recent studies indicated that Tet1 is predominantly expressed in ES cells and plays dual functions in promoting transcription of pluripotency genes and as well as participating in the repression of developmental genes by facilitating recruitment of PRC21-5. These studies further raised the possibility that Tet1 might play a role in somatic cell reprogramming. Here, we provide evidence showing that Tet1 can substitute for pluripotent transcription factors in reprogramming differentiated somatic cells to pluripotent stem cells. Tet1 can replace any one of the four traditional transcription factors including Oct4, Sox2, Klf4 and c-Myc during somatic cell reprogramming. Subsequently, the chimeric mice with germline transmission capacity could be efficiently produced from all induced pluripotent stem (iPS) cell lines reprogrammed by OT (Oct4, Tet1), TSKM (Tet1, Sox2, Klf4, c-Myc), OTK, OTKM and OSTM combinations. Furthermore, the TSKM-reprogrammed iPS cells without using Oct4 could produce viable full-term iPS mice with normal fertility through tetraploid complementation and secondary iPS cells could be induced subsequently from the somatic cells retrieved from the iPS mice. Moreover, we demonstrated that conversion of 5mC into 5hmC in Nanog promoter occurred during reprogramming, which might account in part for the mechanism of Tet1 mediated reprogramming. To our knowledge, our study provides the first evidence demonstrating that DNA modifying enzyme Tet1 can replace the pluripotent transcription factors to reprogram differentiated somatic cells to iPS cells. Gene expression profile of iPS cells and ES cells were generated by Affymetrix Mouse Gene 1.0 ST Array. The Gene expression profile of ES cell R1 was used as control. Three biological repeats were included for each line.
2011-05-07 | E-GEOD-29102 | biostudies-arrayexpress
Project description:The heterochronic genes Lin28a/b and let-7 are well-established regulators of invertebrate development, however their functions in patterning the mammalian body plan remain unexplored. In this study, we discovered a novel function of Lin28/let-7 in controlling caudal vertebrae number during body axis formation. We found that FoxD1-driven overexpression of Lin28a or LIN28B led to a striking increase in caudal vertebrae number, whereas loss of Lin28a stunted tail formation. Accordingly, perturbation of the let-7 microRNA family led to the opposite phenotypes. We further show that overexpressing Lin28a in embryos resulted in increased tail bud cell proliferation, whereas Lin28a KO decreased cellular proliferation. This was accompanied by a transcriptional shift, including down-regulation of the neural marker Sox2, and resulted in a pro-mesodermal phenotype with decreased proportions of neural tissue relative to mesoderm in Lin28a-overexpressing embryos. Strikingly, Lin28a KO and let-7 over-expression demonstrated opposite effects, suggesting that the Lin28/let-7 pathway acts upstream of the signaling pathways controlling the self-renewal and cell fate commitment of neuro-mesodermal progenitors. We propose that Lin28a/b activity controls the pool of caudal progenitors during tail development, promotes their self-renewal potential, is involved in controlling the balance of neural versus mesodermal cell fate decisions, and that progressive down-regulation of Lin28a allows for species-specific vertebral formula to be achieved. These findings suggest that Lin28/let-7 play a role in the regulation of tail length through heterochrony of the body plan.
2019-05-01 | GSE123193 | GEO
Project description:Neural progenitor cells (NPCs) undergo rapid proliferation during neurulation in early development. This rapid growth generates a high demand for mRNA translation in a timing-dependent manner, but its underlying mechanism and functional importance remain poorly understood. Lin28 is an RNA-binding protein with two paralogs, Lin28a and Lin28b, in mammals. Lin28b deletion exhibited no developmental defects in mice, while we previously reported that Lin28a deletion led to microcephaly. Here we found that Lin28a/b double knockout (dKO) mice displayed neural tube defects (NTDs) coupled with reduced proliferation and precocious differentiation of NPCs. We used ribosomal protein 24 hypomorphic mice (Rpl24Bst/+) as a genetic tool to dampen global protein synthesis. In support of the importance of Lin28-mediated translation promotion, Lin28a-/-;Rpl24Bst/+ compound mutants exhibited NTDs resembling those seen in Lin28a/b dKO mice. Furthermore, increased NPC numbers and brain sizes in Lin28a-overexpressing mice were rescued by Rpl24Bst/+ heterozygosity. Mechanistically, RNA-sequencing of polysome sucrose gradient fractions revealed a reduced translation of genes involved in the regulation of cell cycle, ribosome biogenesis, and translation in mutants. Lin28a localizes in the nucleoli of NPCs, and ribosome biogenesis was reduced in dKO and increased in Lin28a-overexpressing NPCs. Together, these results suggest that Lin28-mediated promotion of protein synthesis is essential for NPC maintenance and early brain development.
2019-05-22 | GSE131536 | GEO