Discovery of Klf2 Interactors in Mouse Embryonic Stem Cells by Immunoprecipitation-Mass Spectrometry Utilizing Exogenously Expressed Bait
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ABSTRACT:
PROVIDER: PRJNA716537 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14041431_1.fastq.gz | Fastqsanger.gz | |||
| SRR14041431_2.fastq.gz | Fastqsanger.gz | |||
| SRR14041432_1.fastq.gz | Fastqsanger.gz | |||
| SRR14041432_2.fastq.gz | Fastqsanger.gz | |||
| SRR14041433_1.fastq.gz | Fastqsanger.gz |
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Project description:We report the RNA-seq data of Klf2 knockdowned cells and Snd1 knockdowned cells from mouse embryonic stem cells. Samples were lysed with total RNA prep kit.
2021-03-24 | GSE169406 | GEO
Project description:Krüppel-like factor 2 (Klf2) is a DNA-binding transcription factor that regulates embryonic stem cell-specific gene expression. Transcription cofactors such as p300 acetyltransferases and Erk kinases interact with Klf2, providing an additional layer of transcription regulation in embryonic stem cells. To carry out a thorough survey of the Klf2 interactome in embryonic stem cells and identify novel transcription cofactors, we designed a modified immunoprecipitation-mass spectrometry (IP-MS) method. In this method, recombinant Klf2, expressed and purified from Sf9 insect cells instead of ectopically expressed in cells, was used as bait. Using this modified IP-MS method, we discovered nine Klf2-interacting proteins, including the previously reported Crebbp and p300. These proteins showed at least an 8-fold increase in signal intensity in Klf2 pull-downs compared with controls, with P-values < 0.010. Among the identified Klf2-binding proteins confirmed using our IP-MS workflow was Snd1, which we found to interact directly with Klf2 and function as a transcriptional coactivator of Klf2 to drive Oct4 gene expression. Collectively, our IP-MS protocol may offer a useful tool for identifying novel transcription cofactors in stem cells.
2021-09-20 | PXD022123 | JPOST Repository
Project description:The self-renewing pluripotent state was first captured in mouse embryonic stem cells (mESCs) over two decades ago. The standard condition requires the presence of serum and LIF, which provide growth promoting signals for cell expansion. However, there are pro-differentiation signals which destabilize the undifferentiated state of mESCs. The dual inhibition (2i) of the pro-differentiation Mek/Erk and Gsk3/Tcf3 pathways in mESCs is sufficient to establish an enhanced pluripotent “ground state” which bears features resembling the pre-implantation mouse epiblast. Gsk3 inhibition alleviates the repression of Esrrb, a transcription factor that can substitute for Nanog function in mESCs. The molecular mechanism that is mediated by Mek inhibition is however not clear. In this study, we investigate the pathway through which Mek inhibition operates to maintain ground state pluripotency. We have found that in mESCs, Kruppel-like factor 2 (Klf2) is a protein target of the Mek/Erk pathway; and that Klf2 protein is phosphorylated by Erk2 and subsequently degraded through the proteosome. It is therefore by Mek-inhibition through PD0325901 or 2i that enables the stabilization and accumulation of Klf2 to sustain ground state pluripotency. Importantly, we found that Klf2-null mESCs, while viable under LIF/Serum conditions, cannot be maintained and eventually gradually die within a few passages. Our result thus demonstrates that Klf2 is an essential factor of ground state pluripotency. Collectively, our study defines the Mek/Klf2 axis that cooperates with the Gsk3/Esrrb pathway in mediating ground state pluripotency.
2014-06-04 | E-MTAB-2366 | biostudies-arrayexpress
Project description:This study describes the epigenetic profiling of the novel interactors of H3K4me3, H3K36me3 or H3K9me3. The interactors were ChIP-Seq profiled by their GFP tag in stably transfected HeLa (Kyoto) cells. The interactors include GATAD1, Sgf29, BAP18, TRRAP, PHF8, N-PAC and LRWD1 (including replicates), as well as an GFP ChIP-Seq profile on non-transfected HeLa cells (negative control). Also included are the profiles of the histone modifications themselves (H3K4me3, H3K27me3, H3K9me3, H3K36me3, H3K9/14Ac and H3K79me3) ChIP-Seq profiling of 8 proteins by their GFP tag in stably transfected cells HeLa (Kyoto) cells, 6 replicas, as well as ChIP-Seq profiling of 6 histone modifications in wt HeLa (Kyoto) cells
2010-09-17 | E-GEOD-20303 | biostudies-arrayexpress
Project description:KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell, and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. Embryonic -like globin gene expression is reduced in KLF2-/- embryos compared to wildtype (WT), and E10.5 erythroid cells exhibit abnormal morphology. Other KLF2 target genes were identified by comparing E9.5 KLF2-/- and WT yolk sac erythroid cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression; eighty-nine of these are downregulated in KLF2-/- compared to WT. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. Previously identified erythroid-enriched regulatory genes such as reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT. SOX2, a pluripotency factor in ES cells, is also a KLF2 target in embryonic erythroid cells. We investigated whether reelin, which has an established role in neuronal migration and proliferation, has a role in embryonic erythropoiesis. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter, but reelin mutant mice have no apparent abnormalities in embryonic erythroid morphology or globin gene expression. Timed-pregnant KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo, cryoprotected in 20% sucrose in PBS and frozen in OCT media. A small portion of the embryo tail was used for PCR genotyping. Eight micron KLF2-/- frozen yolk sac sections were obtained and laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 4 different KLF2-/- erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays
2011-03-02 | E-GEOD-27602 | biostudies-arrayexpress
Project description:Our previous single-cell RNA sequencing study in the adult human heart revealed that cardiomyocytes from both the atrium and ventricle display high activities of Krüppel-like factor 2 (KLF2) regulons. However, the role of the transcription factor KLF2 in cardiomyocyte biology remains largely unexplored. We employed transverse aortic constriction surgery in male C57BL/6J mice to develop an in vivo model of cardiac hypertrophy, and generated different in vitro cardiac hypertrophy models in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes. Our results demonstrated a significant reduction in KLF2 expression during the progression of cardiac hypertrophy. In vitro, KLF2 deficiency exacerbates cardiac hypertrophy and enhances hypertrophic reprogramming, while KLF2 overexpression attenuates cardiac hypertrophy and reverses hypertrophic transcriptome reprogramming. Mechanistically, combined RNA-seq and cleavage under targets & tagmentation (CUT&Tag) analysis revealed that KLF2 exerts its protective effects by directly regulating a set of genes associated with cardiac hypertrophy. In vivo, KLF2 overexpression specifically in cardiomyocytes effectively prevents TAC-induced cardiac hypertrophy in mice. Additionally, we found that simvastatin elevates KLF2 expression in cardiomyocytes, which subsequently alleviates cardiomyocyte hypertrophy. This study provides the first evidence that transcription factor KLF2 serves as a negative regulator of cardiac hypertrophy. Our findings highlight the therapeutic potential of enhancing KLF2 expression, particularly through simvastatin administration, as a promising strategy in the treatment of cardiac hypertrophy.
2025-01-01 | GSE283973 | GEO
Project description:On triggering of the T cell receptor CD8 T lymphocytes downregulate expression of the transcription factor KLF2. KLF2 expression remains low as these cells differentiate to Cytotoxic T lymphocytes (CTL) but may be re-expressed depending on the local environmental signals. We used retroviral transduction to enforce KLF2 expression in CTL to determine the impact of it re-expression on the CTL genetic program. T lymphocytes with a transgenic T cell receptor (P14 LCMV) isolated from murine spleens were activated with gp33-41 peptide for 2 days and transduced with empty vector (evGFP) or GFP-KLF2. After differentiation to CTL in culture with Interleukin-2 for 2 further days, cells positive for GFP were isolated by Fluorescence Activated Cell Sorting and the RNA extracted for microarray analysis.
2013-05-31 | E-GEOD-26568 | biostudies-arrayexpress
Project description:KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell, and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. Embryonic -like globin gene expression is reduced in KLF2-/- embryos compared to wildtype (WT), and E10.5 erythroid cells exhibit abnormal morphology. Other KLF2 target genes were identified by comparing E9.5 KLF2-/- and WT yolk sac erythroid cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression; eighty-nine of these are downregulated in KLF2-/- compared to WT. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. Previously identified erythroid-enriched regulatory genes such as reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT. SOX2, a pluripotency factor in ES cells, is also a KLF2 target in embryonic erythroid cells. We investigated whether reelin, which has an established role in neuronal migration and proliferation, has a role in embryonic erythropoiesis. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter, but reelin mutant mice have no apparent abnormalities in embryonic erythroid morphology or globin gene expression.
2011-03-02 | GSE27602 | GEO
Project description:The ERK5 MAP kinase signalling pathway was recently discovered as a driver of naïve pluripotency in mouse Embryonic Stem Cells (mESCs). However, the molecular functions of ERK5 in mESCs have not been investigated. Here, we employ combinatorial mESC proteomics to identify ERK5 target genes and substrates. Global proteomic profiling reveals ZSCAN4 and other 2-cell stage genes as transcriptional targets of the ERK5 pathway. ZSCAN4 expression is specifically induced by ERK5-dependent transcription of the core pluripotency factor KLF2. Additionally, ERK5 directly phosphorylates tandem KLF2 Thr-Pro/Ser-Pro motifs identified by phosphoproteomics to recruit the FBXW7-CUL1 E3 ligase, promoting KLF2 ubiquitylation and degradation. ERK5 phosphorylation of KLF2 thereby provides negative-feedback control to restrain transcriptional induction of ZSCAN4. Our data uncover an auto-regulatory module by which ERK5 co-opts KLF2 to pattern ZSCAN4 expression. This study provides the first molecular insight into ERK5 functions in mESCs, and suggests a novel role for ERK5 signalling in stem cell rejuvenation
2021-11-29 | PXD010581 | Pride
Project description:On triggering of the T cell receptor CD8 T lymphocytes downregulate expression of the transcription factor KLF2. KLF2 expression remains low as these cells differentiate to Cytotoxic T lymphocytes (CTL) but may be re-expressed depending on the local environmental signals. We used retroviral transduction to enforce KLF2 expression in CTL to determine the impact of it re-expression on the CTL genetic program.
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