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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse G1ME cells following retroviral infection and shRNA-mediated knock-down of Suz12 and Ezh2

ABSTRACT: Suz12 in hematopoiesis II. Retroviral Infection and shRNA-mediated knock-down of Suz12 and Ezh2 in mouse G1ME cells.

ORGANISM(S): Mus musculus

SUBMITTER: Carolyn de Graaf

PROVIDER: E-TABM-396 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Polycomb repressive complex 2 (PRC2) restricts hematopoietic stem cell activity.

Majewski Ian J IJ Blewitt Marnie E ME de Graaf Carolyn A CA McManus Edward J EJ Bahlo Melanie M Hilton Adrienne A AA Hyland Craig D CD Smyth Gordon K GK Corbin Jason E JE Metcalf Donald D Alexander Warren S WS Hilton Douglas J DJ

PLoS biology 20080401 4

Polycomb group proteins are transcriptional repressors that play a central role in the establishment and maintenance of gene expression patterns during development. Using mice with an N-ethyl-N-nitrosourea (ENU)-induced mutation in Suppressor of Zeste 12 (Suz12), a core component of Polycomb Repressive Complex 2 (PRC2), we show here that loss of Suz12 function enhances hematopoietic stem cell (HSC) activity. In addition to these effects on a wild-type genetic background, mutations in Suz12 are s ...[more]

PMID: 18416604

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Transcription profiling of the role of Suz12 in mouse hematopoietic progenitors

Project description:Comparison of Gene Expression in Wild type to Erg Mld2/+ Lin-Sca1+Kit+ cells

2008-07-01 | E-TABM-380 | biostudies-arrayexpress

SUZ12 immunoprecipitation followed by PRM of SUZ12 peptides to identify alternatively spliced isoforms of SUZ12 at the protein level.

Project description:Alternative splicing of SUZ12 exon 4 is predicted to generate two alternative isoforms differing by the inclusion or exclusion of 69 nucleotides in exon 4, which we named SUZ12-long (SUZ12-L) and SUZ12-short (SUZ12-S) respectively. At the protein level SUZ12 exon 4 encodes 23 amino acids (aa 129–152 in SUZ12-L) that partially overlap with the WD-binding domain 1 (WDB1, 110–145). To detect the existance of SUZ12-S at the protein levels we performed SUZ12 immunoprecipitation coupled with mass spectrometry (IP-MS) in the WT and ∆ex4 clones to identify SUZ12-S unique peptides. We identified a SUZ12-S specific peptide in both WT #2 and ∆ex4 #1. These observations were confirmed by SUZ12 IP followed by Western blot (WB) (size shift).

2024-03-18 | PXD046742 | Pride

SUZ12 immunoprecipitation followed by protein indentification via mass spectrometry (SUZ12 IP-MS) in mESCs of SUZ12 WT, delta exon 4, KO, and KO rescues with SUZ12-L and SUZ12-S

Project description:Suz12 exon 4 encodes 23 amino acids (aa 129–152 in SUZ12-L) that partially overlap with the WD-binding domain 1 (WDB1, 110–145). We reasoned that exon 4 skipping might alter the structure of SUZ12 and, possibly, PRC2 composition. To explore this possibility, we generated ESCs that lack the Suz12 exon 4 via CRISPR-Cas9–induced deletion. In addition, to rule out any biases due to the expression levels and/or SUZ12 epitope masking, we generated Suz12 knockout (KO) ESCs (herein, KO) in which Suz12 expression was subsequently rescued by re-introducing either the Suz12-L or Suz12-S mouse isoform fused to a triple-Flag tag under the regulation of a CAG promoter (KO+L/S; Figures S2H–S2K). We performed SUZ12 immunoprecipitation coupled with mass spectrometry (IP-MS) in the WT and ∆ex4 clones to compare their interactomes and with a flag antibody in the KO and rescue cell lines. As expected, no peptides corresponding to exon 4 were retrieved in ∆ex4 samples, while the rest of the sequence displayed similar coverage. Comparison of interactors in the two conditions revealed that SUZ12 binding to AEBP2 and JARID2 was strongly reduced in ∆ex4 cells with respect to WT cells, whereas SUZ12 binding to most core components or to PRC2.1-specific factors was unchanged or only slightly increased . These observations were confirmed by SUZ12 IP followed by Western blot (WB). Flag IP-MS in rescue cells confirmed that, while the long isoform was able to correctly form comparable amounts of both PRC2.1 and PRC2.2 subtypes, interaction of the SUZ12-S with PRC2.2-specific factors was drastically reduced.

2024-03-18 | PXD046660 | Pride

The role of FOG-1 in GATA-1 transcriptional regulation of megakaryocyte differentiation

Project description:We explored the role of FOG-1 in GATA-1 transcriptional regulation of megakaryocyte differentiation through expression of wild-type GATA-1 and the FOG-binding mutant of GATA-1 (GATA-1^V205G) in G1ME cells. G1ME cells were derived from Gata1-null mouse ES cells and have both megakaryocyte and erythrocyte differentiation potential upon reconstitution of GATA-1 expression (Stachura 2006). HA-tagged wild-type or mutant GATA-1 were expressed in G1ME cells grown in TPO via retroviral transductions. The cells were sorted for GFP positivity 68 hours post-transduction and then were allowed to recover in normal growth medium for 4h. Total RNA was then isolated using RNeasy kit from Qiagen 72 hours post-transduction.

2012-03-05 | E-GEOD-35695 | biostudies-arrayexpress

Transcriptome profiling of mouse Gata1- megakaryocyte-erythroid progenitors (G1MEs) expressing one of the two isoforms of GATA1: full-length (GATA1fl) or an amino-truncated form of GATA1 (GATA1s).

Project description:We transduced mouse Gata1- megakaryocyte-erythroid progenitors with MIGRI-GFP vector expressing GATA1fl or GATA1s cDNAs. GFP-positive cells expressing one of the two isoforms of GATA1 were isolated by FACS 42 hours following transduction and used for microarray transcriptome analysis. At this time point, there was no apparent difference in the cell surface phenotypes between GATA1fl and GATA1s-expressing cells. Transcriptome data for G1ME/GATA1fl at 42h were deposited previously under GSE14980 (GSM374049, GSM374050, GSM374051), whereas G1ME/GATA1s at 42h are deposited here. We performed differential expression analysis comparing mean expression levels of genes in G1ME cells at 42 hours after transduction with GATA1fl (3 replicates deposited previously under GSE14980: GSM374049, GSM374050, GSM374051) or GATA1s (3 replicates deposited here). All 6 samples were prepared the same way and analyzed on the same platform, Affymetrix Mouse Genome 420 2.0.

2015-01-26 | E-GEOD-62879 | biostudies-arrayexpress

Genome-wide binding analysis of GATA1fl and GATA1s in G1ME cells.

Project description:We used chromatin immunoprecipitation sequencing (ChIP-seq) to compare genome-wide binding profiles of GATA1fl and GATA1s in G1ME cells, which are immortalized, developmentally arrested megakaryocyte-erythroid progenitors (MEPs) derived from in vitro differentiation of murine Gata1- ES cells. Although the truncation in GATA1s leaves the DNA binding domain intact, GATA1s fails to broadly occupy erythroid specific regulatory regions. These observations point to lineage specific co-factor associations essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. We performed ChIP-seq on G1ME cells 42 hours after retroviral transfer of HA-tagged full-length GATA1 (GATA1fl) or GATA1s cDNAs (2 replicates each). At this time point, there was no apparent difference in the cell surface phenotypes between GATA1fl and GATA1s-expressing cells. For each ChIP-seq replicate we obtained a matching input sample (non-ChIP DNA) as a control.

2015-01-26 | E-GEOD-61932 | biostudies-arrayexpress

Genome-wide analysis of chromatin occupancy of GATA1 and its short isoform GATA1s (ChIP-Seq)

Project description:We report ChIP-Seq data for GATA1 and the leukemia-associated short isoform GATA1s in G1ME cells, a Gata1-null cell line with both erythroid and megakaryocytic differentiation potential. We introduced HA-tagged GATA1 or GATA1s into G1ME cells via retroviral transduction. The cells were crosslinked at 48h post-transduction, and an HA antibody was used for chromatin immunoprecipitation (ChIP). ChIP and input samples were sequenced on Illumina GAII or GAIIx high-throughput sequencers. The data reveal a deficiency of chromatin occupancy by GATA1s, especially at genes involved in erythrocyte differentiation and function. Examinaton of chromatin occupancy of GATA1 and GATA1s in G1ME cells cultured in TPO.

2015-02-04 | E-GEOD-64327 | biostudies-arrayexpress

Jarid2 regulates hematopoietic stem cell function by acting with Polycomb Repressive Complex 2

Project description:Polycomb Repressive Complex 2 (PRC2) has been shown to play a key role in hematopoietic stem and progenitor cell (HSPC) function. Analyses of mouse mutants harboring deletions of core components have implicated PRC2 in fine-tuning multiple pathways that instruct HSPC behavior, yet how PRC2 is targeted to specific genomic loci within HSPCs remains unknown. Here we use shRNA-mediated knockdown to survey the function of known PRC2 accessory factors in HSPCs by testing the competitive reconstitution capacity of transduced murine fetal liver cells. We find that similar to the phenotype observed upon depletion of core subunit Suz12, depleting Jarid2 enhances the competitive transplantation capacity of both fetal and adult, mouse and human HSPCs. Gene expression profiling revealed common Suz12 and Jarid2 target genes that are enriched for the H3K27me3 mark established by PRC2. These data implicate Jarid2 as an important component of PRC2 that has a central role in coordinating HSPC function. RNA-seq of jarid knockdown, suz knockdown and control from HSPC in 16 week old mice.

2015-04-02 | E-GEOD-60808 | biostudies-arrayexpress

Genome-wide analysis of the role of FOG-1 in GATA-1 chromatin occupancy

Project description:We report ChIP-Seq data for GATA-1 and the FOG-binding mutant of GATA-1 (GATA-1^V205G) in G1ME cells, a Gata1-null cell line with both erythroid and megakaryocytic differentiation potential. We introduced HA-tagged GATA-1 or V205G into G1ME cells via retroviral transduction. The cells were crosslinked at 48h post-transduction, and an HA antibody was used for chromatin immunoprecipitation (ChIP). ChIP and input samples were sequenced on Illumina GAII high-throughput sequencer. The data reveal GATA-1-specific and V205G-specific bidning sites, indicating that FOG-1 both faacilitates and prohibits GATA-1 chromatin occupancy in a context-dependent manner. Examinaton of chromatin occupancy of GATA-1 anda FOG-binding mutant of GATA-1 in G1ME cells cultured in TPO.

2012-07-30 | E-GEOD-35644 | biostudies-arrayexpress

Histone post translational modification mass sprectrometry SUZ12 exon 4 cell lines

Project description:We investigate the contribution of each SUZ12 isoform on PRC2 activity on chromatin using our different ESC lines. As an additional comparison for ∆ex4 cells (SUZ12-S), we included an ESC clone in which an unsuccessful CRISPR editing event resulted in a mutant allele with nearly constitutive splicing of exon 4 (herein, CSex4) and normal Suz12 expression levels (expressing only SUZ12-L). First, we profiled H3K27 bulk PTMs by WB and observed that, while CSex4 cells showed no major differences to control cells, ∆ex4 cells displayed lower levels of H3K27me2 and -me3, with higher levels of mono-methylation and acetylation. Similar results were obtained when comparing SUZ12-L and SUZ12-S ESC rescue lines. To validate these findings with an antibody-independent technique, and to additionally profile other histone PTMs, we analysed these cells with histone-MS. In line with the previous estimates26, WT cells displayed approximately 85% of H3K27 methylation. This proportion was largely similar in CSex4 cells, while it dropped to ~50% in ∆ex4 cells, with H3K27me2 and -me3 being the most affected modifications. Notably, H3K27me2/3 loss in ∆ex4 cells occurred at histone peptides regardless of their H3K36 methylation status. Moreover, the global H3K36 methylation rates were not affected in either CSex4 or ∆ex4 cells (Figure S4C), and no major changes in methylation or acetylation levels were observed at other residues. Importantly, reintroduction of SUZ12-L in KO cells rescued a higher degree of methylation compared to SUZ12-S. Overall, these results indicate that SUZ12-S alone is unable to maintain global physiological levels of H3K27 methylation; rather SUZ12-L is also necessary for this task.

2024-03-18 | PXD046640 | Pride

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