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Cardiac transcription factors in HL-1 cells: gene expression and genome binding profiling

ABSTRACT: [1] Gene expression profiling in Gata4, Mef2a knockdown in HL-1 cells. HL-1 cells infected with adenovirus expressing either control siRNA or Gata4, and Gata4 & Mef2a siRNA. [2] Genome-wide maps of cardiac transcription factors binding region in HL-1 cells. We performed bio-ChIP-seq using streptavidin beads immunoprecipitation of biotinylated 5 cardiac transcription factors (fbio) and P300 antibody ChIP-seq. We used a dox-inducible dual adenovirus system to express biotinylated Core Cardiac TFs in HL1. One adenovirus expressed the dox-activated reverse tet activator protein (rtTA) and the E. coli biotinylating enzyme BirA from the cardiac specific rat troponin T promoter. The second virus expressed a Core Cardiac TF fused to a C-terminal flag-bio epitope tag, where bio is the 15 amino acid substrate for BirA. This in vivo biotinylation approach permits pulldown of different factors to be performed under identical, stringent conditions, and circumvents limitations posed by available antibodies. We titrated adenovirus and dox doses to express GATA4flbio and MEF2Aflbio at near endogenous levels. The same conditions were then used to express SRFflbio, TBX5flbio, and NKX2-5flbio. Reference: 12. de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E et al. (2003) Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 100: 7480-7485.

ORGANISM(S): Mus musculus

PROVIDER: GSE21529 | GEO | 2011/03/02

SECONDARY ACCESSION(S): PRJNA125947

REPOSITORIES: GEO

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Cardiac transcription factors in HL-1 cells: gene expression and genome binding profiling

Project description:[1] Gene expression profiling in Gata4, Mef2a knockdown in HL-1 cells. HL-1 cells infected with adenovirus expressing either control siRNA or Gata4, and Gata4 & Mef2a siRNA. [2] Genome-wide maps of cardiac transcription factors binding region in HL-1 cells. We performed bio-ChIP-seq using streptavidin beads immunoprecipitation of biotinylated 5 cardiac transcription factors (fbio) and P300 antibody ChIP-seq. We used a dox-inducible dual adenovirus system to express biotinylated Core Cardiac TFs in HL1. One adenovirus expressed the dox-activated reverse tet activator protein (rtTA) and the E. coli biotinylating enzyme BirA from the cardiac specific rat troponin T promoter. The second virus expressed a Core Cardiac TF fused to a C-terminal flag-bio epitope tag, where bio is the 15 amino acid substrate for BirA. This in vivo biotinylation approach permits pulldown of different factors to be performed under identical, stringent conditions, and circumvents limitations posed by available antibodies. We titrated adenovirus and dox doses to express GATA4flbio and MEF2Aflbio at near endogenous levels. The same conditions were then used to express SRFflbio, TBX5flbio, and NKX2-5flbio. Reference: 12. de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E et al. (2003) Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 100: 7480-7485. [1] Gene expression profiling: Identify differentially expressed genes after siRNA Gata4 or Gata4_Mef2a double knockdown in HL-1 cells [2] Genome occupancy profiling: Identify cardiac active enhancers by identified 5 cardiac transcription factors and P300 peaks.

2011-03-02 | E-GEOD-21529 | biostudies-arrayexpress

RNAi knock down in mouse cardiomyocyte cell line HL-1 using siRNAs against Gata4, Mef2a, Nkx2.5 and Srf to identify downstream targets

Project description:HL-1 cells treated with two different siRNAs against Gata4, Mef2a, Nkx2.5 and Srf each in duplicate to identify downstream targets.

2011-01-05 | E-TABM-376 | biostudies-arrayexpress

A native chromatin purification system for epigenomic profiling in C. elegans

2009-12-08 | GSE18898 | GEO

Sequencing of DNA from the biotinylated chromatin from HeLa S3 clonal cell line

Project description:Sequencing shows that macroH2A1-emerin interaction occurs in lamina-associated domains on a genome-wide scale. We decided to verify that the biotinylated signal is indeed enriched in the chromatin domains positioned at the nuclear periphery. In this regard, we purified the biotinylated chromatin from HeLa S3 clonal cell line stably coexpressing BirA-emerin and BAP-macro-H2A1 using proximity utilizing biotinylation native chromatin immunoprecipitation (PUB-nChIP). Instead of analysing the protein fraction, we isolated DNA from the biotinylated chromatin pull down and performed high throughput sequencing in order to get insight into the genome wide distribution of the isolated DNA. Importantly, PUB-nChIP-seq of the DNA purified from HeLa S3 clonal cell line stably coexpressing BirA-emerin and BAP-macro-H2A1 showed high levels of enrichment at the lamina associated domains, which were identified previously in HeLa cells using anti-Lamin B1 and anti-Lamin A chIP-seq (Lund et al., 2015). The enrichment of BirA-emerin labelled BAP-macro-H2A at LADs is visibly clear at both the chromosome level and at individual LADs. To test if this enrichment was significant, the genome was divided into 100kb windows and monte carlo simulation using 10,000 iterations was performed to examine if probes overlapping LADs were significantly different to the genomic average. For both replicates, at both LMNA and LMNB1 LADs, the enrichment of biotinylated BAP-macroH2A1 observed was significant relative to the genome wide average.

2018-08-24 | GSE118996 | GEO

Chromosome localization of cohesin oligomers in mid-M arrested yeast cells

Project description:We report the genomic localization of cohesin oligomers in nocodazole arrested yeast cells. Two alleles of SMC3 were expressed in yeast cells, one fused to BirA enzyme and the other tagged with AviTag. Cohesin oligomers were biotinylated and ChIP with streptavidin beads. As control experiments, cohesin localization on chromosome was determined in strains expresses freely diffusable BirA enzyme, where all Smc3 proteins were biotinylated; non-specific ChIP were determined in strains with no BirA.

2020-08-31 | GSE157155 | GEO

Mapping of Notch1 binding sites in a mouse immature T cell line

Project description:We used an immature mouse T cell line engineered to express a biotinylated form of the cleaved form of Notch1 (ICN1). ICN1-bound sites were precipitated with streptavidin-coated beads and subjected to ChIP-sequencing. Beko cells correspond to a spontaneous T lymphoma immature cell line derived from a TCRb deficient mouse. These cells were engineered to express a biotin-tagged-ICN1 and the bacterial biotin ligase BirA (Bio-ICN1) or just BirA as a control (Bio). Chromatin from both cell lines was subjected to strepatavidin-mediated precipitation and subjected to sequencing with the Illumina GAII sequencer as single end 36 base pair reads.

2014-04-02 | E-GEOD-46114 | biostudies-arrayexpress

APEX proximity labeling of murine NDUFS4 interacting proteins in podocytes

Project description:In this project we used an unbiased approach to identify the proteins that interact with NDUFS4 in the mouse podocytes using the APEX2 proximity labeling system. An immortalized murine podocyte was permanently transduced with a doxycycline (DOX) inducible NDUFS4-APEX2 chimeric construct. The cells were treated with DOX for 72 h to induce NDUFS4-APEX2 expression, followed by H2O2 activation of APEX2 mediated biotinylation of neighboring proteins. Biotinylated proteins were pull down by streptavidin beads and analyzed by LC-MS/MS. Cells without DOX induction or with DOX but without H2O2 activation were used as controls

2024-02-08 | PXD041378 | Pride

A native chromatin purification system for epigenomic profiling in C. elegans

Project description:We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, or streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin to randomly fragmented DNA from embryo nuceli.

2010-05-19 | E-GEOD-18898 | biostudies-arrayexpress

Expression data from mouse liver over-expressing either GFP alone or biotinylated-TRb1 and GFP

Project description:We over-expressed biotinylated-thyroid hormone receptor beta 1 (TRb1) in mouse liver using an adenovirus in order to perform ChIP-seq experiments. These microarrays were performed to determine gene expression changes in response to tri-iodothyronine (thyroid hormone; T3) stimulation. A control GFP adenovirus was used and gene expression from these livers was also done as a comparison. We performed microarrays from Ad-GFP-infected propylthiouracil (PTU)-fed livers injected with either saline or T3 and Ad-TRb1-GFP infected livers injected with either saline or T3. RNA was extracted from livers of biotin ligase (BirA)-expressing mice that had been infected with either Ad-GFP or Ad-TRb1, fed with PTU for 3 weeks followed by saline or T3 injections for 4 consecutive days.

2013-12-18 | E-GEOD-52433 | biostudies-arrayexpress

AP-MS of tissue-specific protein complexes in C. elegans

Project description:We present a method for the tissue-specific purification of protein complexes from the nematode C. elegans, based on cell-type specific biotinylation. To purify proteins from specific C. elegans tissues, we adapted a system based on in vivo biotinylation of a protein of interest. In this approach, a protein of interest is tagged with the 15 amino acid Avi-tag, which can be biotinylated in vivo by the BirA biotin ligase from Escherichia coli. The biotinylated bait protein and any associated proteins are then purified with streptavidin-coated beads, and their identities are determined by mass spectrometry.

2016-07-18 | PXD002139 | Pride

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