Baculovirus-encoded protein expression for epigenomic profiling in Drosophila cells

Bryson, Terri D.; Weber, Christopher M.; Henikoff, Steven

doi:10.4161/fly.4.3.12177

Cited by 5 publications

(5 citation statements)

References 23 publications

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“…Stable Drosophila S2 cell lines were created as described 50 , 51 . We expressed FLAG-tagged H2Av in these lines with baculovirus under the metallothionein promoter as described 52 . Briefly, we infected one 150 cm 2 flask of 6×10 7 BLRP-H2A and BLRP-H2Av cells and one flask of S2 cells with a baculovirus construct encoding FLAG-H2Av for two hours at a multiplicity of infection of 100.…”

Section: Methodsmentioning

confidence: 99%

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H2A.Z nucleosomes enriched over active genes are homotypic

Weber

Henikoff

2010

Nat Struct Mol Biol

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114

108

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Nucleosomes that contain the histone variant H2A.Z are enriched around transcriptional start sites, but the mechanistic basis for enrichment is unknown. A single octameric nucleosome can contain two H2A.Z histones (homotypic) or one H2A.Z and one canonical H2A (heterotypic). To elucidate H2A.Z function, we generated high-resolution maps of homotypic and heterotypic Drosophila H2A.Z (H2Av) nucleosomes. Although homotypic and heterotypic H2A.Z nucleosomes map throughout most of the genome, homotypic nucleosomes are enriched and heterotypic nucleosomes are depleted downstream of active promoters and intron/exon junctions. The distribution of homotypic H2A.Z nucleosomes resembles that of classical active chromatin and shows evidence of disruption during transcriptional elongation. Both homotypic H2A.Z nucleosomes and classical active chromatin are depleted downstream of paused polymerases. Our results suggest that H2A.Z enrichment patterns result from intrinsic structural differences between heterotypic and homotypic H2A.Z nucleosomes following disruption during transcriptional elongation.

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Section: Methodsmentioning

confidence: 99%

“…We prepared chromatin for affinity purification essentially as described 52 , except that we fixed soluble nucleosomes with formaldehyde prior to affinity purification. Briefly, ~3×10 8 cells from each flask were suspended in HM2 (10 mM Hepes, 2 mM MgCl 2 , 0.5 mM phenylmethanesulphonylfluoride (PMSF)) and lysed with 0.6% (v/v) NP-40.…”

Section: Methodsmentioning

confidence: 99%

H2A.Z nucleosomes enriched over active genes are homotypic

Weber

Henikoff

2010

Nat Struct Mol Biol

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114

108

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“…Plasmid p4RG expressing the D. melanogaster NTF and E. coli BirA was constructed as follows: The BLRP sequence was fused to mCherry cDNA using long oligonucleotides as described for C. elegans and ligated into pRMHa3-3xFLAG-H2Av-Adh39UTR (Bryson et al 2010) between 3xFLAG and His2Av. The 1379-bp twi promoter and the 2100-bp RanGap coding sequence were amplified from cn bw genomic DNA using primers 59-AAGCTTCCGCGG CGCGCCGCATGCTGTGCTGAGGGCAGTAAATC-39 and 59-GGTA CCACCGGTCATTTGGTGATCTTGCTTGG-39 for twi, and primers 59-ATGCATGGCATGTCCACCTTTAACTTC-39 and 59-GTCGACA CAAATCGGAAAGTGGGAAA-39 for RanGap, and cloned separately into pCR4-TOPO (Invitrogen).…”

Section: Fly Constructs and Strainsmentioning

confidence: 99%

Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling

Steiner¹,

Talbert²,

Kasinathan³

et al. 2012

Genome Res.

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119

144

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An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue-or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotinlabeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types.

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“…Various transfection procedures have been covered in detail in other reviews (Cherbas & Cherbas, ; Cherbas & Gong, ; Cherbas et al, ). Briefly, the various transformation methods are: (a) calcium phosphate‐DNA coprecipitation (Bourouis & Jarry, ), (b) electroporation (Swevers, Cherbas, Cherbas, & Iatrou, ), (c) baculovirus infection (Bryson, Weber, & Henikoff, ; D. F. Lee, Chen, Hsu, & Juang, ), and the more commonly used lipid‐based delivery methods (Felgner et al, ).…”

Section: Working With Drosophila Cell Linesmentioning

confidence: 99%

Generating and working with Drosophila cell cultures: Current challenges and opportunities

Luhur

Klueg

Zelhof

2018

WIREs Developmental Biology

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The use of Drosophila cell cultures has positively impacted both fundamental and biomedical research. The most widely used cell lines: Schneider, Kc, the CNS and imaginal disc lines continue to be the choice for many applications. Drosophila cell lines provide a homogenous source of cells suitable for biochemical experimentations, transcriptomics, functional genomics, and biomedical applications. They are amenable to RNA interference and serve as a platform for high‐throughput screens to identify relevant candidate genes or drugs for any biological process. Currently, CRISPR‐based functional genomics are also being developed for Drosophila cell lines. Even though many uniquely derived cell lines exist, cell genetic techniques such the transgenic UAS‐GAL4‐based RasV12 oncogene expression, CRISPR‐Cas9 editing and recombination mediated cassette exchange are likely to drive the establishment of many more lines from specific tissues, cells, or genotypes. However, the pace of creating new lines is hindered by several factors inherent to working with Drosophila cell cultures: single cell cloning, optimal media formulations and culture conditions capable of supporting lines from novel tissue sources or genotypes. Moreover, even though many Drosophila cell lines are morphologically and transcriptionally distinct it may be necessary to implement a standard for Drosophila cell line authentication, ensuring the identity and purity of each cell line. Altogether, recent advances and a standardized authentication effort should improve the utility of Drosophila cell cultures as a relevant model for fundamental and biomedical research. This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes

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Baculovirus-encoded protein expression for epigenomic profiling in Drosophila cells

Cited by 5 publications

References 23 publications

H2A.Z nucleosomes enriched over active genes are homotypic

H2A.Z nucleosomes enriched over active genes are homotypic

Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling

Generating and working with Drosophila cell cultures: Current challenges and opportunities

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