Unknown,Transcriptomics,Genomics,Proteomics

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Chromatin position effects assayed by thousands of reporters integrated in parallel (cell lines with mPGK IRs)

ABSTRACT: Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ~1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for cross-talk between neighboring genes, and estimate that enhancers can influence gene expression on average over ~20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation. TRIP assay of mPGK promotor; 11 clonal cell lines, 1 replicate each

ORGANISM(S): Mus musculus

SUBMITTER: Bas van Steensel

PROVIDER: E-GEOD-49807 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Chromatin position effects assayed by thousands of reporters integrated in parallel.

Akhtar Waseem W de Jong Johann J Pindyurin Alexey V AV Pagie Ludo L Meuleman Wouter W de Ridder Jeroen J Berns Anton A Wessels Lodewyk F A LF van Lohuizen Maarten M van Steensel Bas B

Cell 20130801 4

Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ∼1,000-fold var ...[more]

PMID: 23953119

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Project description:Sulfate-reducing bacteria (SRB) colonize the guts of ~50% of humans. We used genome-wide transposon mutagenesis and insertion-site sequencing (INSeq), RNA-Seq, plus mass spectrometry to characterize genetic and environmental factors that impact the niche of Desulfovibrio piger, the most common SRB in a surveyed cohort of healthy USA adults. Gnotobiotic mice were colonized with an assemblage of sequenced human gut bacterial species with or without D. piger and fed diets with different levels and types of carbohydrates and sulfur sources. Diet was a major determinant of functions expressed by this artificial 9-member community and of the genes that impact D. piger fitness; the latter includes high- and low-affinity systems for utilizing ammonia, a limiting resource for D. piger in mice consuming a polysaccharide-rich diet. While genes involved in hydrogen consumption and sulfate reduction are necessary for its colonization, varying dietary free sulfate levels did not significantly alter levels of D. piger, which can obtain sulfate from the host in part via cross-feeding mediated by Bacteroides-encoded sulfatases. Chondroitin sulfate, a common dietary supplement, increased D. piger and H2S levels without compromising gut barrier integrity. A chondroitin sulfate-supplemented diet together with D. piger impacted the assemblage’s substrate utilization preferences, allowing consumption of more reduced carbon sources, and increasing the abundance of the H2-producing Actinobacterium, Collinsella aerofaciens. Our findings provide genetic and metabolic details of how this H2-consuming SRB shapes the responses of a microbiota to diet ingredients, and a framework for examining how individuals lacking D. piger differ from those that harbor it. 8 samples total, 2 gropus of 4 mice: Proximal colon gene expression profiles of gnotobiotic mice colonized with an artificial gut community composed of 8 human gut species (group 1: NoDp) and from mice colonized with the same community plus D. piger (Dp). Mice were fed a HF/HS diet supplemented with 3% chondroitin sulfate. Animals were sacrificed 2 weeks after colonization

Dataset Information

Chromatin position effects assayed by thousands of reporters integrated in parallel (cell lines with mPGK IRs)

Publications

Chromatin position effects assayed by thousands of reporters integrated in parallel.

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