Unknown,Transcriptomics,Genomics,Proteomics

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Identifying genetic determinants needed to establish a human gut symbiont in its habitat

ABSTRACT: The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion-sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals. In vivo selection transforms this population, revealing functions necessary for survival in the gut: we show how this selection is influenced by community composition and competition for nutrients (vitamin B12). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems. Keywords: Other 57 samples analyzed, 1 of these is the reference (input) sample

ORGANISM(S): Bacteroides thetaiotaomicron

SUBMITTER: Andrew Goodman

PROVIDER: E-GEOD-17712 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Identifying genetic determinants needed to establish a human gut symbiont in its habitat.

Goodman Andrew L AL McNulty Nathan P NP Zhao Yue Y Leip Douglas D Mitra Robi D RD Lozupone Catherine A CA Knight Rob R Gordon Jeffrey I JI

Cell host & microbe 20090901 3

The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon ...[more]

PMID: 19748469

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Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.

Project description:The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors which are thought to “sense” deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including Hnf4a and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), Ppara, and Tp53. Motif analysis of cis-regulatory further suggested that Hnf4a and Hnf1a are the main transcriptional regulators in the PT. Available expression data from tissue-specific Hnf4a KO tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. ChIP-seq was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1(Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8(Oat3), and Slc47a1(Mate1). Conversely, overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts (MEFs), sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-specific brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT cellular identity. Hnf4a binding was examined in rat kidneys at three timepoints (E20, P13 and Adult) and p300 binding was examined in adult rat kidney cortex tissue using ChIP-seq. Four corresponding input DNA samples were used as controls for peak calling.

Dataset Information

Identifying genetic determinants needed to establish a human gut symbiont in its habitat

Publications

Identifying genetic determinants needed to establish a human gut symbiont in its habitat.

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