Unknown,Transcriptomics,Genomics,Proteomics

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High-throughput sequencing of sequentially reprogrammed iPS cells reveals key epigenetic modifications correlated with reduced pluripotency of iPS cells [sRNA-seq]

ABSTRACT: Full pluripotency of induced pluripotent stem (iPS) cells has been determined as viable all-iPS mice can be generated through tetraploid complementation. Subsequently, activation of imprinted Dlk-Dio3 gene cluster has been suggested to correlate with the pluripotency of iPS cells1. However, evidence from recent studies has demonstrated that loss of imprinting at the Dlk-Dio3 locus did not correlate strictly with the reduced pluripotency of iPS cells. Therefore, it becomes indispensable to exploit other reliable molecular markers for evaluating the quality of iPS cells accurately. In the present study, we successfully utilize the sequential reprogramming approach and produce all-iPS mice to six generations using iPS cell lines derived from different cell lineages which contain the same proviral integration in the genome. By comparing the global gene expression and epigenetic modifications of both “tetra-on” and corresponding “tetra-off” iPS cell lines established from either mesenchymal or hematopoietic lineages through deep sequencing analysis of mRNA expression, small RNA profiling, histone modifications (H3K4m2, H3K4me3 and H3K27me3) and DNA methylation, very few differences are detected among all the iPS cell lines investigated. However, we find that two imprinted genes, disruption of which correlate with the reduced pluripotency of iPS cells. Therefore, our data not only provide the first demonstration that producing of all-iPS mice to six generations is feasible, but reveal that two imprinted regions can be served as pluripotency markers of iPS cells Examination of the expression small RNA in 13 cell types

ORGANISM(S): Mus musculus

SUBMITTER: Tao Cai

PROVIDER: E-GEOD-36291 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Small RNAs (sRNAs) are hypothesized to contribute to hybrid vigor because they maintain genome integrity, contribute to genetic diversity, and control gene expression. We used Illumina sequencing to assess how sRNA populations vary between two maize inbred lines (B73, Mo17) and their hybrid. We sampled sRNAs from the seedling shoot apex and the developing ear, two rapidly growing tissues that program the greater growth of maize hybrids. We found that parental differences in siRNAs primarily originate from repeat regions. Although the maize genome contains greater number and complexity of repeats compared to Arabidopsis or rice, we confirmed that like these simpler plant genomes, 24-nt siRNAs whose abundance differs between maize parents also show a trend of downregulation following hybridization. Surprisingly, hybrid vigor is fully maintained when 24-nt siRNAs are globally reduced by mutation of the RNA-dependent RNA polymerase2 (RDR2) encoded by modifier of paramutation1 (mop1). We also discovered that 21-22nt siRNAs derived from a number of distinct retrotransposon families differentially accumulate between B73 and Mo17 as well as their hybrid. Thus, maize possesses a novel source of genetic variation for regulating both transposons and genes at a genomic scale, which may contribute to its high degree of observed heterosis. sRNA libraries were derived from RNA isolated from the seedling shoot apex and developing ear tissues from B73, Mo17, B73xMo17 and Mo17xB73. The shoot apex was chosen because it is enriched for meristematic tissue where cell proliferation occurs, rates of organ initiation are determined, and organ size is specified. The developing ear was examined because it is enriched in meristematic tissue and is undergoing rapid growth, and also because the mature ear shows the highest degree of heterosis. Total RNA was isolated and separated on a 15% TBE-Urea polyacrylamide gel. Using a 10-bp ladder, the sRNA fraction representing 10-40-bp was excised. sRNA libraries were prepared according to Lu et al. (2007) or manufacturer's instructitions (Illumina). A combination of Perl scripts and FASTX toolkit scripts were used to remove adapters, collapse identical sequences and count reads per sequence. Supplementary processed data text files contain the distinct sRNA sequences for all of the genotypes analyzed in that experiment. Abundance (reads per million) was calculated for each distinct sequence by dividing the number of reads of distinct sRNA in a library by the total number of sRNA reads for that library and multiplying this by 1 million. Genome builds: B73 genome, maizesequence.org release 4a.53 (October, 2009); Mo17 whole genome shotgun clones.

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High-throughput sequencing of sequentially reprogrammed iPS cells reveals key epigenetic modifications correlated with reduced pluripotency of iPS cells [sRNA-seq]

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