Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse Primary cerebellar granule cell precursors (GCPs) cultures were prepared from P7 Bmi-1-/- or wild type to investigate the role of Bmi1 in Shh-mediated shift in gene expression levels

ABSTRACT: We investigated the role of Bmi1 in Shh-mediated shift in gene expression levels Experiment Overall Design: Primary cerebellar granule cell precursors (GCPs) cultures were prepared from P7 Bmi-1-/- or wild type mice as described before {Messer, 1977 #810}. Cells were grown in Dulbecco MEM high Glucose, supplemented with 10% FCS glutamine, 20 mM KCl and penicillinâstreptomycin for 24 hours. Shh (R&D Systems) if appropriate was added for 24 hours to give at final concentration of 3 mg/ml. Experiment Overall Design: We performed 3 biological replicates for both WT and Bmi-1-/- cultures, and 2 biological replicates for both WT and Bmi-1-/- cultures, treated with Shh. Experiment Overall Design: Total RNA was isolated using RNeasy Mini Kit (Qiagen) and reverse-transcribed into double-stranded cDNA with One-Cycle cDNA Synthesis Kit (Affymetrix Inc., P/N 900431, Santa Clara, CA) and labelled biotin. Biotin-labeled cRNA samples were fragmented randomly to 35â200 bp and hybridized to GeneChipÂ® Mouse Genome 430 2.0. An Affymetrix GeneChip Scanner 3000 (Affymetrix Inc., Santa Clara, CA) was used to measure the fluorescent intensity emitted by the labeled target.

ORGANISM(S): Mus musculus

SUBMITTER: Tatiana Subkhankulova

PROVIDER: E-GEOD-19176 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Three-dimensional genome structure plays an important role in gene regulation. Globally chromosomes are organized into active and inactive compartments, while at the gene level looping interactions connect promoters to regulatory elements. Topologically Associating Domains (TADs), typically several hundred kilobases in size form an intermediate level of organization. Major questions include how TADs are formed and what their relation is with looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb region on chromosome 7 surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Further, we find that these TAD boundaries are present irrespective of expression and looping of genes located between them. In contrast looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell type-specific long-range interactions and gene regulation. We investigated a 2.8 Mb region on Chromosome 7 (hg18 chr7: 115797757-118405450) containing the ENCODE region ENm001 42. The 5C experiment was designed to interrogate looping interactions between HindIII fragments containing transcription start sites (TSSs) and any other HindIII restriction fragment (distal fragments) in the target region. Libraries were generated for five cell lines: Caco2, Calu3, Capan1, GM12878 and HepG2, with two biological replicates for each line. 5C probes were designed at HindIII restriction sites (AAGCTT) using 5C primer design tools previously developed and made publicly available online at our My5C website (http://my5C.umassmed.edu). Probes were designed based on the ENCODE manual region 1 (ENM001) design 25 with additional probes placed throughout the region when appropriate. We also added probes to extend the analysis to include a 700 Kb gene desert region located directly adjacent to ENM001. Probe settings were: U-BLAST, 3; S-BLAST, 100; 15-MER, 3,000; MIN_FSIZE, 250; MAX_FSIZE, 20,000; OPT_TM, 65; OPT_PSIZE, 40. We designed 74 reverse 5C probes, and 605 forward 5C probes.

Dataset Information

Transcription profiling of mouse Primary cerebellar granule cell precursors (GCPs) cultures were prepared from P7 Bmi-1-/- or wild type to investigate the role of Bmi1 in Shh-mediated shift in gene expression levels

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