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Series GSE5834 Query DataSets for GSE5834
Status Public on Jul 01, 2007
Title Active and repressive chromatin in the mammalian genome intersperse without spreading in imprinted gene clusters
Organism Mus musculus
Experiment type Genome binding/occupancy profiling by genome tiling array
Expression profiling by genome tiling array
Summary The Igf2r imprinted cluster is an epigenetic model of cis-acting silencing in which expression of a ncRNA silences multiple genes. Here, we map chromatin profiles on the maternal and paternal chromosome in a 250 kb region. We show that histone modifications associated with expressed and silent genes are mutually exclusive and localize to discrete regions. Expressed genes are modified in two ways; at promoter regions by H3K4me3+H3K4me2+H3K9Ac and on putative regulatory elements by H3K4me2+H3K9Ac. Repressed genes displayed two types of non-overlapping profiles. Type 1 localized to short regions and contained H3K9me3+H4K20me3 and sometimes HP1. Type 2 spanned a large domain containing multiple tissue-specific silent genes and contained H3K27me3 alone. These data identify different forms of repressive chromatin on chromosome arms that resemble constitutive and facultative heterochromatin but are restricted to short regions and interspersed with euchromatin.
Keywords: ChIP-Chip, chromatin profile, genomic imprinting, noncoding RNA
 
Overall design Chromatin immunoprecipitaion (ChIP) was combined with a custom PCR tiling array (Chip) to determine the profile of histone modifications on the maternal and paternal allele of the imprinted Igf2r cluster on mouse chromosome 17. ChIP-Chip experiments were carried out on mouse embryo fibroblast (13.5dpc) cell lines.
The main steps in the array design were (i) design of primers to amplify single copy DNA, (ii) spotting each PCR fragment 8 times on the array.The main steps in the ChIP were: (i) preparation of chromatin by micrococcal nuclease (MNase) digestion, (ii) use of specific antibodies, (iii) amplification of input and ChIP enriched chromatin using two rounds of T7 in vitro transcription, (iv) conversion of amplified RNA to cDNA and labelling with Alexa dyes, (v) hybridisation, washing, scanning. Two biological replicates were performed for each antibody in each cell line. A dye swap was also incorporated in the second hybridisation. A novel method to test gene expression was also employed. PCR fragments lying within the exons of expressed genes gave a significant signal. This method also allowed for the detection of non-coding RNAs. A total of forty hybridizations were performed in the study.
 
Contributor(s) Regha K, Sloane MA, Huang R, Pauler FM, Warczok KE, Melikant B, Radolf M, Martens JH, Jenuwein T, Barlow D
Citation(s) 17679087
Submission date Sep 14, 2006
Last update date Mar 16, 2012
Contact name Denise P. Barlow
E-mail(s) denise.barlow@univie.ac.at
Organization name Center of Molecular Medicine (CeMM)
Department Austrian Academy of Sciences
Street address Dr. Bohrgasse 9/4
City Vienna
ZIP/Postal code 1030
Country Austria
 
Platforms (1)
GPL4317 Custom Genome PCR Tiling Array of the Igf2r Imprinted Gene Cluster
Samples (40)
GSM134582 MEFB1 H3K4me2 Biological Replicate 1
GSM134583 MEFB1 H3K4me3 Biological Replicate 1
GSM134585 MEFB1 H3K9Ac Biological Replicate 1
Relations
BioProject PRJNA97231

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Supplementary file Size Download File type/resource
GSE5834_RAW.tar 17.4 Mb (http)(custom) TAR (of TXT)

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