NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Series GSE42880 Query DataSets for GSE42880
Status Public on Jan 14, 2013
Title MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system
Organism Mus musculus
Experiment type Methylation profiling by high throughput sequencing
Expression profiling by high throughput sequencing
Other
Summary The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the central nervous system (CNS) may differ from those present in embryonic stem cells. Here we present quantitative, genome-wide analysis of 5hmC, 5- methylcytosine (5mC) and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes, and that surprisingly strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG binding protein 2 (MeCP2) as the major 5hmC binding protein in the brain, and demonstrate that MeCP2 binds 5hmC and 5mC containing DNA with similar high affinities. The Rett Syndromecausing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell specific epigenetic mechanism for regulation of chromatin structure and gene expression.
 
Overall design Isolation and sorting was performed as described in (Kriaucionis and Heintz, 2009). Briefly, cerebella were dissected as described above and homogenized in homogenization medium (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail (Roche) using loose (A) and tight (B) glassglass dounce.Homogenate was supplemented with 50% iodixanol (OptiPrep, Axis-Shield, Scotland), 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8; and laid on a 29% iodixanol cushion. GC nuclei from WT and Mecp2-null mice were sorted by selecting a specific FSC/SSC population identified from GC EGFP+ mice. We observed that this FSC/SSC gated population in GC EGFP+ animals resulted >92% of the sorted nuclei being EGFP positive. Thus, the gate selected allows us to enrich the sample from 65-70% to 92-96% on GC cells. 5hmC was pulled down as described (Song et al., 2010). After purification DNA was amplified as described in TruSeq DNA Sample kit. 1-0.5 μg of DNA was used for each experiment. Sonicated DNA was end-repaired following by ligation to Illumina paired end sequencing adapters (Illumina, PE‐ 102‐ 1003).), following by amplification with Illumina primers. Input samples were produced for each cell types in both procedures 5hmC enriched were then sequenced using Illumina platform obtaining more than 50 x10-6, 36- bp single-end reads per sample. Reads were aligned to mm9 mouse genome assembly using Bowtie v0.12.7 (-m1 --best). Two biological replicas were done for each of the cell types
Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 μg/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 × g, 4 °C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 × g to pellet insoluble material. At this point, 30 μL of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4°C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 μg/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA from cerebellum were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturers’ instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 μg of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer.
Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 μg/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 × g, 4 °C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 × g to pellet insoluble material. At this point, 30 μL of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4°C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 μg/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA per IP or input sample were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturers’ instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 μg of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer.
Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 μg/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 × g, 4 °C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 × g to pellet insoluble material. At this point, 30 μL of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4°C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 μg/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) wit
 
Contributor(s) Heintz N, Mellen M
Citation(s) 23260135, 28847947
Submission date Dec 12, 2012
Last update date Oct 11, 2019
Contact name Marian Mellen
Organization name The Rockefeller University
Street address 1230 York Avenue
City New York
ZIP/Postal code 10065
Country USA
 
Platforms (2)
GPL9250 Illumina Genome Analyzer II (Mus musculus)
GPL13112 Illumina HiSeq 2000 (Mus musculus)
Samples (50)
GSM1063317 nd1 TRAPSeq female 1
GSM1063318 Input Cerebellum tissue for nd1 TRAPSeq female 2
GSM1063319 nd1 TRAPSeq female 2
Relations
BioProject PRJNA186546
SRA SRP017936

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE42880_BG_mC_32M.bedGraph.gz 334.4 Mb (ftp)(http) BEDGRAPH
GSE42880_GC_mC_32M.bedGraph.gz 348.3 Mb (ftp)(http) BEDGRAPH
GSE42880_PC_mC_32M.bedGraph.gz 351.2 Mb (ftp)(http) BEDGRAPH
GSE42880_RAW.tar 5.0 Gb (http)(custom) TAR (of WIG)
SRA Run SelectorHelp
Processed data are available on Series record
Processed data provided as supplementary file
Raw data are available in SRA

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap