GSE42880
MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system
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