Chromatin immunoprecipitation of tissue-specific transcriptional regulators in mouse and human liver to measure binding at regulatory regions of homologous genes
Investigation TitleChromatin immunoprecipitation of tissue-specific transcriptional regulators in mouse and human liver to measure binding at regulatory regions of homologous genes
Comment[Submitted Name]Experimental measurement of the conservation of tissue-specific transcriptional control - part a
Experimental Designbinding_site_identification_design, ChIP-chip by array
Experimental Design Term Source REFThe MGED Ontology, EFO
Comment[ArrayExpressReleaseDate]2006-10-03
Comment[AEMIAMESCORE]4
Comment[ArrayExpressAccession]E-TABM-108
Comment[MAGETAB TimeStamp_Version]2010-08-12 15:29:03 Last Changed Rev: 13058
Experimental Factor NameDiseaseState, Organism, Immunoprecipitate
Experimental Factor Typedisease_state, organism, immunoprecipitate
Experimental Factor Term Source REF
Person Last NameOdom
Person First NameDuncan
Person Mid Initials
Person EmailDuncan.Odom@cancer.org.uk
Person Phone
Person Fax
Person Address9 Cambridge Center
Person AffiliationWhitehead Institute
Person Rolessubmitter
Person Roles Term Source REFThe MGED Ontology
Quality Control Typebiological_replicate
Quality Control Term Source REFThe MGED Ontology
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date2006-10-03
PubMed ID17529977
Publication DOI17529977
Publication Author ListOdom DT, Dowell RD, Jacobsen ES, Gordon W, Danford TW, Macisaac KD, Rolfe PA, Conboy CM, Gifford DK, Fraenkel E.
Publication TitleTissue-specific transcriptional regulation has diverged significantly between human and mouse
Publication Statusjournal_article
Publication Status Term Source REF
Experiment DescriptionTo identify experimentally both conserved and evolving aspects of transcriptional regulation, we have measured the binding of key transcriptional regulators, RNA polymerase II and histone methylation in mouse and human liver at regulatory regions of homologous genes. As expected, the set of genes for which regulator binding is conserved is enriched for known liver-specific genes, conserved binding events are associated with sequence motifs in both species, and target genes show conserved gene expression. Strikingly, a significant number of homlogous genes are bound in only one species, and expression of these genes is no more conserved than expected by chance.
Protocol NameP-WMIT-32, P-TABM-518, P-TABM-517, P-MEXP-10250, P-MEXP-2377, P-WMIT-37, P-WMIT-38, P-WMIT-39
Protocol Typegrow, grow, grow, nucleic_acid_extraction, labeling, hybridization
Protocol DescriptionCell cross-linking: Use 5x10<sup>7</sup> - 1x10<sup>8</sup> cells (70-80% confluency for adhesion cells of 8-12 15 cm<sup>2</sup> plates or 175 cm<sup>2</sup> flasks) for each location analysis reaction. <br>1. Add 1/10 volume of fresh 11% formaldehyde solution to plates.<br>2. Swirl plates briefly and let them sit at RT for 10 min. <br>3. Add 1/20 volume of 2.5 M glycine to plates to quench formaldehyde. <br>4. Rinse cells twice with 5 ml 1X PBS. Harvest cells using silicon scraper. <br>5. Spin cells at 4k for 10 min at 4°C.6. Transfer cells to 15ml conical tubes and spin 4k 10 min at 4°C.<br>7. Flash freeze cells in liquid nitrogen and store pellets at -80 °C., Mouse hepatocytes were prepared by direct perfusion of the liver with buffered salt solution, followed by % formaldehyde. After 10 minutes, the tissue was removed and diced in 500 mM glycine buffer to neutralize the formaldehyde. After homogenization, the hepatocytes were run through a sucrose gradient to remove other cell types, and rinsed with PBS., Human hepatocyte purity was calculated following the final low speed centrifugation step of the published hepatocyte preparation used. The cell pellet from two independent human hepatocyte preparations were resuspended in plating media and samples of the cell preparation were spotted onto glass microscope slides, air dried and fixed in formalin. Duplicate slides were exposed to antibodies for low molecular weight cytokeratins (AE1/AE3), albumin or hepatocyte specific antibody HEPR. The results indicate that 93% of the cells in the preparation reacted with antibodies to cytokeratin, albumin and HEPR, indicating that 93% of the cells plated on culture dishes are epithelial (cytokeratin positive) and are parenchymal hepatocytes (HEPR and Albumin positive). Most of the remaining cells were small and appeared to be hematopoietic. Because the hepatocytes used in these studies are not plated before formaldehyde crosslinking, we predict that our major contaminant is 7% hematopoetic cells (S. Strom personal communication)., HepG2 cells were grown in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% FBS (GIBCO, Invitrogen), 1% PEST (GIBCO, Invitrogen) and 1% Glutamine (GIBCO, Invitrogen), at 37 C with 5% CO2. Cells were grown in 175 cm2 bottles until they reach 80-90% confluency., I. Preblock and binding of antibody to magnetic beads 1. wash 100 µL Dynal magnetic beads (per reaction) in 1 ml fresh BSA/PBS 2. collect the beads by spinning at 3000 RPM for 3 minutes. 3. wash beads in 1.5 ml BSA/PBS 2 times, collect the beads with the magnet. 4. Add 6-10 µg of Ab +250µL of PBS/BSA. 5. incubate 4hr to O.N. on a rotating platform at 4∞C. 6. wash beads 3 times in 1.5 Ml PBS/BSA. 7. Resuspend in 100µL PBS/BSA. II. Chromatin Immunoprecipitation 1. Add 100 µL Ab prebound Dynal magnetic beads from step I to cell lysate from prior protocol. 2. Rock 4°C O/N. III. Washing, eluting, and reverse cross-linking 1. Transfer to centrifuge tubes, continue working in the cold room until step 6. 2. Use the magnetic stand to precipitate the beads. 3. Wash 4-8 times with 1 mL wash buffer 4. Wash once with 1 ml TE-plus-50 mM NaCl 5. Spin 3k for 2-3 min and aspirate any residual TE buffer. 6. Add 100 µl of elution buffer. 7. Elute at 65∞C for 10-15 min with brief vortexing every 2 min. 8. Spin down beads 14k for 1 min. 9. Remove all 100 µl of supernatant. 10. Reverse x-link O/N 65∞C. 11. Thaw input, add 3 vol of elution buffer and reverse x-link O/N 65∞C. IV. RNase, Proteinase K 1. Add 1 vol of TE to IP and input fraction. 2. Add RNase A so final is 0.2µg/µL (~5 µL/250 µL rxn). Incubate 37∞C 1-2hr. 3. Add proteinase K so final is 0.2µg/µL (~2.5 µL/250 µL rxn). Incubate 55∞C 2hr. 4. Extract once w/ 1 vol of phenol. 5. Extract once w/ 1 vol of phenol:chl:IA (made by mixing 1 vol of phenol w/ 1 vol of Chloroform:isoamyl alcohol). 6. extract once w/ 1 vol of chl:IA. 4-6. Or instead extract 1x w/ 1 vol phenol:chl:IA using phaselock tubes 7. add 30 µg (1.5 µL) of glycogen. 8. Add 5M NaCl so final is 0.2M (10 µL/250 µL rxn). 9. Add 2 vol of EtOH and incubate -80∞C 30 min. 10. Spin and wash with 500 µL 75% EtOH. 11. Dry and resuspend pellets in 60 µL 10mM Tris HCl pH 8. Save 5 µL of IP sample for checkpoints to the right. Normalize the input/wce fraction to 100 ng/µL using the Nanodrop. Wash buffer (RIPA buffer) 100ml: final concentration 5ml of 1M Hepes (pH 7.6) 50 mM 200µL of 0.5M EDTA 1 mM 7 ml of 10% DOC (Na deoxycholate) 0.7% 10 ml of 10% NP-40 (IPGEL) 1% 10 ml of 5M LiCl or 2.12g powder 0.5 M Elution buffer: 50mM Tris pH8 10mM EDTA 1% SDS , Amplified DNA was labeled and purified using Invitrogen Bioprime random primer labeling kits (immunoenriched DNA was labeled with Cy5 fluorophore, whole cell extract DNA was labeled with Cy3 fluorophore)., Labeled DNA was combined (5 - 6 ug each of immunoenriched and whole cell extract DNA) and hybridized to arrays in Agilent hybridization chambers for 40 hours at 40C., Slides were scanned using an Agilent DNA microarray scanner BA. PMT settings were set manually to normalize bulk signal in the Cy3 and Cy5 channel.
Protocol Parameters
Protocol Hardware
Protocol Software
Protocol Contact
Protocol Term Source REF
SDRF FileE-TABM-108.sdrf.txt
Term Source NameThe MGED Ontology, ncbitax, ArrayExpress, The MGED Ontology, EFO
Term Source Filehttp://mged.sourceforge.net/ontologies/MGEDontology.php, http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html, http://www.ebi.ac.uk/arrayexpress, http://mged.sourceforge.net/ontologies/MGEDontology.php, http://www.ebi.ac.uk/efo/
Term Source Version