This set of custom tracks displays data from an experiment looking at four different ages of mouse liver to observe how different histone modifications (DNA methylation, H3K4me2, and H3K27) change across postnatal development. A ChIP-on-chip tiling array for three mouse chromosomes (5, 12, 15) was used.
Animals. Eight-week-old C57BL/6 breeding pairs of mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were housed according to the American Animal Association Laboratory Animal Care guidelines, and were bred under standard conditions in the Lab Animal Resources Facility at the University of Kansas Medical Center. The use of these animals was approved by the Institutional Animal Care and Use Committee. Breeding pairs were set up at 4:00 pm, and separated at 8:00 am of the following day. The body weight of females was recorded each day to determine pregnancy status. Livers from offspring were collected at the following four ages: day -2 (gestational day 17), 1, 5, and 45, which represent developmental stages of prenatal (day -2), neonatal (day 1 and 5), and young adults (day 45) during liver maturation. Due to the difficulty in distinguishing gender from day -2 to day 5 mice and no sex differentiation in these ages, livers from sufficient offspring of the same litters were pooled at each age to achieve the desired amount of tissue (200-300 mg). Only male mice for the young adult group (day 45) were selected in this study to eliminate gender difference. Livers were frozen immediately in liquid nitrogen, and stored at -80°C.
DNA methylation. Genomic DNA from the liver tissues was isolated by a ChargeSwitch gDNA Mini Tissue Kit (Invitrogen, Carlsbad, CA), and sonicated to an average length of 300-500 bps. An antibody against 5-methyl-cytosine (ab51552 from Abcam Inc. Cambridge, MA) was used for immunoprecipitation of methylated DNA fragments as described previously (Zang et al., 2006; Furomoto et al., 2007). The purified ChIP-enriched fragments were amplified by four linear amplification reactions with Sequenase (USB, Cleveland, OH). After purification, the randomly primed ChIP DNA was amplified for 30 cycles using the fixed sequence primers. The amplified DNAs were purified, quantified, and tested by Q-PCR at the same genomic regions as the original immunoprecipitated DNA to assess quality of the amplification reactions. The amplified DNAs were fragmented, labeled with the DNA Terminal Labeling Kit according to the standard Affymetrix protocol, and hybridized to arrays. DNA without immunoprecipitation was used as control (input) for background hybridization. Arrays were washed and scanned by a GeneChip HT Array Plate Scanner according to Affymetrix standard procedures. The scanned output CEL files were analyzed with Affymetrix tiling analysis software (TAS) to generate binary analysis result (BAR) files that contain the intensities for all probes on the arrays. The intensities were converted to fold changes based on immunoprecipitated signals vs input signals. The results were compiled into browser extensible data (BED) as explained below.
Histone modification. Likewise, histone modification profiles of H3K4me2 and H3K27me3 were established by using a similar protocol but with polyclonal antibodies of Millipore 07-030 (anti-H3K4me2) and 07-449 (anti-H3K27me3), respectively. Liver tissues were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. The tissues were disaggregated with a Tissue Tearor (BioSpec Products, Bartlesville, OK) and chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA was sheared to an average length of 300-500 bp. Genomic DNA (input) was prepared by treating aliquots of chromatin with RNase and proteinase K, followed by phenol and chloroform extractions and ethanol precipitation. An aliquot of chromatin (~30 ?g) was used to perform ChIP as previously described (Wang Z, et al., 2008). ChIP and input DNAs were amplified by whole-genome amplification (WGA) using the GenomePlex WGA Kit (Sigma-Aldrich, St. Louis, MO). Fragmentation, labeling and hybridization, chip washing and scanning were performed as for the DNA methylation assays.
Based on the Affymetrix's recommendation, thresholds for DNA methylation, H3K4me2, and H3K27me3 enrichment were set as 3-fold signal increases, which corresponds to a false discovery rate of less than 0.1. A genomic region with a sequence of >800 bp and an average signal increase greater than the threshold was defined as an enriched interval shown in the BED files. A genomic region with one or more enriched intervals in close proximity to each other (at least one base overlap) at any given age was defined as an active region.
Thanks to Steven Hart in the Department of Pharmacology, Toxicology, and Therapeutics at the University of Kansas Medical Center, Kansas City, KS, for contributing these tracks.
Zhang X et al. (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126:1189-1201.
Furumoto T, et al. (2007) A kinase subunit of the human mediator complex, CDK8, positively regulates transcriptional activation. Genes Cells 12:119-132.
Wang Z, et al. (2008) Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet. 40:897-903.