Cells were grown in BPYE medium with 25% bactopeptone, 20% yeast extract and 5% heat inactivated fetal calf serum. Expression plasmids for Dam-fusion and Dam-only proteins were transfected in parallel by electroporation, and cells were isolated 24 hr later. The Dam enzyme deposits stable adenine-methylation footprings at genomic sites bound by the Dam-fusion.
Genomic DNA was isolated and adenine-methylated fragments were amplified by methylation-specific PCR using Cy-dye labeled random nonamers. To correct for nonspecific binding of Dam and local differences in DNA accessibility, methylated fragments of Kc cells transfected with a Dam-only construct were labeled with a different fluorescent dye.
Isolated DNA was hybridized to NimbleGen Drosophila 385k array rel4 300bp genomic tiling array.
Quantitative DamID genome profiles for 53 chromatin proteins were analyzed to identify distinct combinations of proteins that recur in the genome. Principal component analysis was initially performed to reduce the dimensionality of the data. The first three principal components were then used, which together account for 57.7% of total variance. Doing so, five distinct lobes could be observed in the three-dimensional scatter plot. The result was robust to different quantification methods. Having established that classification into five types properly summarizes the data, a five-state hidden Markov model was fitted onto the first three principal components. Thus, every probed sequence in the genome was assigned one of five exclusive chromatin types. Each of the five chromatin signatures was labeled with a color, and further characterzied by histone modification and gene expression patterns.
GREEN chromatin: Classic (including pericentric) heterochromatin; marked specifically by binding of Su(var)3-9, Su(var)205 (HP1), Lhr and HP6 proteins; H3K9me2 is highly and specifically enriched. Shares markers with other chromatin types that exhibit low levels of transcriptional activity.
BLUE chromatin: PcG (Polycomb Group) chromatin; marked specifically by binding of Pc, E(z), Pcl, and Sce proteins; H3K27me3 is highly enriched. Shares markers with other chromatin types that exhibit low levels of transcriptional activity.
BLACK chromatin: Transcriptionally silent chromatin; genes typically not expressed or expressed at very low levels (in the cell type assayed). Probably dynamic: may be locally remodeled into different chromatin type during tissue-specific expression. Marked by binding of His1, D1, ial, and SuUR proteins; su(Hw), Lam, and eff proteins frequently present; shares these markers with other chromatin types that exhibit low levels of transcriptional activity.
YELLOW chromatin: Transcriptionally active euchromatin; genes usually broadly expressed. Specific to YELLOW chromatin is binding of MRG15 protein; H3K36me3 is enriched. RED and YELLOW chromatin both marked by binding of Rdp3, Sir2, Sin3A, Df31, ash2, and Max proteins; H3K4me2 and H3K79me3 are usually high; H3K9me2 and H3K27me3 are low.
RED chromatin: Transcriptionally active euchromatin; genes typically exhibit developmentally regulated expression. Specific to RED chromatin are binding of brm, Su(var)2-10, MED31, Caf1, EcR, Trl (GAF), and Jra proteins. RED and YELLOW chromatin both marked by binding of Rdp3, Sir2, Sin3A, Df31, ash2, and Max proteins; H3K4me2 and H3K79me3 are usually high; H3K9me2 and H3K27me3 are low.