Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins

Drosophila Polycomb group (PcG) and Trithorax group (TrxG) proteins are responsible for the maintenance of stable transcription patterns of many developmental regulators, such as the homeotic genes. We have used a ChIP-on- Chip approach to map the distribution of several PcG/TrxG proteins as well as histone modifications across the two homeotic complexes ANT-C and BX-C. Our data indicate the colocalization of the Polycomb repressive complex 1 (PRC1) with Trx and the DNA binding protein Pleiohomeotic (Pho) at discrete sequence elements as well as significant chromatin assembly differences in active and inactive regions. Trx binds to the promoters of active genes and noncoding transcripts. Most strikingly, in the active state Pho covers extended chromatin domains over many kilobases. The histone modifications investigated, seem to be mutually exclusive, with histone H3 trimethylated at lysine 27 covering inactive chromatin domains whereas hyperacetylated histone H4 is present only at active genes. Keywords: ChIP-chip, cell line comparison Currently it is unclear how the interplay between DNA elements, histones and their modifications and the PcG/TrxG-chromatin-associated proteins results in a stable gene expression state which is heritable through DNA replication and mitosis. A key to the understanding of the molecular mechanisms is to uncover the chromatin sites of action of the PcG/TrxG proteins and to correlate their binding patterns with the presence of histone modifications as well as with the expression level of their target genes. Here, we generated a DNA tiling array that comprises the BX-C and the ANT-C as a series of overlapping 1 kb PCR fragments. Based on this tiling array we investigated the distribution of three core components of the PRC1 (PC, PH and PSC), the DNA binding protein PHO, TRX, hyperacetylated H4 and H3K27me3 in two different Drosophila embryonic cell lines that show different expression states for the AbdB and Dfd genes. The AbdB domain and the Dfd gene are highly active in SF4 and in Kc cells, respectively, whereas the other HOX genes are silent. Two versions of the tiling array were used in the course of this study. Version 3 contained 10 and Version 4 contained 8 spotted replicates of each PCR fragment. With Version 3 arrays either three or four independent chromatin samples were taken for each antibody with dye swap, i.e. six or eight arrays. Normalization was based on spike-ins and all 10 spot replicates were averaged afterwards. The biological effect was estimated with a regularized linear model which contained a dye effect and treated the technical replicates like a random effect. With Version 4 arrays either four or six biological replicates were used where in every other hybridization Cy3 and Cy5 were interchanged between ChIP and input sample (four or six arrays total). Spike-ins served to determine the normalization parameters which were subsequently used to normalize all intensity measurements on every microarray. All 8 spot replicates were averaged. The statistical model applied to estimate the biological effect included a dye effect. Multiple testing correction was applied and false discovery rates (FDR) (as well as fold-changes) were reported and used for significance thresholding. All analyses were performed with R (www.rproject. org) and Bioconducter (www.bioconductor.org). The package arrayMagic was used for processing, the package vsn for normalisation and the package limma for statistical testing (Buness, A., Huber, W., Steiner, K., Sultmann, H., and Poustka, A. (2005). arrayMagic: two-colour cDNA microarray quality control and preprocessing. Bioinformatics 21, 554-556; Huber, W., von Heydebreck, A., Sultmann, H., Poustka, A., and Vingron, M. (2002). Variance stabilization applied to microarray data calibration and to the quantification of differential expression. Bioinformatics 18 Suppl 1, S96-104; Smyth, G. K., Michaud, J., and Scott, H. S. (2005). Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21, 2067-2075).