In Drosophila, two chromosome-wide gene regulatory systems have been characterized: the dosage compensation system that acts on the male X-chromosome, and the system that we have discovered, POF (Painting of Fourth) – the chromosome-specific regulation of genes located on the 4th chromosome, which is the first example of a chromosome-wide, autosome-specific gene regulatory system that allow the survival of haplo-4 flies. The targeting and mechanisms employed by these systems to deal with chromosomal aneuploidies can be used as a model to delineate intrinsic mechanisms to tolerate aneuploidies. Central to these mechanisms are epigenetic responses in form of histone modifications and both the X-chromosome and the 4th chromosome are known to have chromosome-specific chromatin structures, e.g. the male X-chromosome is enriched in acetylated H4K16 and the 4th is enriched in methylated H3K9. We have investigated additional histone modifications and found a significant reduction in tri-methylated H3K36 on the male X-chromosome and increased enrichment on the 4th chromosome. H3K36 methylation has been implicated in diverse processes, including alternative splicing, DNA repair and recombination, dosage compensation and transcriptional repression. We will further explore the role of H3K36 methylation in transcription regulation and aneuploidy response. In flies, methylation of H3K36 is mediated by at least three different methyltransferases; Ash1, SET2 and MES-4 together with at least three different demethylases; Kdm2, Kdm4A and Kdm4B. In the current project we will use RNA-seq on mutant fly lines (dissected brains) to test how the chromosome-enriched histone modifications methylated H3K36 affect transcription output. In collaboration with Yuri Schwartz's group (UmU) we have established all single mutants as well as double and triple mutants to further delineate division of labor. In total we will test 12 different genetic conditions.