Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4^th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4^th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF). Although deletion heterozygotes and chromosomal aneuploidies have been used in genetic studies for decades, the relationships between chromosome doses and transcript outputs have been difficult to unravel. In other words, the effects of copy changes on the regulation of entire chromosomes or large chromosomal domains are largely unknown. Hence, we studied these relationships in Drosophila using microarrays prepared from flies with a dosage series of chromosomal domains and a dosage series of the 4^th chromosome. We observed significant buffering of expressed genes, i.e., on average they were expressed at >50% of wild-type levels when present in single copies instead of two copies (the normal complement of diploids). This buffering was also seen to be much stronger for differentially expressed genes than ubiquitously expressed genes. Our findings therefore support the presence of chromosome-wide buffering mechanisms. In addition, we found evidence of a chromosome-specific protein POF-mediated mechanism in the buffering of the 4^th chromosome. Overall, our results suggest that a general buffering system acts on most genes present as single copies due to deletions or chromosome losses. Further work on gene buffering effects should make substantial contributions to our understanding of genome-wide gene regulation.

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Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional “peak” in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome. Species where males and females have a different number of sex chromosomes have to equalize the transcriptional output from the genes located on the X chromosome. In Drosophila this mechanism is achieved by a 2-fold up-regulation of the single male X chromosome. Flies also possess an additional chromosome-wide regulatory system that regulates the transcriptional output from genes on the fourth chromosome. In this case the protein Painting of Fourth (POF), together with heterochromatin protein 1 (HP1) bind to the fourth chromosome and fine tune gene expression. By using a high resolution map of POF and HP1 binding, we can show that they bind to the same sequences on the fourth chromosome. We also demonstrate that POF and HP1 bind to active genes with preferences for exon sequences. In gene regulatory mechanisms, including chromosome-wide gene regulation, a simple on/off switch is often not enough. Our findings support the presence of a balancing mechanism in which the dual recruitment of a repressing and a stimulating factor makes the transcription efficiency more stable and less sensitive to fluctuations.