Polycomb group genes (PcG) encode proteins that mediate transcriptional repression. First identified in Drosophila
as genes necessary to maintain the silencing of homeotic genes, it is now evident that PcG proteins have many other targets (reviewed in Simon and Kingston 2009
). Genome-wide studies show that the PcG repressive mark H3K27me3 is associated with hundreds of genes in single cell types and that targets can be cell-type specific (reviewed in Schwartz and Pirrotta 2008
). Although it is evident that PcG proteins can decrease expression levels in addition to completely silencing expression, it is not clear what determines whether a gene will be completely or only partially repressed.
, PcG proteins are associated with Polycomb group response elements (PRE), DNA elements that recruit PcG proteins to the DNA (reviewed in Müller and Kassis 2006
; Ringrose and Paro 2007
). In genome-wide studies, PREs were identified as binding sites for multiple PcG proteins (Schwartz et al. 2006
; Négre et al. 2006
; Tolhuis et al. 2006
). Two functional assays have also been used to identify PREs. In one assay, the PRE is combined in a transgene with regulatory DNA from a gene normally regulated by PcG proteins, where the PRE is required to maintain the “off” transcriptional state (Müller and Bienz 1991
; Hagstrom et al. 1997
). In the other assay, PREs are used to repress expression of the mini-white
reporter gene in transgenic flies. Because mini-white
repression is stronger in flies homozygous for the PRE-mini-white
reporter, this latter assay has been called pairing-sensitive silencing (Kassis 1994).
One of the puzzles of the transgene assays for PREs is that silencing does not occur at every chromosomal insertion site. For example, for the four engrailed
PREs, pairing-sensitive silencing was observed at a frequency of 21–62% of insertion sites (Americo et al. 2002
; Cunningham et al. 2010
). PRE activity is regulated by the expression state of the gene it regulates; thus it follows that PRE activity in transgenes is dependent on the activity of regulatory elements that flank the transgene insertion site.
We have been studying a 181-bp en
DNA fragment that acts as a PRE in several different assays: (1) it represses inappropriate expression in both en
- and Ubx
-reporter genes in embryos (Americo et al. 2002
; Devido et al. 2008
); (2) PcG proteins are associated with it in tissue culture cells, embryos, larvae, and adults (Strutt and Paro 1997
; Négre et al. 2006
; Oktaba et al. 2008
); and (3) it acts as a pairing-sensitive silencing element (Kassis 1994). This fragment contains binding sites for the PRE DNA binding proteins Pho, Pho-like, GAGA factor, and Spps (Americo et al. 2002
; Brown et al. 2005
; Brown and Kassis 2010
). Thus, the 181-bp DNA fragment is clearly a PRE. Therefore, we reasoned that conducting a genetic screen for mutations that alter the activity of this PRE might yield mutations in PcG genes.
We conducted a genetic screen for dominant suppressors of pairing-sensitive silencing by a transgene that contained the 181-bp en
PRE and mini-white
. These mutations were rare; we only obtained nine suppressors among 68,274 genomes screened. None of the mutations affected mini-white
repression of transgenes at all chromosomal insertion sites. This suggests that none of the mutations affects PRE activity directly. Instead, we believe that these mutations affect the expression of genes flanking the transgene insertion site. Consistent with this, two of the dominant suppressors are the same gain-of-function mutation in the gene without children
), which encodes a transcriptional activator. Our data suggest that there is a competition between transcriptional activators and PcG repression and that certain types of activators may be better able to overcome PcG repression.