Genes of the Polycomb group maintain long-term, segment-specific repression of the homeotic genes in Drosophila. DNA targets of Polycomb group proteins, called Polycomb response elements (PREs), have been defined by several assays, but they have not been dissected in their original chromosomal context. An enhanced method of gene conversion was developed to generate a series of small, targeted deletions encompassing the best-studied PRE, upstream of the Ultrabithorax (Ubx) transcription unit in the bithorax complex. Deletions that removed an essential 185-bp core of the PRE caused anterior misexpression of Ubx and posterior segmental transformations, including the conversion of the third thoracic segment toward a duplicate first abdominal segment. These phenotypes were variable, suggesting some cooperation between this PRE and others in the bithorax complex. Larger deletions up to 3 kb were also created, which removed DNA sites reportedly needed for Ubx activation, including putative trithorax response elements. These deletions resulted in neither loss of Ubx expression nor loss-of-function phenotypes. Thus, the 3-kb region including the PRE is required for repression, but not for activation, of Ubx.bithorax ͉ Ultrabithorax ͉ gene conversion
Long-term repression of homeotic genes in the fruit fly is accomplished by proteins of the Polycomb Group, acting at Polycomb response elements (PREs). Here we use gene conversion to mutate specific DNA motifs within a PRE to test their relevance, and we exchange PREs to test their specificity. Previously we showed that removal of a 185 bp core sequence from the bithoraxoid PRE of the bithorax complex results in posteriorly directed segmental transformations. Mutating multiple binding sites for either the PHO or the GAF proteins separately in the core bithoraxoid PRE resulted in only rare and subtle transformations in adult flies. However, when both sets of sites were mutated, the transformations were similar in strength and penetrance to those caused by the deletion of the 185 bp core region. In contrast, mutating the singly occurring binding site of another DNA-binding protein, DSP1 (reportedly essential for PRE-activity), had no similar effect in combination with mutated PHO or GAF sites. Two minimal PREs from other segment-specific regulatory domains of the bithorax complex could substitute for the bithoraxoid PRE core. Our in situ analysis suggests that core PREs are interchangeable, and the cooperation between PHO and GAF binding sites is indispensable for silencing.
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