Trithorax- and Polycomb-group response elements within an Ultrabithorax transcription maintenance unit consist of closely situated but separable sequences.

In Drosophila, two classes of genes, the trithorax group and the Polycomb group, are required in concert to maintain gene expression by regulating chromatin structure. We have identified Trithorax protein (TRX) binding elements within the bithorax complex and have found that within the bxd/pbx regulatory region these elements are functionally relevant for normal expression patterns in embryos and confer TRX binding in vivo. TRX was localized to three closely situated sites within a 3-kb chromatin maintenance unit with a modular structure. Results of an in vivo analysis showed that these DNA fragments (each ;400 bp) contain both TRXand Polycomb-group response elements (TREs and PREs) and that in the context of the endogenous Ultrabithorax gene, all of these elements are essential for proper maintenance of expression in embryos. Dissection of one of these maintenance modules showed that TRX- and Polycomb-group responsiveness is conferred by neighboring but separable DNA sequences, suggesting that independent protein complexes are formed at their respective response elements. Furthermore, we have found that the activity of this TRE requires a sequence (;90 bp) which maps to within several tens of base pairs from the closest neighboring PRE and that the PRE activity in one of the elements may require a binding site for PHO, the protein product of the Polycomb-group gene pleiohomeotic. Our results show that long-range maintenance of Ultrabithorax expression requires a complex element composed of cooperating modules, each capable of interacting with both positive and negative chromatin regulators. Body segment identity in many organisms is achieved, in large part, through the activities of homeotic genes during development. In Drosophila, the establishment and maintenance of their patterns of expression are critical for the determination of the fates of embryonic cells. Two groups of genes, the trithorax group (trxG) (reviewed in reference 19) and the Polycomb group (PcG) (reviewed in references 3, 25, 29, and 38), play a predominant role in maintenance of the on and off states, respectively, of homeotic gene expression during development. It has been proposed that the products of different PcG genes assemble in a multimeric complex only at target genes that are not actively being transcribed, ostensibly locking these genes in an inactive state. This presumably imprints a determined state of the chromatin which could be inherited by the cellular progeny (25). Indeed, several Polycomb-group (PcG) proteins analyzed thus far colocalized at a large number of sites on salivary gland polytene chromosomes, suggesting that they often function together (11, 23, 33). Moreover, it was shown that the Polycomb (Pc) and polyhomeotic products are constituents of a large multimeric protein complex (15). Contrasting with PcG repression is activation by trxG genes. The trxG includes trithorax (trx), brahma (brm), Trithorax-like (Trl), ash1, ash2, and more than 10 additional members, many of which are only minimally characterized. The products of these genes function as transcriptional activators that sustain particular patterns of homeotic gene expression which act antagonistically to those of the PcG. It has been shown that in trx mutant embryos, expression of all bithorax complex (BX-C) genes and several Antennapedia complex (ANT-C) genes are affected in a tissue-, parasegment (PS)-, and promoter-specific fashion (4, 24, 36). Like PcG gene products, those of the trxG have been found at multiple sites on polytene chromosomes, suggesting that targets of these proteins are not limited to the genes of the homeotic complexes. Indeed, it was shown that the region-specific homeotic gene fork head is a direct target gene of trx based on Trithorax protein (TRX) binding on polytene chromosomes (21).