The cis -regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila

Abstract

Abstract Hox genes encode a family of transcription factors that, despite having similar in vitro DNA binding preferences, regulate distinct genetic programs along the metazoan anterior-posterior axis. To better define mechanisms of Hox specificity, we compared and contrasted the ability of abdominal Hox factors to regulate two cis -regulatory elements within the Drosophila embryo. Both the Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox factors form cooperative complexes with the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the distal-less leg selector gene via the DCRE , whereas only Abd-A interacts with Exd and Hth on the RhoA element to activate a rhomboid serine protease gene that stimulates Epidermal Growth Factor secretion. By swapping binding sites between these elements, we found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can also convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE, but only in one orientation. We further show that the orientation and spacing of Hox sites relative to additional transcription factor binding sites within the RhoA and DCRE elements is critical to mediate appropriate cell- and segment-specific output. These results indicate that the interaction between Hox, Exd, and Hth neither determines activation vs repression specificity nor defines Ubx vs Abd-A specificity. Instead the precise integration of Hox sites with additional TF inputs is required for accurate transcriptional output. Taken together, these studies provide new insight into the mechanisms of Hox target and regulatory specificity as well as the constraints placed on regulatory elements to convey appropriate outputs. Author Summary The Hox genes encode a family of transcription factors that give cells within each region along the developing body plan a unique identity in animals from worms to mammals. Surprisingly, however, most of the Hox factors bind the same or highly similar DNA sequences. These findings raise a paradox: How can proteins that have highly similar DNA binding properties perform different functions in the animal by regulating different sets of target genes? In this study, we address this question by studying how two Hox factors regulate the expression of target genes that specify leg development and the making of liver-like cells in the developing fly. By comparing and contrasting how Hox target genes are activated and/or repressed, we found that the same Hox binding sites can mediate either activation or repression in a manner that depends upon context. In addition, we found that a Hox binding site that is normally regulated by only one Hox factor, can also be used by more than one Hox factor swapped into another target gene. These findings indicate that the specificity of a Hox factor to regulate target genes does not rely solely upon DNA binding specificity but also requires regulatory specificity.

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