RNA Polymerase Stalling at Developmental Control Genes in the Drosophila Embryo

7 Corresponding authors 2 The genomic program for development depends on the precise coordination of gene activity in space and time 1. It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter 2. Although there are well-documented examples where Pol II is recruited to a gene but stalls 3-14 , a general role for Pol II stalling in development has not been established. We have performed comprehensive Pol II ChIP-chip assays in Drosophila embryos using a well-studied mutant, Toll 10b , which contains a homogenous population of mesodermal precursor cells 15-21. Three distinct binding behaviors of Pol II were identified: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The striking feature of the ~10% genes that are stalled is that they are highly enriched for developmental control genes that are repressed at the time of the analysis or poised for activation during subsequent stages of development. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development. Pol II stalling is probably best studied at heat shock genes in Drosophila, where Pol II engages in transcription but pauses immediately downstream of the transcriptional start site 3,4,22. Upon activation by heat shock, Pol II is able to rapidly transcribe these genes. Regulation of Pol II activity after recruitment has also been described in bacteria 23 , yeast 13 and mammalian cell lines 3,6-12 , and includes instances where Pol II is found in an inactive pre-initiation complex 24,25. We will collectively refer to inactive Pol II near the transcription start site as stalled Pol II. To determine at which genes Pol II stalling occurs during development, we analyzed global Pol II occupancy in whole Drosophila embryos. While this is one of the few systems where genomics approaches can easily be applied to developmental questions, interpretation is complicated by the occurrence of multiple tissues. To reduce the complexity, we used Toll 10b embryos (2-4 hours after fertilization), a well characterized mutant that contains a homogenous 3 population of mesodermal precursor cells at the expense of neuronal and ectodermal cells 15-21. In Toll 10b mutants, mesodermal genes are uniformly activated while genes required for the development of ectodermal and neural tissues are repressed throughout the embryo 15-17. Previous whole-genome microarray experiments have …