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In an associated manuscript, we have formulated a stochastic model for transcription factor-nucleosome competition to account for how the homeodomain transcription factor Bicoid (Bcd) regulates its targets in a concentration-sensitive manner. More...

In an associated manuscript, we have formulated a stochastic model for transcription factor-nucleosome competition to account for how the homeodomain transcription factor Bicoid (Bcd) regulates its targets in a concentration-sensitive manner. This model has three central assumptions that we tested using ChIP-seq. First, we assume that, following mitotic exit, Bcd has the ability to compete with nucleosomes and bind to DNA independently of the completion of DNA replication. Second, we also assume that transcription becomes immediately detectable following the completion of both Bcd binding and DNA replication, and that therefore the recruitment of RNA Pol II to the promoter likely also occurs independently of these processes. Finally, we assumed that RNA Pol II initiates transcription at Bcd target genes independently of Bcd, whose function is to release initiated RNA Pol II into productive elongation. We did two experiments to test these assumptions. First, we measured binding of RNA Pol II and Bcd to chromatin in control and replication-inhibited embryos. Second, we measured RNA Pol II binding in control and bcd oskar torsolike triple-mutant embryos. We find that RNA Pol II initiates transcription under conditions of DNA replication arrest, but shows decreases within gene bodies, suggesting that entry into productive transcriptional elongation requires completion of DNA replication. We also find that Bcd binds chromatin independently of DNA replication. Finally, we find that in the absence of Bcd, Bcd target genes initiate transcription, but lack elongating RNA Pol II. These ChIP-seq measurements therefore support the assumptions made in our modeling approach. Overall design: ChIP-seq of either RNA Pol II, Bcd, or a negative control (anti myc-tag) in stage 5 Drosophila embryos. Experiment 1: embryos were permeabilized and treated with hydroxyurea for 40 minutes prior to fixation, followed by hand-sorting for embryos staged at late nuclear cycle 13, and processing for ChIP-seq. Experiment 2: wild-type or bcd oskar torsolike mutant embryos were collected and fixed prior to hand-sorting for stage 5 morphology, followed by processing for ChIP-seq. Less...