Because chromatin determines whether information encoded in DNA is accessible to transcription factors, dynamic chromatin states in development may constrain how gene regulatory networks impart embryonic pattern. To determine the interplay between chromatin states and regulatory network function, we performed ATAC-seq on Drosophila embryos during the establishment of the segmentation network, comparing wild-type and mutant embryos in which all graded maternal patterning inputs are eliminated. While during the period between zygotic genome activation and gastrulation many regions maintain stable accessibility, cis-regulatory modules (CRMs) within the network undergo extensive patterning-dependent changes in accessibility. A component of the network, Odd-paired (opa), is necessary for pioneering accessibility of late segmentation network CRMs. opa-driven changes in accessibility are accompanied by equivalent changes in gene expression. Interfering with the timing of opa activity impacts the proper patterning of expression. These results indicate that dynamic systems for chromatin regulation directly impact the reading of embryonic patterning information.

31Because regulatory networks of transcription factors drive embryonic patterning, it is possible that 32 chromatin accessibility states impact how networks interact with information encoded in DNA. To 33 determine the interplay between chromatin states and regulatory network function, we performed 34 ATAC seq on Drosophila embryos over the period spanning the establishment of the segmentation 35 network, from zygotic genome activation to gastrulation. Chromatin accessibility states are 36 dynamic over this period, and establishment of the segmentation network requires maturation of 37 the ground chromatin state. Elimination of all maternal patterning information allows 38 identification of patterning-dependent and -independent dynamic chromatin regions. A significant 39proportion of patterning-dependent accessibility stems from pioneer activity of the pair-rule factor 40Odd-paired (opa). While opa is necessary to drive late opening of segmentation network cis-41 regulatory elements, competence for opa to pioneer is regulated over time. These results indicate 42 that dynamic systems for chromatin regulation directly impact the interpretation of embryonic 43 patterning information. 44In this study, we have investigated how chromatin accessibility states change following 97ZGA and to what extent these changes are dependent on the mechanisms of embryonic patterning. 98We find that the ZGA chromatin state must continue to evolve in order to support the establishment 99 of accessible CRMs within the regulatory network that confers embryonic segmental identities. 100By measuring changes in chromatin accessibility over the one-hour period between ZGA and 101 gastrulation comparing wild-type and mutant embryos in which all graded maternal inputs to 102 patterning are either eliminated or flattened, we define sites that display dynamic regulation of 103 accessibility downstream of either localized pattern-dependent or global patterning-independent 104 cues. We find that although maternal patterning systems are limited in their ability to influence 105 directly chromatin accessibility states, distinct downstream components of zygotic gene regulatory 106 networks make major contributions to patterning-dependent alterations of the chromatin 107 accessibility landscape. We focus on the characterization of one such factor, Odd-paired (Opa), 108 which we demonstrate is both necessary and sufficient to pioneer open chromatin states for a set 109 of cis-regulatory elements critical for the function of the embryonic segmentation network. These 110 results highlight that individual components of gene regulatory networks may operate not simply 111 to activate or repress target gene expression, but to dictate how and when network components 112 interact by controlling dynamic cis-regulatory element accessibility. 113 114 Results 115 The ZGA Chromatin State is Insufficient to Support Embryonic Segmentation 116To estimate the sufficiency of the ZGA chromatin state to support early embryonic 117 development, we scored all known enhancer...

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