Zygotic pioneer factor activity of Odd-paired/Zic is necessary for establishing the Drosophila Segmentation Network

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 over the period spanning 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 segmentation network undergo extensive patterning-dependent changes in accessibility. A component of the network, Odd-paired (opa), is necessary for pioneering accessibility of segmentation network CRMs. While opa is necessary to drive late opening of segmentation network cis-regulatory elements, competence for opa to pioneer is regulated over time. These results indicate that dynamic systems for chromatin regulation directly impact the interpretation of embryonic patterning information. Overall design: This study contains four distinct experiments. Experiment 1: ATAC-seq comparing wild-type and bicoid oskar capicua torsolike Toll[RM9] mutant embryos, staged either at 12 or 72 minutes into nuclear cycle 14. Three biological replicates (individual embryos) were collected per genotype and timepoint and subjected to ATAC library preparation. Samples were subjected to paired-end sequencing on an Illumina HiSeq2500 (2 x 63 bp reads). Experiment 2: ATAC-seq on a collection of embryos produced from a cross between opa[IIP32]/+ adults. Embryos were staged at 72 minutes into nuclear cycle 14. Twenty two embryos in total were collected and subjected to paired-end sequencing on an Illumina HiSeq 2500 (2 x 63 bp reads). Experiment 3: ChIP-seq for myc-tagged Opa. 200 cellular blastoderm stage embryos were collected from opa-myc homozygous adults and ChIPped with an anti-myc antibody. The control sample was an equivalent number of staged 'wild-type', i.e., not-myc-tagged embryos. The wild-type genotype is w[1118]. Three independent biological replicates were performed for experimental and control samples. ChIP-seq libraries were subjected to single-end sequencing on an Illumina HiSeq4000 (1 x 50 bp reads). Experiment 4: ATAC-seq comparing wild-type and maternal misexpression of odd-paired (tub>opa), staged at 12 minutes into nuclear cycle 13. Six biological replicates (individual embryos) were collected per genotype and subjected to ATAC library preparation. Samples were subjected to paired-end sequencing on an Illumina NextSeq500 (2 x 36bp reads).