The transition from a specified germ cell to a population of pluripotent cells occurs rapidly following fertilization. During this developmental transition, the zygotic genome is largely transcriptionally quiescent and undergoes significant chromatin remodeling. In Drosophila, the DNA-binding protein Zelda (also known as Vielfaltig) is required for this transition and for transcriptional activation of the zygotic genome. Open chromatin is associated with Zelda-bound loci, as well as more generally with regions of active transcription. Nonetheless, the extent to which Zelda influences chromatin accessibility across the genome is largely unknown. Here we used formaldehyde-assisted isolation of regulatory elements to determine the role of Zelda in regulating regions of open chromatin in the early embryo. We demonstrate that Zelda is essential for hundreds of regions of open chromatin. This Zelda-mediated chromatin accessibility facilitates transcription-factor recruitment and early gene expression. Thus, Zelda possesses some key characteristics of a pioneer factor. Unexpectedly, chromatin at a large subset of Zelda-bound regions remains open even in the absence of Zelda. The GAGA factor-binding motif and embryonic GAGA factor binding are specifically enriched in these regions. We propose that both Zelda and GAGA factor function to specify sites of open chromatin and together facilitate the remodeling of the early embryonic genome.

Background: Zelda initiates widespread transcription of the zygotic genome during embryogenesis. Results: Zelda binds DNA using C-terminal zinc fingers and activates transcription through a low-complexity domain. Conclusion: Transcriptional activation by Zelda is conserved in insects and uses domains we have identified to bind cisregulatory regions and drive gene expression. Significance: We provide the first insights into the functional domains of the essential activator Zelda.

It has been suggested that transcription factor binding is temporally dynamic, and that changes in binding determine transcriptional output. Nonetheless, this model is based on relatively few examples in which transcription factor binding has been assayed at multiple developmental stages. The essential transcription factor Grainy head (Grh) is conserved from fungi to humans, and controls epithelial development and barrier formation in numerous tissues. Drosophila melanogaster, which possess a single grainy head (grh) gene, provide an excellent system to study this conserved factor. To determine whether temporally distinct binding events allow Grh to control cell fate specification in different tissue types, we used a combination of ChIP-seq and RNA-seq to elucidate the gene regulatory network controlled by Grh during four stages of embryonic development (spanning stages 5-17) and in larval tissue. Contrary to expectations, we discovered that Grh remains bound to at least 1146 genomic loci over days of development. In contrast to this stable DNA occupancy, the subset of genes whose expression is regulated by Grh varies. Grh transitions from functioning primarily as a transcriptional repressor early in development to functioning predominantly as an activator later. Our data reveal that Grh binds to target genes well before the Grh-dependent transcriptional program commences, suggesting it sets the stage for subsequent recruitment of additional factors that execute stage-specific Grh functions.

Single-nucleotide polymorphisms (SNPs) are preferred over microsatellite markers in many evolutionary studies, but have only recently been applied to studies of parentage. Evaluations of SNPs and microsatellites for assigning parentage have mostly focused on special cases that require a relatively large number of heterozygous loci, such as species with low genetic diversity or with complex social structures. We developed 120 SNP markers from a transcriptome assembled using RNA-sequencing of a songbird with the most common avian mating system-social monogamy. We compared the effectiveness of 97 novel SNPs and six previously described microsatellites for assigning paternity in the black-throated blue warbler, Setophaga caerulescens. We show that the full panel of 97 SNPs (mean H = 0.19) was as powerful for assigning paternity as the panel of multiallelic microsatellites (mean H = 0.86). Paternity assignments using the two marker types were in agreement for 92% of the offspring. Filtering individual samples by a 50% call rate and SNPs by a 75% call rate maximized the number of offspring assigned with 95% confidence using SNPs. We also found that the 40 most heterozygous SNPs (mean H = 0.37) had similar power to assign paternity as the full panel of 97 SNPs. These findings demonstrate that a relatively small number of variable SNPs can be effective for parentage analyses in a socially monogamous species. We suggest that the development of SNP markers is advantageous for studies that require high-throughput genotyping or that plan to address a range of ecological and evolutionary questions.

Diffuse midline gliomas and posterior fossa type-A ependymomas contain the highly recurrent histone H3 K27M mutation and the H3 K27M-mimic EZHIP, respectively. In vitro, H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3K27me3 levels, however residual peaks of H3K27me3 remain at CpG islands through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with an allosterically activated form of PRC2 in vivo. The formation of H3 K27M-and EZHIP-PRC2 complexes occurs at CpG islands containing H3K27me3 and impedes PRC2 and H3K27me3 spreading. While EZHIP is not found outside of placental mammals, we find that expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved molecular mechanism. Our results highlight the mechanistic similarities between EZHIP and H3 K27M in vivo and provide mechanistic insight for the retention of residual H3K27me3 in tumors driven by these oncogenes.