Project description:The transcription factor Grainyhead primes epithelial enhancers for spatiotemporal activation by displacing nucleosomes [ATAC-seq Grh mutants and Human GRHL]
Project description:Transcriptional enhancers function as docking platforms for combinations of transcription factors to control gene expression. How enhancer sequences determine nucleosome occupancy, transcription factor recruitment, and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains we found that SNPs affecting Grainyhead binding sites causally determine the accessibility of epithelial enhancers. We show that deletion or ectopic expression of Grh causes loss or gain of DNA accessibility, respectively. However, while Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs, GRHL1/2/3, function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers, but not for their activation. Our data support the model that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.
Project description:Transcriptional enhancers function as docking platforms for combinations of transcription factors to control gene expression. How enhancer sequences determine nucleosome occupancy, transcription factor recruitment, and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains we found that SNPs affecting Grainyhead binding sites causally determine the accessibility of epithelial enhancers. We show that deletion or ectopic expression of Grh causes loss or gain of DNA accessibility, respectively. However, while Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs, GRHL1/2/3, function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers, but not for their activation. Our data support the model that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.
Project description:Transcriptional enhancers function as docking platforms for combinations of transcription factors to control gene expression. How enhancer sequences determine nucleosome occupancy, transcription factor recruitment, and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains we found that SNPs affecting Grainyhead binding sites causally determine the accessibility of epithelial enhancers. We show that deletion or ectopic expression of Grh causes loss or gain of DNA accessibility, respectively. However, while Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs, GRHL1/2/3, function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers, but not for their activation. Our data support the model that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.
Project description:Transcriptional enhancers function as docking platforms for combinations of transcription factors to control gene expression. How enhancer sequences determine nucleosome occupancy, transcription factor recruitment, and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains we found that SNPs affecting Grainyhead binding sites causally determine the accessibility of epithelial enhancers. We show that deletion or ectopic expression of Grh causes loss or gain of DNA accessibility, respectively. However, while Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs, GRHL1/2/3, function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers, but not for their activation. Our data support the model that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.
Project description:Transcription factor/enhancer interactions determine cell specific gene expression. Here, we followed enhancers during differentiations of embryonic stem (ESCs) to epiblast like cells (EpiLCs). There were highly dynamic changes in histone lysine 27 acetylation at enhancer sites throughout the genome. These sites were enriched for a Foxd3 binding motif, a forkhead transcription factor essential in early embryonic development. Surprisingly, Foxd3 occupied largely mutually exclusive sites in the ESCs versus EpiLCs. Foxd3 bound to nucleosome occupied regions, simultaneously evicting the histones while inhibiting full gene expression through the recruitment of histone deacetylases. Knockout of Foxd3 resulted in hyperacetylation and transcriptional upregulation of neighboring genes, many of which were further upregulated at later stages of differentiation. These data show that Foxd3 primes enhancer sites during pregastrulation by removing nucleosomes, yet suppresses neighboring histone hyperacetylation. Such a mechanism may be common to many transcription factors that prepare enhancers for later gene activation during development. Total RNA obtained Foxd3 knockout embryonic stem cells (ESCs) and epiblast-like cells (EpiLCs) (treated with 1uM tamoxifen for 36h to induce knockout) compared to wild-type controls
Project description:A central question in biology is how enhancers are made accessible. The Drosophila embryo is a good model system to study this question as the gene regulatory networks regulating early developmental events have been well characterized. Zelda (Zld) is a uniformly distributed transcription factor (TF) integral to these networks, acting prior to and in collaboration with the patterning TFs to regulate target enhancers. Here we test the hypothesis that Zld directs TF binding, examplified by Dorsal (Dl) which patterns the dorsoventral axis, across the genome by displacing nucleosomes at enhancers. By performing ChIP-seq and MNase-seq experiments on early embryos with or without Zld, we demonstrated that early enhancers are characterized by an intrinsically high nucleosome barrier, which is overcome by Zld, and that without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that enhancers are initially specified across the genome by the binding of Zld, which locally decreases nucleosome occupancy, thereby assisting TFs in accessing their binding motifs and promoting transcriptional activity. Zld, Dl, Pol II ChIP-seq and MNase-seq profiles comparing 2-3h wild-type (wt) and zld- embryos, and MNase-seq profiles comparing 2-4h wt and gd7 embryos, all in 2 replicates
