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Blythe, S. A.

Publications and source records attributed to Blythe, S. A..

12 recordsLinked to original sources

A reaction-diffusion framework for de novo Polycomb spreading

Eukaryotic organisms rely on post-translational modifications to chromatin to maintain stable patterns of gene silencing. These modifications include trimethylation at histone H3 lysine 27 (H3K27me3), which is deposited by Polycomb Repressive Complex 2 (PRC2) and accumulates on the genome during embryogenesis. While this process underlies the proper specification of cell types, we lack the ability to quantitatively predict the de novo establishment of Polycomb states. The kinetics of H3K27 methylation is difficult to quantify in vivo, and further, the network of molecular interactions that influences Polycomb states is complex. Here, leveraging the Drosophila embryonic system, we measure H3K27me3 dynamics with ChIP-seq and extract the rate the modification spreads along chromatin in vivo. To provide a mechanistic explanation for this rate, we build a reaction-diffusion framework that models how PRC2 establishes states of gene silencing de novo. The reaction-diffusion system recapitulates experimental observations in wild-type and mutant embryos, and suggests that PRC2 can diffuse in 1D along chromatin at a rate enhanced by Polycomb Repressive Complex 1. Through this work, we define a minimal set of parameters that dictate in vivo Polycomb dynamics, and provide evidence that the early embryo creates a super-charged environment for epigenetic modification.

developmental biology↗

Lower-order methylation states underlie the maintenance and re-establishment of Polycomb modifications in Drosophila embryogenesis

Polycomb Group (PcG) proteins regulate the chromatin composition of an embryo by facilitating the mono, di, and tri-methylation of Histone H3 Lysine 27 (H3K27me1/2/3). For the zygote to inherit an H3K27 methylation blueprint from its mother, PcG-modified states established during oogenesis must persist through early embryogenesis until the onset of large-scale zygotic transcription (Zygotic Genome Activation, ZGA). However, questions have persisted regarding the relative contributions of two molecular mechanisms to the propagation of H3K27 methylation through early development: 1) allosteric regulation of the H3K27 methyltransferase Enhancer of Zeste (E(z)) by existing H3K27me2/3, and 2) nucleation of E(z) activity at chromatin by DNA binding factors. Here, we investigate how allostery and nucleation contribute to H3K27 methylation dynamics in early Drosophila embryogenesis by developing and experimentally validating a mathematical model. This model incorporates measurements of the nuclear concentration dynamics of E(z) and the Polycomb Response Element binding factor Pleiohomeotic (Pho), as well as the dilution of epigenetic modifications at DNA replication with the incorporation of histones to nascent chromatin. With stochastic simulations and in vivo experiments, we assert that allosteric regulation of E(z) maintains a PcG-imprint on maternal chromosomes in the form of lower-order H3K27 methylation states (H3K27me1/2), that de novo establishment of H3K27 methylation at paternal chromosomes relies on nucleation of E(z) activity by Pho, and that broad H3K27me3 domains at both maternal and paternal chromosomes are re-established at ZGA. This work provides a mechanistic explanation for the inheritance of Polycomb states in contexts of intense cellular proliferation.

developmental biology↗

Nucleation-dependent propagation of Polycomb modifications emerges during the Drosophila maternal to zygotic transition

During zygotic genome activation (ZGA) in Drosophila, broad domains of Polycomb-modified chromatin are rapidly established across the genome. Here, we investigate the spatial and temporal dynamics by which Polycomb group (PcG) histone modifications, H3K27me3 and H2Aub, emerge during early embryogenesis. Using ChIP-seq and live imaging of CRISPR-engineered GFP-tagged PcG components, we show that PRC2-dependent H3K27me3 accumulates adjacent to a subset of E(z)-bound prospective Polycomb Response Elements (PREs) beginning in nuclear cycle 14 (NC14), with patterns indicative of nucleation followed by spreading. Surprisingly, PRE-binding factors Pho, Combgap, and GAGA-factor are excluded from interphase nuclei prior to NC10 despite nuclear localization of E(z) throughout early interphases. Loss-of-function studies further demonstrate that GAGA-factor is largely dispensable for PcG domain establishment, whereas the pioneer factor Zelda is required for proper deposition of H3K27me3 and H2Aub at a subset of Polycomb domains. The role of Zelda at Polycomb domains is context-dependent; a subset of targets requires Zelda not for E(z) recruitment, but instead to license an E(z)-loaded PRE to deposit H3K27me3. Our findings support a model where licensing of PcG domains is an initial step in the regulatory processes governing Polycomb-regulated developmental genes. Impact StatementEpigenomics and quantitative imaging are used to investigate the re-establishment of histone modifications associated with the Polycomb group of epigenetic regulators during the Drosophila maternal-to-zygotic transition.

developmental biology↗

Rapid transcriptional response to a dynamic morphogen by time integration

During development, cells must interpret extracellular signals with speed and accuracy. While morphogen gradients pattern tissues, how cells respond to dynamic morphogens remains unclear. Here, we investigate how dorsal patterning in the Drosophila embryo is specified by a rapidly evolving BMP gradient. Using a live reporter of BMP pathway activity and nascent transcription reporters, we find that gene expression is best predicted by time integration of BMP signaling, rather than instantaneous levels. However, in sog mutant embryos with broad BMP activity, integration alone fails to predict gene expression outside the normal domain. We show that the transcription factor Zen lowers the signaling threshold required for activation, enabling integration to drive rapid transcriptional responses even at low BMP levels. Together, these results suggest that cells interpret dynamic morphogen signals through the combined action of temporal integration and spatial competence, providing a framework for robust pattern formation on fast developmental timescales.

developmental biology↗

Conservation of symmetry breaking at the level of chromatin accessibility between fly species with unrelated anterior determinants

Establishing the anterior-posterior (AP) body axis is a fundamental process during embryogenesis, and the fruit fly, Drosophila melanogaster, provides one of the best-known case studies. But for unknown reasons, different species of flies (Diptera) establish the AP axis through unrelated, structurally distinct anterior determinants (ADs). The AD of Drosophila, Bicoid (Bcd), initiates symmetry-breaking during nuclear cleavage cycles (NCs) when ubiquitous pioneer factors, such as Zelda (Zld), drive zygotic genome activation (ZGA) at the level chromatin accessibility by nucleosome depletion. While Bcd engages in a concentration-dependent competition with nucleosomes at the loci of a small set of transcription factor (TF) genes that are expressed in the anterior embryo, it remains unknown whether unrelated ADs of other fly species function in the same way and target homologous genes. We have examined the symmetry-breaking mechanism of a moth fly, Clogmia albipunctata, in which a maternally expressed transcript isoform of the pair-rule segmentation gene odd-paired serves as AD. We provide a de novo assembly and annotation of the Clogmia genome and describe how Clogmias orthologs of zelda (Cal-zld) and odd-paired (Cal-opa) affect chromatin accessibility and gene expression. Our results suggest direct roles of Cal-zld in opening and closing chromatin during nuclear cleavage cycles (NCs) and show that during the early phase of ZGA maternal Cal-opa activity promotes chromatin accessibility and anterior expression at Clogmias homeobrain and sloppy-paired loci. These genes are not known as key targets of Bcd but may serve a more widely conserved role in the initiation of anterior pattern formation given their early anterior expression and function in head development in insects. We conclude that the ADs of Drosophila and Clogmia differ in their target genes but share the mechanism of concentration-dependent nucleosome depletion.

developmental biology↗

Bicoid-nucleosome competition sets a concentration threshold for transcription constrained by genome replication

Transcription factors (TFs) regulate gene expression despite constraints from chromatin structure and the cell cycle. Here we examine the concentration-dependent regulation of hunchback by the Bicoid morphogen through a combination of quantitative imaging, mathematical modeling and epigenomics in Drosophila embryos. By live imaging of MS2 reporters, we find that, following mitosis, the timing of transcriptional activation driven by the hunchback P2 (hb P2) enhancer directly reflects Bicoid concentration. We build a stochastic model that can explain in vivo onset time distributions by accounting for both the competition between Bicoid and nucleosomes at hb P2 and a negative influence of DNA replication on transcriptional elongation. Experimental modulation of nucleosome stability alters onset time distributions and the posterior boundary of hunchback expression. We conclude that TF-nucleosome competition is the molecular mechanism whereby the Bicoid morphogen gradient specifies the posterior boundary of hunchback expression.

developmental biology↗

Localization of the pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression

Abstract/SummaryThe eukaryotic genome is organized to enable the precise regulation of gene expression required for development. This organization is established during early development when the embryo transitions from a fertilized germ cell to the totipotent zygote. To understand the factors and processes that drive genomic organization, we focused on the pioneer factor GAGA factor (GAF) that is required for early embryonic development in Drosophila. GAF transcriptionally activates the zygotic genome and is localized to subnuclear foci. We show that this non-uniform distribution is driven by binding to the highly abundant GA-satellite repeats. At GA-repeats, GAF is necessary to form heterochromatin and silence transcription. Thus, GAF is required to establish both active and silent regions. We propose that foci formation enables GAF to have opposing transcriptional roles within a single nucleus. Our data support a model in which modulation of the subnuclear concentration of transcription factors acts to organize the nucleus into functionally distinct domains that are essential for the robust regulation of gene expression.

developmental biology↗

Adaptable and Efficient Genome Editing by sgRNA-Cas9 Protein Co-injection into Drosophila

Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than color. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.

genetics↗

Quantitative dissection of transcription in development yields evidence for transcription factor-driven chromatin accessibility

Thermodynamic models of gene regulation can predict transcriptional regulation in bacteria, but in eukaryotes chromatin accessibility and energy expenditure may call for a different framework. Here we systematically tested the predictive power of models of DNA accessibility based on the Monod-Wyman-Changeux (MWC) model of allostery, which posits that chromatin fluctuates between accessible and inaccessible states. We dissected the regulatory dynamics of hunchback by the activator Bicoid and the pioneer-like transcription factor Zelda in living Drosophila embryos and showed that no thermodynamic or non-equilibrium MWC model can recapitulate hunchback transcription. Therefore, we explored a model where DNA accessibility is not the result of thermal fluctuations but is catalyzed by Bicoid and Zelda, possibly through histone acetylation, and found that this model can predict hunchback dynamics. Thus, our theory-experiment dialogue uncovered potential molecular mechanisms of transcriptional regulatory dynamics, a key step toward reaching a predictive understanding of developmental decision-making.

biophysics↗

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

Because regulatory networks of transcription factors drive embryonic patterning, it is possible that chromatin accessibility states impact how networks interact with information encoded in DNA. 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, from zygotic genome activation to gastrulation. Chromatin accessibility states are dynamic over this period, and establishment of the segmentation network requires maturation of the ground chromatin state. Elimination of all maternal patterning information allows identification of patterning-dependent and -independent dynamic chromatin regions. A significant proportion of patterning-dependent accessibility stems from pioneer activity of the pair-rule factor Odd-paired (opa). 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.

developmental biology↗

Loss of Competence in early development is mediated by loss of chromatin accessibility

As development proceeds, inductive cues are interpreted by competent tissues in a spatially and temporally restricted manner. While key inductive signaling pathways within competent cells are well-described at a molecular level, the mechanisms by which tissues lose responsiveness to inductive signals are not well understood. Localized activation of Wnt signaling before zygotic gene activation in Xenopus laevis leads to dorsal development, but competence to induce dorsal genes in response to Wnts is lost by the late blastula stage. We hypothesize that loss of competence is mediated by changes in histone modifications leading to a loss of chromatin accessibility at the promoters of Wnt target genes. We use ATAC-seq to evaluate genome-wide changes in chromatin accessibility across several developmental stages. Based on overlap with p300 binding, we identify thousands of putative cis-regulatory elements at the gastrula stage, including sites that lose accessibility by the end of gastrulation and are enriched for pluripotency factor binding motifs. Dorsal Wnt target gene promoters are not accessible after the loss of competence in the early gastrula while genes involved in mesoderm and neural crest development maintain accessibility at their promoters. Inhibition of histone deacetylases increases acetylation at the promoters of dorsal Wnt target genes and extends competence for dorsal gene induction by Wnt signaling. Histone deacetylase inhibition, however, is not sufficient to extend competence for mesoderm or neural crest induction. These data suggest that chromatin state regulates the loss of competence to inductive signals in a context-dependent manner.

developmental biology↗

Concentration Dependent Chromatin States Induced by the Bicoid Morphogen Gradient

In Drosophila, graded expression of the maternal transcription factor Bicoid (Bcd) provides positional information to activate target genes at different positions along the anterior-posterior axis. We have measured the genome-wide binding profile of Bcd using ChIP-seq in embryos expressing single, uniform levels of Bcd protein, and grouped Bcd-bound targets into four classes based on occupancy at different concentrations. By measuring the biochemical affinity of target enhancers in these classes in vitro and genome-wide chromatin accessibility by ATAC-seq, we found that the occupancy of target sequences by Bcd is not primarily determined by Bcd binding sites, but by chromatin context. Bcd drives an open chromatin state at a subset its targets. Our data support a model where Bcd influences chromatin structure to gain access to concentration-sensitive targets at high concentrations, while concentration-insensitive targets are found in more accessible chromatin and are bound at low concentrations.

developmental biology↗