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 by the Bicoid morphogen through a combination of quantitative imaging, mathematical modeling and epigenomics in embryos. By live imaging of MS2 reporters, we find that, following mitosis, the timing of transcriptional activation driven by the P2 ( P2) enhancer directly reflects Bicoid concentration.

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

The eukaryotic genome is organized to enable the precise regulation of gene expression. This organization is established as the embryo transitions from a fertilized gamete to a 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 development in Drosophila.

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 by the activator Bicoid and the pioneer-like transcription factor Zelda in living embryos and showed that no thermodynamic or non-equilibrium MWC model can recapitulate transcription.

Zygotic pioneer factor activity of Odd-paired/Zic is necessary for late function of the 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 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.

Rapid Dynamics of Signal-Dependent Transcriptional Repression by Capicua.

Optogenetic perturbations, live imaging, and time-resolved ChIP-seq assays in Drosophila embryos were used to dissect the ERK-dependent control of the HMG-box repressor Capicua (Cic), which plays critical roles in development and is deregulated in human spinocerebellar ataxia and cancers. We established that Cic target genes are activated before significant downregulation of nuclear localization of Cic and demonstrated that their activation is preceded by fast dissociation of Cic from the regulatory DNA. We discovered that both Cic-DNA binding and repression are rapidly reinstated in the absence of ERK activation, revealing that inductive signaling must be sufficiently sustained to ensure robust transcriptional response.

Chromatin accessibility and histone acetylation in the regulation of competence in early development.

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.

Concentration dependent chromatin states induced by the bicoid morphogen gradient.

In , 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.

Establishment and maintenance of heritable chromatin structure during early embryogenesis.

During embryogenesis, the initial chromatin state is established during a period of rapid proliferative activity. We have measured with 3-min time resolution how heritable patterns of chromatin structure are initially established and maintained during the midblastula transition (MBT). We find that regions of accessibility are established sequentially, where enhancers are opened in advance of promoters and insulators.

Coordinating Cell Cycle Remodeling with Transcriptional Activation at the Drosophila MBT.

During the maternal-to-zygotic transition (MZT), major changes in cell cycle regulation coincide with large-scale zygotic genome activation. In this chapter, we discuss the current understanding of how the cell cycle is remodeled over the course of the Drosophila MZT, and how the temporal precision of this event is linked to contemporaneous alterations in genome-wide chromatin structure and transcriptional activity. The cell cycle is initially lengthened during the MZT by activation of the DNA replication checkpoint but, subsequently, zygotically supplied factors are essential for establishing lasting modifications to the cell cycle.

Zygotic genome activation triggers the DNA replication checkpoint at the midblastula transition.

A conserved feature of the midblastula transition (MBT) is a requirement for a functional DNA replication checkpoint to coordinate cell-cycle remodeling and zygotic genome activation (ZGA). We have investigated what triggers this checkpoint during Drosophila embryogenesis. We find that the magnitude of the checkpoint scales with the quantity of transcriptionally engaged DNA.

Posttranslational control of Cdc25 degradation terminates Drosophila's early cell-cycle program.

In most metazoans, early embryonic development is characterized by rapid mitotic divisions that are controlled by maternal mRNAs and proteins that accumulate during oogenesis. These rapid divisions pause at the midblastula transition (MBT), coinciding with a dramatic increase in gene transcription and the degradation of a subset of maternal mRNAs. In Drosophila, the cell-cycle pause is controlled by inhibitory phosphorylation of Cdk1, which in turn is driven by downregulation of the activating Cdc25 phosphatases.

Regulation of primitive hematopoiesis by class I histone deacetylases.

Background: Histone deacetylases (HDACs) regulate multiple developmental processes and cellular functions. However, their roles in blood development have not been determined, and in Xenopus laevis a specific function for HDACs has yet to be identified. Here, we employed the class I selective HDAC inhibitor, valproic acid (VPA), to show that HDAC activity is required for primitive hematopoiesis.

Prepatterning embryonic development: tabula scripta?

Recent work has raised the possibility that chromatin modifications pre-set embryonic patterns of gene expression. In this issue of Developmental Cell, Lindeman et al. (2011) support this observation and describe how the pattern of several chromatin marks evolves over the transition from maternal to zygotic control of development.

beta-Catenin primes organizer gene expression by recruiting a histone H3 arginine 8 methyltransferase, Prmt2.

An emerging concept in development is that transcriptional poising presets patterns of gene expression in a manner that reflects a cell's developmental potential. However, it is not known how certain loci are specified in the embryo to establish poised chromatin architecture as the developmental program unfolds. We find that, in the context of transcriptional quiescence prior to the midblastula transition in Xenopus, dorsal specification by the Wnt/beta-catenin pathway is temporally uncoupled from the onset of dorsal target gene expression, and that beta-catenin establishes poised chromatin architecture at target promoters.

Mesodermal Wnt signaling organizes the neural plate via Meis3.

In vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined.

Chromatin immunoprecipitation in early Xenopus laevis embryos.

Chromatin immunoprecipitation (ChIP) is a powerful method for analyzing the interaction of regulatory proteins with genomic loci, but has been difficult to apply to studies on early embryos due to the limiting amount of genomic material in these samples. Here, we present a comprehensive technique for performing ChIP on blastula and gastrula stage Xenopus embryos. We also describe methods for optimizing crosslinking and chromatin shearing, verifying antibody specificity, maximizing PCR sensitivity, and quantifying PCR results, allowing for the use of as few as 50 early blastula stage embryos (approximately 5x10(4) cells) per experimental condition.