Protein-intrinsic properties and context-dependent effects regulate pioneer factor binding and function.

Chromatin is a barrier to the binding of many transcription factors. By contrast, pioneer factors access nucleosomal targets and promote chromatin opening. Despite binding to target motifs in closed chromatin, many pioneer factors display cell-type-specific binding and activity. The mechanisms governing pioneer factor occupancy and the relationship between chromatin occupancy and opening remain unclear. We studied three Drosophila transcription factors with distinct DNA-binding domains and biological functions: Zelda, Grainy head and Twist. We demonstrated that the level of chromatin occupancy is a key determinant of pioneering activity. Multiple factors regulate occupancy, including motif content, local chromatin and protein concentration. Regions outside the DNA-binding domain are required for binding and chromatin opening. Our results show that pioneering activity is not a binary feature intrinsic to a protein but occurs on a spectrum and is regulated by a variety of protein-intrinsic and cell-type-specific features.

Setting the stage for development: the maternal-to-zygotic transition in Drosophila.

The zygote has a daunting task ahead of itself; it must develop from a single cell (fertilized egg) into a fully functioning adult with a multitude of different cell types. In the beginning, the zygote has help from its mother, in the form of gene products deposited into the egg, but eventually, it must rely on its own resources to proceed through development. The transfer of developmental control from the mother to the embryo is called the maternal-to-zygotic transition (MZT). All animals undergo this transition, which is defined by two main processes-the degradation of maternal RNAs and the synthesis of new RNAs from the zygote's own genome. Here, we review the regulation of the MZT in Drosophila, but given the broad conservation of this essential process, much of the regulation is shared among metazoans.

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. GAF transcriptionally activates the zygotic genome and is localized to subnuclear foci. This non-uniform distribution is driven by binding to highly abundant GA 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 the subnuclear concentration of transcription factors acts to organize the nucleus into functionally distinct domains essential for the robust regulation of gene expression.

Low-level repressive histone marks fine-tune gene transcription in neural stem cells.

Coordinated regulation of gene activity by transcriptional and translational mechanisms poise stem cells for a timely cell-state transition during differentiation. Although important for all stemness-to-differentiation transitions, mechanistic understanding of the fine-tuning of gene transcription is lacking due to the compensatory effect of translational control. We used intermediate neural progenitor (INP) identity commitment to define the mechanisms that fine-tune stemness gene transcription in fly neural stem cells (neuroblasts). We demonstrate that the transcription factor FruitlessC (FruC) binds cis-regulatory elements of most genes uniquely transcribed in neuroblasts. Loss of fruC function alone has no effect on INP commitment but drives INP dedifferentiation when translational control is reduced. FruC negatively regulates gene expression by promoting low-level enrichment of the repressive histone mark H3K27me3 in gene cis-regulatory regions. Identical to fruC loss-of-function, reducing Polycomb Repressive Complex 2 activity increases stemness gene activity. We propose low-level H3K27me3 enrichment fine-tunes gene transcription in stem cells, a mechanism likely conserved from flies to humans.

From neurons to sperm, our bodies are formed of a range of cells tailored to perform a unique role. However, organisms also host small reservoirs of unspecialized 'stem cells' that retain the ability to become different kinds of cells. When these stem cells divide, one daughter cell remains a stem cell while the other undergoes a series of changes that allows it to mature into a specific cell type. This 'differentiation' process involves quickly switching off the stem cell programme, the set of genes that give a cell the ability to keep dividing while maintaining an unspecialized state. Failure to do so can result in the differentiating cell reverting towards its initial state and multiplying uncontrollably, which can lead to tumours and other health problems. While scientists have a good understanding of how the stem cell programme is turned off during differentiation, controlling these genes is a balancing act that starts even before division: if the program is over-active in the 'mother' stem cell, for instance, the systems that switch it off in its daughter can become overwhelmed. The mechanisms presiding over these steps are less well-understood. To address this knowledge gap, Rajan, Anhezini et al. set out to determine how stem cells present in the brains of fruit flies could control the level of activity of their own stem cell programme. RNA sequencing and other genetic analyses revealed that a protein unique to these cells, called Fruitless, was responsible for decreasing the activity of the programme. Biochemical experiments then showed that Fruitless performed this role by attaching a small amount of chemical modifications (called methyl groups) to the proteins that 'package' the DNA near genes involved in the stem cell programme. High levels of methyl groups present near a gene will switch off this sequence completely; however, the amount of methyl groups that Fruitless helped to deposit is multiple folds lower. Consequently, Fruitless 'fine-tunes' the activity of the stem cell programme instead, dampening it just enough to stop it from overpowering the 'off' mechanism that would take place later in the daughter cell. These results shed new light on how stem cells behave ­ and how our bodies stop them from proliferating uncontrollably. In the future, Rajan, Anhezini et al. hope that this work will help to understand and treat diseases caused by defective stem cell differentiation.

Reprogramming of three-dimensional chromatin organization in the early embryo.

Chromatin organization within the three-dimensional (3D) nuclear space is important for proper gene expression and developmental programming. This organization is established during the dramatic reprogramming that occurs in early embryonic development. Thus, the early embryo is an ideal model for examining the formation and dynamics of 3D chromatin structure. Advances in high-resolution microscopy and single-nucleus genomic analyses have provided fundamental insights into the mechanisms driving genome organization in the early embryo. Here, we highlight recent findings describing the dynamics and driving mechanisms for establishing 3D chromatin organization and discuss the role such organization has on gene regulation in early embryonic development.

Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function.

Chromatin is a barrier to the binding of many transcription factors. By contrast, pioneer factors access nucleosomal targets and promote chromatin opening. Despite binding to target motifs in closed chromatin, many pioneer factors display cell-type specific binding and activity. The mechanisms governing pioneer-factor occupancy and the relationship between chromatin occupancy and opening remain unclear. We studied three Drosophila transcription factors with distinct DNA-binding domains and biological functions: Zelda, Grainy head, and Twist. We demonstrated that the level of chromatin occupancy is a key determinant of pioneering activity. Multiple factors regulate occupancy, including motif content, local chromatin, and protein concentration. Regions outside the DNA-binding domain are required for binding and chromatin opening. Our results show that pioneering activity is not a binary feature intrinsic to a protein but occurs on a spectrum and is regulated by a variety of protein-intrinsic and cell-type-specific features.

The control of transcriptional memory by stable mitotic bookmarking.

To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is mediated, in part, by transcription factors (TF) mitotic bookmarking. However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that GAGA pioneer factor (GAF) acts as a stable mitotic bookmarker during zygotic genome activation. We show that, during mitosis, GAF remains associated to a large fraction of its interphase targets, including at cis-regulatory sequences of key developmental genes with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we report that GAF binding establishes competence for rapid activation upon mitotic exit.

Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila.

During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is correlated with differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network driven by cell-type-specific enhancers.

Pioneering the developmental frontier.

Coordinated changes in gene expression allow a single fertilized oocyte to develop into a complex multi-cellular organism. These changes in expression are controlled by transcription factors that gain access to discrete cis-regulatory elements in the genome, allowing them to activate gene expression. Although nucleosomes present barriers to transcription factor occupancy, pioneer transcription factors have unique properties that allow them to bind DNA in the context of nucleosomes, define cis-regulatory elements, and facilitate the subsequent binding of additional factors that determine gene expression. In this capacity, pioneer factors act at the top of gene-regulatory networks to control developmental transitions. Developmental context also influences pioneer factor binding and activity. Here we discuss the interplay between pioneer factors and development, their role in driving developmental transitions, and the influence of the cellular environment on pioneer factor binding and activity.

GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo.

Following fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are essential for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal-to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were required for this transition. We identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is necessary to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. We demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer-like factors, and we propose that as development proceeds control is gradually transferred from Zelda to GAF.

Most cells in an organism share the exact same genetic information, yet they still adopt distinct identities. This diversity emerges because only a selection of genes is switched on at any given time in a cell. Proteins that latch onto DNA control this specificity by activating certain genes at the right time. However, to perform this role they first need to physically access DNA: this can be difficult as the genetic information is tightly compacted so it can fit in a cell. A group of proteins can help to unpack the genome to uncover the genes that can then be accessed and activated. While these 'pioneer factors' can therefore shape the identity of a cell, much remains unknown about how they can work together to do so. For instance, the pioneer factor Zelda is essential in early fruit fly development, as it enables the genetic information of the egg and sperm to undergo dramatic reprogramming and generate a new organism. Yet, it was unclear whether additional helpers were required for this transition. Using this animal system, Gaskill, Gibson et al. identified GAGA Factor as a protein which works with Zelda to open up and reprogram hundreds of different sections along the genome of fruit fly embryos. This tag-team effort started with Zelda being important initially to activate genes; regulation was then handed over for GAGA Factor to continue the process. Without either protein, the embryo died. Getting a glimpse into early genetic events during fly development provides insights that are often applicable to other animals such as fish and mammals. Ultimately, this research may help scientists to understand how things can go wrong in human embryos.

H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2.

Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. Our results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.

Establishment of chromatin accessibility by the conserved transcription factor Grainy head is developmentally regulated.

The dramatic changes in gene expression required for development necessitate the establishment of cis-regulatory modules defined by regions of accessible chromatin. Pioneer transcription factors have the unique property of binding closed chromatin and facilitating the establishment of these accessible regions. Nonetheless, much of how pioneer transcription factors coordinate changes in chromatin accessibility during development remains unknown. To determine whether pioneer-factor function is intrinsic to the protein or whether pioneering activity is developmentally modulated, we studied the highly conserved, essential transcription factor Grainy head (Grh). Prior work established that Grh is expressed throughout Drosophila development and is a pioneer factor in the larva. We demonstrated that Grh remains bound to mitotic chromosomes, a property shared with other pioneer factors. By assaying chromatin accessibility in embryos lacking maternal and/or zygotic Grh at three stages of development, we discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. Our data reveal that the pioneering activity of Grh is temporally regulated and likely influenced by additional factors expressed at a given developmental stage.

Tissue-Specific DNA Replication Defects in Drosophila melanogaster Caused by a Meier-Gorlin Syndrome Mutation in Orc4.

Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct tissue-specific developmental defects. Genes encoding members of the origin recognition complex (ORC) and additional proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, MCM5, and DONSON) are mutated in individuals diagnosed with MGS. The essential role of ORC is to license origins during the G1 phase of the cell cycle, but ORC has also been implicated in several nonreplicative functions. Because of its essential role in DNA replication, ORC is required for every cell division during development. Thus, it is unclear how the Meier-Gorlin syndrome mutations in genes encoding ORC lead to the tissue-specific defects associated with the disease. To begin to address these issues, we used Cas9-mediated genome engineering to generate a Drosophila melanogaster model of individuals carrying a specific Meier-Gorlin syndrome mutation in ORC4 along with control strains. Together these strains provide the first metazoan model for an MGS mutation in which the mutation was engineered at the endogenous locus along with precisely defined control strains. Flies homozygous for the engineered MGS allele reach adulthood, but with several tissue-specific defects. Genetic analysis revealed that this Orc4 allele was a hypomorph. Mutant females were sterile, and phenotypic analyses suggested that defects in DNA replication was an underlying cause. By leveraging the well-studied Drosophila system, we provide evidence that a disease-causing mutation in Orc4 disrupts DNA replication, and we propose that in individuals with MGS defects arise preferentially in tissues with a high-replication demand.

Structural Features of Transcription Factors Associating with Nucleosome Binding.

Fate-changing transcription factors (TFs) scan chromatin to initiate new genetic programs during cell differentiation and reprogramming. Yet the protein structure domains that allow TFs to target nucleosomal DNA remain unexplored. We screened diverse TFs for binding to nucleosomes containing motif-enriched sequences targeted by pioneer factors in vivo. FOXA1, OCT4, ASCL1/E12α, PU1, CEBPα, and ZELDA display a range of nucleosome binding affinities that correlate with their cell reprogramming potential. We further screened 593 full-length human TFs on protein microarrays against different nucleosome sequences, followed by confirmation in solution, to distinguish among factors that bound nucleosomes, such as the neuronal AP-2α/ß/γ, versus factors that only bound free DNA. Structural comparisons of DNA binding domains revealed that efficient nucleosome binders use short anchoring α helices to bind DNA, whereas weak nucleosome binders use unstructured regions and/or ß sheets. Thus, specific modes of DNA interaction allow nucleosome scanning that confers pioneer activity to transcription factors.

Grainyhead-like Protein 2: The Emerging Role in Hormone-Dependent Cancers and Epigenetics.

In mammals, the grainyhead-like transcription factor (GRHL) family is composed of three nuclear proteins that are responsible for driving epithelial cell fate: GRHL1, GRHL2, and GRHL3. GRHL2 is important in maintaining proper tubulogenesis during development and in suppressing the epithelial-to-mesenchymal transition. Within the last decade, evidence indicates both tumor-suppressive and oncogenic roles for GRHL2 in various types of cancers. Recent studies suggest that GRHL2 may be especially important in hormone-dependent cancers, as correlative relationships exist between GRHL2 and various steroid receptors, such as the androgen and estrogen receptors. Acting as a pioneer factor and coactivator, GRHL2 may directly affect steroid receptor transcriptional activity. This review will highlight recent discoveries of GRHL2 activity in cancer and in maintaining the epithelial state, while also exploring recent literature on the role of GRHL2 in hormone-dependent cancers and epigenetics.

Continued Activity of the Pioneer Factor Zelda Is Required to Drive Zygotic Genome Activation.

Reprogramming cell fate during the first stages of embryogenesis requires that transcriptional activators gain access to the genome and remodel the zygotic transcriptome. Nonetheless, it is not clear whether the continued activity of these pioneering factors is required throughout zygotic genome activation or whether they are only required early to establish cis-regulatory regions. To address this question, we developed an optogenetic strategy to rapidly and reversibly inactivate the master regulator of genome activation in Drosophila, Zelda. Using this strategy, we demonstrate that continued Zelda activity is required throughout genome activation. We show that Zelda binds DNA in the context of nucleosomes and suggest that this allows Zelda to occupy the genome despite the rapid division cycles in the early embryo. These data identify a powerful strategy to inactivate transcription factor function during development and suggest that reprogramming in the embryo may require specific, continuous pioneering functions to activate the genome.

Publisher Correction: Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs.

The original version of this Article contained an error in Fig. 4a, in which the "=" sign of the equation was inadvertently replaced with a "-" sign. This has been corrected in the PDF and HTML versions of the Article.

Mechanisms regulating zygotic genome activation.

Following fertilization, the two specified gametes must unite to create an entirely new organism. The genome is initially transcriptionally quiescent, allowing the zygote to be reprogrammed into a totipotent state. Gradually, the genome is activated through a process known as the maternal-to-zygotic transition, which enables zygotic gene products to replace the maternal supply that initiated development. This essential transition has been broadly characterized through decades of research in several model organisms. However, we still lack a full mechanistic understanding of how genome activation is executed and how this activation relates to the reprogramming of the zygotic chromatin architecture. Recent work highlights the central role of transcriptional activators and suggests that these factors may coordinate transcriptional activation with other developmental changes.

Optogenetic Inactivation of Transcription Factors in the Early Embryo of Drosophila.

The early embryo of Drosophila melanogaster exists as a rapidly dividing syncytium of nuclei that are transcriptionally silent. Maternally deposited factors are required to awaken the genome and assist in the transition from maternal to zygotic control of development. Because many of these essential factors are maternally deposited and the early nuclear divisions are so rapid, it has been difficult to assess the functional role of transcription factors at discrete points in early embryonic development. To address this issue, we have developed an optogenetic system that can rapidly and reversibly inactivate transcription factors with nuclear-cycle resolution. The temporal precision enabled by this technique will allow a mechanistic understanding of how transcription factors function together to control genome activation and patterning in the early embryo and is likely broadly applicable to factors throughout embryogenesis.