BIOLOGICAL OVERVIEW

Grainy head (Grh) is a conserved transcription factor (TF) controlling epithelial differentiation and regeneration. To elucidate Grh functions, embryonic Grh targets were identified by ChIP-seq and gene expression analysis. Grh was shown to control hundreds of target genes. Repression or activation correlates with the distance of Grh binding sites to the transcription start sites of its targets. Analysis of 54 Grh-responsive enhancers during development and upon wounding suggests cooperation with distinct TFs in different contexts. In the airways, Grh repressed genes encode key TFs involved in branching and cell differentiation. Reduction of the POU-domain TF, Vvl, (ventral veins lacking) largely ameliorates the airway morphogenesis defects of grh mutants. Vvl and Grh proteins additionally interact with each other and regulate a set of common enhancers during epithelial morphogenesis. It is concluded that Grh and Vvl participate in a regulatory network controlling epithelial maturation (Yao, 2017).

Grh controls epithelial development and regeneration in multiple organisms. ChIP-seq data provide a broad view of Grh-binding to its targets in all Grh-expressing tissues. The analysis of Grh-dependent regulatory sequences indicates that the majority of the 5599 peaks that include the consensus Grh-binding sequence identify true Grh targets. Hitherto, analysis of Grh targets in development focused on proteins involved in epidermal barrier formation, adhesion molecules and junctional proteins. Identification of functional Grh targets in the airways adds large groups of proteins involved in lipid metabolism, cell signaling and TFs. This suggests additional functions of Grh in tubulogenesis that might explain several of its additional roles. For example, the phenotype of grh mutants in the airways includes the selective expansion of the epithelial apical membranes, a phenotype that has not been detected in any of the mutants affecting junctional proteins or the formation and modification of the apical extracellular barrier. Definition of new Grh targets during airway maturation provides a rich resource for future studies addressing how Grh controls epithelial morphogenesis (Yao, 2017).

A prevalent group of Grh targets in the epidermis and airways includes genes involved in innate immune responses ranging from pattern recognition receptors to effectors. Interestingly, several putative GRHL2 targets in human bronchial epithelial cells, such as serpins and chitinase 3-like proteins, have been implicated in immune responses. Analysis of PGRP-LC reveals a direct role of Grh in endowing epithelial cells the ability to combat infections. Although the PGRP-LC reporter expression was not inducible by wounding or bacterial injection, it remains possible that Grh also directly controls the activation of epithelial immune responses upon infection. Indeed, partial inactivation of Grh by RNAi in adult flies resulted in increased morbidity and mortality upon bacterial infection (Yao, 2017).

Analysis of 47 new Grh-activated enhancers in epithelial development suggests the presence of distinct, tissue-specific Grh co-factors in the control of target genes in different epithelial cell types. The activation of some of these reporters upon injury expands the repertoire of Grh-activated enhancers and is in line with previous models proposing wound-induced interactions of Grh with other factors. These interactions could be induced by post-translational modifications of Grh or its co-factors by Rolled and other kinases downstream of Stit receptor kinase signaling and might facilitate the activation of transcription by Grh pre-bound to chromatin (Yao, 2017).

The ChIP-seq and gene expression analysis also reveal a potential role for Grh as a repressor. Such a repressor function of Grh is consistent with previous studies addressing Grh function on individual targets in flies and mammals. A higher correlation of PcG-binding sites and repressive chromatin marks were found around the Grh-binding sites of repressed targets as compared with the binding sites of activated genes. The positioning of Grh-binding sites relative to the TSS of repressed versus activated or unaffected genes also differs: Grh-binding sites are usually further from the TSS in repressed target genes. This observation is supported by the analysis of vvl ds3 and vvl 1.8, the only two identified repressible enhancers, which are located more than 2 kb from the vvl TSS. The difference in the structure of the repressed and activated Grh enhancers suggests that Grh repression might require chromatin looping and involve co-repressors. Further work is needed to elucidate a potential direct function of Grh in transcriptional repression (Yao, 2017).

A characteristic group of Grh targets in the airways includes TFs involved in epithelial cell differentiation. This resembles the complex regulatory functions of Grh during neuronal specification. For instance, in neuroblasts, Grh demarcates the last time window for TF expression by repressing Castor. In intermediate neural progenitors (INPs), Grh is detected in the 'middle-aged' INPs and overlaps with the expression of the TFs Dichaete and Eyeless. The three TFs cross-regulate each other. Similar cross-talk between Grh and its TF targets might specify and maintain epithelial differentiation. Since reduction of vvl in grh mutants largely ameliorates the tube elongation defects, the direct or indirect repression of genes encoding TFs is likely to be a crucial function of Grh in the airways. The shared expression pattern of Vvl and Grh, their binding to a set of common enhancers and their ability to form complexes suggest that they collectively control tube growth during airway maturation. Given their co-expression in other contexts, they might also co-operate during neural cell specification and epithelial immune responses (Yao, 2017).

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, the highly conserved, essential transcription factor Grainy head (Grh) was studied. Prior work established that Grh is expressed throughout Drosophila development and is a pioneer factor in the larva. This study 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, it was discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. These data reveal that the pioneering activity of Grh is temporally regulated and likely influenced by additional factors expressed at a given developmental stage (Nevil, 2020).

Previous work had demonstrated that Grh was both necessary and sufficient for chromatin accessibility in the larval eye disc and that GRHL2 similarly had a pioneering function in mammalian cell culture (Chen, 2018; Jacobs, 2018). This study tested the requirement for maternally and zygotically encoded Grh in determining regions of open chromatin in embryo. Maternally encoded Grh was not required for chromatin accessibility at stage 5, and neither maternally nor zygotically encoded Grh was required at stage 6, when zygotic grh is normally expressed. Nonetheless, Grh motifs, among other sequence motifs, are enriched at sites that become accessible during gastrulation. In contrast to gastrulating embryos, this study established that Grh activity is important for determining chromatin accessibility later in embryonic development. This is also the developmental time point at which Grh is essential for viability. Despite this role in determining chromatin accessibility in the late embryo and larvae, the loss of Grh binding at a single locus in larval tissue did not decrease chromatin accessibility at this site. Thus, it is proposed that the pioneering activity of Grh is not required at all stages of development nor at all Grh-bound cis-regulatory elements. Instead, in these tissues or at these loci other factors may compensate for the loss of Grh (Nevil, 2020).

Although pioneer factors are defined by their ability to establish gene regulatory networks by binding to cis-regulatory modules and promoting chromatin accessibility, recent evidence suggests that pioneer factors vary in their capacity to accomplish this task. FOXA1, which is known to displace nucleosomes and to bind mitotic chromatin, is redirected to previously unoccupied sites upon activation of the TNFα pathway. Similarly, OCT4 binding to the genome is dynamic and is modulated by a cohort of transcription factors, including OTX2, to compete with nucleosomes at enhancers, and SOX2 requires PARP1 to reshape nucleosomal DNA to access 26% of its sites in vivo. Thus, the pioneering roles of these transcription factors are regulated in a tissue-specific or temporal manner. By assaying the conserved, essential transcription factor Grh at multiple stages of development, this study demonstrated that the role of Grh in determining cis-regulatory modules depends on developmental stage. Although Grh functions as a pioneer factor at a subset of enhancers in Drosophila eye imaginal discs and in mammalian epiblast stem cells (Chen, 2018; Jacobs, 2018), the current data show that this activity is not required to establish or maintain chromatin accessibility in the early embryo. Together, these data suggest that there is context specificity to Grh pioneering activity and support a model in which pioneer-factor activity is regulated by additional factors, expression of which is variable across development (Nevil, 2020).

The conditions that lead to context-specific Grh-pioneering activity remain unknown, but the combinatorial action of other transcription factors could provide temporal robustness to chromatin remodeling. By examining changes in chromatin accessibility during gastrulation, this study has identified factors that may function to define cis-regulatory regions at this stage of development. Among the top motifs associated with gains in accessibility during gastrulation are binding motifs for Odd-paired, Dichaete and Forkhead-like transcription factors. The mammalian orthologs of these Drosophila transcription factors mark cis-regulatory regions. Zic2, the mammalian ortholog of Opa, occupies enhancers prior to OCT4 binding and thus differentiation, suggesting that Zic2 has a role in marking cis-regulatory regions. Forkhead- like factors in Drosophila are the homologs of FOXA1, a pioneer factor that actively displaces nucleosomes. Dichaete is a Drosophila member of the Group B Sox transcription factors. In mammals, the Group B Sox protein SOX2 is known to be a pioneer factor essential for development. In Drosophila, both opa and D are upregulated upon zygotic genome activation and both are required for embryonic development. Opa is required for temporally regulated changes in expression of multiple pair-rule genes and functions both directly to induce spatiotemporal changes in expression and to modify the role of additional factors. D binds to core promoters and enhancers in the embryo and is required for proper gene expression of thousands of genes. Indeed, recent evidence has shown that Opa is required for hundreds of regions of accessible chromatin in the gastrulating embryo, demonstrating that individual factors can drive chromatin accessibility at this stage of development (Koromila, 2019 preprint; Soluri, 2019 preprint). Opa, Grh and D are all factors that are broadly expressed at the transition to gastrulation and are required for proper gene expression. Together with the current data demonstrating an enrichment for their binding motifs at regions of chromatin that become accessible at gastrulation, this suggests that these factors collaborate to determine the cis-regulatory regions. Given the role of Opa in pioneering regions of chromatin accessibility, it is possible that Opa, and perhaps D, compensate for the loss of Grh at a subset of co-occupied regions at this time in development. However, later in development Grh is required broadly to establish chromatin accessibility (Jacobs, 2018), suggesting that these and other factors can no longer compensate. Indeed in the larval brain, D, Opa and Grh constitute a non-redundant temporal cascade regulating neuroblast fate in the larval brain. This supports a model in which the requirement for pioneer-factor activity in determining cis- regulatory regions is not strictly inherent to the protein, but is dependent on the developmental stage in which the protein is acting (Nevil, 2020).

During mitosis, chromatin condensation leads to the removal of many transcription factors from their interphase binding sites, but recent studies have indicated that a subset of factors remain bound to mitotic chromatin (Raccaud, 2018; Raccaud, 2019). Although not a unique property of pioneer transcription factors, mitotic chromatin occupancy is correlated with the ability of pioneer factors to bind nucleosomal DNA and may allow these factors to re- establish transcriptional networks rapidly following mitosis. This study has shown that Grh binds to mitotic chromatin in the gastrulating embryo. However, at the same time in development Grh is not essential for defining regions of chromatin accessibility. Thus, these data separate the pioneering activity and mitotic chromatin-binding activities of Grh. Although the mechanisms and consequences of the retention of Grh on mitotic chromosomes in the early embryo are unclear, this ability may be related to its surprisingly stable binding profiles during embryogenesis as assayed by ChIP-seq (Nevil, 2017). It was previously demonstrated that Grh binding is stable across days of development (Nevil, 2017). By contrast, this study shows that Grh activity in defining cis-regulatory regions is regulated during development. This analysis suggests that a number of factors that may have pioneering roles at gastrulation compensate for loss of Grh. Furthermore, the results demonstrate that Grh remains bound to chromatin during mitosis, but that this function is not directly related to its pioneering function. Together, these data support a model in which pioneering activity is not a static property of the protein but is rather regulated and context dependent (Nevil, 2020).

Drosophila Grainyhead specifies late programmes of neural proliferation by regulating the mitotic activity and Hox-dependent apoptosis of neuroblasts

The Drosophila central nervous system is generated by stem-cell-like progenitors called neuroblasts. Early in development, neuroblasts switch through a temporal series of transcription factors modulating neuronal fate according to the time of birth. At later stages, it is known that neuroblasts switch on expression of Grainyhead (Grh) and maintain it through many subsequent divisions. The function of this conserved transcription factor is to specify the regionalised patterns of neurogenesis that are characteristic of postembryonic stages. In the thorax, Grh prolongs neural proliferation by maintaining a mitotically active neuroblast. In the abdomen, Grh terminates neural proliferation by regulating the competence of neuroblasts to undergo apoptosis in response to Abdominal-A expression. This study shows how a factor specific to late-stage neural progenitors can regulate the time at which neural proliferation stops, and identifies mechanisms linking it to the Hox axial patterning system (Cenci, 2005).

Thoracic neuroblasts normally continue dividing into pupal stages, stopping at ~120 hours, by which time ~100 adult-specific neurons have been generated. By compromising grh function, it was observed that neurogenesis ceases two days prematurely, at ~72 hours. This limits the average size of neuroblast clones to ~30 cells, indicating that Grh is required to generate 70% of all adult-specific neurons in the thorax (Cenci, 2005).

Four lines of evidence are provided suggesting that the underlying basis for premature cessation of thoracic proliferation in grh mutant clones is reduced mitotic activity of the neuroblast, most probably followed by Hox-independent apoptosis. (1) Although grh mutant neuroblasts are present at 72 hours they are mitotically inactive; (2) by 96 hours, no recognisable grh mutant neuroblasts remain; (3) inhibiting cell-death effector caspases by misexpressing P35 rescues the loss of grh mutant neuroblasts; (4) although misexpression of Hox proteins in thoracic neuroblasts induces apoptosis, Ubx, the resident Hox protein of the posterior thorax, remains excluded from grh mutant neuroblasts at 72 hours. Importantly, the role of Grh in maintaining mitotically-active neuroblasts is not a general 'housekeeping' function but is specific for their age. Thus, wild-type neuroblasts in the early embryo are Grh-negative yet viable and actively dividing. This observation suggests that the late switch to Grh-dependency involves additional factors. These could be intrinsic to the neuroblast or provided by a glial-cell niche. Consistent with the niche idea, neuroblast divisions within the postembryonic brain require DE-cadherin-dependent interactions between glia and neural cells (Cenci, 2005).

In the central abdomen, it has been found that, at 72 hours, many neuroblasts downregulate Grh and become TUNEL positive. When the neuroblast death pathway is blocked in H99 clones, Grh expression continues in mitotically active neuroblasts long after the 72-hour stage. This indicates that abdominal neuroblasts remain in Grh-positive mode during their final division and that Grh is only downregulated after the onset of apoptosis. Moreover, loss of Grh activity leads to the failure of neuroblasts to undergo apoptosis. As these persistent neuroblasts not only survive but also remain actively engaged in the cell cycle, they generate a 3.5-fold excess of cells within each abdominal neuroblast lineage. Together, these findings identify Grh as a terminal neuroblast factor that is an essential component of the abdomen-specific 'stop' programme (Cenci, 2005).

Two different interactions with the Hox gene AbdA underlie the dramatic reversal of Grh function from pro-proliferative in the thorax to anti-proliferative in the abdomen. (1) Grh acts upstream of AbdA to maintain its late phase of expression, and (2) it functions in parallel with AbdA to activate apoptosis. Although the functional significance of grh-dependent AbdA maintenance is not clear, it may be that efficient neuroblast apoptosis requires AbdA levels to remain high for a significant proportion of the interval separating initial AbdA upregulation and the TUNEL-positive stage. More definitively, epistasis tests were used to show that Grh, acting in parallel with AbdA activity, is essential for abdominal neuroblast apoptosis. Thus, when the AbdA-maintenance deficit is rescued using hs-AbdA, neuroblast death remains blocked. Since AbdA is not required to activate neuroblast Grh expression, Grh and AbdA must work in parallel to activate apoptosis. Together with the finding that AbdA is required to activate H99 gene activity, this study demonstrates that inputs from Grh and AbdA are both essential to activate proapoptotic genes and thus trigger neuroblast apoptosis. Whereas the late upregulation of AbdA provides a timing cue to schedule the onset of apoptosis, the much broader phase of Grh expression defines the period of neuroblast competence to respond appropriately to it (Cenci, 2005).

The restricted temporal pattern of Grh expression ensures that competence to undergo AbdA-dependent apoptosis, rather than some other AbdA-dependent output, is only installed at late stages. Consistent with this, neuroblasts in the early embryo that are AbdA positive but Grh negative go on to generate substantial embryonic lineages. Low levels of expression from UAS-grh transgenes make it difficult to test whether Grh is sufficient to confer apoptotic competence to these early embryonic neuroblasts. In the late embryo, however, neuroblasts have already switched on Grh, and, within the central abdomen, all but three undergo abdA-dependent death The observation that reduced neural grh function leads to supernumerary postembryonic neuroblasts positioned outside the vm, vl and dl rows, raises the possibility that Grh is required for all developmentally programmed neuroblast apoptosis (Cenci, 2005).

Cooperation between Su(H) and Grainyhead

Cell-cell signaling mediated by Notch is critical during many different developmental processes for the specification or restriction of cell fates. Currently, the only known transduction pathway involves a DNA binding protein, Suppressor of Hairless [Su(H)] in Drosophila and CBF1 in mammals, and results in the direct activation of target genes. It has been proposed that in the absence of Notch, Su(H)/CBF1 acts as a repressor and is converted into an activator through interactions with the Notch intracellular domain (Morel, 2000). It has also been suggested that the activation of specific target genes requires synergy between Su(H) and other transcriptional activators. An assay has been designed that allows a direct test of these hypotheses in vivo. The results clearly demonstrate that Su(H) is able to function as the core of a molecular switch, repressing transcription in the absence of Notch and activating transcription in the presence of Notch. In its capacity as an activator, Su(H) can cooperate synergistically with a DNA-bound transcription factor, Grainyhead. These interactions indicate a simple model for Notch target-gene regulation that could explain the precision of gene activation elicited by Notch signaling in different developmental fate decisions (Furriols, 2001).

Activation of Notch by its ligands promotes proteolytic processing, releasing an intracellular fragment (N^icd) that embodies most functions of the activated receptor. There is substantial in vivo and in vitro evidence demonstrating that Su(H) and its homologs in other species are required for the activation of Notch target genes, such as the Enhancer of split/HES genes, and it is proposed that Su(H) DNA binding proteins cooperate with N^icd to promote transcription. However, Su(H) and N^icd are relatively ineffectual at activating Enhancer of split [E(spl)] genes in ectopic locations and it appears that their capacity to promote transcription of specific target genes requires synergistic interactions with other enhancer-specific factors. Cell transfection assays have also revealed a potential repressive role for the mammalian homolog of Su(H), CBF1, and indicate that in the absence of N^icd, Su(H)/CBF1 could recruit a corepressor complex to shut off target genes. Recent work in Drosophila has supported this model through the analysis of single-minded, one target gene whose expression is derepressed in animals that lack Su(H) function (Furriols, 2001).

An assay has been designed that allows investigation of whether this is a general mechanism by first testing whether Su(H) can mediate repression of a heterologous activator, and second, whether it can synergize with the same activator in the presence of N^icd to promote transcription (Furriols, 2001).

In order to assess whether Su(H) is able to function as a repressor as well as an activator, it was necessary to target it to a well-defined enhancer that independently confers widespread expression. Through work on the Grainyhead (Grh) transcription factor, a palindromic binding site (Gbe) has been defined that, when combined in three copies with a minimal promoter, confers expression throughout the imaginal discs, epidermis, and trachea of the Drosophila larvae. Since Su(H) is expressed ubiquitously, it was anticipated that when Su(H) sites are combined with Gbe, Su(H) would cooperate with Grh to yield high levels of expression in the cells where Notch is active (e.g., dorsal/ventral boundary, interveins in the wing imaginal disc) and would prevent Grh-mediated activation in cells where Notch is inactive (e.g., larval epidemis, where there is no evidence for Notch activity based on expression patterns of known Notch target genes (Furriols, 2001).

The Su(H) binding sites used were the paired sites derived from the regulatory region of the Enhancer of split m8 gene, which is primarily expressed in association with proneural clusters in the imaginal discs. On their own, two pairs of Su(H)_m8 sites only give extremely limited activity; patchy expression was detected at the wing disc dorsal/ventral boundary and the tracheal branchpoints [Su(H)_m8]. In contrast, the Su(H)_m8 sites have a dramatic effect when combined with three copies of Gbe [Gbe+ Su(H)_m8]. In the imaginal discs, strong activation is detected in a pattern reminiscent of the most widely expressed Notch target gene, Enhancer of split mß [E(spl)mß]. Expression also occurs at tracheal branchpoints in a similar manner to E(spl)mß suggesting that this, too, is a site of Notch activity (Furriols, 2001).

The activation was coupled with apparent inhibition of Gbe-driven expression in some patches in the discs that correspond to the places where E(spl)mß is also silent. More definitive, however, is the effect in the epidermis and the trachea. The widespread expression throughout these tissues that is normally elicited by Gbe is shut off, while the activation at the tracheal branchpoints is enhanced. Similar results were obtained using a single copy of the paired Su(H)_m8 site. This construct has virtually no expression on its own but give an E(spl)mß-like pattern in the discs with Gbe and in two out of six lines represses Gbe-derived expression in the epidermis and trachea. Overall, the patterns obtained with Gbe+ Su(H)_m8 indicate first that Grh and Su(H) can cooperate synergistically to confer high levels of transcription in places known to have Notch activity and second that Su(H) is able to repress the Grh activation function in regions without Notch activity. Intriguingly, the resulting pattern strongly resembles that of E(spl)mß, although, since no evidence is as yet available that Grh normally confers this expression, Su(H) may synergize with a different activator on this E(spl)mß enhancer. It is important to note, however, that neither the synergy nor the repressive effects imply direct interactions between Su(H) and the DNA-bound activators. Based on the experiments with CBF-1, it is likely that Su(H) exerts its effects through the recruitment of cofactors, which probably include chromatin-modifying enzymes such as histone deacetylases (Furriols, 2001).

To confirm that the effects of adding the paired Su(H)_m8 sites to Gbe are due to the activity and not simply to the length of the Su(H) sequences inserted, a similar construct was generated in which the Su(H)_m8 sites had been mutated by substituting critical bases in the recognition sequence. The resulting transgene [Gbe+ Su(H)_MUT] has an expression pattern similar to the parental Gbe sites alone, although the levels of expression are reduced. Since these mutations restore the widespread activity of the enhancer, it must be the Su(H) sequence per se that confers the activation and repression detected with Gbe+ Su(H)_m8 (Furriols, 2001).

If this interpretation is correct and the E(spl)mß -like expression from Gbe+ Su(H)_m8 reflects a synergistic interaction between Su(H)/N^icd and Grh, this expression should be dependent on Notch and Su(H). Reducing Su(H) activity [Su(H)^SF8] in clones of cells in the wing disc leads to an autonomous loss of the high levels of expression from the mutant cells. Likewise, reducing Notch activity using a temperature sensitive combination (N^ts1/N^55e11) at the nonpermissive temperature also eliminates the intervein pattern and reduces the dorsal/ventral boundary expression of Gbe+ Su(H)_m8. Thus, the strong disc expression requires Notch and Su(H) and, as it is not seen with the Su(H)_m8 sites alone, must involve cooperation with Gbe-bound protein (Furriols, 2001).

Similar experiments were carried out to assess whether repression depends on Su(H). In this case, it would be anticipated that mutations in Su(H) should cause derepression, restoring Gbe-mediated epidermal expression, whereas mutations in Notch should not. In Su(H) mutant animals [Su(H)^SF8/Su(H)^AR9], there is widespread expression from Gbe+ Su(H)_m8 throughout the epidermis and tracheal cells, and the strong activation at tracheal branchpoints is lost. In contrast, there is no expression in the epidermal cells or most tracheal cells when Notch function is reduced. In the latter case, the activity at the branchpoints is reduced, as in Su(H) mutants, consistent with this being Notch-dependent activation, but there is no derepression in the other tracheal cells. Clearly, the transgene can be expressed in a similar pattern to the parental Gbe when there is little or no Su(H) protein present, confirming, therefore, that Su(H) is critical for the repression. In contrast, reducing Notch activity has no effect on repression. This differential highlights the fact that mutations in Notch and Su(H) are unlikely to have the same consequences on many target genes, as shown recently for singleminded. Since nonconsonance in phenotypes has been taken to indicate that certain Notch functions are independent of Su(H), it will be important to reevaluate these phenotypes, taking into consideration the possibility that Su(H) mutations can lead to derepression of target genes (Furriols, 2001).

If Su(H) has the ability to function as a molecular switch, the silencing of Gbe+ Su(H)_m8 expression in the epidermis should be alleviated by ectopic activation of Notch in this tissue. To test this, hsN^icd flies, which have the intracellular domain of Notch (N^icd) under the control of the heat-shock promoter, were used. Exposure to 37°C induces ubiquitous expression of N^icd, which is a constitutively active fragment of Notch, and under these conditions Gbe+ Su(H)_m8 confers expression throughout the epidermis and the trachea. In the presence of N^icd, therefore, the silencing is alleviated, and the transgene becomes activated in all the places where Grh is present (Furriols, 2001).

These data indicate that in the absence of Notch activation, Su(H) is capable of binding to its cognate sites and repressing transcription. Notch activation can alleviate the repression so that Su(H) is able to cooperate with other DNA-bound activators, like Grh, to promote transcription. These results are in agreement with recent models and strongly suggest that this is a general mechanism through which Su(H) acts at native targets. Thus, Su(H) is capable of acting as the pivot in a sensitive switch that would ensure that Notch target genes can be poised but silent until Notch is activated. For example, the E(spl) genes, which mediate the inhibitory effect of Notch during lateral inhibition, appear to be targets of proneural proteins. However, E(spl) genes are not expressed in the cells that are selected to be neural, even though proneural proteins accumulate at highest levels in these cells. According to the model, Su(H) would be able to suppress activators like the proneural proteins until Notch is activated. As soon as levels of Notch are sufficient to overcome Su(H)-mediated repression, the synergistic interactions with activators would lead to a sharp transition in the expression of E(spl) genes. The potent effect of combining Su(H) and Grh also gives a precedent for the way that individual target genes might respond to Notch in specific contexts, if each involves a different transregulator cooperating with Notch. This demonstrates the potential for designing specific molecular assays for Notch activity in different cellular contexts. By replacing the Gbe sites with elements that respond to other activators, it should be possible to generate a transcriptional readout for Notch activity in any cell type (Furriols, 2001)

The transcription factor odd-paired regulates temporal identity in transit-amplifying neural progenitors via an incoherent feed-forward loop

Neural progenitors undergo temporal patterning to generate diverse neurons in a chronological order. This process is well-studied in the developing Drosophila brain and conserved in mammals. During larval stages, intermediate neural progenitors (INPs) serially express Dichaete (D), grainyhead (Grh) and eyeless (Ey/Pax6), but how the transitions are regulated is not precisely understood. In this study a method was developed to isolate transcriptomes of INPs in their distinct temporal states to identify a complete set of temporal patterning factors. This analysis identifies odd-paired (opa), as a key regulator of temporal patterning. Temporal patterning is initiated when the SWI/SNF complex component Osa induces D and its repressor Opa at the same time but with distinct kinetics. Then, high Opa levels repress D to allow Grh transcription and progress to the next temporal state. It is proposed that Osa and its target genes opa and D form an incoherent feedforward loop (FFL) and a new mechanism allowing the successive expression of temporal identities (Abdusselamoglu, 2019).

Temporal patterning is a phenomenon where NSCs alter the fate of their progeny chronologically. Understanding how temporal patterning is regulated is crucial to understanding how the cellular complexity of the brain develops. This study presents a novel, FACS-based approach that enabled isolation of distinct temporal states of neural progenitors with very high purity from Drosophila larvae. This allowed a study the transitions between different temporal identity states. odd-paired (opa), a transcription factor that is required for INP temporal patterning, was identified. By studying the role of this factor in temporal patterning, a novel model is proposed for the regulation of temporal patterning in Drosophila neural stem cells (Abdusselamoglu, 2019).

Two different roles are established of the SWI/SNF complex subunit, Osa, in regulating INP temporal patterning. Initially, Osa initiates temporal patterning by activating the transcription factor D. Subsequently, Osa regulates the progression of temporal patterning by activating opa and ham, which in turn downregulate D and Grh, respectively. The concerted, but complementary action of opa and ham ensures temporal identity progression by promoting the transition between temporal stages. For instance, opa regulates the transition from D to Grh, while ham regulates the transition from Grh to Ey. It is proposed that opa achieves this by repressing D and activating grh, as indicated by the lack of temporal patterning in D and opa-depleted INPs. Loss of opa or ham causes INPs to lose their temporal identity and overproliferate. Moreover, it is proposed that D and opa activate Grh expression against the presence of ham, which represses Grh expression. As D and opa levels decrease as INPs age and become Grh positive, ham is capable of repressing Grh later on in temporal patterning. This explains how opa and ham act only during specific stages even though they are expressed throughout the entire lineage (Abdusselamoglu, 2019).

An open question pertains to the fact that the double knock-down of opa and ham, as well as that of D and opa, failed to recapitulate the Osa phenotype. Even though opa and ham RNAi caused massive overproliferation in type II lineages, no Dpn+ Ase- ectopic NB-like cells (as occurs in Osa mutant clones) were detected. It is proposed that this is caused by D expression, which is still induced even upon opa/ham double knockdown, but not upon Osa knock-down, where D expression fails to be initiated. Thus, the initiation of the first temporal identity state may block the reversion of INPs to a NB-state. In the future, it will be important to understand the exact mechanisms of how opa regulates temporal patterning (Abdusselamoglu, 2019).

This study further demonstrates that Osa initiates D expression earlier than opa expression. Osa is a subunit of SWI/SNF chromatin remodeling complex, and it guides the complex to specific loci throughout the genome, such as the TSS of both D and opa. The differences in timing of D and opa expression may be explained by separate factors involved in their activation. Previous work suggests that the transcription factor earmuff may activate . However, it remains unknown which factor activates opa expression. One possibility is that the cell cycle activates opa, since its expression begins in mINPs, a dividing cell unlike imINPs, which are in cell cycle arrest (Abdusselamoglu, 2019).

It is proposed that balanced expression levels of D and opa regulate the timing of transitions between temporal identity states. Indeed, Osa initiates D and opa, the repressor of D, at slightly different times, which could allow a time window for D to be expressed, perform its function, then become repressed again by opa. Deregulating this pattern, for example by overexpressing opa in the earliest INP stage, results in a false start of temporal patterning and premature differentiation. This elegant set of genetic interactions resembles that of an incoherent feedforward loop (FFL). In such a network, pathways have opposing roles. For instance, Osa promotes both the expression and repression of D. Similar examples can be observed in other organisms, such as in the galactose network of E. coli, where the transcriptional activator CRP activates galS and galE, while galS also represses galE. In Drosophila SOP determination, miR-7, together with Atonal also forms an incoherent FFL. Furthermore, mammals apply a similar mechanism in the c-Myc/E2F1 regulatory system (Abdusselamoglu, 2019).

The vertebrate homologues of opa consist of the Zinc-finger protein of the cerebellum (ZIC) family, which are suggested to regulate the transcriptional activity of target genes, and to have a role in CNS development. In mice, during embryonic cortical development, ZIC family proteins regulate the proliferation of meningeal cells, which are required for normal cortical development. In addition, another member of the ZIC family, Zic1, is a Brn2 target, which itself controls the transition from early-to-mid neurogenesis in the mouse cortex. Along with these lines, it has been shown that ZIC family is important in brain development in zebrafish. Furthermore, the role of ZIC has been implicated in variety of brain malformations and/or diseases. These data provide mere glimpses into the roles of ZIC family proteins in neuronal fate decisions in mammals, and this study offers an important entry point to start understanding these remarkable proteins (Abdusselamoglu, 2019).

These findings provide a novel regulatory network model controlling temporal patterning, which may occur in all metazoans, including humans. In contrast to existing cascade models, this study instead shows that temporal patterning is a highly coordinated ensemble that allows regulation on additional levels than was previously appreciated to ensure a perfectly balanced generation of different neuron/glial cell types. Together, these results demonstrate that Drosophila is a powerful system to dissect the genetic mechanisms underlying the temporal patterning of neural stem cells and how the disruption of such mechanisms impacts brain development and behavior (Abdusselamoglu, 2019).

Earlier Descriptions of Grh Biology

Interest in grainy head function will vary, depending on one's developmental perspective. For the biologists who take a classical approach, studying morphological or phenotypic results caused by mutation, grainy head is not the most exciting gene in the lab. But over the past half dozen years, a second developmental perspective has gained adherents, and made meticulous, if understated, contributions to an understanding of developmental processes by revealing the inner workings of gene regulation. Grainy head has been used as a tool to reveal the structure of the general transcription apparatus involved in gene regulation, one cog in a vast machine used to transcribe genes into messenger RNA, into proteins, and ultimately, into the mature organism. The difference in perspective is the difference between wanting to know how the engine operates versus where the engine will carry the organism. Both are valid. morphology: reads like shopping list of disparate activities Grainy head is synthesized during oogenesis and deposited in the developing oocyte to await the call to action. Grainy head maternal mRNA contributes to ventral repression during early embryogenesis.

The earliest involvement of GRH in development must be in the regulation of tailless downstream of Torso. Here GRH acts as a cofactor with GAGA (Liaw, 1995). GRH also acts as a cofactor of the transcription factor Dorsal in the repression of dpp and zerknüllt as mentioned above. As a repressor GRH acts as a downstream target of both Torso and Toll receptor tyrosine kinase pathways. Activation of either pathway results in a phosphorylation cascade; MAP kinase is responsible for attaching the phosphates to GRH. Said another way, GRH is a substrate of MAP kinase. (Liaw, 1995 and Huang, 1995).

The role of GRH in tailless regulation is both critical and complex. grainy head mutants show an enlargment of the tailless range of transcription. GRH and GAGA/Trithorax-like act at the Torso response element of tailless. GAGA is a transcriptional activator, but the GRH-GAGA interaction produces repression. It is concluded that GAGA is involved in relief of GRH-caused repression. Signaling through Torso results in the phosphorylation of GRH and its consequent inactivation (Liaw, 1995).

Overexpression of grainy head in the postblastoderm embryo results in a phenotype consistent with its role in the repression of dpp and zerknüllt later in embryogenesis (Huang, 1995). In spite of all these observed regulatory interactions, mutants of grainy head have only a minimal effect on fly morphology, in particular, an effect on cuticular morphology.

In contrast to its role as a repressor, as originally characterized, GRH is also a transcriptional activator: Grainy head regulates genes involved in epidermal development, including Ultrabithorax, engrailed, and fushi tarazu. An incredible amount of work has been expended in understanding the mechanism by which GRH acts as a transcriptional activator. This work has paid off grandly in an understanding of the basic apparatus for transcriptional activation. Studies of the role of GRH in transcriptional activation have resulted in the isolation of coactivators associated with the TATA-binding protein that mediates transcriptional activation (Dynlacht, 1991).

The activities of Grainyhead and other members of the family appear to be modulated so that they can participate in different developmental processes. The structure and function of mRNAs from the Drosophila grainyhead gene have been examined. Alternate splicing is responsible for generating a neuroblast-specific isoform of the protein. The N transcript of grh is expressed in larval epidermis, trachea, and foregut as well as in imaginal discs and optic lobes. However the mRNA detected by the O-specific fragment is expressed in the CNS, where the O-specific fragment is found at high levels in neuroblasts but not in the developing optic lobes. Similar results have been achieved in embryos, where O mRNA is predominantly detected in the neuroblasts of the CNS (Uv, 1997).

A mutation which abolishes the O isoform results in pupal and adult lethality and in the absence of Grh protein from neuroblasts. In other places, such as the epidermis, head skeleton, trachea, anterior spiracles, discs, and the foregut, Grh levels appear normal. The specific mutant has been localized to a base pair change in exon 5. Reporter genes containing different Grainyhead binding sites exhibit tissue-specific patterns of expression that correlate with the Grainyhead isoforms, suggesting that the alternate splicing serves to alter the repertoire of target genes controlled in the neuroblasts. Two Grh binding elements (be1 and be2) derived from the Dopa decarboxylase gene direct grh expression to different cells. be1 activity is detected in the optic lobes, neuroblasts and tracheal cells, whereas reporter be2 is detected only in the neuroblasts. The expression of both reporters is dependent on Grh function, implying that Grh can activate expression of reporter be2 in a tissue-specific manner: this correlates with the expression pattern of the O isoform of GRH. Since the be1 and be2 sites differ (be1 is palindromic and be2 is nonpalindromic), it is suggested that be1 and be2 represent targets for homo- and heterodimers of Grh, respectively, and that the O isoform preferentially forms heterodimers with an unknown partner. Similarly, regulation of globin genes by mammalian Grh homolog CP2 requires complex with a second, tissue-specific factor, which is as yet not cloned but which confers tissue and stage specificity on CP2 function (Uv, 1997).

GENE STRUCTURE

Four distinct classes of c DNAs have been described (N, N', O, and O'). There are two sites of variation. One is a small intron within exon 12 which remains unspliced in some cDNA variants (e.g., N and O) and codes for an extra 30 amino acids. The other consists of exons 4 and 5, which are present in the rarer large cDNAs (O and O') and lead to an inserion of 810 bp. It appears that exons 4 and 5 are always inserted together (Uv, 1997).

cDNA clone length - Three cDNAs representing alternative splicing are present (Bray, 1989).

Bases in 5' UTR -930 for N transcript

Exons -16

Bases in 3' UTR - 729 for N transcript

PROTEIN STRUCTURE

Structural Domains

Grainyhead belongs to a recently identified group of transcription factors that share a 250-amino-acid domain required for binding to DNA and a carboxy-terminal dimerization domain. grainy head has a basic helix-loop-helix motif (Bray, 1989), and a novel isoleucine-rich activation motif, distinct from the glutamine-rich activation domain of Sp1 (Attardi, 1993a). NTF-1 has an unusually large, unique DNA-binding and dimerization domain, as well as a novel, isoleucine-rich activation domain. This 56-amino-acid activation region fails to interact with the putative Sp1 coactivator, dTAFII110, and thus appears to use a mechanism distinct from the glutamine-rich activation domain of Sp1 (Attardi, 1993b).

The regions in the Drosophila tissue-specific transcription factor Grainyhead have been mapped that are required for DNA binding and dimerization. These functional domains correspond to regions conserved between Grainyhead and the vertebrate transcription factor CP2, which has similar activities. The identified DNA-binding domain is large (263 amino acids) but contains a smaller core that is able to interact with DNA at approximately 400-fold lower affinity. The major dimerization domain is located in a separate region of the protein and is required to stabilize the interactions with DNA. The data also suggest that Grainyhead activity can be modulated by an N-terminal inhibitory domain (Uv, 1994; full text of article).

date revised: 23 October 2023

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