BIOLOGICAL OVERVIEW

Su(H) is an integral part of the Notch signaling pathway and is neurogenic, like Notch. Su(H) protein is both cytoplasmic and nuclear. In the cytoplasm it interacts with the transmembrane Notch receptor that receives neurogenic signals from outside the cell. Su(H) then carries the Notch signal into the nucleus. Once there, Su(H) acts as a transcription factor regulating neurogenesis. The target of Su (H) is the Enhancer of split complex of genes which acts to suppress neural development. This suppressive function of the E(spl) complex is at the heart of the lateral inhibition function of the Notch pathway.

The names for Drosophila genes often run counter-intuitive to their actual function. One example of this perversity is the gene Hairless. One would assume a gene named "hairless" acts to restrict the number of bristles. Just the opposite is true. It seems that Hairless functions to enhance the formation of adult sense organs. Only when mutated does Hairless 's name match the results of its (in)activity, that is, the loss of adult kinesthetic sense organs. A fully functioning Hairless, novel in sequence, acts to oppose the antineural effect of Su(H), that is, the tendency of Su(H) to inhibit neurogenesis. Hairless physically interacts with Su(H), interferring with Su(H)'s ability to activate transcription of the Enhancer of split complex. And here too the naming is perverse. Enhancer of split genes oppose neurogenesis, yet they are grouped correctly among the neurogenic genes. Thus Hairless can be considered a neural patterning gene, carrying out its normal function by direct inhibition of Su(H) (Brou, 1994). One can speculate whether or not Hairless also interacts positively with proneural genes to enhance the neural fate.

Supressor of Hairless, also known as CSL (CBF-1/SuH/Lag-1), is the nuclear effector of the Notch signaling pathway and is 68% conserved with the human ortholog CBF-1. It is a three domain protein consisting of an N-terminal Rel homology region (NTD), a Beta-trefoil domain (BTD), and a C-terminal Rel homology region (CTD). The NTD and BTD mediate interactions with DNA, while the BTD and CTD mediate interactions with the intracellular domain of Notch and the NTD and CTD mediate interactions with the transcriptional coactivator Mastermind

Aspects of Drosophila development seem to resemble development of C. elegans more and more at every turn. The assumption of alternative sister cell fates in asymmetric division and the role of cleavage plane in determining positional fate of cells are two common aspects that keep cropping up. Su(H) expression in sensory organ precursors (SOP) has an integral role in establishment of alternative cell fates, and also indicates the importance cleave plane in determining cell fate. SOPs divide once to give rise to pIIb and pIIa progeny. PIIb give rise to neuron and sheath (glial theocogen) cells and pIIa give rise to shaft (stimulus receptive) and socket cells. These four progeny of SOPs make up the mechanosensory bristle of the adult fly.

Specific accumulation of Su(H) in the socket cell, which is the receiver of lateral inhibition, is delayed, suggesting that the differential expression of Su(H) is a consequence of a pre-existing asymmetry. In any case, a high level of Su(H) is both necessary and sufficient for socket cell fate (Schweisguth, 1994).

Division of pIIa occurs before division of pIIb, resulting in a three-cell stage. The orientation of these three-cell clusters along the anteroposterior axis and the position of the socket cell within this cluster is reproducibly defined in the developing notum. The presumptive socket cell is always the most posterior of the three cells. It is probable that this alignment is primarily determined by the orientation of the cleavage plane. It is not yet known what factors are responsible for the positional clues determining cleavage plane (Gho, 1996).

A new hypomorphic numb mutant not only displays a double-socket phenotype, due to a hair cell to socket cell transformation, but also a double-sheath phenotype, due to a neuron to sheath cell transformation. This provides direct evidence that numb functions in the neuron/sheath cell lineage as well. These results, together with the observation from immunofluorescence analysis that Numb forms a crescent in the dividing IIa and IIb cells suggest that asymmetric localization of Numb is important for the cell fate determination in all three asymmetric cell divisions in the sensory organ lineage. In the hair/socket cell lineage, but not the neuron/sheath cell lineage, a Suppressor of Hairless mutation acts as a dominant suppressor of numb mutations whereas Hairless mutations act as enhancers of numb. Therefore Su(H) and Hairless are required for determining the hair/socket cell lineage but not the neuron/sheath cell lineage. The neuron/sheath cell lineage is not affected by the loss or increase of Su(H) activity. Epistasis analysis indicates that Suppressor of Hairless acts downstream of numb. Results from in vitro binding analysis show a physical interaction between Numb and Hairless and suggest that the genetic interaction between numb and Hairless may occur through direct protein-protein interaction. These studies reveal that Suppressor of Hairless is required for only a subset of the asymmetric divisions that depend on the function of numb and Notch. Since numb and Notch are involved in an antagonistic manner in all three asymmetric divisions of the sensory organ precursor lineage, and Su(H) is involved in only a subset of these, there must exist a Su(H)-idependent Notch signaling that has not yet been well characterized (Wang, 1997).

Su(H) can undergo a functional switch from a repressor to an activator

Notch signal transduction appears to involve the ligand-induced intracellular processing of Notch, and the formation of a processed Notch-Suppressor of Hairless complex that binds DNA and activates the transcription of Notch target genes. This suggests that loss of either Notch or Su(H) activities should lead to similar cell fate changes. However, previous data indicate that, in the Drosophila blastoderm embryo, mesectoderm specification requires Notch but not Su(H) activity. The determination of the mesectodermal fate is specified by Single-minded (Sim), a transcription factor expressed in a single row of cells abutting the mesoderm. The molecular mechanisms by which the dorsoventral gradient of nuclear Dorsal establishes the single-cell wide territory of sim expression are not fully understood. Notch activity is required for sim expression in cellularizing embryos. In contrast, at this stage, Su(H) has a dual function. Su(H) activity is required to up-regulate sim expression in the mesectoderm, and to prevent the ectopic expression of sim dorsally in the neuroectoderm. Repression of sim transcription by Su(H) is direct and independent of Notch activity. Conversely, activation of sim transcription by Notch requires the Su(H)-binding sites. Thus, Notch signaling appears to relieve the repression exerted by Su(H) and to up-regulate sim transcription in the mesectoderm. A model is proposed in which repression by Su(H) and derepression by Notch are essential to allow for the definition of a single row of mesectodermal cells in the blastoderm embryo (Morel, 2000).

Proneural enhancement by Notch overcomes Suppressor-of-Hairless repressor function in the developing Drosophila eye

The receptor protein Notch plays a conserved role in restricting neural-fate specification during lateral inhibition. Lateral inhibition requires the Notch intracellular domain to coactivate Su(H)-mediated transcription of the Enhancer-of-split Complex. During Drosophila eye development, Notch plays an additional role in promoting neural fate independent of Su(H) and E(spl)-C, and this finding suggests an alternative mechanism of Notch signal transduction. Genetic mosaics were used to analyze the proneural enhancement pathway. Proneural enhancement involves upregulation of proneural gene expression in single cells that will become neurons. In Drosophila eye development, Notch (N) is required for proneural enhancement in addition to lateral inhibition. The molecular mechanism of proneural enhancement has not been determined. As in lateral inhibition, the metalloprotease Kuzbanian, the EGF repeat 12 region of the Notch extracellular domain, Presenilin, and the Notch intracellular domain are required. By contrast, proneural enhancement becomes constitutive in the absence of Su(H), and this leads to premature differentiation and upregulation of the Atonal and Senseless proteins. Ectopic Notch signaling by Delta expression ahead of the morphogenetic furrow also causes premature differentiation. It is concluded that proneural enhancement and lateral inhibition use similar ligand binding and receptor processing but differ in the nuclear role of Su(H). Prior to Notch signaling, Su(H) represses neural development directly, not indirectly through E(spl)-C. During proneural enhancement, the Notch intracellular domain overcomes the repression of neural differentiation. Later, lateral inhibition restores the repression of neural development by a different mechanism, requiring E(spl)-C transcription. Thus, Notch restricts neurogenesis temporally to a narrow time interval between two modes of repression (Li, 2001).

In the developing eye, lateral inhibition restricts the proneural gene atonal (ato) to individual R8 photoreceptor cells, which found each ommatidium. Earlier, ato must first have reached levels of activity sufficient to sustain expression by autoregulation, in conjunction with its bHLH heterodimer partner encoded by daughterless (da) and with a zinc-finger protein encoded by senseless (sens). Such 'proneural enhancement' depends on N and Dl but not on Su(H) or E(spl)-C. Clones of cells mutant for the E(spl)-C or for Su(H) lead to neural hyperplasia because they lack lateral inhibition, but clones of cells mutant for N or Dl show reduced neural differentiation because they lack proneural enhancement. These divergent phenotypes show that proneural enhancement occurs by a mechanism distinct from that of lateral inhibition (Li, 2001).

Mosaic analysis with Notch pathway mutations have been used to elucidate the mechanism of proneural enhancement. Requirements similar to those of canonical N signaling for processed forms of Dl, Notch EGF repeats 10-12, and proteolytic processing of the N intracellular domain have been found. Proneural enhancement is independent of any Su(H)-mediated gene activation but is mimicked by the complete absence of Su(H) protein, and this indicates that proneural enhancement depends on the disruption of Su(H)-mediated gene repression (Li, 2001).

The phenotypes of other mutations can be compared to the E(spl) or N phenotypes. A neurogenic mutant phenotype indicates a role in lateral inhibition, not in proneural enhancement. A hyponeural phenotype indicates a requirement in proneural enhancement (Li, 2001).

The neurogenic phenotype of the metalloprotease kuz suggests that processed Dl might be important for lateral inhibition and that unprocessed, transmembrane Dl may not be sufficient. It is unknown what form of Dl is required for proneural signaling. Clones mutant for kuz show neural hyperplasia. The distribution of R8 cells labeled by Boss antibody is intermediate between the distributions of clones null for E(spl) and for N. This indicates either partial loss of lateral inhibition or a weak proneural phenotype that still permits some neurogenesis to occur. Ato expression was examined to distinguish these possibilities. In kuz clones, Ato protein appears at the same time as it does in neighboring wild-type regions, but it remains at a low level. Posterior to the furrow, small clusters of R8 cells express Ato at a higher level, but many fewer cells do so than in E(spl) clones. This shows that proneural enhancement is affected in kuz mutant clones, but to a lesser degree than in N null clones, so that more cells go on to take the R8 cell fate. An intermediate phenotype associated with small clusters of R8 cells results in combination with the kuz lateral-inhibition defect. This is consistent with a role for processed Dl in proneural enhancement as well as in lateral inhibition, although it is important to note that kuz might have roles besides Dl processing. Such roles might include other aspects of N function (Li, 2001).

EGF repeats 10-12 bind Dl and are important during lateral inhibition because a glutamic acid-to-valine substitution in EGF repeat 12 in the N^M1 mutant is embryonic lethal and neurogenic. Clones of N^M1 mutant cells in the eye affect proneural enhancement and lateral inhibition, as does kuz, and this finding indicates that Dl interacts with the EGF repeat 12 region of N for proneural enhancement as well as for lateral inhibition (Li, 2001).

Clones mutant for the psn mutation were examined to test whether the novel proneural pathway requires proteolytic processing of N. Clones of psn exhibit an intermediate phenotype. Small patches of R8 cells differentiate, as in N^M1 or kuz clones but unlike in E(spl) clones. Ato expression initiates normally but never elevates to the same levels seen in the wild type. Lateral inhibition is deficient in psn clones as judged by the loss of E(spl) expression [E(spl) mDelta], so the intermediate psn phenotype indicates an effect on proneural enhancement in addition (Li, 2001).

In lateral inhibitory signaling, the processed intracellular domain enters the nucleus. Clones mutant for the N^CO mutation were examined to test whether proneural enhancement is also mediated by the released intracellular domain or, alternatively, by other parts of the processed protein. In place of Gln-1865, N^CO encodes a termination codon that truncates the N intracellular domain close to the transmembrane domain. Eye clones of N^CO almost completely lack R8 cells or other neurons. Ato expression is greatly reduced, and only rare R8 cells form posterior to the furrow. Expression of the Senseless protein, a marker for Ato activity, is also greatly reduced, and this finding confirms the failure to establish high levels of Ato expression and function. These results show that the N intracellular domain is required for proneural enhancement. Similar results were obtained with N^60g11, which truncates the intracellular domain carboxy-terminal to the ankyrin/CDC repeats (Li, 2001).

It is noteworthy that the N^CO phenotype is 'stronger' than clones of the N protein null, in which occasional patches of neurogenesis are seen. If this is attributed to the dominant-negative effect of the protein encoded by N^CO, then residual neurogenesis in N null clones must reflect residual N protein, perhaps persisting from before the mitotic recombination event (Li, 2001).

The N intracellular domain converts nuclear Su(H) protein from a transcriptional repressor into a transcriptional activator during lateral inhibition. What is its role in proneural enhancement? It has been concluded that proneural enhancement does not require Su(H) based on the neurogenic phenotype of Su(H) mutant clones. However, the original Su(H) mutants seem not to have eliminated the Su(H) repressor function. Recently, deletion alleles of the Su(H) gene have been recovered that eliminate all Su(H) function (Li, 2001).

Clones homozygous for the Su(H)^Delta47 allele are neurogenic, as described previously for other alleles. In addition, however, Su(H)^Delta47 mutant cells differentiate prematurely. Ato expression begin earlier in Su(H)^Delta47 clones than in neighboring tissue, and it soon reaches high levels. The senseless gene is expressed in response to ato activity. Senseless is also expressed prematurely in Su(H)^Delta47 clones. Daughterless protein is ubiquitous but upregulated in ato-expressing cells of the furrow. It was hard to see premature elevation of Daughterless in Su(H)^Delta47 clones, and this must be subtle if it occurs (Li, 2001).

Premature differentiation in Su(H)^Delta47 clones might be explained if Su(H) normally antagonizes proneural enhancement. Then, in the total absence of Su(H) protein, Ato would enhance prematurely and initiate eye differentiation. Accelerated differentiation would in turn accelerate the progress of the morphogenetic furrow, induce Atonal expression more anteriorly, and begin the cycle again. To investigate the effect of N signaling on this Su(H) function, Dl was misexpressed ahead of the morphogenetic furrow. A transposon insertion in the hairy gene provided GAL4 protein expression. Ato expression is expanded anteriorly throughout the domain of h expression in h^GAL4; UAS-Dl eye discs. The sca gene, which is expressed in response to ato activity, is also expressed more anteriorly in response to ectopic Dl. Neural differentiation begins normally in the most posterior part of h^GAL4;UAS-Dl eye discs but becomes progressively disorganized more anteriorly as differentiation accelerates (Li, 2001).

The similiarity between activating N signaling ahead of the morphogenetic furrow and deleting Su(H) indicates that N signaling overcomes repression mediated by Su(H). If Su(H) antagonizes proneural enhancement by activating gene transcription, activating N ahead of the furrow should have released the N intracellular domain, elevated gene transcription, and antagonized morphogenetic furrow progression and differentiation, opposite that of what was observed (Li, 2001).

Different forms or complexes of N intracellular domain might be required to antagonize Su(H)-mediated repression during proneural enhancement from those that coactivate Su(H)-mediated gene transcription. The possible role of bib, mam, and neur in proneural enhancement has not been assessed. The bib gene encodes a transmembrane protein required for lateral inhibition in embryonic neurogenesis. Ommatidia that are mutant for bib contain occasional extra photoreceptor cells, and some ommatidia have multiple R8 cells. Ato expression begins and progresses normally, but posterior to the morphogenetic furrow small clusters of two or three cells, instead of single cells as in the wild type, often retain Ato expression. Sections through the adult retinas often reveal ommatidia with extra photoreceptor cell rhabdomeres, both of the R8/R7 small rhabdomere class and of the larger R1-R6 outer photoreceptor class. Since bib affects lateral inhibition only slightly, it is possible that an equally subtle requirement for bib in proneural enhancement might be undetected in these experiments (Li, 2001).

These findings suggest a model for proneural enhancement. The release of N intracellular domain in response to Dl derepresses genes that are repressed by Su(H). The relevant targets do not require Su(H)-mediated transcriptional activation, so deletion of Su(H) mimics N signaling. The mechanism contrasts with lateral inhibition. N signaling provides N intracellular domain as a coactivator for Su(H), which is essential for the transcription of E(spl)-C. Lateral inhibition cannot proceed in the absence of Su(H) because blocking repression by Su(H) is not sufficient for E(spl)-C transcription (Li, 2001).

The ato gene could be a direct target of proneural enhancement. ato regulatory sequences have been examined for activity control regions, but possible repression sites have not been assessed. Another candidate is daughterless, which encodes a bHLH heterodimer partner of Ato that is required for Ato function in eye development. A third candidate is senseless, a zinc finger protein that enhances and maintains proneural gene expression. Expression of ato and sens is prematurely elevated in the absence of Su(H), which is consistent with regulation by Su(H)-R. However, each might depend on Su(H)-R only indirectly because elevated expression of ato or sens requires the function of all three genes (Li, 2001).

Why does proneural enhancement precede lateral inhibition if both depend on Su(H) and nuclear N intracellular domain? (1) Multiple lines of evidence indicate that proneural enhancement requires less N activation than does lateral inhibition. These include the greater sensitivity of lateral inhibition to the N^ts mutation, nonautonomous rescue of proneural enhancement by Dl over distances for which lateral inhibition go unrescued, and neurogenesis in N mutant clones due to undetectably low levels of N protein (which is eliminated by dominant-negative protein from the N^CO allele). Therefore proneural enhancement is expected to occur sooner in response to N signaling. (2) The evolving transcriptional regulation of ato changes sensitivity to lateral inhibition over time. Even recombinant N intracellular domain expression does not prevent initial ato expression ahead of the furrow, but ato is exquisitely sensitive later when its expression depends on autoregulation (Li, 2001).

The main result of this study is that neural development in the Drosophila eye depends on two functions of the N intracellular domain in response to ligand binding: (1) N relieves Su(H)-mediated repression to enhance ato expression and function and to permit neurogenesis (proneural enhancement); (2) later, another pathway requires N to coactivate Su(H)-dependent E(spl) transcription (lateral inhibition). No genes or regions of N have yet been found to be required to affect one function but not the other. By means of these two functions stimulated by the same ligand, N signaling coordinates the upregulation of ato in proneural cells and represses ato in cells not specified as neural precursor cells, and N restricts neural patterning to a narrow time interval between two distinct modes of repression (Li, 2001).

An enhancer composed of interlocking submodules controls transcriptional autoregulation of Suppressor of Hairless

Positive autoregulation is an effective mechanism for the long-term maintenance of a transcription factor's expression. This strategy is widely deployed in cell lineages, where the autoregulatory factor controls the activity of a battery of genes that constitute the differentiation program of a postmitotic cell type. In Drosophila, the Notch pathway transcription factor Suppressor of Hairless activates its own expression, specifically in the socket cell of external sensory organs, via an autoregulatory enhancer called the ASE. This study shows that the ASE is composed of several enhancer submodules, each of which can independently initiate weak Su(H) autoregulation. Cross-activation by these submodules is critical to ensure that Su(H) rises above a threshold level necessary to activate a maintenance submodule, which then sustains long-term Su(H) autoregulation. Our study reveals the use of interlinked positive-feedback loops to control autoregulation dynamically and provides mechanistic insight into initiation, establishment, and maintenance of the autoregulatory state (Liu, 2014).

Positive autoregulation by a gene encoding a DNA-binding transcription factor is a widely utilized mechanism for insuring the long-term maintenance of the factor's expression, well after the signals and other factors that initiated this activity are no longer present. One common setting in which such prolonged, stable expression of a transcription factor may be especially advantageous is a postmitotic, differentiated cell type. Here, the autoregulatory factor can function as a 'terminal selector,' responsible for driving the coexpression of a battery of downstream genes that constitute the cell's differentiation program (Liu, 2014).

Suppressor of Hairless (Su(H)) is an ancient, highly conserved protein that acts as the transducing transcription factor for the Notch cell-cell signaling pathway. Functioning in this role, which dates at a minimum to the last common ancestor of demosponges and eumetazoa, Su(H) participates in a huge variety of conditional cell fate specification events in virtually all metazoans (Liu, 2014).

It came as a surprise, then, to find that Su(H) has been coopted in Drosophila for a very different role: acting as an essential regulator of the differentiation of the socket cell, a nonneuronal component of external sensory organs in the fly. Regulation of Notch pathway target genes by Su(H) requires only a low basal level of the protein, which is present broadly or ubiquitously. But in Drosophila, Su(H) is in addition very highly expressed specifically in socket cells, beginning soon after the birth of the cell via the division of the pIIa secondary precursor and continuing stably thereafter. This high level of transcript and protein accumulation from Su(H) is driven by a dedicated transcriptional control module, the autoregulatory socket enhancer (ASE). The ASE lies downstream of the gene, includes eight high-affinity Su(H) binding sites, and mediates a direct positive autoregulation activity specifically in socket cells (Liu, 2014).

Although the fate of the socket cell is specified by Notch signaling, the ASE plays no role in this-indeed, the enhancer's activity does not commence until after the cell's fate has already been determined. Rather, mechanosensory organs in a fly lacking the ASE display severely impaired mechanotransduction, evidently due to defects in socket cell differentiation. In addition, the socket cell autoregulatory activity of Su(H), acting in concert with the socket cell-specific transcription factor Sox15, is also required to prevent deployment of an alternative differentiative program, that of the shaft cell, the socket cell's sister. This is accomplished by repressing expression of shaven (sv), which encodes a high-level regulator of shaft cell differentiation (Liu, 2014).

A previous study revealed that the ASE is initially activated by the Notch signaling event that specifies the socket cell fate. Here, a low level of Notch-stimulated Su(H) functions cooperatively with certain 'local activators' that are expressed in both the socket and shaft cells. By contrast, the long-term maintenance of high-level Su(H) autoregulation was found to be independent of Notch (Liu, 2014).

Despite the prevalence of positive autoregulation by transcription factor-encoding genes as a developmental control strategy, the specific mechanisms by which the autoregulatory state is initiated, established, and maintained have not been studied in detail. The Su(H) ASE was chosen as a model for investigating this question. Dissecting a direct transcriptional autoregulatory activity necessitates separately analyzing the associated enhancer's ability to respond to the normal wild-type context, with its normal level of the autoregulatory factor (e.g., by examining the expression of reporter transgene variants in a wild-type background) and its ability to establish the autoregulatory state (e.g., by examining levels of the autoregulatory factor generated by genomic rescue construct variants in a genetic background lacking the function of the autoregulatory gene). These two capabilities were studied in detail in exploring the ASE's mechanism of operation (Liu, 2014).

Counter to the expectation that the ASE functions as a single modular enhancer unit, this investigation reveals that it is instead composed of several overlapping structural and functional elements that are referred to as enhancer submodules, each of which can independently become active in the differentiating socket cell. Moreover, because each of the ASE's submodules contains one or a few Su(H) binding sites, together they form several interlinked positive feedback loops with the Su(H) gene. Interestingly, not all of the ASE's autoregulatory submodules respond to Su(H) equally: although some are activated by a low level of Su(H), others require much higher levels. As a result, the different submodules are deployed in succession: first to initiate a low-level autoregulatory activity, then to establish the autoregulatory state by exceeding a threshold level of Su(H), and finally to 'lock down' a permanent high-level maintenance function. A coherent model is proposed that explains how the ASE rapidly translates an initiating Notch pathway input signal into a highly elevated and irreversible level of Su(H) expression, specifically in the developing socket cell. It is suggested that enhancer subfunctionalization via enhancer submodules may be a generalizable mechanism for integrating inputs from a suite of dynamically expressed trans-regulators into a stable gene expression state (Liu, 2014).

This study has systematically dissected the functional organization of the autoregulatory socket enhancer (ASE) of the Drosophila Su(H) gene, which controls the long-term transcriptional autoactivation of Su(H) specifically in the socket cell of external sensory organs. Based on these experiments, a dynamic model is proposed to explain how Su(H) autoregulation is controlled (Liu, 2014).

It is suggested that, within a short time window (0–2 hr) after the division of the pIIa secondary precursor cell, the ASE receives certain 'local activator' inputs in both postmitotic daughter cells via the YZW fragment; at the same time, the ASE is silenced by the repressive activity of basal levels of Su(H), via the two flanking clusters of high-affinity Su(H) binding sites, S2-S6 and S7-S9. Next, the incoming Notch signal that specifies the socket cell fate converts Su(H) into a transcriptional activator in this cell; Su(H) then synergizes with the early inputs on YZW to activate two enhancer submodules, ASE5Y and ZW3S, to trigger a rapid rise in Su(H) transcription. Simultaneously, YZW may contribute independently to the activity of Su(H). The activation of these three submodules marks the initiation of the ASE-Su(H) autoregulatory loop (Liu, 2014).

Later, about 4 hr after the socket cell is born, the accumulated level of Su(H) rises above a certain threshold and feeds back on the ASE to activate two Su(H) response elements, ASE5 and W3S, the activities of which are strictly Su(H) concentration dependent. Moreover, at least for ASE5, Su(H) must synergize with two other activator inputs that are mediated by the A-motif and several high-affinity Vvl binding motifs. The activation of ASE5 and W3S marks the time when the ASE-Su(H) autoregulatory loop is fully established (Liu, 2014).

Finally, as the socket cell differentiates, the factors acting upon Y and Z disappear from the socket cell, leading to the inactivation of the three submodules (ASE5Y, YZW, and ZW3S) that are responsible for the initiation of Su(H) autoregulation. However, one of the Su(H) response elements, ASE5, remains strongly active and maintains the autoregulatory loop into adulthood. The other Su(H) response element, W3S, is silent at this stage, evidently due to a late repression function of the adjacent sequences (Liu, 2014).

Another important dynamic element is embodied in the above model. Su(H) is seen to play an essential role in all three phases of Su(H) autoregulation in the socket cell-initiation, establishment, and maintenance. In the first step, initiation, Su(H) is already present at its low basal level; here, it is fully dependent on Notch signaling, which converts it from a repressor to an activator. But in the long-term maintenance phase, when Su(H) is present at a high level in the socket cell, it acts independently of Notch, possibly employing a distinct coactivator. Thus, the transition from low-level initiation to high-level maintenance in Su(H) autoregulation involves a transition from a Notch-dependent to a Notch-independent mode of action for Su(H) and the ASE (Liu, 2014).

An enhancer module is typically defined as a discrete genomic fragment capable of driving a specific pattern of gene transcription (with respect to location, time, and level). Most studies of enhancer function have accordingly treated each such module as a unit and have focused on its integrative capacity; namely, the enhancer's ability to synthesize multiple transcription factor inputs, both positive and negative, into a single transcriptional output (Liu, 2014).

Detailed functional analysis of the ASE has revealed a more complex picture that emphasizes enhancer substructure. This study has found that different component fragments of the full ASE have distinct functional roles to play in the initiation, establishment, and maintenance of cell-type-specific Su(H) autoregulation. One might perhaps conceive of these component fragments as discrete enhancer modules themselves, but as this study has shown, their boundaries often overlap (for example, ASE5 and W3S are respectively embedded within ASE5Y and ZW3S), and in any case they all help generate the same output -- continuous, elevated expression of Su(H) in the socket cell. Therefore, it is suggested that it is conceptually more useful to think of the ASE as a single enhancer composed of several enhancer submodules that are functionally distinct, but not clearly separable physically. In a highly dynamic manner, each submodule makes an important contribution to the overall spatial, temporal, and quantitative cis-regulatory activity of the full enhancer. Conversely, the ASE's architecture gives it the ability to integrate these multiple regulatory contributions into a stably progressing temporal pattern of Su(H) expression in the differentiating socket cell, a context in which both the external stimuli and the internal gene expression profile undergo dramatic changes. Such a sophisticated integration capacity has been observed previously for the control of some genes by a cohort of separate enhancer modules, but this study shows that even a single enhancer may employ this strategy using a compact set of submodules (Liu, 2014).

Finally, it is suggested that dividing the ASE's various activities among multiple functionally connected, and partially redundant, submodules confers on the enhancer considerable evolutionary flexibility to fine-tune almost every aspect of its function (time, space, and level). The successive employment of distinct submodules allows the ASE's activity at different stages to be modified separately by mutational changes within individual submodules. Likewise, the partially redundant functions of the ASE5Y and ZW3S initiation submodules allow each to undergo separate changes in a context in which the ASE's activity is buffered by the other submodule (Liu, 2014).

A particularly notable feature of the ASE is that each of its submodules contains one or a few Su(H) binding sites. Because each submodule can activate Su(H) transcription independently, and at the same time can respond to direct activation by Su(H), the reciprocal interaction between the ASE and Su(H) comprises not one, but several, interlinked positive feedback loops. Furthermore, due in part to the requirement for different levels of Su(H) for sub-module activation, the feedback loops between the ASE and Su(H) are interlinked in three distinct layers. The first linkage occurs among the ASE's three initiation submodules (ASE5Y, YZW, and ZW3S), which cooperate to drive Su(H) expression above its basal level. The second linkage is between the initiation submodules and the two Su(H) response elements (ASE5 and W3S), because the activities of the former group are responsible for generating the high level of Su(H) required to activate the latter. The third linkage occurs between the two Su(H) response elements themselves: when they are activated by high levels of Su(H), each then acts as a discrete Su(H) autoregulatory enhancer to reinforce the other's activity via the feedback input from Su(H). The logic of this system is evident: once a certain threshold level of Su(H) expression is established by the linked initiation submodules of the ASE, it can be quickly 'locked down' by the linked autoregulatory functions of the Su(H) response elements (Liu, 2014).

Recent studies have suggested that, in comparison with single positive feedback loops, interlinked positive feedback loops with different time constants are less sensitive to background noise and more effective at mediating rapid irreversible gene activation. It is suggested that the ASE represents a transcriptional implementation of this paradigm. For example, when the function of one of the ASE's initiation submodules is removed, Su(H) autoregulation can still be established, but the process is either fast-and-reversible (e.g., RC-ASE3) or slow-and-irreversible (e.g., RC-ASE5Y). Thus, whereas a simpler module might indeed be capable of establishing Su(H) autoregulation per se, the interlinked feedback loops of the ASE ensure that this happens rapidly and robustly, in a switch-like manner (Liu, 2014).

Activation of the Notch signaling pathway in vivo elicits changes in CSL nuclear dynamics

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, this study developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or 'assisted' loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which is proposed to confer sensitivity to small input changes and facilitate timely shut-down (Gomez-Lamarca, 2018).

Until recently, most existing models have portrayed CSL as a molecule with long DNA residence that serves as a static platform for exchange between NICD and co-repressors. This analysis, using a combination of FRAP and single-molecule tracking (SMT) to measure Su(H) dynamics, reveals a very different story and highlights two important characteristics. First, in Notch-OFF conditions, Su(H) normally undergoes very transient DNA residency, despite the fact that it is important for repression of the target loci. This implies that prolonged binding is not a prerequisite for repression. It also argues against a model where co-factors are exchanged while CSL remains bound to DNA. Second, in Notch-ON conditions, there is a striking enrichment of Su(H) at E(spl)-C, its primary target locus, where its dwell time is significantly increased. These changes in CSL-binding dynamics, can enable a sensitive and accurate response to NICD at its target sites (Gomez-Lamarca, 2018).

This study has found that NICD enhances both Su(H) recruitment and residence time at its target locus E(spl)-C, via a combination of mechanisms. One key step is that NICD-Su(H) complexes induce local changes in chromatin, which requires Trr (MLL3/4), a long-range co-activator that can contribute to chromatin opening. Notably, the consequence of NICD-induced chromatin opening is that it renders the target enhancers more accessible for additional complexes, regardless of whether they contain NICD or Hairless. Since binding of Hairless and NICD to Su(H) are mutually exclusive, it is likely that these represent discrete activator (Su(H)-NICD) and repressor (Su(H)-Hairless) complexes, although this study has not formally shown that Hairless recruitment relies on Su(H). This enhanced recruitment by NICD resembles that described for the glucocorticoid receptor and other factors, referred to as 'assisted loading,' whereby the binding of one protein complex helps the binding of another. It is proposed that the localized chromatin remodeling brought about by Su(H)-NICD reduces obstacles (e.g., moves nucleosomes) to facilitate DNA binding, i.e., effectively increasing K_ON. Such indirect cooperativity would render the response very sensitive to signal levels (Gomez-Lamarca, 2018).

A second aspect helps explain how the transiently bound Su(H)-NICD complexes can successfully activate transcription. Although at genomic locations with paired binding motifs the dimerization of NICD could enhance binding, the data argue that the presence of Mam itself confers a longer dwell time to the activator complex, most likely by favoring contacts with additional chromatin-associated factors, such as Mediator complex. One candidate to mediate these effects was CBP, a histone acetyltransferase that interacts with Mam and is necessary for its ability to stimulate transcription. However, inhibiting CBP or depleting the Mediator subunit Med7 only slightly modified the Su(H) dynamics, suggesting that each makes at best a modest contribution to the change in its behavior. As neither manipulation fully replicated the effects of Mam inhibition/depletion, despite preventing transcriptional activation, it is likely that they also act at a later step in the initiation process. Thus Mam is likely to exert its early effects on Su(H) recruitment through a combination of other chromatin factors besides CBP. The interaction of the tripartite Su(H)-NICD-Mam complex with these chromatin factors, although still transient, could confer a probabilistic switch between an inactive state and an active state, by leaving a longer-lasting modification or reorganization of the chromatin template or initiation complex (Gomez-Lamarca, 2018).

The fact that the Su(H)-NICD activator complex also enhances recruitment of Hairless co-repressor complexes was entirely unexpected based on prevailing models, and has several important consequences. First, it will bring opposing enzymatic activities (e.g., both histone acetyl-transferases and histone deacetylases), which could create a covalent modification cycle with switch-like properties, potentially further sensitizing responses to Notch. Second, enhanced recruitment of Hairless would ensure that genes are rapidly turned off after the signal decays, the switch operating in the converse direction when NICD levels decrease. Such 'facilitated repression,' where transcriptional activators promote global chromatin decondensation to facilitate loading of repressors, has also been described during circadian gene regulation where it operates as an amplitude rheostat (Gomez-Lamarca, 2018).

In conclusion, in vivo analysis of the mechanisms underlying the transcriptional response to Notch signaling reveal the fundamental importance of changes in DNA-binding dynamics and highlight how different mechanisms combine to enhance Su(H) recruitment and dwell time at E(spl)-C in Notch-ON cells. Whether both mechanisms operate at all Notch-regulated loci remains to be established, but they will likely be relevant for most genes where CSL occupancy was found to increase in Notch-ON conditions. Furthermore, this new insight into Notch signaling leads to a proposal that similar changes in the dynamics of nuclear effectors may also operate to deliver proper transcriptional outputs of other key signaling pathways (Gomez-Lamarca, 2018).

GENE STRUCTURE

date revised: 12 January 2023 

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