BIOLOGICAL OVERVIEW

The gap genes hunchback, giant, knirps and Krüppel function in concert to subdivide the anterior-posterior axis of the early embryo. They form an interactive system whose functional domains are set, at least in part, by their effects on one another. Krüppel expression forms a single broad band, like a belt, around the middle of the embryo. Two broad stripes of knirps expression lie adjacent to the anterior and posterior edges of the Krüppel band. Two stripes of giant expression abut the two knirps domains, and consequently lie even closer to the embryo's terminal ends.

Even when functioning as transcription factors, these genes do not necessarily operate independently. All transcription factors form multiprotein complexes when bound to promoters, and many interact when in the free, unbound state. Krüppel can physically associate with Knirps and Hunchback to form heterodimers (Sauer, 1995), and often their binding sites on promoters interdigitate (Langland, 1995 and Gutjahr, 1994). Further refuting hopes for functional simplicity, these proteins may act either as inhibitors or activators. Knirps, for example, activates hairy stripe 6 (Langeland, 1994) and represses stripe 7 (Pankratz, 1990). Despite the interactive associations among these three transcription factors, they belong to three distinctly different molecular families: Knirps is a steroid receptor; Krüppel, a zinc finger, and Giant, a basic leucine zipper. There are no simple answers here.

Knirps is considered a short range repressor. Rather than functioning competitively to block the binding of inducers, such repressors are thought to engender local changes in chromatin structure resulting in enhancer silencing. Human CtBP attenuates transcriptional activation and tumorigenesis mediated by the adenovirus E1A protein. CtBp is known to interact with a conserved sequence in the adenovirus E1A protein, Pro-X-Asp-Leu-Ser-X-Lys (P-DLS-K). Mutations in this sequence eliminate E1a-CtBP interactions so that CtBP no longer inhibits E1A mediated transcriptional activation and tumorigenesis in mammalian cell cultures. The P-DLS-K sequence is present in the repression domains of two unrelated short-range repressors, Knirps and Snail; the latter protein also contains the related sequence P-DLS-R. The P-DLS-K sequence is essential for the interaction of these proteins with Drosophila CtBP (dCtBP). A P-element-induced mutation in dCtBP exhibits gene-dosage interactions with a null mutation in knirps, which is consistent with the occurrence of Knirps-dCtBP interactions in vivo. Mutation of the D-DLS-K motif in Knirps, results in a Knirps protein that cannot mediate repression. These observations suggest that CtBP and dCtBP are engaged in an evolutionarily conserved mechanism of transcriptional repression, which is used in both Drosophila and mammals. Mammalian and Drosophila CtBP may mediate repression through the enzymic modification of chromatin because both proteins are related to D-isomer 2-hydroxy acid dehydrogenases. Despite this rather unexpected homology, immunolocalization assays indicate that the Drosophila CtBP protein accumulates in nuclei. Perhaps CtBP and dCtBP cause local changes in chromatin structure by introducing subtle changes in core histones. Alternatively, it is possible that CtBPs are components of an enzymatic cascade that modulates the activities of histone deacetylases or other co-repressor proteins (Nibu, 1998).

The knirps and knirps-related genes organize the development of the second wing vein in Drosophila. The position of the L2 wing vein is determined by a chain of known developmental events, beginning with the primary subdivision of the wing imaginal disc into anterior versus posterior lineage compartments. The subdivision of body segments such as the wing primordium into anterior and posterior compartments, in turn, can be traced back to early A/P patterning in the blastoderm stage embryo. To summarize these events briefly:

1. The posterior compartment fate is defined by expression of engrailed (en), which activates expression of the short-range Hedgehog (Hh) signal in posterior compartment cells and prevents posterior compartment cells from responding to Hh.
2. Secreted Hh travels a short distance (6-8 cells) into the anterior compartment where it initiates a sequence of signaling events, culminating in the activation of several Hh target genes including decapentaplegic.
3. Dpp synthesized in this narrow strip of cells travels significant distances in both the anterior and posterior directions to activate expression of Dpp target genes, such as the neighboring spalt-major (salm) and spalt-related (salr) genes, in a threshold-dependent fashion.
4. Juxtaposition of salm expressing and salm non-expressing cells induces expression of the rhomboid (rho) gene in a stripe 1-2 cells wide, corresponding to the L2 vein primordium.
5. rho then promotes differentiation of all longitudinal veins during late larval and early pupal development by potentiating signaling through the Egf-R/RAS pathway (Lunde, 1998 and references).

Evidence is presented that kni and knrl link A/P positional information in larval wing imaginal discs to morphogenesis of the second longitudinal wing vein (L2). kni and knrl are expressed in similar narrow stripes corresponding to the position of the L2 primordium. The kni and knrl L2 stripes abut the anterior border of the broad central expression domain of the Dpp target gene spalt major. Evidence is provided that radius incompletus (ri), a well-known viable mutant lacking the L2 vein, is a regulatory mutant of the kni/knrl locus. In ri mutant wing discs, kni and knrl fail to be expressed in the L2 primordium. In addition, the positions of molecular breakpoints in the kni/knrl locus indicate that the ri function is provided by cis-acting sequences upstream of the kni transcription unit. Consistent with kni and knrl playing a role in L2 vein formation, kni and knrl are expressed in similar narrow stripes corresponding to the position of the L2 primordium. kni-expressing cells abut the anterior border of strong sal-lacZ expression and express little or no detectable lacZ. For convenience, these kni expressing cells are referred to as salm non-expressing cells. Consistent with the genetic evidence that ri is a regulatory mutant of the kni/knrl locus, the L2 stripes of kni and knrl expression are absent in ri mutant discs. However, outside the wing pouch of ri discs, kni and knrl are expressed normally. In support of the genetic evidence suggesting that ri is a cis-acting regulatory allele of the kni/knrl locus, ri function has been mapped to a region lying immediately upstream of the kni transcription unit (Lunde, 1998).

Knirps targets rhomboid, which is required for the formation of the L2 vein. ri function is required to initiate expression of the vein-promoting gene rho in the L2 primordium, but is not essential for rho expression in other vein stripes. As would be expected if the kni/knrl locus acted upstream of rho, initiation of kni expression in the L2 primordium precedes that of rho. Another early marker for the L2 vein primordium is down-regulation of the key intervein gene blistered (bs). In ri mutants, down-regulation of Bs in L2 is not observed. Consistent with the kni/knrl locus functioning upstream of both rho and Egf-receptor signaling, kni and knrl are expressed normally in rho ^ve; vn ¹ double mutant wing discs. rho ^ve; vn ¹ mutants, which lack rho expression in vein primordia and have reduced levels of the Egf-R ligand encoded by the vn gene, are devoid of veins. Rescue of ri mutants by a ubiquitously expressed kni transgene also suggests that kni controls rho expression (Lunde, 1998).

The salm transcription factor has been shown to function upstream of rho in the L2 primordium; rho expression in L2 has been shown to be induced at the boundary between salm expressing cells and salm non-expressing cells (Sturtevant, 1997). The L2 vein primordium abuts salm-expressing cells but is comprised largely of salm non-expressing cells (Sturtevant, 1997). Like rho, expression of kni in the L2 primordium abuts the anterior edge of the broad salm expression domain in wild-type third instar wing discs, and is displaced along with the anterior border of salm expression in hedgehog ^Moonrat (hh ^Mrt) wing discs. In hh ^Mrt wing discs, the anterior limit of the salm expression domain on the ventral surface is frequently shifted forward, relative to the border on the dorsal surface. Associated with the asymmetry in sal-lacZ expression, the dorsal and ventral components of the kni L2 stripe are driven out of register. The coordinate shift of salm and kni expression is consistent with salm functioning upstream of kni. Strong ectopic expression of either salm or spalt related using the GAL4/UAS system eliminates kni and knrl expression, and leads to the production of small wings lacking the L2 and L5 veins. The loss of kni and knrl expression in discs mis-expressing salm or salr and the subsequent elimination of L2 presumably results from obscuring the sharp boundary of endogenous salm and salr expression. Clonal analysis also indicates that salm acts upstream of kni/knrl. salm - clones generated in the anterior compartment between L2 and L3 induce ectopic forks of the L2 vein, which lie along the inside edge of the salm - clones. In contrast, salm - clones produced in corresponding positions of ri mutant wings never induce L2 forks. However, other phenotypes associated with salm - clones, such as ectopic islands of triple row bristles at the margin, are observed with regularity in an ri background. It is proposed that a short-range diffusible signal X, functioning downstream of salm and salr induces expression of kni/knrl along the anterior border of the salm expression domain (Lunde, 1998).

To determine the importance of restricting kni expression to the L2 primordium, the GAL4/UAS system was used to mis-express kni or knrl at high levels in various patterns. The GAL4-MS1096 line drives expression of UAS-target genes ubiquitously throughout the dorsal surface of third instar wing discs, and weakly on the ventral surface in the anterior region of the disc. GAL4-MS1096-driven expression of either the UAS-kni or UAS-knrl transgenes eliminates expression of vein markers such as rho, the provein/proneural gene caupolican (caup), the lateral inhibitory gene Delta (Dl), and the proneural gene achaete on the dorsal surface of the wing disc. In contrast, these vein markers are expressed in normal patterns on the ventral surface, albeit at reduced levels, presumably reflecting the weak expression of GAL4 in ventral cells of GAL4-MS1096 discs. In addition, modulated expression of blistered (bs), which is lower in vein than intervein cells of wild-type discs, also disappears on the dorsal surface of GAL4-MS1096 wing discs. Thus, strong expression of kni or knrl on the dorsal surface of wing discs eliminates expression of both vein and intervein markers. Similarly, when GAL4-71B is used to drive UAS-kni or UAS-knrl expression in a central domain slightly broader than that of salm, distinctions between vein and intervein cells are eliminated within the region of GAL4 expression. In these discs, vein and intervein markers are expressed normally in the L5 primordium, which lies outside of the GAL4-71B expression domain. These data reveal that ectopic kni or knrl expression does not simply favor vein over intervein cell fates. Since strong uniform kni or knrl mis-expression is required to eliminate veins, higher levels of kni/knrl activity are necessary to inhibit vein formation than are required to induce expression of rho in or near the L2 primordium (Lunde, 1998).

In addition to activating rho expression, kni and knrl also are likely to positively autoregulate. Patterned mis-expression of kni using the GAL4/UAS system induces corresponding expression of the knrl gene and vice versa. Since kni and knrl appear to share cis-regulatory elements in third instar larval wing discs and during other stages of development, the reciprocal cross-regulation observed between kni and knrl is likely to reflect an autoregulatory function for these genes. kni function does not appear to be necessary for activating knrl expression in the L2 primordium, however, since elimination of kni function in large kni - clones covering both the dorsal and ventral components of L2 does not lead to any loss of the L2 vein. Another consequence of high level ectopic kni expression is strong down-regulation of salm expression. Since kni and knrl are normally expressed immediately adjacent to the anterior salm border, suppression of salm expression by kni appears to sharpen the anterior salm border and refine the position of the L2 primordium. Thus, the kni/knrl locus functions downstream of spalt major (salm) and upstream of genes required to initiate vein-versus-intervein differentiation. Mis-expression experiments suggest that kni and knrl expressing cells inhibit neighboring cells from becoming vein cells. kni and knrl are likely to refine the L2 position by positively auto-regulating their own expression and by providing negative feedback to repress salm expression (Lunde, 1998).

It is possible to trace formation of the L2 vein that occurs during the larval stage back to early A/P patterning that occurs during segmentation in the embryo. This chain of events leads to activation of the kni and knrl genes in narrow stripes at the anterior edge of the salm expression domain, thus linking positional information to morphogenesis. It is proposed that salm activates expression of a short-range signal X, which induces expression of kni and knrl in adjacent salm non-expressing cells. Since Kni and Knrl are members of the steroid hormone receptor superfamily, it is possible that the signal X could be a lipid-soluble factor, which binds and activates Kni and Knrl. However, given the minimal sequence conservation between Kni and Knrl in the putative ligand binding regions of these proteins, this direct form of signaling seems unlikely. Once activated, kni and knrl organize formation of the L2 primordium. kni and knrl are proposed to organize development of the L2 vein primordium through a variety of concerted actions. A key target gene activated by kni and knrl in the L2 primordium is the vein-promoting gene rho, which potentiates signaling through the Egf-receptor/RAS pathway. Because low levels of ubiquitous kni and knrl expression preferentially promote vein development near the location of L2, another activity provided at the anterior boundary of the salm expression domain is likely to act in parallel with the kni and knrl genes to define the position of the L2 primordium. This parallel genetic function may be supplied by the signal X, hypothesized to induce kni and knrl expression in salm non-expressing cells (Lunde, 1998).

kni and knrl are also likely to suppress vein development in neighboring intervein cells since strong uniform mis-expression of kni and knrl eliminates veins. This result could be explained if kni and knrl normally activate expression of a signal that suppresses vein development in neighboring intervein cells. Such a lateral inhibitory function presumably restricts formation of the L2 primordium to a narrow linear array of cells. To account for the fact that kni and knrl do not turn themselves off in L2 as a consequence of the proposed lateral inhibitory signaling, it is imagined that these cells are refractory to the lateral inhibitory mechanism. Alternatively, the hypothetical signal X, which promotes kni and knrl expression in cells adjacent to the salm expression domain, might continue to exert an inductive influence that overrides lateral inhibitory signaling in the L2 primordium. This possibility is consistent with low levels of ubiquitous kni expression rescuing rho expression in the vicinity of the normal L2 primordium in ri mutants. Although the nature of the proposed lateral inhibitory mechanism is unknown, the Notch signaling pathway is an obvious candidate, since loss of Notch function during late larval stages results in the formation of much broadened rho expressing stripes. Since Delta is unlikely to be the ligand mediating lateral inhibition, due to its absence in the L2 primordium, another Notch ligand might be activated in response to kni and knrl to suppress the vein fate in neighboring cells. It is also possible that a different type of signaling pathway is involved in this process. Finally, kni and knrl are likely to maintain and sharpen the anterior salm border through a combination of autoactivation and negative feedback on salm expression. Kni and Knrl may repress salm expression directly or could function indirectly through an intermediate tier of regulation. The ability of ectopic kni or knrl expression to suppress expression of salm as well as vein markers, but not to suppress expression of genes involved in defining the A/P organizing center (i.e. hh, dpp and ptc), is consistent with kni and knrl functioning at the last step in defining positional information required for placement of the L2 primordium. It will be interesting to determine whether there are genes functioning analogously to kni and knrl, that specify the positions of other longitudinal veins along the A/P axis of wing imaginal discs (Lunde, 1998).

The model proposed for activating expression of kni and knrl in a narrow stripe of cells is analogous to the earlier induction of dpp in a narrow stripe of anterior compartment cells by the short-range Hh signal emanating from the posterior compartment. In both cases a domain-defining gene (i.e. en or salm) activates expression of a short-range signal (i.e. Hh or X), while preventing these same cells from responding to the signal. According to such a genetic wiring diagram, only cells that are immediately adjacent to cells producing the short-range signal are competent to respond to it. This set of constraints restricts the expression of target genes to narrow stripes or sharp lines. An exquisite example of linear gene activation is the initiation of single minded expression in a single row of mesectodermal cells abutting the snail expression domain in the mesoderm of blastoderm embryos. Direct mechanisms contribute to activating sim in this precise pattern, because snail represses sim expression in ventral cells and Dorsal and Twist collaborate to define a relatively sharp threshold for activating sim that extends a short distance beyond the snail border. However, these direct transcriptional mechanisms alone do not seem sufficient to explain the absolutely faithful linear path of sim expression in a single row of cells along the irregular contour of snail expressing mesodermal cells. Perhaps communication between snail expressing cells and their immediate dorsal neighbors plays a role in achieving the invariant registration of the sim and snail expression patterns. In support of a role for cell-cell communication in this process, initiation of sim expression in the blastoderm embryo requires signaling through the Notch/Delta/E(spl) pathway. Furthermore, in the mesoderm, ubiquitously supplied maternal Delta protein is rapidly retrieved from the surface in the form of multi-vesicular bodies, which is typical of ligands involved in active signaling. Thus, Snail may regulate expression of some co-factor required for membrane bound Delta to productively activate the Notch signaling pathway in adjacent cells, which are free to respond by activating sim expression. It is noteworthy that in each of three cases considered above, products of entirely distinct domain-defining genes (e.g. En, Salm and Sna) induce the linear expression of genes in adjacent cells by activating production of short-range signals (e.g. Hh, X, Dl) while simultaneously suppressing response to those signals. The width of the target gene stripes presumably depends on the range of the signal and on the level of signal required to activate expression of specific genes. Thus, Hh activates expression of the target gene dpp in a domain 6-8 cells wide; the hypothetical factor X acts more locally to induce expression of kni and knrl in a stripe 2-3 cells wide, and the putative (activated) form of membrane tethered Delta induces sim expression in a single row of abutting mesectodermal cells. Perhaps this ‘for export only’ signaling mechanism is a general scheme for drawing lines in developing fields of cells (Lunde, 1998 and references).

Groucho corepressor functions as a cofactor for the Knirps short-range transcriptional repressor

Despite the pervasive roles for repressors in transcriptional control, the range of action of these proteins on cis regulatory elements remains poorly understood. Knirps has essential roles in patterning the Drosophila embryo by means of short-range repression, an activity that is essential for proper regulation of complex transcriptional control elements. Short-range repressors function in a local fashion to interfere with the activity of activators or basal promoters within approximately 100 bp. In contrast, long-range repressors such as Hairy act over distances >1 kb. The functional distinction between these two classes of repressors has been suggested to stem from the differential recruitment of the CtBP corepressor to short-range repressors and Groucho to long-range repressors. Contrary to this differential recruitment model, this study reports that Groucho is a functional part of the Knirps short-range repression complex. The corepressor interaction is mediated via an eh-1 like motif present in the N terminus and a conserved region present in the central portion of Knirps. This interaction is important for the CtBP-independent repression activity of Knirps and is required for regulation of even-skipped. This study uncovers a previously uncharacterized interaction between proteins previously thought to function in distinct repression pathways, and indicates that the Groucho corepressor can be differentially harnessed to execute short- and long-range repression (Payankaulam, 2009).

Groucho mediates the CtBP-independent repression activity of Knirps. The essential logic of Drosophila blastoderm transcription cascade is reliant on the short range of gap repressors proteins such as Knirps, Kruppel, and Giant acting on modular enhancers. Thus, the functional features of these repressors, which set them apart from long-range acting proteins such as Hairy, have been of special interest. Earlier studies suggested that the distinction between these classes of repressors may be attributed to differential recruitment of the CtBP corepressor to short-range repressor and Groucho to long-range repressors. The genetic and physical interactions of CtBP and Hairy were contradictory to this simple model, but further work has indicated that CtBP may not in fact serve as a Hairy corepressor, but as an antagonist of Groucho. In another case, the Brinker transcription factor can interact with Groucho and CtBP in vitro, but appears to rely on Groucho for repression of many target genes, whereas CtBP has a minor role. Significantly, the range of Brinker repression has never been elucidated. Foreshadowing this study, it has been shown that Slp1 acts as a gap type regulator of pair-rule genes in the early embryo. The short-range nature of this regulation was apparent on eve, hairy, run, ftz, prd, and odd when the Slp1 protein was expressed in a ventral pattern. Consistent with a role for Groucho, a mutant form of the Slp1 protein lacking the eh1 motif was reported to be inactive, but this assay was complicated by the brief temporal window of repression. This study shows that the well characterized short-range repressor Knirps physically and functionally interacts with Groucho, and this interaction is pivotal for the CtBP-independent repression potential of Knirps. These findings are definitely not consistent with the differential recruitment model of short- and long-range repression. Instead the results suggest an alternative model, that Groucho functions distinctly in the context of short- and long-range repression (Payankaulam, 2009).

One possible explanation for the diverse function of Groucho may involve oligomerization. Recent studies have shown that Groucho and its homolog can form oligomeric structures that have been proposed to spread along DNA. Mutations that block Groucho oligomerization in vitro compromise the activity of this protein in vivo in the imaginal disc. Thus, Groucho oligomerization has been assumed to be critical for its function and potentially related to the long-range activity of repressors such as Hairy. However, it seems likely that in the context of Knirps repressor complex, Groucho does not spread, because repression effects are clearly short range. Possibly the mode of recruitment dictates whether Groucho oligomerizes or not. It is hypothesized that the distinct eh1-like repression motifs in Knirps interact with Groucho in a unique conformation to restrain Groucho from spreading and, thus, from mediating long-range repression. Crystal structures of the WD domain of human Groucho homolog TLE1 bound to either WRPW or eh1 peptide revealed that these peptides adopt different conformations on the corepressor binding surface. Such differences may affect the ability of Groucho to oligomerize. Other components in the Knirps corepressor complex may also control Groucho oligomerization. An 'optional oligomerization' by Groucho model may explain earlier studies that found Hairy does not always cause dominant silencing of nearby enhancers. Also, hypomorphic alleles of Groucho have been identified that appear to compromise oligomerization but still retain some activity (Payankaulam, 2009).

What role might Groucho have in Knirps-mediated repression? As shown previously, the CtBP-independent repression activity of Knirps is critical for full activity on some endogenous enhancers, underscoring the importance of Knirps-Groucho association. The histone deacetylase Rpd3 is recruited by Groucho, and is also a part of the Knirps repression complex. CtBP proteins are known to interact with histone deacetylases; thus, both CtBP and Groucho may recruit Rpd3 cooperatively. The deacetylase activity may then augment Groucho-histone interactions, bringing about local modification of the chromatin, resulting in enhanced repressor output. Consistent with the cooperative recruitment of Rpd3, the purification of Knirps complexes indicated that Rpd3 associates preferentially with the full-length protein, and not the CtBP-independent domain alone. Therefore, in the context of Kni 1-330, Groucho may use another HDAC protein or rely on its HDAC-independent repression activity. The functional importance of this association may be to achieve quantitatively correct levels of Knirps activity, suggesting a similarity of function of these two corepressors. For example, in the context of the composite eve promoter, both of these activities can have roles in repressing enhancers of differential sensitivity (Payankaulam, 2009).

In conclusion, this study provides compelling evidence that Groucho can mediate short-range repression; thus, the long- and short-range effects of transcriptional repression do not appear to be a simple function of differential recruitment of distinct corepressors. Not only does this change the perspective of Groucho, it changes the perspective of different repressor proteins. It appears that long-range repressors such as Hairy and short-range repressors such as Knirps may function as modulators of the repression range of common machinery (Payankaulam, 2009). Interestingly, Knirps protein sequences from different insect genomes indicate that Groucho binding by Knirps may be an ancestral trait, because the Groucho-binding eh1-like motif is present in Drosophila species as well as Tribolium and Apis. In contrast, the critical CtBP-interacting residues are present only in Drosophila, suggesting that the acquisition of an additional corepressor may be a derived trait, possibly as a part of the remodeling of embryonic gene circuitry associated with the unique syncytial environment (Payankaulam, 2009).

Deciphering a transcriptional regulatory code: modeling short-range repression in the Drosophila embryo

Systems biology seeks a genomic-level interpretation of transcriptional regulatory information represented by patterns of protein-binding sites. Obtaining this information without direct experimentation is challenging; minor alterations in binding sites can have profound effects on gene expression, and underlie important aspects of disease and evolution. Quantitative modeling offers an alternative path to develop a global understanding of the transcriptional regulatory code. Recent studies have focused on endogenous regulatory sequences; however, distinct enhancers differ in many features, making it difficult to generalize to other cis-regulatory elements. This study applied a systematic approach to simpler elements and presents the first quantitative analysis of short-range transcriptional repressors, which have central functions in metazoan development. Fractional occupancy-based modeling uncovered unexpected features of these proteins' activity that allow accurate predictions of regulation by the Giant, Knirps, Krüppel, and Snail repressors, including modeling of an endogenous enhancer. This study provides essential elements of a transcriptional regulatory code that will allow extensive analysis of genomic information in Drosophila melanogaster and related organisms (Fakhouri, 2010).

In this study, by using a reductionist analysis of short-range repression, a relatively untouched, yet central aspect of gene regulation was explored in Drosophila. Earlier qualitative studies highlighted the extreme distance dependence of short-range repressors, and comparative analysis has shown many instances of evolutionary plasticity of regulatory regions controlled by these proteins. Knowing that transcription factors influence each other in a local manner permitted the identification of novel enhancers, based on the clustering of binding sites. Yet, clustering studies alone do not provide the basis for predicting evolutionary changes that reshape transcriptional output, or predicting activity of coregulated enhancers. For example, the original hypothesis that the affinity and or number of Bicoid-binding sites dictates the output of regulated genes has been replaced by an understanding that other, as-yet unknown features, seem to have more decisive functions (Fakhouri, 2010).

Earlier modeling studies focused on endogenous enhancers, which have complex arrangements of transcription factor-binding sites. The curret studies focused on detecting quantitative differences resulting from subtle differences in binding sites, allowing modeling with a tractable number of parameters. A common block of Dorsal and Twist activator sites was used, allowing a focus on changes made in the number and arrangement of repressor sites; clearly, differences in affinity, number, and arrangement of activator sites also have decisive functions in dictating transcriptional output; thus, future modeling efforts will need to integrate these elements as well. The tight focus on short-range repressors with the analysis of a relatively small number of reporter genes provided sufficient data for robust estimation of important parameters. From the comparison of repression by other short-range repressors, it is likely that the analysis of Giant can guide studies of other similarly acting repressors, including Krüppel, Knirps, and Snail (Fakhouri, 2010).

Relating to transcriptional regulatory code, this study uncovered specific quantitative features that seem to apply to short-range repressors in a general context. A complex non-linear quenching relationship was found that suggests that within the range of activity, Giant, and probably other short-range repressors, have an optimum distance of action that may reflect steric constraints. Multiple formulations of the model generated very similar predictions, suggesting that this non-linear distance function is a real feature of the system. Consistent with this notion, an earlier study of transcription factor-binding sites in Drosophila enhancers discovered an overall preference of Krüppel sites to be found 17 bp from Bicoid activator sites, which may be an indication that other short-range repressors also have preferred distances for optimal activity (Fakhouri, 2010).

The similar quenching efficiencies for repressors acting adjacent to Dorsal or Twist activator sites were an additional significant finding. The similar effect on disparate activator proteins indicates that the effects of short-range repression are general, and are likely to be translatable to distinct contexts. Earlier empirical tests had already pointed in this direction; for example, insertion of ectopic-binding sites for Knirps and Krüppel into rho NEE sequences is sufficient to induce repression, although these proteins do not usually cross-regulate. In addition, short-range repressors can counteract a variety of transcriptional activation domains with similar efficiency, suggesting that specific protein-protein contacts are not essential. In one area quantitative differences were found between parameters derived from the synthetic gene modules and the endogenous regulatory regions. The importance of homotypic cooperativity predicted for Snail sites in the context of the rho NEE was overall much higher than that found for Giant, Krüppel, and Knirps sites acting on the synthetic gene constructs; this might be an example in which the individual proteins do exhibit different context dependencies perhaps because the proteins differ in level of stickiness. Alternatively, the distance between the Snail sites in question, 23 bp, might facilitate cooperative interactions much more than the closely apposed spacing used in the genes genes used in this study, in which steric interference may have an opposing function (Fakhouri, 2010).

In modeling mutant forms of the endogenous rho NEE, several important features of the architecture of this regulatory region were uncovered. This enhancer seems to use redundancy in use of Snail to mediate repression; based on earlier experiments, it seems that even a single Snail site is sufficient to mediate repression. Such redundancy may provide the correct dynamical response, with a swift repression of rho at an early enough time in which Snail levels are still low, or it may ensure that gene output is robust to environmental and genetic noise (Fakhouri, 2010).

The rho NEE modeling also highlighted features of transcriptional activators. Activator-scaling factors for Dorsal were reproducibly lower than those of Twist, and this was apparent for several different assumptions of expression level. The relative differences in contribution to activation can be explained by examination of the structure of the enhancer; contribution by the low intrinsic values of Dorsal is amplified by strong cooperativity with Twist, setting up a chain of interacting weak sites that together are highly active. Experimental evidence bears out these conclusions: isolated Dorsal sites tested on reporter genes mediate relatively weak activation, and a rho NEE lacking Twist sites, but containing four Dorsal sites, is similarly compromised (Fakhouri, 2010).

Earlier studies suggested that many developmental enhancers, including those regulated by short-range repressors, may possess a flexible 'billboard' design, in which individual factors or small groups of proteins would independently communicate with the promoter region, so that the net output of an enhancer would reflect the cumulative set of contacts over a short time period. Such a view of enhancers would account for the evolutionary plasticity observed in regulatory sequences. No DNA-scaffolded superstructure, reflecting the formation of a unique three-dimensional complex, would be necessary in this scenario. Yet, the modeling suggests that the rho NEE might involve communication between relatively distant-binding sites, through sets of cooperative interactions. In this case, it is possible that such distant interactions might be compatible with a flexible structure, if many distinct configurations of binding sites provide such a cooperative network. Current studies have indeed highlighted potential frameworks involving Dorsal and interacting factors on same classes of enhancer. Application of a transcriptional regulatory code integrating activities of activators and repressors is a critical next step to illuminate enhancer design and evolution (Fakhouri, 2010).

Diverse spatial expression patterns emerge from unified kinetics of transcriptional bursting

How transcriptional bursting relates to gene regulation is a central question that has persisted for more than a decade. This study measure nascent transcriptional activity in early Drosophila embryos and characterize the variability in absolute activity levels across expression boundaries. Boundary formation follows a common transcription principle: a single control parameter determines the distribution of transcriptional activity, regardless of gene identity, boundary position, or enhancer-promoter architecture. The underlying bursting kinetics were inferred, and the key regulatory parameter was identified as the fraction of time a gene is in a transcriptionally active state. Unexpectedly, both the rate of polymerase initiation and the switching rates are tightly constrained across all expression levels, predicting synchronous patterning outcomes at all positions in the embryo. These results point to a shared simplicity underlying the apparently complex transcriptional processes of early embryonic patterning and indicate a path to general rules in transcriptional regulation (Zoller, 2018).

A multitude of processes influence eukaryotic transcription rates. It is not clear which events might be more likely than others to determine the kinetics of bursting-either globally or in a gene specific manner, nor is it known how bursting kinetics compare across endogenous genes over a range of expression levels. Quantitative bursting measurements reveal that all gap gene (hunchback, knirps, Kruppel and giant) expression boundaries arise from the same underlying kinetics regardless of the differences in regulatory elements. Thus, from the complex combination of diverse interactions specific to each gene emerges a simple, common strategy for transcriptional regulation (Zoller, 2018).

The recognition of shared regulation surfaced only upon development of a highly precise single-molecule method of quantification. Conclusions about bursting depend heavily upon understanding sources and extent of measurement error and minimizing variability from extrinsic sources. Extrinsic processes, such as cell growth and division, DNA duplication, and mRNA transport and decay, can significantly affect the apparent variability between cells and thus also bursting rates. These effects were minimized by measuring transcription at nascent sites in an endogenous system with synchronized cell divisions. Moreover, explicit quantification of measurement error resulted in a noise model that significantly constrained the inference framework. All these approaches are generally applicable to enable precise quantification in any system (Zoller, 2018).

The fundamental mean-cumulant relationships uncovered in this study demonstrate that a single-parameter distribution globally determines transcriptional activity. Employing the telegraph model, this study found that the modulation of mean occupancy (η) predicts mean mRNA synthesis rates comparable with previous measurements and reproduces the distribution of nascent activity, whereas kini and τ_n (see Terminology and Parameterization of Transcription Rates) are conserved. The global behavior observed is surprising, given that bursting is generally believed to be gene and promoter specific. Multiple factors and processes, including enhancer-promoter interactions, chromatin context, nucleosome occupancy, Pol II pausing, and transcription factor interactions, all impinge on bursting rates. It remains to be determined whether the same processes are modulated in the same manner or, conversely, whether different regulatory strategies have converged to generate identical transcriptional activity across genes (Zoller, 2018).

These observations raise the question of whether the common transcriptional bursting kinetics carry a functional advantage. In early embryos, the precise positioning of cell fates requires minimizing variability between nuclei, which is achieved by a combination of long mRNA lifetimes permitting accumulation and spatial averaging through the syncytial cytoplasm. In principle, modulating kini (Pol II initiation rate) at a constitutive promoter would generate the theoretical minimal (Poisson) transcriptional noise at all levels. The fact that neither constitutive activity (η≤0.85) nor Pol II saturation (k_elo/k_ini~215 bp >;> Pol II footprint) is ever observed suggests that some constraint prohibits this system from maintaining a continuous active state and/or it is not straightforward to alter k_ini. Instead, a constant switching correlation time suggests that this value is important in facilitating robust patterning. It is proposed that both expression timing and noise minimization jointly constrain switching rates (Zoller, 2018).

The mechanistic origins of the conserved parameters are unknown. One possibility is that protein-DNA affinities have been individually selected to confer the switching rates that were observe. However, it is unclear how transient transcription factor interactions, usually on the order of seconds, could generate bursts on the order of minutes. Another possibility is that the fast transcription factor binding kinetics are masked by the slower dynamics of common general factors involved in the transcription process. In fact, recent evidence suggests that mediator and TATA-binding protein binding, as well as the core promoter and its shape, play a key role in bursting. Alternatively, processes of potentially even slower dynamics, such as long-range enhancer-promoter interactions, chromatin modification, or Pol II pausing, may determine common bursting kinetics (Zoller, 2018).

The observed constancy of τ_n (switching correlation time; see Terminology and Parameterization of Transcription Rates) will guide further modeling and identification of the molecular mechanisms. This constancy is connected to the binomial noise level. Extensions of the two-state model must provide similar filtering of the binomial noise, which will restrict the possible class of models. For example, two particular extensions of the two-state model were tested. One possibility is a three-state model consisting of a two-step reversible activation. Alternatively, a model with an additional noise term, such as input noise stemming from input transcription factor diffusion, could explain dual modulation of switching rates observed under the two-state model. However, distinguishing these models will require live imaging (Zoller, 2018).

The common transcriptional parameters of the gap genes highlight a form of complexity reduction: despite the variety of upstream regulatory elements, all expression boundaries result from similar bursting kinetics. Whether this signature results from an underlying molecular simplicity has yet to be determined. Regardless of the mechanistic means by which these similarities are achieved, the convergence suggests the general constraints that limit the range of permitted bursting rates and/or minimize transcription variability. The unexpected conservation of the initiation rate and the correlation time might indicate a path to general rules in transcriptional regulation. It is now possible to inquire about the breadth of these generalities and whether they apply to the same gene expressed in different cell types, to the transcriptome as a whole, or even across organisms. Indeed, it appears plausible that other classes of genes share similarly constrained bursting kinetics. The methods utilized in this study are applicable in a variety of systems and permit the discovery of the molecular mechanism(s) conferring unified transcription kinetics (Zoller, 2018).

GENE STRUCTURE

Exons - three

Bases in 3' UTR - 508

PROTEIN STRUCTURE

Amino Acids - 429

Structural Domains

Knirps is a zinc finger steroid/thyroid orphan receptor-type transcription factor (Nauber, 1988).

knirps and knirps-related are closely linked in the 77E1,2 region on the left arm of the third chromosome. They encode nuclear hormone-like transcription factors containing almost identical Cys2/Cys2 DNA-binding zinc finger motifs which bind to the same target sequence (Rothe, 1994).

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3." Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).

date revised: 12 April 2022

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