BIOLOGICAL OVERVIEW

tramtrack was isolated on the basis of its binding to the promoter of fushi tarazu The action of TTK on the ftz promoter is repressive. When the two TTK binding sites on the "zebra element" of the ftz promoter are mutated, transcription is directed to very early embryos, well before the onset of normal ftz. Expression is seen as early as stage 2, with no hint of a striped pattern. However, ttk is not responsible for the striped pattern of FTZ.

tramtrack is expressed downstream of Notch in the peripheral nervous system (PNS). The PNS consists of external sense organs and internal chordotonal neurons (stretch receptors). In external sense organs, a single sensory organ precursor cell (SOP) divides to produce two progeny, one of which (IIb) gives rise to a neuron and a glial cell while the other (IIa) produces two non-neuronal support cells. Loss of numb transforms IIb (the neural precursor) into IIa (the non-neural precursor). Overexpression of numb has the opposite effect: IIa becomes IIb. A similar transformation is apparent in chordotonal neurons. Numb protein, transferred asymmetrically into SOP IIb binds to the intracellular domain of Notch, thereby inhibiting Notch signaling preventing the expression of tramtrack. This biases the Delta-Notch machinery during cell-cell communication resulting in a neural phenotype in SOP IIb (Guo, 1996).

TTK is responsible for the effects of numb, repressing neural cell fate in support cells. Numb is asymmetrically distributed to neuronal cells during cell divisions that give rise to the PNS. When numb is mutant Notch functions in neurons and these cells are transformed into support cells. When numb is overexpressed, support cells get a share of Numb protein, TTK fails to be activated, and the support cells adopt a neural fate (Guo, 1995).

The normal pattern of expression of ttk has been analyzed, as well as the effect of ttk overexpression at different steps of the tactile bristle lineage. Throughout the period of emergence of macrochaete precursors, a low level expression of ttk is observed in the nuclei of epidermal cells, but not in the nuclei of the precursor cells. After division of the mother cell, neither of the two daughter cells are labeled. Thus, ttk is expressed neither in the precursor nor, just after division, in its progeny. At a later stage, one of the two daughter cells begins to express ttk in the nucleus. A little later, the second daughter expresses ttk, and still later, the second mother cell divides, but only one of its progeny expresses ttk. ttk is never expressed in cells having a neural potential; in cells where ttk is expressed, there is a delay between division and the onset of expression. The ectopic expression of ttk before some stage of the cell cycle can block further cell division. This expression transforms neural into non-neural cells, suggesting that ttk acts as a repressor of neural fate at each step of the lineage. These results suggest that ttk is probably not involved in setting up the mechanism that creates an asymmetry between sister cells, but rather in the implementation of that choice (Raemaekers, 1997).

The developmental hierarchy of the PNS can therefore be expressed as follows: cut determines external sensory organ fate. In the absence of cut, external sensory cells are transformed into chordotonal (ch) organs. Genes like numb, prospero and ttk affect both internal/chordotonal neurons and external sensory cells. BarH1 and BarH2 however are expressed only in external sensory cell organs and are involved in the fate of the two types of external sensilla: campaniform and trichord. Therefore genes like cut determine the fate of a whole block of cells, internal and external, while other genes like numb and prospero work in both organ types to select neural fate. Still other genes, BarH1/BarH2 fine tune the process at just one site. tramtrack is even further down the hierarchy, carrying out the orders of Notch.

Tramtrack (Ttk) proteins are transcriptional repressors and inhibitors of the neuronal fate of cells such as photoreceptors. TTK RNA is alternatively spliced, giving rise to two proteins. One protein is 69 kDa and a second is 88 kDa and possesses an alternative set of zinc fingers, having a DNA binding specificity distinct from that of the first (Read, 1992b). Evidence is provided that one of the Ttk proteins, Ttk69, plays a positive and autonomous role in promoting or maintaining differentiation of photoreceptor neurons at the late stages of Drosophila eye development. Consistent with this notion, the Ttk69 protein, but not Ttk88, is expressed in all photoreceptor cells during pupal stage. Thus, Ttk69 appears to play a dual function by serving negative and positive regulatory roles at different stages of photoreceptor development (Lai, 1999).

Using the FRT/FLP recombination system, clones of ttk-lethal mutations were generated that are known to remove ttk69 function. These recessive ttk mutations caused degeneration of the corneal lens (which is secreted by the underlying cone cells and primary pigment cells) and failure of photoreceptor development. Rhabdomeres of photoreceptors are not observed in the clones, but residual cellular structures in the mutant ommatidia are still recognizable. Near the boundary of ttk- clones, no genetically mosaic ommatidia are ever observed. Although these ommatidia do not contain a full complement of photoreceptor cells, there are no genetically ttk minus photoreceptors. This observation demonstrates that the ttk function is autonomously required for photoreceptor cell development. Similar adult eye phenotypes have also been observed in seven EMS-induced ttk loss-of-function mutations. The results of these clonal analyses in the adult eye suggest that ttk also plays a positive role during eye development. The positive and autonomous function of ttk69 in photoreceptor cells is not required during larval development but rather at the late pupal stage (Lai, 1999).

It is unlikely that ttk88 is responsible for this positive function, because previous work has demonstrated that specific loss of ttk88 results in the formation of ectopic photoreceptor cells (and overexpression of ttk88 inhibits neural development). Instead, ttk69 might be responsible for the positive function. This would be unexpected because ectopic expression of ttk69 inhibits photoreceptor cell formation in the eye. Another possibility is that ttk69 and ttk88 together are responsible for the positive function. To resolve this issue, expression of Ttk69 and Ttk88 isoforms was examined in ttk- clones in third instar larval eye discs using isoform-specific antibodies. ttk- clones were marked with a cell-autonomous lacZ reporter that exhibits ubiquitous expression in all cells behind the morphogenetic furrow. Among the ttk mutations used in this clonal analysis, the ttk^rM730 hypomorphic mutation was caused by a P-element insertion about 1.2 kb upstream of the first exon, resulting in the loss of both ttk69 and ttk88 expression. Normally, both Ttk69 and Ttk88 proteins are found in all four cone cells in the larval eye disc. Another ttk mutation, ttk^1e11, carries a deletion in the translated region of ttk69 and has been considered a null allele for both ttk69 and ttk88. Indeed, no Ttk69 protein can be detected in ttk^1e11 cells in third instar larval eye discs. However, the expression of ttk88 is not dramatically affected. Complementation tests were then carried out to confirm the finding that only ttk69 function is removed in ttk^1e11 mutation. If indeed ttk^1e11 has a specific loss of ttk69, one might expect that it would complement ttk¹, because only ttk88 function has been removed in the ttk¹ mutation. It appears that ttk^1e11 does effectively complement ttk¹ mutant eye phenotype, because up to 94% of the ommatidia in ttk¹/ttk^1e11 flies are wild type. In contrast, there are only 65% of normal ommatidia in ttk¹/ttk^rM733 flies and most mutant ommatidia contain ectopic photoreceptors. ttk¹ homozygotes contain ~50% - 60% normal ommatidia. These data further confirm that there is a relatively specific loss of ttk69 in ttk^1e1 mutation, and the mutant eye phenotype observed in the adult eye clones must be mainly caused by the loss of ttk69 function. Contrary to the gain-of-function data, whereby ttk69 is an inhibitor of photoreceptor cell fate, evidence presented here reveals a positive function for ttk69 in photoreceptor cell development. Thus, ttk69 might play a dual function as both a positive and negative regulator in this process (Lai, 1999).

It is unclear at this moment how ttk69 might act to promote photoreceptor differentiation at the late stages of eye development. One possible scenario is that ttk69 could be involved in activating expression of genes required for terminal differentiation of photoreceptors. Rh genes might be one of the targets of ttk69. Supporting this hypothesis, Ttk69 protein has been found to bind specifically to the Rhodopsin upstream sequence 4A (RUS4A) element (Z.-C. Lai, M. E. Fortini and G. M. Rubin, unpublished data cited in Lai, 1999), which is essential for Rh4 gene expression in a subset of R7 photoreceptors. Interestingly, the absence of Rh gene expression leads to a similar neural degeneration phenotype as seen in the ttk69- adult eye. This would suggest a potential role for ttk69 in initiating and maintaining late differentiation events in eye development. However, ectopic expression of ttk69 in the adult retina reduces expression of the Rh4-lacZ gene. It suggests that Ttk69 is intrinsically a transcriptional repressor required for its neural inhibitory activity. Other mechanisms convert Ttk69 into a positive factor required for terminal differentiation of photoreceptors. The conversion of ttk69 from a neural inhibitor into a factor positively required for development of photoreceptor neurons poses an intriguing developmental mechanism. It appears that Ttk69 acts intrinsically as a transcriptional repressor, mediating its neural inhibitory function. Many transcriptional repressors act by competing with activators for DNA sequences. They may also interact directly with activators or the transcriptional machinery to turn off transcription. A number of transcriptional repression systems utilize corepressors to prevent transcription. As a transcriptional repressor, Ttk69 may utilize such mechanisms to inhibit inappropriate photoreceptor cell fate in early eye development. Interestingly, Ttk69 is expressed in photoreceptor cells at later stages, where Ttk69 activity might be changed through protein modification, availability of cofactors, or changes in the context of the target gene promoter. Consequently, Ttk69 becomes a positive regulator critical for photoreceptor development (Lai, 1999 and references).

Tramtrack regulates different morphogenetic events during Drosophila tracheal development

Tramtrack (Ttk) is a widely expressed transcription factor, the function of which has been analysed in different adult and embryonic tissues in Drosophila. So far, the described roles of Ttk have been mainly related to cell fate specification, cell proliferation and cell cycle regulation. Using the tracheal system of Drosophila as a morphogenetic model, a detailed analysis of Ttk function was undertaken. Ttk is autonomously and non-autonomously required during embryonic tracheal formation. Remarkably, besides a role in the specification of different tracheal cell identities, it was found that Ttk is directly involved and required for different cellular responses and morphogenetic events. In particular, Ttk appears to be a new positive regulator of tracheal cell intercalation. Analysis of this process in ttk mutants has unveiled cell shape changes as a key requirement for intercalation and has identified Ttk as a novel regulator of its progression. Moreover, Ttk was defined as the first identified regulator of intracellular lumen formation and; it is autonomously involved in the control of tracheal tube size by regulating septate junction activity and cuticle formation. In summary, the involvement of Ttk in different steps of tube morphogenesis identifies it as a key player in tracheal development (Araújo, 2007).

As with the transcription factors Trh and Vvl, which are involved in orchestrating early events of tracheal development, Ttk plays a role in orchestrating several late tracheal events. Ttk69 has been found to act mostly as a repressor. This study identified Ttk targets that appear to be negatively regulated (such as mummy (mmy), encodes a UDP-N-acetylglucosamine pyrophosphorylase enzyme required for the synthesis of the building blocks of chitin, and escargot (esg) whereas others appear to be positively regulated (such as polychaetoid (pyd) and branchless (bnl). In this latter case, Ttk might be converted into a positive regulator, as already described during photoreceptor development (Araújo, 2007).

This study identified multiple tracheal requirements for Ttk. Interestingly, most of them depend on Ttk regulating events downstream of cell fate specification, at the level of cellular responses. Additionally, a few other requirements depend on cell fate specification, as has been described for most other functions of Ttk in other developmental situations. For instance, Ttk regulates fusion cell specification by acting as a target and mediator of Notch, as occurs during sensory organ development and oogenesis. Such regulation of Ttk by N might be post-transcriptional, as occurs during sensory organ development. Remarkably, it was found that, although Ttk is sufficient to repress esg expression in fusion cells, it might not be the only esg- and fusion fate-repressor, because absence of Ttk does not increase the number of Esg-positive cells, as does downregulating N. Other N targets might be redundant with Ttk, and such redundancy could reinforce N-mediated repression of fusion fate in positions in which inductive signals (such as Bnl, Dpp and Wg) are very high, particularly near the branch tips (Araújo, 2007).

Cell rearrangements during development are common to most animals and ensure proper morphogenesis. During tracheal development, many branches grow and extend by cell intercalation. Several cellular and genetic aspects of tracheal intercalation have been well described. However, targets of Sal (which inhibits intercalation) are currently unknown (Araújo, 2007).

This study identified Ttk as a new and positive regulator of intercalation. Ttk is involved in cell junction modulation by transcriptionally regulating pyd, the only junctional protein shown, so far, to affect intercalation. In fact, modulation of AJs has been proposed to play a role during intercalation. However, Pyd cannot be the only Ttk effector of intercalation, because the pyd mutant phenotype is much weaker than that of ttk mutants. Accordingly, it was found that, in ttk mutants, cells in branches that usually intercalate remain paired and cuboidal, and appear unable to change shape and elongate. Although other explanations could account for the impaired intercalation detected in ttk mutants, it is proposed that inefficient cell shape changes represent the main cause, and might prevent the proper accomplishment of several events, such as the sliding of cells, formation of a first autocellular contact and zipping up, thereby blocking intercalation. Hence, it is proposed that cell shape changes, particularly cell elongation, are an obligate requisite for different steps of intercalation. Other targets of Ttk might presumably be regulators or components of the cytoskeleton involved in cell shape changes. It is relevant to point out here that Ttk has also been proposed to regulate morphogenetic changes required for dorsal appendage elongation (Araújo, 2007).

How does Ttk relate to the known genetic circuit (Sal-dependent) involved in intercalation? Being a transcription factor, Ttk initially appeared as an excellent candidate to participate in this genetic network by regulating sal and/or kni expression. However, both these genes to be normally expressed in ttk mutants, and several differences were detected in the intercalation phenotype of ttk loss versus sal upregulation. For instance, although both situations block intercalation, cells expressing sal, unlike those lacking ttk, are still able to undergo a certain change in shape, from cuboidal to elongated. Therefore, the results fit a model in which Ttk acts in a different and parallel pathway to Sal during intercalation. Consistent with this model, it was found that Ttk is not sufficient to promote intercalation on its own, because its overexpression cannot overcome the inhibition of intercalation imposed by Sal in the DT. Finally, genetic interactions also favour this model, because it was found that: (1) ttk overexpression did not rescue lack of intercalation produced by sal overexpression (even though it rescued the intercalation defects of ttk mutants), and (2) absence of sal (by means of the constitutive activation of the Dpp pathway) does not overcome the intercalation defects of ttk mutants. Therefore, it is proposed that Ttk promotes intercalation by endorsing changes in cell shape, but absence of Sal is still required to allow other aspects of intercalation to occur (Araújo, 2007).

Tube size regulation is essential for functionality. It was found that Ttk is involved in such regulation. Tube expansion and extension relies on a luminal chitin filament that assembles transiently in the tracheal tubes. The metabolic pathway that leads to chitin synthesis involves several enzymes, among which are Mmy and krotzkopf verkehrt (Kkv, a Chitin synthase). In addition, other proteins are known to participate in the proper assembly and/or modification of the chitin filament, such as Knk, Rtv, Verm and Serp. SJs are also required to regulate tube size and it was proposed that they exert this activity, at least partly, via the control of the apical secretion of chitin modifiers. The current results revealed that ttk acts as a key gene in tube size control, playing at least two roles: it regulates chitin filament synthesis and septate junction (SJ) activity (Araújo, 2007).

SJ regulation by Ttk appears functional rather than structural: mild defects were detected in the accumulation of only some SJ markers and there was a loss of the transepithelial diffusion barrier, whereas accumulation of other markers and SJ localisation remained apparently unaffected. It is speculated that Ttk transcriptionally controls one or several SJ components that contribute to maintain the paracellular barrier and to control a specialised apical secretory pathway. As a result, chitin binding proteins such as Verm or Serp are not properly secreted (Araújo, 2007).

It was also found that mmy is transcriptionally regulated by Ttk. mmy tracheal expression positively depends on a mid-embryonic peak of the insect hormone 20-hydroxyecdysone. Therefore, it is proposed that Ttk and ecdysone exert opposing effects on chitin synthesis. Excess of mmy mRNA results in the abnormal deposition of the chitin filament, as occurs in ttk mutants. Defects in chitin deposition might lead to the irregular organisation of taenidia and the faint larval cuticle observed in ttk mutants. Strikingly, Ttk is also required for normal chorion production, which represents another specialised secreted layer (Araújo, 2007).

ttk mutants are defective in the formation of terminal and fusion branches. These defects are due, in part, to non-autonomous, secondary and/or pleiotropic effects of ttk. For instance, ttk mutants exhibit a dorsal closure defect, which prevents the approach and fusion of contralateral dorsal branches. Additionally, terminal and fusion branches depend on correct cell type specification, which did not reliably occur in ttk mutants. For instance, DSRF (Blistered) was missing in some presumptive terminal cells of ttk mutants, impairing terminal branch formation. These tracheal cell identity specification defects might be related to non-autonomous requirements of ttk. For instance, DSRF is not properly expressed in ttk mutants because of an abnormal expression of its regulator, Bnl (Araújo, 2007).

It is important to note that, in spite of these non-autonomous and cell fate specification defects, two pieces of evidence indicate that ttk also plays a specific and autonomous role in the formation of terminal and fusion tubes. First, markers for fusion and terminal cell specification were expressed in many tracheal cells of ttk mutants, but yet most of these cells did not form terminal or fusion branches. Second, only the tracheal expression of ttk in ttk mutants (but not the constitutive activation of the btl pathway, which regulates the terminal and fusion identity) was able to restore the formation of terminal branches (Araújo, 2007).

A common feature of terminal and fusion branches is that they both display intracellular lumina that lack detectable junctions. The cellular events that precede the formation of fusion and terminal branches differ, but the mechanisms by which their intracellular lumina form has been proposed to be comparable. It was found that, in ttk mutants, terminal and fusion cells engage in the correct cellular changes before intracellular lumen formation. However, neither of these two cell types finalised the cellular events leading to tube formation. It has been proposed that the lumen of terminal and fusion branches forms by the coalescence of intracellular vesicles that use a 'finger' tip provided by the neighbouring stalk cell as a nucleation point. Interestingly, it was found that vesicles containing luminal material are less abundant in ttk mutants. These observations suggest a new role for Ttk in the formation of intracellular lumina in distinct cell types. Intracellular lumen formation also occurs in other branched tubular structures, such as in vertebrate endothelial cells and in the excretory cell of Caenorhabditis elegans, presumably by the coalescence of vesicles. Importantly, a crucial role for vesicle formation and their fusion during intracellular tube formation has been demonstrated (Araújo, 2007).

ttk is the first gene described to be involved in intracellular lumen formation during tracheal development. Possible targets of Ttk might be genes related to the apical surface and the underlying cytoskeleton, because several of these genes are involved in C. elegans excretory canal formation. Additionally, genes involved in intracellular vesicle trafficking might also be good candidates. In this respect, several abnormalities have been detected in ttk mutants that might reflect defects in vesicle trafficking (Araújo, 2007).

A switch in tissue stem cell identity causes neuroendocrine tumors in Drosophila gut

Intestinal stem cells (ISCs) are able to generate gut-specific enterocytes, as well as neural-like enteroendocrine cells. It is unclear how the tissue identity of the ISC lineage is regulated to confer cell-lineage fidelity. This study shows that, in adult Drosophila midgut, loss of the transcriptional repressor Tramtrack in ISCs causes a self-renewal program switch to neural stem cell (NSC)-like, and that switch drives neuroendocrine tumor development. In Tramtrack-depleted ISCs, the ectopically expressed Deadpan acts as a major self-renewal factor for cell propagation, and Sequoia acts as a differentiation factor for the neuroendocrine phenotype. In addition, the expression of Sequoia renders NSC-specific self-renewal genes responsive to Notch in ISCs, thus inverting the differentiation-promoting function of Notch into a self-renewal role as in normal NSCs. These results suggest an active maintenance mechanism for the gut identity of ISCs, whose disruption may lead to an improper acquisition of NSC-like traits and tumorigenesis (Li, 2020).

In addition to the nervous system, neuroendocrine (NE) cells are found in many non-neural tissues and can develop neoplasias that are known as NE tumors (NETs). The NE cells in non-neural tissues display characteristics that are typical of neurons, such as membrane excitability and hormone secretion, yet many of these NEs are generated from adult stem cells of endodermal origin. It is, thus, intriguing how the tissue identity of stem cells is regulated and controlled to safeguard cell-lineage fidelity (Li, 2020).

The intestinal epithelium in adult Drosophila midgut is maintained by intestinal stem cells (ISCs)-the multipotent cells that are capable of generating both absorptive enterocytes (ECs) and secretary enteroendocrine cells (EEs). EEs are neural-like cells and are able to secrete sets of hormone peptides that are similar to those secreted by NE cells in the Drosophila brain. While the initiation of EC generation is driven by Delta (Dl)/Notch-mediated lateral inhibition between the two immediate stem cell daughters, the initiation of EE generation occurs at the stem cell level, with a transient expression of the proneural gene Scute (Sc) that induces ISCs to self-renew and to generate an EE progenitor cell (EEP). Each EEP then divides one more time before terminal differentiation to yield a pair of EEs. Sc encodes a basic helix-loop-helix (bHLH) transcription factor and belongs to the achaete-scute gene complex (AS-C), a Drosophila proneural gene cluster that is expressed in neural progenitor cells and is important for the development of the embryonic central nervous system and sensory organs of both larva and adult. Thus, it appears that there is a transient activation of neural-like programs in ISCs that directs EE generation from ISCs (Li, 2020).

Tramtrack (Ttk, or Ttk69 isoform), which encodes a BTB-domain-containing transcriptional repressor, acts as a master repressor of the differentiation of EEs from ISCs. Depletion of ttk in ISCs causes derepression of AS-C genes including Sc and Asense (Ase). The continuous expression of Sc and Ase directs ISCs to continuously generate EEs, leading to the formation of EE-like tumors, or NETs. One intriguing observation from the ttk-depleted ISCs is that continuous derepression of the differentiation-promoting factors does not compromise ISC maintenance, but continuous overexpression of Sc in normal ISCs will cause regional ISC loss over time. This study characterized the ttk-depleted ISCs and, surprisingly, found that the original self-renewal program of ISCs had switched to a neuroblast-like self-renewal program that is responsible for NET tumorigenesis (Li, 2020).

The results reveal an ISC-to-NB switch in the tissue stem cell self-renewal program that drives NET development from ttk- depleted ISCs. Loss of ttk causes the derepression of NB-specific transcription factors, including dpn and seq, and the concomitant loss of ISC-specific factors. The ectopically expressed Dpn acts as a major self-renewal factor for the self-duplication of tumor cells, while Seq has a 'selector' function in selecting Notch target genes by recruiting Su(H) to the enhancer regions of NB-specific genes, thereby rendering these genes responsive to Notch in the tumor cells. In addition, Seq also has a role in NE differentiation by inducing AS-C gene expression. The cooperative function of Dpn and Seq leads to the activation of a NB-like self-renewal program as well as a NE differentiation program and the continuous activation of these two programs leading to NET development from ISCs (Li, 2020).

There are two types of NBs in the central brain of Drosophila larva: type I and type II. Dpn is specifically expressed in the type II but not the type I NBs. Interestingly, Notch appears to be more important in type II NBs than in type I NBs. Thus, it appears that the ectopically activated self-renewal program in ISCs described in this study is more similar to that used in the type II NBs. Compared to the type I NB lineage, the type II NB lineage goes through one extra type of transient amplification progenitors before terminal cell fate specification, indicating that this type II-like self-renewal program, if hijacked by tumor cells, could potentially be more potent to initiate tumorigenesis (Li, 2020).

As the loss of a single factor, Ttk, in ISCs is sufficient for the switch of tissue stem cell program and for the subsequent development of NETs in the midgut, Ttk could be viewed as a specific class of stem cell factors, which is proposed in this study as a tissue identity maintenance (TIM) factor; such factors function to safeguard the tissue identity of stem cells. ISCs not only give rise to ECs that function to digest and absorb nutrients but also give rise to neural-like EE cells. Conceivably, ISCs may need to use a basal or transient neural-like program in order to endow their capacity to generate EEs, as the proneural factor Sc is transiently expressed in ISCs, and this transient expression initiates EE generation. In this context, a stem cell identity factor like Ttk may be necessary to enable ISCs to maintain a gut identity and thereby prevent excessive acquisition of NSC-like traits (Li, 2020).

The Ttk protein is characterized by having a BTB domain in addition to a zinc-finger DNA-binding domain, and although there is no direct protein ortholog of Ttk in mammals, BTB-domain-containing transcription factors are found in all eukaryotes, including mammals. Moreover, alteration of Notch activity as well as increased expression of proneural genes are also known to occur in mammalian models of NETs and in human NETs. Thus, it is possible that there are TIM factors that function in stem cells in other tissues and organisms and that 'stem cell identity switch' could be a common mechanism underlying NET formation and, possibly, other stem-cell-mediated tumorigenesis (Li, 2020).

GENE STRUCTURE

Base pairs in 5' UTR - 257

Base pairs in 3' UTR - 897

PROTEIN STRUCTURE

TTK RNA is alternatively spliced, giving rise to two forms. One is a protein of 69 kDa that binds the fushi tarazu promoter. A second is a protein of 88 kDa with an alternative set of zinc fingers, having a DNA binding specificity distinct from that of the first protein (Read, 1992b). The N-terminal 288 amino acid region is common in the two proteins. While the first of the two zinc fingers in each protein do not bear substantial identity, the second fingers are greater than 50% identical (Harrison, 1990).

Amino Acids - The 69 kDa form has 641aa and the 88 kDa form has 811aa.

Structural Domains and BTB domain proteins

The zinc-fingers of both the 88 kDa and the 69 kd Tramtrack protein are C2H2 type. There are in addition a BTB domain and a Pest domain, confering rapid protein turnover. The two proteins share an N-terminal POZ domain, also known as a BTB domain; this is, a conserved protein-interaction motif (Harrison, 1990).

A novel zinc finger protein, ZID (standing for zinc finger protein with interaction domain) was isolated from humans. ZID has four zinc finger domains and a BTB domain, also know ans a POZ (standing for poxvirus and zinc finger) domain. At its amino terminus, ZID contains the conserved POZ or BTB motif present in a large family of proteins that include otherwise unrelated zinc fingers, such as Drosophila Abrupt, Bric-a-brac, Broad complex, Fruitless, Longitudinals lacking, Pipsqueak, Tramtrack, and Trithorax-like (GAGA). The POZ domains of ZID, TTK and TRL act to inhibit the interaction of their associated finger regions with DNA. This inhibitory effect is not dependent on interactions with other proteins and does not appear dependent on specific interactions between the POZ domain and the zinc finger region. The POZ domain acts as a specific protein-protein interaction domain: The POZ domains of ZID and TTK can interact with themselves but not with each other, or POZ domains from ZF5, or the viral protein SalF17R. However, the POZ domain of TRL can interact efficiently with the POZ domain of TTK. In transfection experiments, the ZID POZ domain inhibits DNA binding in NIH-3T3 cells and appears to localize the protein to discrete regions of the nucleus (Bardwell, 1994).

The BTB/POZ domain defines a conserved region of about 120 residues; it has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RARalpha (retinoic acid receptor alpha) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. The BTB/POZ domain from PLZF (PLZF-BTB/POZ) has been overexpressed, purified, characterized, and crystallized. Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a Kd below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet. Crystals of the PLZF-BTB/POZ have been prepared that are suitable for a high resolution structure determination using x-ray crystallography. The data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/POZ domain function (X. Li, 1997).

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein-protein interaction motif found at the N terminus of 5%-10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism for linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 A resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and it is expected that the full-length PLZF protein exists as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins (Ahmad, 1998).

Each 30-residue zinc finger motif folds to form an independent domain with a single zinc ion tetrahedrally coordinated beween an irregular, antiparallel, two stranded ß-sheet and a short alpha-helix. Each zinc finger of mouse Zif268 (which has three fingers) binds to DNA with the amino terminus of its helix angled down into the major groove. An important contact between the first of the two histidine zinc ligands and the phosphate backbone of the DNA contributes to fixing the orientation of the recognition helix. Although the two fingers of Drosophila Tramtrack interact with DNA in a way very similar to those of Zif268, there are important differences. Tramtrack has an additional amino-terminal ß-strand in the first of the three zinc fingers. The charge-relay zinc-histidine-phosphate contact of Zif268 is substituted by a tyrosine-phosphate contact. In addition, for TTK, the DNA is somewhat distorted with two 20 degree bends. This distortion is correlated with changes from the rather simple periodic pattern of amino base contacts seen in Zif268 and finger 2 of TTK (Klug, 1995 and references).

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-a-brac tramtrack broad complex/pox viruses and zinc fingers). The LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation (Dhordain, 1997).

The bcl-6 proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center (GC) B and T cells and required for GC formation and antibody affinity maturation. Deregulation of bcl-6 expression by chromosomal rearrangements and point mutations of the bcl-6 promoter region are implicated in the pathogenesis of B-cell lymphoma. The signals regulating bcl-6 expression are not known. Antigen receptor activation leads to BCL-6 phosphorylation by mitogen-activated protein kinase (MAPK). Phosphorylation, in turn, targets BCL-6 for rapid degradation by the ubiquitin/proteasome pathway. These findings indicate that BCL-6 expression is directly controlled by the antigen receptor via MAPK activation (Niu, 1998).

MAPK is a ubiquitous, evolutionarily conserved signal transducer that is activated by heterogeneous signals that originate from the cell membrane and are transduced to MAPK via RAS proteins. Accordingly, POZ/zinc finger proteins represent a large family of highly conserved transcription factors, including Drosophila cell fate regulators such as Tramtrack and Broad-complex, as well as human cancer-associated proteins such as BCL-6 and PLZF. These molecules have strong structural (POZ and ZF domains), as well as functional homologies since they are transcriptional repressors that control cell differentiation. Most notably, POZ/zinc finger proteins also carry possible MAPK phosphorylation sites and PEST sequences in approximately the same position as those carried by BCL-6. In Drosophila, degradation of TTK88, a POZ/zinc finger inhibitor of neural-cell differentiation, has been shown to be mediated by MAPK. Thus, degradation of POZ/zinc finger transcription factors may represent a general mechanism by which the RAS/MAPK pathway controls cell function and differentiation (Niu, 1998 and references).

Virtually all diffuse large cell lymphomas and a significant fraction of follicular lymphomas contain translocations and/or point mutations in the 5' non-coding region of the putative oncogene BCL-6, that are presumed to deregulate the expression of BCL-6. BCL-6 encodes a Cys2-His2 zinc finger transcriptional repressor with a POZ domain at its amino-terminus. The POZ (or BTB) domain, a 120-amino-acid motif, mediates homomeric and, in some proteins, heteromeric POZ-POZ interactions. In addition, the POZ domain is required for transcriptional repression of several proteins, including BCL-6. Using a yeast two-hybrid screen, N-CoR and SMRT have been identified as BCL-6 interacting proteins. Both N-CoR and SMRT, which were originally identified as co-repressors for the unliganded nuclear thyroid hormone and retinoic acid receptors, are components of large complexes containing histone deacetylases. The interaction between BCL-6 and these co-repressors is also detected in the more physiologically relevant mammalian two-hybrid assay. The POZ domain is necessary and sufficient for interaction with these co-repressors. BCL-6 and N-CoR co-localize to punctate regions of the nucleus. Furthermore, when BCL-6 is bound to its consensus recognition sequence in vivo, it can interact with N-CoR and SMRT. In vitro POZ domains from a variety of other POZ domain-containing proteins (including the transcriptional repressor PLZF, as well as ZID, GAGA and a vaccinia virus protein, SalF17R) also interact with varying affinities with N-CoR and SMRT. BCL-6 POZ domain mutations that disrupt the interaction with N-CoR and SMRT no longer repress transcription. In addition, these mutations no longer self associate, suggesting that self interaction is required for interaction with the co-repressors and for repression. More recently N-CoR has also been implicated in transcriptional repression by the Mad/Mxi proteins. The demonstration that N-CoR and SMRT interact with the POZ domain containing proteins indicates that these co-repressors are likely involved in the mediation of repression by multiple classes of repressors and may explain, in part, how POZ domain containing repressors mediate transcriptional repression (Huynh, 1998).

The J element is a novel DNA sequence involved in the regulated expression of class II major histocompatibility complex genes. DPA, a J element binding protein, contains 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The H/C motif is a stretch of seven amino acids connecting the final histindine of one finger to the first cysteine of the next finger. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box). This N-terminal region shares sequence homology with Drosophila proteins Tramtrack, Broad Complex and Kelch, a structural component of ring canals. It has been suggested that the TAB is a protein-protein interaction domain (Sugarawa, 1994).

Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. It is proposed that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes (Geyer, 2003).

These results identified BTB/POZ proteins as components of Pcu3p/Pip1p ubiquitin ligase complexes. Four pieces of evidence suggest that BTB/POZ domain proteins are functionally equivalent to the SKP1/F box adaptor dimers determining the substrate specificity of CUL1-associate SCF complexes: (1) all three BTB/POZ proteins present in the fission yeast genome interact with Pcu3p/Pip1p complexes; (2) BTB/POZ domains are structurally related to SKP1; (3) N-terminal residues invariably conserved in all CUL3 homologs, including Pcu3p, cluster in the same region of CUL1 that mediates its interaction with SKP1/F box adaptor dimers. Both the Btb3p/Pcu3p interaction and Pcu3p-dependent Btb3p degradation depend on the integrity of this conserved N-terminal region. (4) Btb3p is ubiquitylated in vitro in a Pcu3p-dependent manner, a finding reminiscent of CUL1-dependent ubiquitylation and degradation of F box proteins. Taken together, these findings strongly suggest that the BTB/POZ domain proteins ubiquitously present in eukaryotes define a family of substrate-specific adaptors for CUL3. Since fission yeast encodes three different BTB/POZ domain proteins, all of which interact with Pcu3p and Pip1p, it may form a minimum of three distinct RING/cullin 3/BTB complexes (Geyer, 2003).

date revised: 10 December 2002

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