Gene name - prospero
Cytological map position - 86E1
Function - transcription factor
Symbol - pros
Genetic map position - 3-
Classification - homeodomain
Cellular location - nuclear and cytoplasmic
|Recent literature||Takashima, S., Aghajanian, P., Younossi-Hartenstein, A. and Hartenstein, V. (2016). Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells. Dev Biol [Epub ahead of print]. PubMed ID: 27321560
Proliferating intestinal stem cells (ISCs) generate all cell types of the Drosophila midgut, including enterocytes, endocrine cells, and gland cells (e.g., copper cells), throughout the lifetime of the animal. Among the signaling mechanisms controlling the balance between ISC self-renewal and the production of different cell types, Notch (N) plays a pivotal role. This study investigates the emergence of ISCs during metamorphosis and the role of N in this process. Precursors of the Drosophila adult intestinal stem cells (pISCs) can be first detected within the pupal midgut during the first hours after onset of metamorphosis as motile mesenchymal cells. pISCs perform 2-3 rounds of parasynchronous divisions. The first mitosis yields only an increase in pISC number. During the following rounds of mitosis, dividing pISCs give rise to more pISCs, as well as the endocrine cells that populate the midgut of the eclosing fly. Enterocytes do not appear among the pISC progeny until around the time of eclosion. The "proendocrine" gene prospero (pros), expressed from mid-pupal stages onward in pISCs, is responsible to advance the endocrine fate in these cells; following removal of pros, pISCs continue to proliferate, but endocrine cells do not form. Conversely, the onset of N activity that occurs around the stage when pros comes on restricts pros expression among pISCs. Loss of N abrogates proliferation and switches on an endocrine fate among all pISCs. These results suggest that a switch depending on the activity of N and pros acts at the level of the pISC to decide between continued proliferation and endocrine differentiation.
|Li, Y., Pang, Z., Huang, H., Wang, C., Cai, T.
and Xi, R. (2017). Transcription
factor antagonism controls enteroendocrine cell specification from intestinal stem cells. Sci Rep 7: 988. PubMed ID: 28428611
The balanced maintenance and differentiation of local stem cells is required for homeostatic renewal of tissues. In the Drosophila midgut, the transcription factor Escargot (Esg) maintains undifferentiated states in intestinal stem cells, whereas the transcription factors Scute (Sc) and Prospero (Pros) promote enteroendocrine cell specification. However, the mechanism through which Esg and Sc/Pros coordinately regulate stem cell differentiation is unknown. By combining chromatin immunoprecipitation analysis with genetic studies, this study shows that both Esg and Sc bind to a common promoter region of pros. Moreover, antagonistic activity between Esg and Sc controls the expression status of Pros in stem cells, thereby, specifying whether stem cells remain undifferentiated or commit to enteroendocrine cell differentiation. These data therefore reveal transcription factor antagonism between Esg and Sc as a novel mechanism that underlies fate specification from intestinal stem cells in Drosophila.
|Tripathi, B. K., Das, R., Mukherjee, A. and Mutsuddi, M. (2017). Interaction of Spoonbill with Prospero in Drosophila: Implications in neuroblast development. Genesis 55(9). PubMed ID: 28722203
Identification of Spoon as a suppressor of SCA8 associated neurodegeneration provides a hint about its role in neuronal development and maintenance. However, a detailed molecular characterization of spoon has not yet been reported. This study describes spatial expression pattern of Spoon during Drosophila development. Quantitative real time-PCR and fluorescent RNA-RNA in situ hybridization indicate that Spoon is expressed at relatively high levels in larval brain and photoreceptors of eye-antennal discs. Immunostaining reveals that Spoon is subcellularly localized in the cytoplasm and is also membrane bound. Strong expression is also seen in adult ovary and testes. Spoon immunostaining exhibits unique pattern of expression in larval brain. Spoon in the neuroblasts colocalizes with Prospero, a transcription factor regulating genes involved in neuroblast self-renewal or cell-cycle control. Co-immunoprecipitation suggests that Spoon and Prospero reside in the same protein complex. Using Drosophila model of SCA8 RNA neuropathy this study has also shown that loss of Prospero hinders the suppression of SCA8 associated neurodegeneration by Spoonbill, suggesting Prospero and Spoon might genetically interact and function together. This study presents Spoon as a novel interacting partner of Prospero and this might be critical in determining the polarized localization of cell fate determinants.
|Shaw, R. E., Kottler, B., Ludlow, Z. N., Buhl, E., Kim, D., Morais da Silva, S., Miedzik, A., Coum, A., Hodge, J. J., Hirth, F. and Sousa-Nunes, R. (2018). In vivo expansion of functionally integrated GABAergic interneurons by targeted increase in neural progenitors. EMBO J. Pubmed ID: 29728368
A central hypothesis for brain evolution is that it might occur via expansion of progenitor cells and subsequent lineage-dependent formation of neural circuits. This study reports in vivo amplification and functional integration of lineage-specific circuitry in Drosophila Levels of the cell fate determinant Prospero were attenuated in specific brain lineages within a range that expanded not only progenitors but also neuronal progeny, without tumor formation. Resulting supernumerary neural stem cells underwent normal functional transitions, progressed through the temporal patterning cascade, and generated progeny with molecular signatures matching source lineages. Fully differentiated supernumerary gamma-amino butyric acid (GABA)-ergic interneurons formed functional connections in the central complex of the adult brain, as revealed by in vivo calcium imaging and open-field behavioral analysis. These results show that quantitative control of a single transcription factor is sufficient to tune neuron numbers and clonal circuitry, and provide molecular insight into a likely mechanism of brain evolution.
|Wu, D., Wu, L., An, H., Bao, H., Guo, P., Zhang, B., Zheng, H., Zhang, F., Ge, W., Cai, Y., Xi, Y. and Yang, X. (2018). RanGAP-mediated nucleocytoplasmic transport of Prospero regulates neural stem cell lifespan in Drosophila larval central brain. Aging Cell: e12854. PubMed ID: 30549175
By the end of neurogenesis in Drosophila pupal brain neuroblasts (NBs), nuclear Prospero (Pros) triggers cell cycle exit and terminates NB lifespan. This study reveals that in larval brain NBs, an intrinsic mechanism facilitates import and export of Pros across the nuclear envelope via a Ran-mediated nucleocytoplasmic transport system. In rangap mutants, the export of Pros from the nucleus to cytoplasm is impaired and the nucleocytoplasmic transport of Pros becomes one-way traffic, causing an early accumulation of Pros in the nuclei of the larval central brain NBs. This nuclear Pros retention initiates NB cell cycle exit and leads to a premature decrease of total NB numbers. These data indicate that RanGAP plays a crucial role in this intrinsic mechanism that controls NB lifespan during neurogenesis. This study may provide insights into understanding the lifespan of neural stem cells during neurogenesis in other organisms.
How do two cells, the progeny from a single cell division, develop different fates? This is the fundamental question of developmental biology. prospero and numb provide clues to one possible mechanism.
When Prospero and Numb proteins are first made, they congregate in the cell's cytoplasm, but unlike many proteins they are not distributed uniformly throughout the cell. Rather, they form a cresent pattern at one end of the cell, just under the cell membrane, between the membrane and the cell's centrioles (Hirota, 1995, Spana, 1995a and 1995b, and Knoblich, 1995). The cell is said to be polarized; that is, one end is somehow different from the other end.
Is the centriole the immediate cause of Prospero and Numb localization? One might consider that the two centrioles are not identical, and that one directs Prospero and Numb localization? But there are other elements involved in cell polarity, and their involvement must be considered. The cytoskeleton and the cell membrane are also involved. Perhaps the cell membrane acts as a tether for Prospero and Numb. In fact, the region directly under the cell membrane, the cortex is known to be highly structured with subcortical cytoskeletal elements.
Action of Inscuteable, a cytoskeleton associated protein, suggests an involvement of cytoskeleton in providing cues for Prospero and Numb localization. Inscuteable localizes opposite the pole to which Prospero and Numb localize. In inscuteable mutants both Prospero and Numb form cresents, but the position of both the spindle and the position of Prospero and Numb is disrupted, that is, Prospero and Numb do not localize preferentially localize. Cytochalasin D, which disrupts Actin filaments, eliminates Inscuteable cresents and results in incorrect Prospero crescent positioning, while treatment with colcemid, which destroys microtubules (and consequently the spindle) does not effect Inscuteable localization (Kraut, 1996).
In yeast the site of the previous bud determines the cell polarity. Does the same thing happen in the fly? It seems not. Instead Inscuteable localizes to the apical cortex of ectodermal cells and neuroblasts (the apical direction is towards the surface and is opposite to the basal pole), irrespective of the orientation of their previous division, suggesting that the localization responds to the apical-basal polarity.
It appears that there must be an organizer to provide positional information for both the orientation of the mitotic spindle and asymmetric localization of Numb and Prospero (Knoblich, 1995). The dynamics of Inscuteable suggests that the microfilamental network, organized with respect to apical-basal polarity, acts to organize both the Numb and Prospero by acting through Inscuteable. Likewise Inscuteable acts to organize the microtubule network and thus the plane of cell division. For more information an apical-basal polarity, see the Crumbs site.
Relevant to this discussion is a structure found oogenesis called the fusome. Fusomes consist of cytoskeletal proteins, alpha-Spectrin, ß-Spectrin, Hu-li tai shao (an adducin-like protein) and Ankyrin. Of particular interest is the association of fusomes with the pole of the mitotic spindle (Lin, 1995). During the first cystoblast (cystoblasts are derived from germ line stem cells) division, fusome material is associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing fusome always associates with the pole of each mitotic spindle that remains in the mother cell, and only extends through the newly formed ring canals after each division is completed. The association of fusome proteins with the mitotic spindle indicates a direct interaction between cytoskeletal components and only one pole of the mitotic spindle. Surely this must have something to do with the underlying mechanism of asymmetric cell division.
As a cell divides, Prospero and Numb are asymmetrically distributed to the two progeny. The cell receiving the two proteins has a different fate from that of its sister. In the cell receiving both Prospero and Numb ( the ganglion mother cell), Prospero migrates from its cytoplasmic position into the nucleus, where they assumes the role of transcription factor, thus establishing the neural fate of the cell in which it is found. Numb function is to antagonize Notch signaling, and has been shown to directly interact with the cytoplasmic domain of Notch (Guo, 1996). The second progeny, the one that does not receive Prospero or Numb, is in effect, a new neuroblast. This cell will continue to divide, generating new ganglion mother cells and new neuroblasts (Knoblich, 1995, Rhyu, 1995 and Spana, 1995) A special sequence in Numb and Prospero protein is responsible for localization of the protein in the cortex, immediately adjacent to the cell membrane (Hirata, 1995). With what protein does Numb and Prospero interact? An answer to this question will still not shed light on how apical-basal polarity is established, but it will help in understanding how Prospero and Numb become asymmetrically distributed.
A similar asymmetrical system of Prospero distribution operates in other cells expressing prospero, such as the precursors of glia (Spana, 1995).
Specification of cell fate in the adult sensory organs is known to be dependent on intrinsic and extrinsic signals. Prospero (Pros) acts as an intrinsic signal for the specification of cell fates within the mechanosensory lineage. The sensory organ precursors divide to give rise to two secondary progenitors: PIIa and PIIb. Pros is expressed in PIIb, which gives rise to the neuron and thecogen cells. Pros expression was first detected among the secondary progenitors in the nucleus of the more anteriorly located cell (PIIb). Interestingly, this was the first cell to divide in all cases examined. Prior to cell division, Pros becomes uniformly distributed on the cortical membrane and throughout the cytosol. Unlike the findings in the central nervous system, Pros is not asymmetrically localized in cells at any stage of the lineage. Asymmetric crescents of Pros immunoreactivity are not observed even after blocking mitosis with colcemid. These data suggest that Pros is expressed first in the nucleus and then generally in the cytosol of PIIb (Reddy, 1999).
Loss of Notch function generated using either a conditional mutant allele or by misexpressing Numb protein results in the ectopic expression of Pros in PIIa. This observation is consistent with a PIIa-to-PIIb conversion by Notch loss of function or nb gain of function. It is not clear whether the apparent negative regulation of Pros by Notch is a direct effect or merely reflects the altered fate of the cells that is caused by other molecular factors. Pros misexpression is sufficient for the transformation of PIIa to PIIb fate (Reddy, 1999).
Pros is not asymmetrically localized in PIIb; following division of this cell, the protein is detected in both the progeny. This is strikingly different from findings in the CNS where Pros, together with Numb and Miranda, is localized asymmetrically in the neuroblasts and inherited by the GMC. Following division of PIIb, Pros is inherited by nuclei of both progeny. Expression in one of the siblings decays rapidly and this cell differentiates as the neuron. Pros immunoreactivity is sustained in the thecogen cell possibly due to de novo synthesis. The requirement for pros function in PIIb precludes analysis of its later role after division. In experiments where Pros was misexpressed in all four cells of the sensory organ, a conversion of external to internal cells is observed, consistent with a PIIa-to-PIIb transformation. Neuronal cells form normally despite the fact that they express the thecogen cell marker. Similarly, Notch loss of function and Numb gain of function results in a conversion of all four cells of the lineage to neurons. Pros is expressed in all these cells under these conditions. These observations together demonstrate that pros activity is not sufficient for identity of the thecogen cell and that neuronal cell differentiation can occur normally despite Pros expression. The elucidation of pros function in the thecogen cell awaits the availability of a hypomorphic mutant allele that can allow loss of function in the thecogen cell without affecting the secondary progenitors. Pros has been shown to be expressed in several CNS- as well as PNS-associated glial cells and it is possible that it plays a role in the later differentiation and/or function of these cells (Reddy, 1999).
In the normal mechanosensory lineage, Notch is involved in the binary choice between thecogen and neuron. In this lineage Notch signaling is experienced by the cell that ultimately becomes the thecogen cell. This is distinct from the scenario at the secondary progenitor stage, where the cell that experiences Notch signaling does not express Pros. This means that, unlike in PIIb, Notch signaling does not downregulate Pros in the thecogen cell. There are several possible explanations for this finding. One possibility is that Pros protein is merely partitioned to the daughters of the PIIb after division. It therefore serves no role in the binary choice of thecogen versus neuron but is synthesized de novo after these cells have acquired their identity. At this later time point, the thecogen cell is no longer experiencing the Notch signal. Another possibility lies in the different effector mechanisms utilized for Notch activity. In three instances (during lateral signaling, PIIa-PIIb choice, as well as in the PIIa lineage) Notch activation results in release of Su(H) from its binding site on the cytoplasmic domain of Notch and its translocation to the nucleus. Su(H) protein can be detected in the nucleus of the socket cell; however, the role of Su(H) cannot be seen in the PIIb lineage. Extra copies of Su(H) do not produce thecogen-to-neuron transformations, suggesting that Notch signaling in the thecogen/neuron choice occurs by a Su(H)-independent mechanism. Thus the regulation of Pros expression could be mediated by Notch through a Su(H)-independent event (Reddy, 1999 and references).
Regulated transcription of the prospero gene in the Drosophila eye provides a model for how gene expression is specifically controlled by signals from receptor tyrosine kinases. prospero is controlled by signals from the Egfr receptor and the Sevenless receptor. A direct link is established between Egfr activation of a transcription enhancer in prospero and binding of two transcription factors that are targets of Egfr signaling. Binding of the cell-specific Lozenge protein is also required for activation, and overlapping Lozenge protein distribution and Egfr signaling establishes expression in a subset of equivalent cells competent to respond to Sevenless. Sevenless activates prospero independent of the enhancer and involves targeted degradation of Tramtrack, a transcription repressor (Xu, 2000).
Thus, Egfr signaling is required to activate pros expression in the R7 equivalence group but is restricted from activating pros expression in other cells by the distribution of the transcription factor Lz. The transcriptional effectors of the Egfr pathway combinatorially interact with Lz at an eye-specific pros enhancer to restrict enhancer activity to the R7 equivalence group. It is suggested that this mechanism is a primary means by which pros transcription is restricted to the R7 equivalence group. This combinatorial mechanism supposes that Egfr signaling inactivates Yan and activates Pnt, but modification of these transcription factors is not sufficient to activate the enhancer. Lz is also required to activate the enhancer. The only cells that contain Lz, activated Pnt, and inactivated Yan are R1, R6, R7, and cone cells. Thus, the enhancer is activated in a subset of Egfr-responsive cells. A similar combinatorial mechanism regulates shaven expression in cone cells. shaven expression requires both Lz and Egfr-induced regulation of Yan and Pnt. However, Notch signaling through Su(H) is also required for shaven expression in cone cells. This third input may explain why shaven has a more restricted expression pattern than pros, given that cone cells receive a robust Notch signal (Flores, 2000). In muscle and cardiac cells, RTK signaling is similarly integrated with other signal inputs and tissue-restricted transcription factors to regulate enhancer activity of the even skipped gene (Halfon, 2000). Thus, differential expression of genes in response to an RTK/Ras signal appears to be controlled by each gene's capacity to bind and be regulated by different combinations of transcription factors (Xu, 2000).
A model is presented for the regulatory inputs into prospero. (1) In eye progenitor cells, the presence of Yan represses pros transcription through its binding to the enhancer and competitively excluding Pnt from binding to the same sites. (2) Lz begins to be produced in progenitor cells after the first wave of photoreceptor differentiation. However, Lz alone cannot activate the enhancer in progenitor cells that have not received a Spitz signal. (3) When a progenitor cell receives a Spitz signal, Egfr is activated. This inactivates Yan, allowing activated Pnt to bind to the enhancer. At the morphogenetic furrow, the enhancer is inactive despite Egfr-stimulated cells containing inactive Yan and active Pnt since progenitor cells in this region do not contain Lz, which is also required for enhancer activity. Hence, photoreceptors R2, R3, R4, R5, and R8 do not express pros. It is only in cells that receive a Spitz signal and contain Lz that the combination of Lz and Pnt bound to the enhancer activate the enhancer. (4) Ttk88 reduces the level of pros transcription through a mechanism independent of the eye enhancer. This repression may not be strong enough to block the eye enhancer in the R7 equivalence group but acts to limit its level of transcription. (5) When a progenitor cell receives both a Spitz and Boss signal, stronger or longer signal transduction induces Ttk88 inactivation. This Egf represses pros transcription and leads to a specific increase of Pros in R7 cells (Xu, 2000).
The ETS factors Yan and Pnt have been implicated as substrates for activated MAPK, whose activities are modified upon phosphorylation. Both Yan and Pnt bind to the same sites in the pros eye enhancer except for one site that is Yan-specific. Their effects on enhancer activity are antagonistic; Yan represses while Pnt activates. One model is that Yan represses transcription by outcompeting Pnt for their binding sites, thereby preventing Pnt from activating transcription. This model is attractive since it has been found that Yan has a 100-fold greater affinity than Pnt for ETS factor binding sites in vitro. If this difference between purified fusion proteins in vitro is extrapolated to the fly eye, it would explain how Yan can outcompete Pnt and repress transcription. Results from mutagenesis of the binding sites is also consistent with this model. Mutated binding sites cause the enhancer to be inactive, which is the result predicted if Yan merely prevents Pnt from interacting with those sites. If Yan were actively repressing transcription in a manner dependent upon binding, then mutated binding sites would cause derepression and ectopic expression. Although a model where the binding sites are obligatory for both active repression by Yan and activation by Pnt cannot be excluded, the competitive binding model is the simplest one consistent with these data (Xu, 2000).
From these data it is proposed that two RTKs, Egfr and Sev, regulate pros by activating the Ras1 intracellular pathway in R7 cells, but these RTKs regulate pros differentially. Egfr regulates pros by modifying Yan and Pnt, which act directly through the eye-specific enhancer. The Egfr signal in R7 cells appears to occur before Sev, and it sufficiently inactivates Yan and activates Pnt to switch on the enhancer before the Sev signal. This sufficiency is demonstrated in sev mutants where enhancer activity in R7 cells is no different from wild-type. In contrast, the Sev signal in R7 cells is not sufficient to switch on the enhancer in the absence of the Egfr signal since the enhancer is inactive in Egfr mutant R7 cells (Xu, 2000).
Sev regulates pros in R7 cells by inactivating Ttk88, which otherwise represses pros through sequence elements distinct from the eye-specific enhancer. This is demonstrated by finding that overproduced Ttk88 blocks Sev from activating pros, and Sev can regulate the eye-specific enhancer only if it is linked with Ttk88 binding sites. It is not clear if the Sev signal is sufficient to inactivate Ttk88 without an Egfr input since the assay for Ttk88 activity is a reporter gene that includes the eye-specific enhancer. It is quite possible that Ttk88 inactivation in R7 cells requires both Sev and Egfr signals, since Ebi, acting downstream of Egfr to promote Ttk88 degradation, and Phyl/Sina, acting downstream of Sev, are both required to inactivate Ttk88 in R7 cells (Xu, 2000).
How do these RTKs selectively regulate particular transcription factors and thereby regulate different aspects of pros transcription? The most attractive model is that RTK selection reflects the timing or intensity of each signal. If it is timing, then there must be a time period of competence during which a factor is sensitive to any RTK signal, and the time period is different for each factor. Alternatively, the intensity of a signal may dictate which transcription factor activities are sensitive. For example, Yan and Pnt activities may be insensitive to signal strength that is less than or equal to the level achieved by Sev but not Egfr within R7 cells. Ttk88 activity may be insensitive to signal strength that is less than or equal to the level achieved by Sev or Egfr alone but not the combination of the two within R7 cells. Signal 'strength' may be determined by the level of Ras pathway activity or the length of time that the Ras pathway is active. Sensitivity of transcription factors might be set either by the affinities of these factors for binding sites in a gene such as pros, or by the ability of factors to be substrates for RTK-stimulated modification. Given that Yan and Pnt are modified by a very different mechanism from Ttk88, substrate sensitivity is a possible determinant. In summary, RTK signals may provide specificity to gene regulation based on quantitative variation in which threshold transcription responses are set by transcription factors that have different sensitivities to RTK signal strength (Xu, 2000).
Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).
To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).
Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).
Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).
Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).
A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).
The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).
Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).
In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).
The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.
The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).
Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).
To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).
To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).
In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).
Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).
The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).
The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).
Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).
S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).
To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).
Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).
Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).
Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).
Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).
Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).
The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).
It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).
Bases in 5' UTR - 1089
Exons - two
Bases in 3' UTR - 1097
The Prospero transcript is alternatively spliced to encode two proteins: ProsL protein (1403 amino acids, predicted 165 kDa) and ProsS protein (1374 amino acids, predicted 160 kDa). The extra 29 amino acids in ProsL are at the beginning of the homeodomain (Chu-LaGraff, 1991).
Prospero has a novel homeodomain and three PEST domains which confer susceptability to rapid degradation. There is also a CAX (opa) repeat, a common transcription activation domain (Doe, 1991 and Vaessin, 1991).
Prospero promotes neural differentiation in Drosophila, and its activity is tightly regulated by modulating its subcellular localization. Prospero is exported from the nucleus of neural precursors but imported into the nucleus of daughter cells, which is necessary for their proper differentiation. Prospero has a highly divergent putative homeodomain adjacent to a conserved Prospero domain; both are required for sequence-specific DNA binding. The structure of these two regions consists of a single structural unit (a homeo-prospero domain), in which the Prospero domain region is in position to contribute to DNA binding and also to mask a defined nuclear export signal that is within the putative homeodomain region. It is proposed that the homeo-prospero domain coordinately regulates Prospero nuclear localization and DNA binding specificity (Ryter, 2002).
The C-terminal 163 amino acids of Drosophila Prospero (residues 1241-1403) were overexpressed, purified, and crystallized. The X-ray structure was determined at 2.05 Å resolution. Despite classification as highly divergent based on sequence comparisons, residues 1241-1303 were predicted to be capable of assuming a canonical homeodomain structure. In fact, structural analysis shows that this region does assume an overall fold very similar to homeodomains but with one striking difference. In every homeodomain structure determined to date, the so-called DNA recognition helix is either at the extreme C terminus of the protein or leads into a flexible linker of variable length that connects to another essentially independent domain. In Prospero, the recognition helix (alpha3) connects the putative homeodomain and the Prospero domain as a single structural unit. The region corresponding to the Prospero domain can be described as a four-helix bundle (alpha3–alpha6). Residues 1314-1326 are disordered and residues 1368-1370 and 1391-394 form short 310 helices. While interactions between the homeo- and Prospero domain regions occur primarily within the hydrophobic core, the C-terminal residues of the Prospero domain region make additional contacts with the homeodomain region by extending into a cleft between helices alpha1 and alpha2 (Ryter, 2002).
A structural comparison of the homeodomain region (HD) alone reveals that, while possessing a highly divergent class sequence, the structure assumes a fold most similar to the Drosophila Engrailed homeodomain. A detailed residue-by-residue analysis was possible. A majority of the core residues seen in the homeodomain region are either invariant or conserved hydrophobic with respect to the Engrailed homeodomain sequence or homeodomain consensus sequence, indicating that this region possesses a core consistent with a standard homeodomain hydrophobic core. Variations occur primarily at the loop between alpha1 and alpha2 and in the middle of alpha3. In these regions, residues that would be on the surface of a typical homeodomain are replaced by bulky hydrophobic groups that interact with the hydrophobic core of the Prospero domain. Within the loop, Phe1261 and Trp1262, along with the invariant Phe1260, create a very hydrophobic turn that protrudes into this core region. In the middle of alpha3, Phe1298, Tyr1299, Tyr1300, and Met1303 contribute to the hydrophobic core of the Prospero domain four-helix bundle (Ryter, 2002).
The Prospero domain region (PD) is composed of a four-helix bundle (alpha3-alpha6). Consecutive helices are antiparallel and all helices interact via ridges in grooves. The up-down-up-down topology is the simplest found among four-helix bundles and in this case is right turning. The central residues of this bundle, representing the alpha-carbons in closest proximity, are Ala1307 of alpha3 and Phe1361 of alpha5. The ~50° crossing angle between the helix axes is also the most common angle found in globular proteins. The core consists of hydrophobic residues throughout that are either invariant or highly conserved throughout the Prospero/Prox1 protein family. As noted above, a number of residues within the homeodomain region also contribute to this hydrophobic core (Ryter, 2002).
The homeodomain and the Prospero domain regions do not just touch each other; rather, they interact quite extensively. If the two regions are considered as separate entities, then 22% (1499 Å2) of the surface area of the homeodomain region contributes to the interface with the Prospero domain region and, vice versa, 35% of the surface of the Prospero domain region contributes to the same interface. These fractions are significantly higher than the average value of 15% that is observed for the surface area involved in the interfaces of typical protein dimers. It confirms that the homeodomain and Prospero domain regions combine to form a distinct structural unit (Ryter, 2002).
There are extensive hydrophobic core contacts that occur between the HD and PD regions. A hydrophobic cleft on the HD is formed between Trp1262 and Tyr1274 of the strand just C-terminal to alpha1 and alpha2, respectively. SeMet1259 forms the floor of this cleft, with Val1263 and Val1270 flanking one end. Seated in the cleft are Val1389, Pro1390, and Phe1393 of the PD region. This cleft is flanked at the opposite end by a hydrogen-bonding interaction between His1252 and Asp1277. Phe1275, of the strand C-terminal to alpha2, positions Lys1255 of alpha1 in an orientation optimal for a number of hydrogen-bonding interactions. These include direct interactions with backbone carbonyls of residues 1274 and 1393, thereby creating contacts between HD alpha1 and alpha2, as well as the PD region (Ryter, 2002).
There are interesting similarities (see 1mijA at Structural Neighbours in PDB90 and structural alignments) between the homeo-prospero domain (HPDP structure) and that of the MATa1/MATalpha2 homeodomain heterodimer bound to DNA (Li, 1998). In the HPD structure, the C terminus of the PD region (residues 1388-1396) extends into a cleft between alpha1 (residues 1250-1259) and alpha2 (residues 1268-1274) of the HD region. This rather lengthy segment includes a 310 helix (residues 1391-1394) and contacts the HD region on the face opposite its DNA binding surface. This interaction between HD and PD is stabilized by hydrophobic interactions, as well as a number of hydrogen bonds. In the homeodomain heterodimer complex between MATa1 and MATalpha2, the C terminus of MATalpha2 also extends away from the body of the domain and similarly makes hydrophobic and hydrogen bond interactions within a groove formed between alpha1 and alpha2 of MATa1. This binding groove on the homeodomain MATa1 corresponds to that within the HD region of Prospero. In addition, the polypeptide chain that binds within the respective grooves in both cases adopts an alpha-helical or 310-helical conformation (Ryter, 2002).
The MATa1/MATalpha2 homeodomain complex is required for cell type-specific transcriptional repression in yeast. The Prospero HPD is also required for regulating transcription, but may have an additional function in controlling the subcellular localization of Prospero. Demidenko (2001) utilized molecular dissection of the HPD with expression of chimeric proteins in mammalian and insect cultured cells to show that residues 1248–1261, encompassing alpha1, contain a nuclear export signal (NES). In the absence of any portion of the PD region, Prospero is exported from the nucleus via the Exportin pathway and subsequently found in the cytoplasm, while presence of this region blocks nuclear export and allows Prospero to accumulate in the nucleus. The HPD structure strongly suggests that it is the extreme C terminus of the PD region that sterically prevents access to the nuclear export signal (Ryter, 2002).
Interestingly, previous analysis of Prospero during asymmetric cell division has shown its phosphorylation state correlates with subcellular localization. Cytoplasmic Prospero is highly phosphorylated compared to nuclear Prospero, which raises the possibility that phosphorylated HPD may have a more open conformation in which the NES is exposed (Ryter, 2002).
In a structural comparison, the structure of the HD region was shown to be most similar to the Engrailed homeodomain. It is therefore possible to use the structural superposition matrix to model the HPD structure in complex with DNA. Three regions of potential protein-DNA contact are suggested. (1) The DNA recognition helix alpha3 appears to contact the major groove of the DNA; (2) the N-terminal arm appears to contact the minor groove of the DNA, and (3) unexpectedly, the PD region appears to contact the DNA backbone via residues within or close to the N terminus of helix alpha6. Engrailed homeodomain residues Asn51 (Pros HD Asn1294) and Arg53 (Pros HD Arg1296) are invariant and presumably contact the DNA in a similar fashion. In the Engrailed structure, Ile47 (Pros HD Lys1290) and Lys50 (Pros HD Ser1293) also contact the bases in the major groove to provide binding site discrimination. Pros HD Glu1297 appears poised in the major groove available for possible water-mediated contacts. Additionally, there are a number of residues that are not only invariant between the Engrailed homeodomain and the Prospero HD region, but are also positioned to make similar contacts with the DNA phosphate backbone as seen in the Engrailed homeodomain structure. In particular, Engrailed homeodomain Arg53 (Pros HD Arg1296) and Tyr25 (Pros HD Tyr1265) are predicted to interact directly with one strand of the DNA backbone in a similar fashion, while Trp48 (HD Trp1291) could interact with the opposite strand (Ryter, 2002).
In the Engrailed homeodomain structure, the N-terminal arm (residues 5-9) fits into the DNA minor groove, supplementing the contacts made by the recognition helix. In the Prospero HPD-DNA model, the N-terminal arm has a somewhat different alignment but could easily move so as to contact the DNA. A sequence comparison reveals predominantly conservative differences between the Engrailed and the Prospero sequences in the N-terminal arm, except at Engrailed homeodomain Arg5 (Pros HD Ser1245), a position shown to be important for binding site discrimination (Ryter, 2002).
This structural comparison also suggests that the Prospero HD region may contact DNA as well. Lys1380, -1376, and -1375, found within or close to the N terminus of the alpha6 helix, are all poised to potentially interact with the DNA phosphate backbone. The basic nature of this region is further manifest in the negative electrostatic potential of the molecular surface. Tyr1379 and Ser1373 could possibly interact with the DNA backbone either directly or via hydrogen bond interactions. All these residues are conserved to a high degree in the Prospero/Prox class homeobox proteins. Of course, final confirmation of the HPD-DNA interactions discussed above must await the structure determination of the HPD structure in complex with its DNA binding site (Ryter, 2002).
Therefore, instead of forming an essentially independent unit, the Prospero domain is shown to join together with the homeodomain to form a larger structural unit that has been named the 'homeo-prospero domain'. Model building suggests that this larger structural unit serves in part to align the Prospero domain region on the DNA target. Also, the Prospero domain region is positioned in such a way that it is able to mask a defined nuclear export signal that is within the homeodomain region (Ryter, 2002).
Subcellular localization of the transcription factor Prospero is dynamic. For example, the protein is cytoplasmic in neuroblasts, nuclear in sheath cells, and degraded in newly formed neurons. The carboxy terminus of Prospero, including the homeodomain and Prospero domain, plays roles in regulating these changes. The homeodomain has two distinct subdomains, which exclude proteins from the nucleus, while the intact homeo/Prospero domain masks this effect. One subdomain is an Exportin-dependent nuclear export signal requiring three conserved hydrophobic residues, which models onto helix 1. Another, including helices 2 and 3, requires proteasome activity to degrade nuclear protein. Finally, the Prospero domain is missing in pros(I13) embryos, thus unmasking nuclear exclusion, resulting in constitutively cytoplasmic protein. Multiple processes direct Prospero regulation of cell fate in embryonic nervous system development (Bi, 2003).
date revised: 10 November 2000
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.