InteractiveFly: GeneBrief

split ends : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

split ends (spen) encodes nuclear 600 kDa proteins that contain RNA recognition motifs and a conserved C-terminal sequence. These features define a new protein family, Spen, which includes the vertebrate MINT transcriptional regulator. Zygotic spen mutants affect the growth and guidance of a subset of axons in the Drosophila embryo. Removing maternal and zygotic protein elicits cell-fate and more general axon-guidance defects that are not seen in zygotic mutants. The wrong number of chordotonal neurons and midline cells are generated, and defects have been identified in precursor formation and EGF receptor-dependent inductive processes required for cell-fate specification. The number of neuronal precursors is variable in embryos that lack Spen. The levels of Suppressor of Hairless, a key transcriptional effector of Notch required for precursor formation, are reduced, as are the nuclear levels of Yan, a transcriptional repressor that regulates cell fate and proliferation downstream of the EGF receptor. Thus, defects in Notch and EGFR signaling are likely to be at least partially responsible for the cell-fate defects. spen is therefore the first nuclear link between these two signaling pathways (Kuang, 2000).

spen is required for normal migration and survival of midline glial cells (MGCs); embryos lacking spen have CNS defects strikingly reminiscent of those seen in mutants of several known components of the Egfr signaling pathway. In addition, spen interacts synergistically with the RTK effector pointed. Using MGC-targeted expression, it was found that increased Ras signaling rescues the lethality associated with expression of a dominant-negative spen transgene. Therefore, spen encodes a positively acting component of the Egfr/Ras signaling pathway (Chen, 2000). It is proposed that Spen proteins regulate the expression of key effectors of signaling pathways required to specify neuronal cell fate and morphology (Wiellette, 1999; Chen, 2000; Kuang, 2000). spen also has a homeotic mutant phenotype, and plays two important segment identity roles. One is to promote sclerite development in the head region, in parallel with Hox genes; the other is to cooperate with Antennapedia and teashirt to suppress head-like sclerite development in the thorax (Wiellette, 1999).

Worms, flies and vertebrates contain distinct genes that encode either large (>300 kDa) or smaller (<95 kDa) Spen-like proteins. These proteins contain RNA recognition motifs (RRMs) and a conserved C-terminal sequence, the SPOC domain, but little other homology. RRMs were identified originally in RNA binding proteins involved in mRNA splicing, stability and translation, and more recently as DNA binding motifs in several transcription factors. These transcription factors include MINT, which binds via the RRMs to GT-rich DNA sequences (Newberry, 1999) and contains the C-terminal SPOC domain. The MINT protein is proteolytically processed in vivo into 110 kDa N-terminal and 250 kDa C-terminal fragments (Newberry, 1999). The MINT RRM-containing N-terminal domain both suppresses FGF activation of the rat osteocalcin promoter and activates the HSV thymidine kinase promoter (Newberry, 1999). Thus, depending on the promoter, the RRM-containing fragment can act as a repressor or an activator of transcription. These data and structural similarities between the MINT and Spen RRM domains suggest that spen may function as a transcription factor, rather than as an RNA binding protein. Spen could be required for Su(H) and yan transcription, or affect the levels of Su(H) and Yan proteins by another mechanism. Intriguingly, the C-terminal fragment of MINT binds to the Msx2 homeobox protein in vitro, though not via the SPOC domain, and may coregulate the expression of genes required for craniofacial development (Newberry, 1999). Thus, the C-terminal domain of spen is likely to be important for interactions with transcription factors and key signaling pathways regulating cell fate (Kuang, 2000).

To investigate the developmental role of maternally contributed Spen, germ line clones of spen alleles were generated in virgin females by standard methods and the females crossed to heterozygous spen mutant males. Females containing germ line clones of spen were crossed to heterozygous spen mutant males. Since these females can only lay eggs that lack Spen in the germ line, half of the resulting embryos lack both maternally and zygotically contributed Spen. Most experiments were performed on embryos derived from spen³ germline clone X heterozygous spen⁵ or spen^poc361 germline clone X heterozygous spen³ crosses, since these alleles produce no detectable full-length Spen protein. Embryos that lack both maternal (M) and zygotic (Z) Spen protein were identified by the absence of Spen staining (Kuang, 2000).

Such embryos are referred to as MZspen embryos; embryos that lack only zygotic spen expression are referred to as Zspen mutant embryos and still express Spen protein until late stage 12. The molecular basis of the spen^poc231 and spen^poc361 EMS-generated alleles has not been determined, but spen^poc231 produces detectable, though not functional, protein and spen^poc361 does not. The severity of mutant phenotypes was comparable among protein null alleles, and was only slightly weaker in embryos derived from spen^poc231 germline clones (Kuang, 2000).

Midline development is defective in MZspen embryos. The Single minded (Sim) transcription factor is expressed by midline glial cells and their precursors. MZspen embryos have approximately twice as many Sim-positive cells. Embryos that lack maternal and zygotic Su(H) are neurogenic, and are missing some midline cells, but MZspen embryos are not neurogenic, and produce extra midline cells. Refinement of proneural clusters still occurs in the absence of Spen, but occurs less reproducibly. These differences between spen and Su(H) suggest that Spen affects another pathway required for neuronal and midline cell fate. EGFR/Ras signaling is required for chordotonal (ch) neuron and midline development; ectopic activation of ras1 produces extra midline cells, and spen enhances mutations in the Ras pathway (Rebay, 2000). Therefore, whether cells can send and receive EGF signals in the absence of Spen was investigated. The cells at the midline normally express Rhomboid, a membrane protein that potentiates EGF signaling. Consequently, the EGFR, Ras and the MAPK cascade are activated in midline cells and cells immediately flanking the midline, as assayed with an antibody that detects activated MAPK. Rhomboid expression at the midline and MAPK cascade activation in adjacent cells appear normal in the absence of Spen. In the absence of Spen, Rhomboid expression in the ch proneural cluster, and MAPK signaling in the cluster and neighboring cells also appears normal. Thus, spen does not appear to affect early steps in EGFR signaling (Kuang, 2000).

Nonetheless, defects in MAPK interactions with nuclear targets such as the Yan transcriptional repressor could contribute to the cell-fate phenotypes. spen mutants enhance activated yan (Rebay, 2000), whose protein product is resistant to MAPK inactivation (Rebay, 1995). Therefore, Yan protein levels and localization were examined in MZspen embryos. In stage-10 wild-type embryos, Yan protein is detected in most nuclei in the developing CNS, but not in those of midline cells or in cells immediately adjacent to the midline. Levels of Yan appear to be relatively uniform outside this domain, and the boundary between cells that express Yan and the midline region appears relatively sharp. However, in MZspen embryos, Yan is either absent or present at reduced levels in many cells that would normally express Yan. Abnormal reductions in Yan levels are also observed surrounding ch proneural clusters. These results suggest that spen is required to maintain or establish the nuclear levels of Yan in many cells in the embryo. The data suggest that spen functions downstream of the MAPK cascade or in parallel to regulate Yan, rather than upstream (Kuang, 2000).

spen is required for normal migration and survival of midline glial cells. spen germline clones were generated using the ovo^D-FLP/FRT system. Embryos lacking both maternal and zygotic spen will be referred to as 'spen mutants'. These mutants exhibit moderate defects in CNS morphology, as visualized by anti-Elav antibody, a pan-neuronal marker. In stage 16 spen mutant embryos, the space separating the two longitudinal halves of the CNS is reduced compared with the wild type and, in some segments, the two sides are completely collapsed across the midline. Because of this collapse, the midline neurons are difficult to detect; however, labeling with the antibody 22C10 reveals that the ventral midline neurons are present and have no obvious defects in their projections. Because such collapsed CNS phenotypes might be indicative of defects in the midline, spen mutant embryos were examined for expression of the MGC-specific marker Slit. Initial determination of the MGCs appears normal in spen mutants. The first detectable defects occur at late stage 12/early stage 13 when the MGCs normally initiate their migration. In the wild type, the glial cells migrate in a tightly packed configuration along the dorsal surface of the ventral nerve cord, whereas in spen mutants, the glia migrate aberrantly and become spread out in a more diffuse pattern. The results of these analyses differ from a recent report that excessive numbers of MGCs are initially specified in spen mutants. Using the Slit-lacZ nuclear enhancer trap marker to count the MGCs, comparable numbers of MGCs in the wild type and in spen mutants were detected up until stage 13, and a reduction in MGC number in spen mutants beginning at stage 14. Thus, in these spen mutants, which appear to be genetic and protein nulls, normal numbers of MGCs are initially specified, a phenotype consistent with what has been reported for other Egfr pathway mutants (Chen, 2000).

By stage 16, in a wild-type embryo, the Slit-positive MGCs have migrated and elongated to ensheathe the anterior commissure (AC) and posterior commissure (PC) axons, thereby maintaining proper separation and bundling. In similarly staged spen mutant embryos, the MGCs had not properly migrated or wrapped themselves around the commissure bundles. In addition, while apoptosis reduces the number of MGCs in wild type embryos from ~8 per segment at stage 13 down to only ~3 per segment by stage 16-17, in spen mutants this reduction is even more drastic, leaving only 1-2 MGCs per segment. Thus, in spen mutants, although initiation of MGC differentiation appears normal, the later aspects of glial development, including migration, wrapping, and survival or maintenance of the MGC fate, are defective. To confirm that spen is expressed in the MGCs at stage 13 when its function is required, embryos carrying the MGC-specific enhancer trap line AA142, were double labeled with anti-beta-galactosidase and anti-Spen antibodies. The highest level of spen expression is seen in the MGCs (Chen, 2000).

Because defects in glial cell development are likely to perturb organization of the CNS, spen mutant embryos were labeled with the antibody BP102, which highlights all axon tracts in the CNS. As predicted, the AC and PC axon bundles are not properly organized or separated and, in some segments, are completely fused. In addition, the two longitudinal connectives appear closer together than normal and are occasionally fully collapsed across the midline. Staining with the anti-Fasciclin II (FasII) antibody, which highlights a distinct set of three axon bundles in each longitudinal branch, further clarifies this phenotype. These longitudinal axon tracts never cross the midline in a wild-type embryo. In contrast, the FasII-positive axons cross and recross the midline in spen mutants, producing a fragmented and disorganized longitudinal axonal array (Chen, 2000).

In Drosophila, the genes rhomboid, Star, pointed and spitz, all positively acting components of the Egfr pathway, share a characteristic CNS phenotype similar to that of spen mutants. Specifically, whereas the proper number of MGCs are initially specified, they later migrate abnormally and eventually degenerate and die. The phenotypic similarities between spen and the Egfr pathway genes, as well as the isolation of spen as an enhancer of an activated yan allele, are consistent with the hypothesis that spen may be a positively acting factor in the Egfr/Ras signaling pathway. To explore this possibility, it was investigated whether spen and the RTK pathway effector pointed interact synergistically in the midline. The expectation was that a reduction in activity of a proven positive effector of the Egfr pathway, such as pnt, should dominantly enhance the spen phenotype. Embryos lacking maternal spen can be partially rescued by zygotic spen expression from a paternally inherited wild-type allele (this genotype is referred to as spen/+). Stage 15-17 spen/+ embryos appear phenotypically wild type, with only ~4% of the embryos exhibiting CNS defects. Embryos heterozygous for a pnt loss-of-function mutation (pnt/+) have no apparent dominant defects. Reducing the pnt dosage in the spen/+ background increases the frequency of axonal defects to ~25%. The predominant phenotype is reduced separation between the two longitudinal axon pathways and a single inappropriate crossing of the midline by one of the FasII-positive axon tracts. This dose-sensitive interaction between pnt and spen strongly supports a role for spen as either a positively acting component of the Egfr pathway or as a component of a parallel pathway synergizing with Egfr during MGC development (Chen, 2000).

To investigate further the connection between spen and Egfr/Ras signaling in the MGCs, a putative dominant-negative spen transgene was generated that truncates the carboxy-terminal ~1500 amino acids, including the highly conserved SPOC domain. When transfected into S2 cultured cells, this construct (SpenDeltaC) is expressed at high levels and localizes to the nucleus just as is found for the endogenous wild-type spen protein. Ubiquitous expression of SpenDeltaC is unable to rescue the lethality or phenotypes associated with spen mutants, implying an essential function for the conserved carboxy-terminal SPOC domain. To determine whether SpenDeltaC might behave as a dominant-negative mutation, the Slit-Gal4 driver was used to induce high levels of expression specifically in the MGCs. MGC-specific expression of SpenDeltaC results in completely penetrant lethality. In contrast, and consistent with the lack of primary neuronal defects associated with spen mutants, pan-neural expression of SpenDeltaC using the Elav-Gal4 driver does not compromise the viability or patterning of the fly. To test the hypothesis that the Slit-Gal4/SpenDeltaC lethality might be due to compromised RTK/Ras pathway signaling, a determination was made of whether increasing the level of Egfr/Ras pathway signaling, specifically in the MGCs, could compensate for the reduction in spen function associated with expression of the dominant-negative SpenDeltaC transgene. Whereas Slit-Gal4-driven expression of either an activated Ras^V12 or the SpenDeltaC transgene results in lethality, flies expressing both Ras^V12 and SpenDeltaC in the MGCs are viable and appear normally patterned. The mutual suppression is extremely penetrant, since over 50% of the expected class of flies was recovered. Similar, but less penetrant, rescue was obtained when SpenDeltaC and a secreted form of the Egfr ligand Spitz were coexpressed in the MGCs. Together, these results strongly suggest that spen functions autonomously in the MGCs, acting either downstream of or in parallel to Ras as a positive effector or regulator of RTK signaling (Chen, 2000).

Although the molecular mechanisms underlying spen function in the RTK/Ras pathway remain to be elucidated, given its membership of the RRM family, one possibility is that spen might directly regulate the processing and/or stability of specific transcripts to generate functionally distinct protein isoforms in response to, or required for, Ras signaling events. Post-transcriptional regulation of gene expression allows quick responses to external or developmental signals, and RRM family members have been shown to mediate many different cellular processes, including mRNA splicing, stabilization, localization and transport. Two attractive potential targets of such activity in the CNS are the Ras pathway effector pointed and the zinc finger transcription factor tramtrack. Both genes produce alternatively spliced transcripts and are required in the MGCs. The synergistic interactions detected between spen and pointed make pointed a particularly appealing candidate. A third possibility, given that spen was isolated as an enhancer of an activated yan allele, is that spen might function to destabilize yan transcripts in response to RTK-initiated signals. In this model, spen would contribute a second level of post-translational regulation that would reinforce the transient mitogen-activated protein (MAP) kinase signal that downregulates Yan protein, thereby stabilizing release from the Yan-mediated block to differentiation. In all these scenarios, spen could either function in parallel to the Ras/MAP kinase cascade, or could itself be directly regulated or activated by the pathway (Chen, 2000).

Taken together, it is suggested that spen affects cell-fate specification by disrupting at least two key signaling pathways: Notch and EGFR. (1) spen is required for normal Su(H) expression throughout the embryo during neurogenesis. As a result, Notch-mediated lateral inhibition is defective and abnormal numbers of neuronal precursors are specified in embryos that lack Spen. These precursors likely retain their correct identity; ch precursors still express Atonal and form ch neurons. (2) Cells that lack spen often do not express normal nuclear levels of Yan, which antagonizes EGFR signaling to control cell proliferation and fate. Hence, some cells that lack spen may respond aberrantly to an EGF signal. Though spen is ubiquitous, other cells achieve apparently normal levels of Yan in the absence of Spen, suggesting that not all Yan expression requires Spen (Kuang, 2000).

Defects in Su(H) and Yan protein expression occur in embryos that lack spen and can themselves cause the number of neurons and midline cells to vary. Whether combination of these two effects completely explains the observed variability in cell numbers remains to be tested; loss of either Su(H) or Yan causes modest reductions in midline glia, rather than the increase observed in embryos that lack Spen. However, reductions in Notch levels can partially suppress hypomorphic Yan phenotypes during eye development. In the embryo, reducing Notch activity may lead to forming extra lch precursors; lowering Yan levels may affect the recruitment of other cells to become neurons, or the ability of precursors to become neurons. Reduced Su(H) expression in embryos that lack spen may therefore partially counter the effect of lowering Yan (Kuang, 2000).

Notch and Ras/MAPK signaling are also required for the development of other tissues. The dependence of Su(H) and Yan expression on spen is not restricted to the nervous system, and may contribute to defects in muscle and tracheal development observed in MZspen embryos (B. Kuang and P. Kolodziej, unpublished data, cited in Kuang, 2000). The muscle and trachea phenotypes may parallel those observed in the nervous system, in that many muscle- and trachea-specific markers are normally expressed in the absence of Spen, but the number of cells and their morphology in these tissues is likely altered. Defects in head and cuticle development observed in MZspen embryos may also reflect these defects in signaling pathways. Notch and spen mutants are both enhancers of Deformed mutants (Florence, 1998), and yan and spen head development defects overlap. Further study of spen's role in the development of these other tissues will clarify whether these phenotypes result from defective Notch and Ras/MAPK signaling (Kuang, 2000).

The data do not exclude other possible explanations for the cell-fate defects such as effects on cell division or fate transformation within a lineage. spen mutants enhance the rough eye phenotype caused by the ectopic expression of dE2F and its heterodimeric partner dDP and suppress the rough eye phenotype caused by p21 CIP1 (Staehling-Hampton, 1999). dE2F/dDP ectopic expression causes extra cell divisions and p21 CIP1 has the opposite effects. Effects on Yan levels or activity probably do not explain the genetic interactions between spen and these cell-cycle regulators because the dE2F/dDP phenotype is not sensitive to yan gene dosage (Staehling-Hampton, 1999). Thus, spen may act on other molecules that regulate the decision to proliferate or differentiate. These possibilities notwithstanding, the expression of many highly specific markers for cell fate appears relatively normal in MZspen embryos, once defects in Su(H) and Yan expression are considered (Kuang, 2000).

Spenito and Split ends act redundantly to promote Wingless signaling

Wingless (Wg)/Wnt signaling directs a variety of cellular processes during animal development by promoting the association of Armadillo/β-catenin with TCFs on Wg-regulated enhancers (WREs). Split ends (Spen), a nuclear protein containing RNA recognition motifs (RRMs) and a SPOC domain, is required for optimal Wg signaling in several fly tissues. Spenito (Nito), the only other fly protein containing RRMs and a SPOC domain, acts together with Spen to positively regulate Wg signaling. The partial defect in Wg signaling observed with spen RNAi is enhanced by simultaneous knockdown of nito while it is rescued by expression of nito in wing imaginal discs. In cell culture, depletion of both factors causes a greater defect in the activation of several Wg targets than RNAi of either spen or nito alone. These nuclear proteins are not required for Armadillo stabilization or the recruitment of TCF and Armadillo to a WRE. Loss of Wg target gene activation in cells depleted for spen and nito was not dependent on the transcriptional repressor Yan or Suppressor of Hairless, two previously identified targets of Spen. It is proposed that Spen and Nito act redundantly downstream of TCF/Armadillo to activate many Wg transcriptional targets (Chang, 2008).

Although Drosophila, Spen and Nito are the only two proteins containing both RRMs and a SPOC domain, the sequence similarity of these domains is low and they are unrelated outside of these motifs. Spen and Nito show higher conservation to their respective orthologs in humans than to each other, suggesting that they evolved functional specificity after a duplication event (Jemc, 2006). Consistent with this, Spen and Nito have been shown to act antagonistically in the developing fly eye. Overexpression of nito disrupts eye development, and this phenotype is enhanced by a reduction in spen gene activity. Conversely, a small eye phenotype caused by overexpression of spen^DN is enhanced with nito overexpression and suppressed with nito RNAi (Jemc, 2006). Since Spen has been shown to regulate Notch and EGFR signaling pathways in the eye, it may be that Spen and Nito have opposing functions in these pathways (Chang, 2008).

In this study strong evidence is provided for functional redundancy between spen and nito in the context of Wg signaling. Single and double RNAi analysis indicated that both genes positively regulate the pathway in the fly eye, the wing imaginal disc and in Kc cells. Expression of nito can rescue the spen RNAi phenotype in the wing disc, strongly suggesting that Nito and Spen perform a similar biochemical function in the Wg pathway. There is a report concluding that loss ofspen has no role in Wg signaling in the developing wing (Mace, 2004). While the discrepancy may be due to the use of different Wg signaling readouts, it is likely that redundancy with nito can explain the negative results obtained with spen mutant clones in that study (Chang, 2008).

In humans, there is evidence that the spen and nito homologs possess both similar and distinct functional activities in the Notch pathway. SHARP can repress Notch signaling through interaction with RBP-Jκ, acting as a transcriptional corepressor. OTT1 can also bind to RBP-Jκ, but this interaction can lead to either repression or activation of a Notch/RBP-Jκ reporter gene, depending on the cell line used (Ma, 2007). Taken together with these data and those of Jemc (2006), the data suggest that Spen and Nito share some biochemical activities but also have distinct, antagonistic properties, depending on the molecular context (Chang, 2008).

In addition to acting redundantly in Wg signaling, it was found that spen and nito are required for the activity of pro-apoptotic factors Hid and Rpr in the fly eye. The fact that the suppression is greatest when both spen and nito are inhibited suggests that they act redundantly, though this requires further study and the molecular relationship between Spen, Nito and apoptosis is not clear (Chang, 2008).

Previous work has demonstrate that spen and nito are required for Arm*, a constitutively active form of Arm, to activate Wg signaling, suggesting that they act downstream of Arm stabilization. In Kc cells, spen, nito depletion did not effect the formation of a TCF-Arm complex on the nkd intronic WRE, even though they are required for activation of nkd expression by Wg signaling. Taken together, these data indicate that Spen and Nito act downstream or in parallel of TCF and Arm to promote transcriptional activation of Wg targets (Chang, 2008).

These results are consistent with those recently reported for SHARP in human cells (Feng, 2007). SHARP was required for maximal activation of Wnt transcriptional targets and reporter constructs, acting downstream of β-catenin stabilization. SHARP could potentiate the ability of Lef1 to activate transcription, independently of Lef1's ability to bind to β-catenin (Feng, 2007). Whether there is a direct biochemical interaction between SHARP and Lef1 is not known (Chang, 2008).

Interestingly, SHARP expression was elevated in several types of carcinomas with constitutively active Wnt/β-catenin signaling, suggesting that it is part of a positive feedback loop regulating the pathway (Feng, 2007). This circuit does not appear to be present in flies, where spen and nito are ubiquitously expressed (Chang, 2008).

Consistent with a role in transcription, Spen and Nito contain domains that could be involved in DNA binding. The murine Spen counterpart Mint has been shown to bind to a G/T-rich element in the FGF-responsive minimal enhancer of the OC promoter via its RRMs. While the recognition site is not well defined, it is interesting to note that there are several G/T-rich regions near some of the functional TCF sites in nkd intronic WRE. Whether the RRMs of Nito or Spen can recognize these sequences is currently being explored (Chang, 2008).

While Spen and Nito may play a direct positive role in the activation of transcriptional targets by Wg signaling, it is also possible that the functional requirement is indirect. The fact that loss of spen elevates Notch signaling in the fly eye fits with the vertebrate data showing that SHARP, Mint and OTT1 can associate with RBP-Jκ and inhibit its ability to activate Notch target genes. However, RNAi inhibition of the fly RBP-Jk homolog Su(H) did not restore activation of Wg targets to spen, nito-depleted cells. This suggests that activation of Notch signaling is not the mechanism by which Spen and Nito promote Wg signaling (Chang, 2008).

Upstream of Su(H), Notch signaling has been reported to repress Wg signaling in flies and fly cell culture. However, this cross-talk has been shown to occur at the level of Arm stabilization. Since Spen and Nito do not effect Arm protein levels, this mechanism appears not be involved in the promotion of Wg signaling by Spen and Nito (Chang, 2008).

Spen is also required to downregulate protein levels of the Ets-domain transcriptional repressor Yan in the eye and wing imaginal discs. Could an increase in Yan levels explain the block in Wg signaling observed when spen and nito are depleted? This does not appear to be the case, since inhibition of yan does not reverse the spen, nito requirement for Wg signaling in Kc cells. In addition, overexpression of Yan in the wing imaginal disc does not affect the expression of the Wg target Sens, which is highly dependent on spen and nito (Chang, 2008).

In contrast to Su(H) and yan, depletion of gro does reverse the spen, nito defect in Wg activation of nkd. Gro is a transcriptional corepressor that is thought to bind to TCF in the absence of Wg signaling and is known to be important in silencing nkd expression. In vitro, binding of β-catenin and TLE1 or TLE2 (vertebrate homologs of Gro) to TCF is mutually exclusive. β-Catenin and TLE1 binding to WREs in cells is also exclusive. This suggests a model where Gro displacement by Arm is defective in spen, nito-depleted cells, leading to reduced activation of nkd. However, it was found that Arm recruitment to the nkd WRE is not affected by spen, nito knockdown. In addition to this discrepancy, Gro has no apparent role in Spen, Nito regulation of CG6234 by the Wg pathway. This suggests that Gro displacement is not the major mechanism by which Spen/Nito function in Wg signaling (Chang, 2008).

One interesting aspect of Spen–Nito regulation of Wg signaling is that all targets of the pathway do not appear to require these proteins for activation. Originally it was shown that Spen is required for Wg function in several imaginal discs, but not in embryos. This could be explained by redundancy with Nito, but ubiquitous knockdown of both genes throughout the embryonic epidermis caused no detectable defect in Wg signaling. In the wing imaginal discs, spen and nito clearly are required for Sens expression, but two other Wg targets, Distal-less and fz3 are expressed normally under the RNAi conditions used. In Kc cells, several Wg targets required spen and nito for activation, but hth did not. These results have to be interpreted cautiously, because residual spen and/or nito activity may be sufficient for regulation of some Wg targets. However, the data do support a model where Spen and Nito regulate Wg-mediated transcriptional activation in a gene-specific manner (Chang, 2008).

In mice, disruption of Mint or OTT1 results in early embryonic lethality. It is interesting to note that a conditional deletion of OTT1 in the hematopoietic compartment caused a defect in pro/pre-B cell differentiation. Loss of Lef1 results in poor survival/growth of pro-B cells. While the phenotypes are not identical, this could be due to redundancy between Lef1 and other TCFs or between Mint and OTT1. The existence of floxed alleles of Mint and OTT1 should allow the relationship between these factors and the Wnt/β-catenin pathway to be more fully explored in the mouse (Chang, 2008).

An autonomous metabolic role for Spen

Preventing obesity requires a precise balance between deposition into and mobilization from fat stores, but regulatory mechanisms are incompletely understood. Drosophila Split ends (Spen) is the founding member of a conserved family of RNA-binding proteins involved in transcriptional regulation and frequently mutated in human cancers. This study found that manipulating Spen expression alters larval fat levels in a cell-autonomous manner. Spen-depleted larvae had defects in energy liberation from stores, including starvation sensitivity and major changes in the levels of metabolic enzymes and metabolites, particularly those involved in beta-oxidation. Spenito, a small Spen family member, counteracted Spen function in fat regulation. Finally, mouse Spen and Spenito transcript levels scaled directly with body fat in vivo, suggesting a conserved role in fat liberation and catabolism. This study demonstrates that Spen is a key regulator of energy balance and provides a molecular context to understand the metabolic defects that arise from Spen dysfunction (Hazegh, 2017).

This work provides the first detailed investigation of a fat regulatory role for Spen in any organism, and the first evidence that Nito also functions in this process. Spen depletion in the fat body drastically increased stored fat. Spen has been implicated in multiple pathways involved in endocrine signaling, including Notch, Wingless, and nuclear receptor signaling. This study found it unlikely that nuclear receptor pathways are relevant to the fat regulatory role this study defines, because upon Spen depletion or overexpression consistent changes in the expression of genes that are targets of those pathways were not observed. Furthermore, the lack of phenotypes involving fat storage per se upon overexpression of C-terminal Spen-SPOC^only domain argues against a role for Wg signaling, in which the same construct has potent dominant negative effects. Conversely, whereas a C-terminally truncated version of mSpen has little effect on Notch signaling, the strong fat phenotypes resulting from Spen-ΔSPOC overexpression suggest that Spen does not regulate fat via the Notch pathway (Hazegh, 2017).

Notably, Spen KD larvae also exhibited behavioral changes (increased food intake, decreased locomotion) that may have contributed to the fat increase. Thus, in addition to direct roles in fat accumulation within fat storage cells, Spen may be involved in a cross-talk pathway between the FB and the brain. However, a model is strongly support wherein increased food intake is instead an attempt to compensate for a condition of 'perceived starvation' resulting from an inability to access energy stores. Similarly, a lack of available energy may restrict locomotion. This hypothesis is further strengthened by the observation that Spen overexpression was sufficient to deplete stored fat but did not cause opposing behavioral phenotypes (Hazegh, 2017).

Mosaic analysis confirmed an autonomous role for Spen in FB cells. Spen KD in clones throughout the FB showed a striking increase in LD size. Larger LDs normally have lower surface tension, and the stored fat is easier to access. LD remodeling in WT animals is a highly regulated process involving specific factors, some of which were identified in a genome-wide RNAi screen in cultured Drosophila S2 cells. Notably, RNAseq data revealed that the products of several LD-regulating genes were significantly altered by Spen depletion, including l(2)01289 (~7-fold decreased), CG3887 (1.3-fold decreased), and eIF3-S9 (1.5-fold increased). While it is unclear if these changes are direct effects of Spen depletion, they may explain why LDs in Spen KD larvae are large yet apparently inaccessible, resulting in starvation sensitivity (Hazegh, 2017).

Consistent with the observed changes in FB cell and LD morphology and starvation sensitivity, changes in metabolites and gene expression in Spen KD larvae pointed to a drastic defect in lipid catabolism. Defects in β-oxidation were the most obvious, in part because the opposite effects were observed upon FB-restricted Spen overexpression. Spen depletion led to a decrease in the levels of free and acyl-conjugated carnitine, as well as of transcripts of three of the four enzymes necessary to break down acyl-carnitines into free fatty acids. Three lipases were also downregulated, which likely further contributes to an inability to convert energy stored as TAGs into usable forms. While an apparent upregulation of gluconeogenesis is evident, as supported by alterations in aspartate and PEPCK expression, these processes may be unable to completely compensate for decreased trehalose utilization, and these defects may contribute to the lethargy phenotype resulting from Spen KD. Consequently, surviving the loss of Spen may require breakdown of protein into free amino acids in order to anaplerotically replenish the TCA cycle, consistent with changes in expression of proteases, the observed decrease in many free amino acids, as well as increases in protein catabolism and collagen turnover markers (N-acetylmethionine and hydroxyproline). Of note, sustained proteolysis is a marker of aging and inflammation, a phenotype that has been previously associated with decreased locomotion in human and mouse models of physical activity, suggesting potential future ramifications of Spen’s role in metabolism with respect to aging/inflammation research. Finally, the observed decrease in glycogen levels upon Spen KD supports a model wherein glycogen is used as a carbohydrate source (in lieu of decreased levels of trehalose) to fuel glycolysis. The overall metabolic defects described in this study are distinctly different from what has been observed upon manipulation of other fat regulators (e.g., Sir2), suggesting that Spen operates in a previously undescribed pathway (Hazegh, 2017).

The results with Spen and Nito truncations provide additional mechanistic insight into how these proteins function in fat regulation. Overexpressing Spen-ΔSPOC reversed the phenotype of full-length Spen overexpression, and instead resulted in similar phenotypes to Spen depletion. Nito-ΔC overexpression had the same effects: larvae arrested development and FB clones mimicked starvation even when dietary nutrients were abundant. Overexpression of the Spen-SPOC^only construct had no effect on FB cells, as was the case for Nito-ΔN. Thus only Spen harboring the RRMs and the SPOC domain was able to promote fat depletion when overexpressed. Conversely, only truncated forms of Spen or Nito that retain the RRMs dominantly perturbed both FB cell viability and organismal resistance to starvation (Hazegh, 2017).

Recent studies of X chromosome inactivation found that mSpen RRMs mediate binding to the lncRNA Xist. Rbm15 (mNito) also binds Xist, and is required for N⁶-methyladenosine (m⁶A) modification of that lncRNA, which is in turn required for its ability to repress X chromosome transcription. Nito is a subunit of the Drosophila m⁶A methyltransferase complex and is required for RNA binding by that complex; Nito knockdown severely decreases global m⁶A modification of mRNA (Lence, 2016). Interestingly, the m⁶A demethylase FTO/ALKBH9 was the first human obesity susceptibility gene identified by genome-wide association studies, but the relevant nucleic acid target(s) remain unknown. This work provides the first hint that an RNA bound by Spen and/or Nito may be a key FTO substrate (Hazegh, 2017).

These findings lead to a model for Spen and Nito function in the regulation of fat storage. Spen binds via its RRMs to one or more RNAs and, via recruitment of other factors, promotes the expression of enzymes key for mobilization of energy stored as fat (e.g. lipases). The mechanism of activation may be direct or indirect, and via alternative splicing, activation/repression of transcription, or effects on RNA stability and/or translation. Moreover, RNA binding partners may be mRNA or non-coding RNA. Future work will be required to make these distinctions. It is proposed that the Spen SPOC domain is critical for this function, but undefined domains in between the N-terminal RRMs and C-terminal SPOC domain are also important, and these are not shared with Nito. It is proposed that Nito binds via its RRMs the same or a largely overlapping set of RNAs, and also recruits additional factors via its SPOC domains, but–either because it fails to recruit specific factors recruited by Spen, or because it recruits other factors not recruited by Spen-Nito ultimately inhibits/represses the same energy-storage-mobilizing enzymes that are activated by Spen. Overexpressed Spen or Nito fragments containing RRMs sequester target RNAs away from endogenous full-length Spen and the other effectors of fat storage control. Finally, the findings in mouse adipose tissue that mSpen and mNito both increase in expression when a HFD drives fat accumulation lead to the belief that in WT animals Nito acts as a counterbalance to Spen in order to fine-tune fat storage. Future studies delving into more mechanistic details may lead to treatments for obesity and related metabolic disorders that result from perturbation of the pathway that was elucidated here (Hazegh, 2017).

The large and small SPEN family proteins stimulate axon outgrowth during neurosecretory cell remodeling in Drosophila

Split ends (SPEN) is the founding member of a well conserved family of nuclear proteins with critical functions in transcriptional regulation and the post-transcriptional processing and nuclear export of transcripts. In animals, the SPEN proteins fall into two size classes that perform either complementary or antagonistic functions in different cellular contexts. This study shows that the two Drosophila representatives of this family, SPEN and Spenito (NITO), regulate metamorphic remodeling of the CCAP/bursicon neurosecretory cells. CCAP/bursicon cell-targeted overexpression of SPEN had no effect on the larval morphology or the pruning back of the CCAP/bursicon cell axons at the onset of metamorphosis. During the subsequent outgrowth phase of metamorphic remodeling, overexpression of either SPEN or NITO strongly inhibited axon extension, axon branching, peripheral neuropeptide accumulation, and soma growth. Cell-targeted loss-of-function alleles for both spen and nito caused similar reductions in axon outgrowth, indicating that the absolute levels of SPEN and NITO activity are critical to support the developmental plasticity of these neurons. Although nito RNAi did not affect SPEN protein levels, the phenotypes produced by SPEN overexpression were suppressed by nito RNAi. It is proposed that SPEN and NITO function additively or synergistically in the CCAP/bursicon neurons to regulate multiple aspects of neurite outgrowth during metamorphic remodeling (Gu, 2017).

The SPEN family has been evolutionarily conserved, with representatives from protists and plants to animals. The family includes mouse MINT (Msx2-interacting nuclear target protein) and human SHARP (SMRT/HDAC1 associated repressor protein). SPEN, SHARP, and MINT are unusually large proteins of ~3575 to 5500 amino acids in length. In addition to SPEN, the Drosophila genome contains one other SPEN-like gene, spenito (nito), that encodes a much smaller, 793 amino acid protein . Although SPEN and NITO share conserved N-terminal RRMs and the SPOC domain, their overall sequence similarity is only 28%, suggesting that SPEN and NITO may function similarly in some processes, but differently in others. For example, SPEN and NITO display functional antagonism during eye development (Jemc, 2006) but act synergistically in regulating Wingless signaling in wing imaginal discs and cultured Kc cells (Chang, 2008). In addition, NITO was identified as a splicing factor involved in Sxl regulation and sex determination in Drosophila, while SPEN had no such effect (Yan, 2015). Several studies in vertebrates also suggest that the relationship between these two proteins is context-dependent (Chang, 2008). Similar to SPEN, NITO is broadly expressed in Drosophila tissues (Chang, 2008). Therefore, this study investigated the function of NITO and its possible interactions with SPEN in the context of remodeling of the CCAP/bursicon neurons (Gu, 2017).

Several previous studies have examined the role of SPEN during neuronal differentiation. In embryos, SPEN contributes to neuronal cell fate specification and regulates the growth, pathfinding, and fasciculation of PNS and CNS axons. SPEN also regulates proliferation and differentiation of Drosophila photoreceptor neurons. In addition to its role in neuronal differentiation, SPEN has also been shown to modify late-onset, progressive neurodegeneration in a model for spinocerebellar ataxia in the mature Drosophila retina. This study reports that SPEN regulates developmental plasticity in mature neurons (Gu, 2017).

SPEN overexpression specifically inhibited neurite outgrowth during metamorphic remodeling of the CCAP/bursicon cells, with little or no effect on the larval morphology of these neurons. The reasons for the stage-dependence of SPEN activity in these cells are unknown, but one intriguing possibility is that the stage-dependence results from a direct or indirect link to the ecdysone titer during metamorphosis. This is suggested by work on the human SPEN ortholog, SHARP. SHARP expression is steroid-inducible. In addition, the RRM domains of SHARP interact directly with the steroid receptor RNA cofactor SRA, which acts as a scaffold to bring together nuclear receptors, corepressors, and coactivators. SHARP inhibits the transcriptional activity of SRA-stimulated estrogen and glucocorticoid receptors. No Drosophila ortholog of SRA has yet been identified. Nevertheless, in future studies, it will be of interest to examine interactions between SPEN and signaling by the ecdysone receptor (EcR) during neuronal remodeling. (Gu, 2017).

The overexpression of spen and nito produced axon growth and branching phenotypesthat were qualitatively similar (although not identical) to the loss-of-function effects of both genes. Such similarities in GOF and LOF phenotypes are often observed in systems where the stoichiometric ratio of gene products to the concentration of other cellular components is important. Thus, the current observations indicate that SPEN may need to be maintained at a specific level, or within an expression window, to properly regulate axonal outgrowth. Recent studies observed strong genetic interactions between spen and multiple factors controlling Myosin II activity. Notably, many cellular movements, including growth cone migration and branching, depend upon dynamic cytoskeletal rearrangements that can be disrupted by either increasing or decreasing the stability or function of cytoskeletal components. Thus, the interaction between SPEN and Myosin II provides one possible explanation for the similar responses of the CCAP/bursicon neurons to reduced versus enhanced levels of SPEN activity. Interestingly, SPEN also interacts genetically with crinkled (Myosin VIIA) in controlling wing vein development and wing bristle positioning, suggesting a more general function of SPEN in cytoskeletal rearrangements. If SPEN and NITO regulate common cellular processes, then this explanation may also apply to NITO (Gu, 2017).

The current results showed that NITO and SPEN act additively or synergistically to regulate CCAP/bursicon cell remodeling. This finding is in agreement with other studies showing that SPEN and NITO are both positive regulators of Wg signaling in developing wing discs and Drosophila Kc cells, and of programmed cell death in the eye disc induced by the pro-apoptotic factors Head involution defective and Reaper. In contrast, SPEN and NITO are antagonistic in regulating photoreceptor number, rhabdomere morphology, and regular ommatidial spacing in the adult eye. Furthermore, SPEN and NITO may play completely different functions in some situations, as NITO regulates Sxl level and its alternative splicing, while SPEN has no such effects (Yan, 2015). Thus, the interaction between SPEN and NITO depends on the cellular context. In mammals, the large and small SPEN-like proteins display similar context-dependence in the regulation of Notch signaling. The large SPEN family proteins MINT and SHARP both suppress Notch signaling by competing with the N intracellular domain (NICD) for binding to the core transcription factor RBP-J. The NITO ortholog, RBM15, also complexes with RBP-J, but it either stimulates or represses expression of a reporter in different cell lines. Therefore, in Drosophila and in vertebrates, large and small SPEN family proteins act redundantly or synergistically in some cellular contexts and antagonistically in others, or they perform completely different roles (Gu, 2017).

Recent insights into the molecular interactions with SPEN family proteins provide some clues to mechanisms underlying these context-dependent differences in function. In different systems, the large SPEN family proteins function as either transcriptional corepressors or coactivators. For example, human SHARP and RBM15 were identified as crucial factors required for the long non-coding RNA Xist-mediated silencing of X-chromosomes by directly interacting with the Xist, recruiting nuclear corepressor, SMRT, activating histone deacetylase3 (HDAC3), and deacetylating histones to exclude Pol II and repress transcription. Other studies have revealed functions of the SPEN proteins in transcription activation. MINT enhances transcriptional activation at the osteocalcin promoter and associates with an actively phosphorylated and processive form of RNA polymerase II. SHARP enhances beta-catenin/T cell factor (TCF)-mediated transcription. In differentiating Drosophila hemocytes, SPEN binds to many target gene promoters in association with a known activating histone modification pattern (Gu, 2017).

The small SPEN proteins have not been shown to function as transcriptional coactivators or corepressors and instead play important roles in alternative splicing, selection of alternative polyadenylation sites, and nuclear export of mRNAs. However, both the large and small SPEN proteins may function together in protein complexes that first associate with nascent transcripts. These messenger ribonucleoprotein (mRNP) complexes are dynamic structures that participate in transcription, pre-mRNA splicing, and nuclear export, and they change in protein composition to permit nuclear export once pre-mRNAs are completely spliced. Importantly, several studies have detected associations among the large and small SPEN proteins in these structures . Given the strong evolutionary conservation of the large and small SPEN proteins, similar interactions are likely to explain some of the overlapping functions of SPEN and NITO during neuronal remodeling in Drosophila (Gu, 2017).

Spen limits intestinal stem cell self-renewal

Precise regulation of stem cell self-renewal and differentiation properties is essential for tissue homeostasis. Using the adult Drosophila intestine to study molecular mechanisms controlling stem cell properties, this study identified the gene split-ends (spen) in a genetic screen as a novel regulator of intestinal stem cell fate (ISC). Spen family genes encode conserved RNA recognition motif-containing proteins that are reported to have roles in RNA splicing and transcriptional regulation. This study demonstrates that spen acts at multiple points in the ISC lineage with an ISC-intrinsic function in controlling early commitment events of the stem cells and functions in terminally differentiated cells to further limit the proliferation of ISCs. Using two-color cell sorting of stem cells and their daughters, spen-dependent changes in RNA abundance and exon usage were identified and potential key regulators were found downstream of spen. This work identifies spen as an important regulator of adult stem cells in the Drosophila intestine, provides new insight to Spen-family protein functions, and may also shed light on Spen's mode of action in other developmental contexts (Andriatsilavo, 2018).

The mechanisms by which ISCs undergo asymmetric and symmetric divisions are still not completely understood. In a genetic screen, this study has identified spen as an essential regulator of adult stem cells in Drosophila. The data indicate that in the absence of spen activity, stem cells have aberrant high levels of Delta (Dl) protein and fail to properly commit into EB daughter cells resulting in large increase in numbers of ISC-like cells and that this activity is cell autonomous roles in the ISC. Furthermore, spen acts in EBs to limit ISC numbers, and in EBs, EEs and ECs cells to suppress ISC proliferation. Therefore, in spen mutant tissue, stem cell autonomous and non-autonomous mechanisms that act to drive ISC proliferation, increased ISC numbers, and aberrant accumulation of Dl protein in ISCs. While spen mutants are dependent on Akt and InR for their growth, they are not as sensitive to a reduction in EGFR activity as wild-type control clones are. These findings argue that Spen does not act as a general regulator of Notch signaling in all tissues, and is upstream of, or parallel to, Notch pathway activation to promote intestinal stem cell commitment. Through a transcriptomic analysis, this study has identified genes controlled by spen in ISCs and EBs, both at the transcript and exon level, providing candidate ISC regulators downstream of spen. Critically, this work shows that the RNA binding protein Spen is an important regulator of asymmetric fate outcomes of ISC division and its proliferation (Andriatsilavo, 2018).

It is interesting that spen has both stem cell-autonomous and non-autonomous activities to regulate stem cell numbers and proliferation. The knockdown of spen in ISCs and EBs can expand stem cell numbers, whereas the knockdown in EEs and ECs led to a dramatic increase in the number of dividing ISCs, but did not change the density of ISCs. It is believed, therefore, that the growth of spen mutant clones is influenced by both cell autonomous and non-autonomous processes: spen inactivation in the stem cell leads to more ISC symmetric self-renewing divisions, however, EB, EE and EC cells are still produced. The mutant EB, EE, and EC cells can further drive proliferation of the extra ISCs through non-autonomous regulation. It is possible that the non-autonomous regulation of ISC proliferation detected upon downregulation of spen in EEs and ECs could be due to stress induced in the tissue owing to disruption of numerous spen targets genes in these cells. Importantly, these findings highlight the complex process of stem cell deregulation arising in tumor contexts whereby inactivation of a tumor suppressor genes may have numerous functions in different cells within a tumor that collaborate to drive tumorigenesis (Andriatsilavo, 2018).

One intriguing effect of spen inactivation is the high levels of accumulation of the Notch ligand Dl at the plasma membrane and in intracellular vesicles. RNAseq data revealed that Dl mRNA levels or exon usage are not affected, suggesting a regulation at the protein level, perhaps during its endocytic trafficking steps. This phenotype of Dl protein accumulation is reminiscent of those occurring upon Dl trafficking perturbation, such as in neuralized mutants. A previous study has shown genetic interaction of spen and regulators of trafficking of Notch ligands. Indeed, in the developing Drosophila eye disc, spen genetically interacts with the endocytic adaptor Epsin/liquid facets (lqf), which promotes Dl trafficking and internalization facilitating Dl activation. Spen, however, is likely not a general regulator of protein trafficking as the localization of the membrane protein Sanpodo occurred normally in spen mutant clones in the intestine. Therefore, Spen likely regulates Dl trafficking in a tissue-specific manner and indirectly through transcriptional or post-transcriptional control of a downstream gene (Andriatsilavo, 2018).

In addition, the data indicated that the knockdown of Akt and InR activity could reduce the number of Dl+ ISCs produced in the spen mutant background. The InR pathway has previously been implicated in regulating symmetric cell divisions of the ISC during adaptive growth. Thus, it appears as if Akt and InR are also facilitating symmetric cell division in the spen mutant context. These data also show that spen mutant stem cells are insensitive to a reduction of EGFR signaling upon overexpression of EGFR-DN, suggesting that spen and EGFR pathway have opposing roles in regulating ISC proliferation. While a decrease in EGFR signaling reduces ISC proliferation, spen inactivation, in contrast, leads to an increase in proliferation. spen has been shown to genetically interact with the EGFR signaling pathway during Drosophila embryogenesis and during eye development. Nevertheless, in these contexts, spen potentiates EGFR signaling. Thus, spen may have a tissue specific effect regarding EGFR pathway regulation. The precise link between spen and EGFR signaling regulation is nevertheless still unclear. Indeed, while a constitutive active form of Ras can rescue the lethality caused by a spen dominant negative form in embryo, spen seems to act downstream or in parallel of Ras activation during photoreceptor specification. Additionally, spen loss of function in cone cells during pupal eye development affects the EGF receptor ligand Spitz level, which suggest a role at the level of the EGF receptor activation. Further studies will be required to better understand the link between spen and the EGFR signaling pathway in the regulation of intestinal stem cell regulation, as well as in other developmental contexts (Andriatsilavo, 2018).

In addition to describing a new function of spen in adult stem cell regulation, the data raise a number of interesting questions about the function of spen and its downstream target genes in stem cell regulation. Importantly, this study demonstrates that inactivation of a large SPEN family member also results in considerable alteration of exon usage, like the smaller family members (spenito in flies, RBM15 and RBM15b in mouse). Thus, spen activity is important for transcriptional and post-transcriptional regulation in Drosophila, although it cannot be excluded that some of these effects might be indirect. Interestingly, in absence of spen function, altered exon usage of spenito was detected in EBs, raising the possibility of feedback control between family members, reminiscent of the plant SPEN family member FPA (Andriatsilavo, 2018).

Little is known about the impact on intestinal stem cell self-renewal of RNA binding proteins. A recent study found that inactivation of Tis11, encoding a regulator of RNA stability, led to larger clones than controls. The Tis11 phenotype differs markedly from that of spen as it appears not to affect cell fate decisions since ISCs do not accumulate as in spen mutant clones. Importantly, this study provides a data set that opens new perspectives for future investigation on the relationship between Spen and RNA regulation at transcriptional and post-transcription levels. Interestingly, the data also reveals that Spen co-regulates several genes encoding proteins that are involved in similar biological processes, such as cytoplasmic ribosomal proteins, immune response pathway and Imd pathway genes, and genes involved in Chitin metabolism. This may explain previously reported spen phenotypes in other tissues (Andriatsilavo, 2018).

Recent studies have highlighted important, essential functions of Spen family proteins from X-inactivation in mammals to sex determination and fat metabolism in flies. This family of proteins is also frequently mutated in cancers, though mechanistically the role SPEN family proteins in cancers is not understood. This work demonstrates that spen inactivation causes the deregulation of numerous genes and results in alteration of the stem cell fate acquisition. In mammals, RBM15 has a critical function in promoting hematopoietic stem cell return to quiescence. These findings raise the possibility that conserved functions of SPEN family proteins may help restrict stem cell activity required for cancer prevention (Andriatsilavo, 2018).

DEVELOPMENTAL BIOLOGY

Embryonic

Antibodies were raised against a common fragment of the Spen proteins (amino acids 3203-3714) to assay the expression pattern and subcellular localization. Immunostaining of whole-mount embryos shows that Spen antigen is expressed in most or all cell types, and is concentrated in nuclei. Spen protein staining is first detectable prior to cellularization in stage 3 embryonic nuclei, and is present in all blastoderm cells, including the pole cells. The protein is expressed throughout the rest of embryogenesis, and is concentrated in nuclei. At later embryonic stages (9 through 14), nuclear Spen staining appears to be most abundant in CNS and epidermal cells, but most tissues exhibit nuclear staining at detectable levels (Wiellette, 1998).

Antibodies against two different C-terminal epitopes reveal that Spen proteins are nuclear and expressed in most, if not all, cells. The Spen proteins are detectable as early as cellular blastoderm and are ubiquitously nuclear during early development. Spen expression in the CNS appears to be at higher levels than in the surrounding epidermis after stage 15. Spen is also expressed in non-neuronal cells, most likely glia, within the CNS. Spen proteins are also detected in muscle nuclei. Maternal protein persists in spen mutant embryos until the onset of terminal neuronal differentiation (Kuang, 2000).

EFFECTS OF MUTATION

Split ends suppresses activated Yan

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Ectopic expression of a constitutively active protein (yan^ACT) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, a genetic screen was performed to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan^ACT. Approximately 190,000 mutagenized flies were screened, and 260 enhancers of Yan (EY) and 90 suppressors were obtained. Among the previously known genes recovered are four RTK pathway components [rolled (MAPK), son-of-sevenless, Star, and pointed], and two genes (eyes absent and string) that have not been implicated previously in RTK signaling events. Mutations in five previously uncharacterized genes were also recovered. One of these, split ends, has been characterized molecularly and shown to encode a member of the RRM family of RNA-binding proteins (Rebay, 2000).

The isolation of spen as an enhancer of yan^ACT suggests it may play a role as a positive regulator of the RTK/Ras pathway. Preliminary results indicate spen is a nuclear protein broadly expressed in most tissues and enriched in neuronal lineages. It is currently not known whether spen functions upstream or downstream of yan. One possibility is that spen might regulate the stability of the yan transcript. It has been postulated that the mechanism for downregulating yan activity involves post-translational modifications of the protein, namely phosphorylation by activated MAPK, that subsequently targets yan for degradation. Such post-translational regulation of yan would presumably need to be reinforced at the transcriptional and/or translational level. Thus, spen might play a role in destabilizing yan mRNA in response to Ras signaling. This would be consistent withthe isolation of mutations in spen as enhancers of yan^ACT. Alternatively, spen could be transcriptionally regulated by yan, and could play a role in splicing, stability, or transport of other downstream effector genes. Future phenotypic, genetic, and biochemical characterization of spen will be necessary to understand its role in Ras/yan signaling events (Rebay, 2000).

Affects on PNS development

In MZspen embryos, the number of many PNS and CNS cell types is altered, and the development of other organs is affected. In order to analyze the molecular basis of these defects, focus was placed on the well-marked development of the lateral chordotonal (lch) sensory neurons. Wild-type embryos contain a cluster of five lch neurons in each abdominal hemisegment. In MZspen embryos, this number varies from none to six, and is typically four. Clusters containing the normal number are often disorganized (Kuang, 2000).

Other mutations have been previously shown to affect lch neuron number by affecting either precursor formation or EGF receptor signaling. In wild-type development, three Atonal-expressing cells become lch precursors. These cells activate EGFR signaling in two neighboring ectodermal cells, recruiting these cells to the lch neuronal fate. Mutants that block either precursor formation or recruitment therefore produce characteristic numbers of lch neurons. atonal, which blocks lch proneural precursor formation, produces occasionally one lch neuron. Mutations in the EGF/Spitz group, which block the recruitment process, produce only three lch neurons. Gain-of-function mutations in the EGF pathway yield 6-7 lch neurons. By contrast, the lch neuron phenotypes in MZspen embryos spans the range observed in loss-of-function mutations that affect precursor formation or recruitment and gain-of-function mutations in EGFR signaling (Kuang, 2000 and references therein).

The variable number of lch neurons could therefore reflect defects in precursor formation, EGFR signaling or another pathway. The formation and refinement of the chordotonal (ch) proneural cluster was followed in wild-type and MZspen embryos using an antibody directed against Atonal, the helix-loop-helix transcription factor that specifies ch neuronal fate. Atonal is initially expressed in the nuclei of a patch of ectodermal cells in each abdominal hemisegment, the proneural cluster. Atonal expression becomes progressively restricted to the precursor cells that contribute to both lateral and ventral chordotonal clusters. At any stage of this dynamic process, abdominal hemisegments in wild-type embryos contain similar numbers of Atonal-expressing cells. By contrast, in MZspen embryos, abdominal hemisegments contain variable numbers of these ch precursor cells. The variability in precursor number suggests possible defects in Notch signaling because Notch is required to generate the appropriate numbers of precursors from proneural clusters. Notch expression is normal in embryos lacking Spen, but expression of Su(H), the key known transcriptional effector of Notch signaling, is dramatically reduced throughout the embryo. Not surprisingly, the expression of Enhancer of Split Complex (En(Spl)-C) proteins, which depends on Su(H), is also reduced in the embryos that lack Spen (Kuang, 2000).

Axon guidance phenotypes

The cell-fate changes in MZspen embryos are not observed in embryos that lack only zygotically contributed Spen. Maternally contributed spen can be detected in zygotic mutant embryos until late stage-12, at which point most cell-fate decisions have been made. No defects are detected in zygotic spen mutant embryos with respect to neuronal cell fate as assayed with mAbs that recognize CNS neuronal subsets [anti-Eve, anti-Engrailed, anti-Ftz, PNS neurons (22C10), glial development (anti-Repo and anti-Sim), and muscle development (anti-myosin heavy chain and anti-connectin)]. spen¹ affects the growth and guidance of a subset of CNS and sensory axons (Kolodziej, 1995), but whether the apparent specificity of these phenotypes reflects residual protein function has not been determined. spen³ and spen⁵ are predicted to produce proteins one-sixth and one-half the size of spen respectively, and so could be nulls. Whether residual spen function is present in zygotic mutant embryos was investigated by comparing CNS and motor axon development in wild-type, spen³ and spen⁵ zygotic mutant embryos, and in embryos that lack both maternal and zygotic Spen. In zygotic mutant embryos, defects were identified in the elongation and pathfinding of axons in a subset of longitudinal CNS axon tracts, in the intersegmental nerve b (ISNb) and segmental nerve a (SNa) motor axon pathways, in the transverse nerve (TN), but not in the commissural CNS axon tracts, nor in the intersegmental nerve (ISN) motor axon pathways. The frequency of motor axon defects is similar in spen³ homozygous embryos and embryos heterozygous for spen³ and a chromosomal deficiency that removes the 21B region, suggesting that spen³ is a null allele. Defects in axon extension and guidance are more pronounced when maternally contributed spen is also removed, indicating that maternally contributed spen provides residual function in zygotic mutants (Kuang, 2000).

mAb 1D4 reveals three parallel longitudinal axon tracts that extend continuously in the CNS on each side of the ventral midline in wild-type late stage-16 or stage-17 embryos. In spen mutant embryos, the outermost fascicle is discontinuous in some segments, and these axons occasionally invade the middle fascicle. Axon defects are more severe, and extend to all axon tracts in the CNS in spen mutant embryos derived from spen germ line clones (Kuang, 2000).

mAb 1D4 also labels motor axons. In each abdominal hemisegment of the Drosophila embryo, approximately 30 motor axons innervate 30 muscle fibers in a stereotyped pattern. Development was examined of the intersegmental nerve (ISN), which innervates the dorsal muscles, the ISNb, which innervates ventral muscles, and the SNa, which innervates the lateral muscles. The ISN appears normal in zygotic spen mutants. In late stage-16 wild-type embryos, the ISNb has defasciculated from the ISN and forms three connections with ventral muscles: at muscles 12 and 13 and the cleft between muscles 6 and 7. In late stage-16 zygotic spen mutant embryos, most ISNb motor axons defasciculate from the ISN, but stall short of their ventral muscle targets. In the 4%-18% of cases where three connections can be detected in spen mutant embryos, they appear generally smaller than wild type. The SNa motor axons are also abnormal in spen mutant embryos. In stage-16 wild-type embryos, the SNa motor axons have traversed the ventral muscle field and bifurcated just above the ventral muscles. The dorsal branch extends away from the ventral muscles, and the lateral branch extends posteriorly, roughly parallel to the dorsal edge of muscle 12. In stage-16 zygotic spen embryos, most SNa motor axons either stall near the initial entry into the lateral muscle field, or make shorter than wild-type dorsal or lateral extensions (Kuang, 2000).

In MZspen embryos, defective in both maternal and zygotic spen, the development of all motor axon pathways is defective. Motor axons exit the CNS, pick the correct pathways, but fail to innervate their muscle targets. In the cases of the SNa and ISNb motor axons, these axons must first cross the midline, and the occasional absence of these axon tracts may reflect midline defects. However, in hemisegments where distinct ISN, SNa and ISNb fascicles are observed, the motor axons still fail to reach target muscle, and even occasionally cross over segmental boundaries. Some of these defects may also reflect the disorganized and missing muscle fibers in these embryos. Thus, spen appears not to be required for motor axons to distinguish dorsal, lateral and ventral muscles, but is required for later steps in motor axon development in the neuron, muscle or both (Kuang, 2000).

split ends homeotic mutant phenotype

Split ends (Spen) is a protein that acts in parallel with Hox proteins to regulate different segmental morphologies. spen plays two important segment identity roles. One is to promote sclerite development in the head region, in parallel with Hox genes; the other is to cooperate with Antennapedia and teashirt to suppress head-like sclerite development in the thorax. Without spen and teashirt functions, Antennapedia loses its ability to specify thoracic identity in the epidermis. Spen is the only known homeotic protein with RNP binding motifs: this indicates that splicing, transport, or other RNA regulatory steps are involved in the diversification of segmental morphology. Other studies have identified spen as a gene that acts downstream of Raf to suppress Raf signaling in a manner similar to the ETS transcription factor Aop/Yan. This raises the intriguing possibility that the Spen RNP protein might integrate signals from both the Raf and Hox pathways (Wiellette, 1999).

Loss of zygotic spen function results in embryonic lethality that is associated with a loss of the anterior portion of the H-piece, and with a kinked median tooth. The anterior parts of the H-piece are derived from the ventral maxillary segment, and are dependent on Dfd function. Head development is profoundly disrupted in maternal/zygotic spen mutants. These mutant embryos have non-involuted heads that are missing many head sclerites, including the base of the mouth hooks, median tooth, anterior regions of the lateralgraten, and the dorsal bridge. The ventral arms and vertical plates are also strongly reduced. Many of these spen-dependent sclerites are also dependent on head Hox genes such as labial (lab), and Sex combs reduced (Scr); gap/homeotic genes such as empty spiracles (ems) and orthodenticle (otd), and other head patterning genes. In the maternal/ zygotic mutants, the trunk region of the embryos appears to be largely unaffected. The denticle belts and posterior structures such as filzkorper appear normally shaped, although these structures, as well as the remnants of head skeleton (and ecopic head-like sclerites, see below), are somewhat less pigmented/ sclerotized than normal. The overall low level of sclerotization may be due to embryonic death in maternal/zygotic mutants before the cuticle is fully developed (Wiellette, 1999).

Approximately 50% of the embryos that are zygotic or maternal/zygotic spen mutants also develop sclerites in the thoracic segments. These sclerites appear most frequently in the second and third thoracic segments (T2 and T3), and overlap the anterior/posterior compartment boundary. The expressivity of this phenotype ranges widely. The weakest phenotypes exhibit a row of small chunks of ectopic sclerotic material, usually limited to T2 and T3. Strong phenotypes show broad bands of sclerotic material in all thoracic segments, often accompanied by sclerotic patches in lateral regions of the abdominal segments. No matter how extensive, the sclerites do not form in the fields of denticle belts, nor do they change the overall size of a segment. In small patches, the ectopic material is variably sclerotized, scar-like, and not recognizable as any other embryonic structure (Wiellette, 1999).

When manifest in broad patches, the sclerotic material is brown and striated, reminiscent of the brown, striated appearance of the ventral arms, vertical plates and dorsal arms of the head skeleton. It is concluded that one role of spen is to suppress the production of head-type sclerotization from ventral thoracic cells, and it is needed to a lesser extent for this function in lateral abdominal cells (Wiellette, 1999).

This spen minus homeotic phenotype is not specific to alleles isolated on the basis of Dfd interaction. spen was also isolated in a screen for suppressors and enhancers of an activated Raf construct. Dickson (1996) isolated three alleles of E(Raf)2A. These alleles do not complement the lethality of spen alleles that generate homeotic phenotypes, and the E(Raf)2A alleles also exhibit ectopic head-like sclerites, as do 11 additional alleles of spen that enhance the rough eye phenotype generated by ectopic expression of Cyclin E in the eye (Wiellette, 1999 and references therein).

Production of ectopic head-like sclerotic material in the trunk indicates that loss of spen function might result in de-repression of head patterning genes. The cuticular phenotypes were examined of embryos doubly mutant for spen and Distal-less, cnc, lab, Dfd, buttonhead, otd and ems, all of which have been shown to be required for determining head-specific pathways. All of the double mutant combinations show the same degree of thoracic sclerotization as spen single mutants. The transcription patterns of Dll, cnc, lab, Dfd, ems, otd and apontic were examined in spen mutant embryos and found to be indistinguishable from wild type. Therefore it is concluded that spen does not repress ectopic head-like cuticle by repressing the expression or function of these known head-determining genes. Finally, ectopic expression of Dfd and lab in spen mutant embryos does not alter the degree or character of the ectopic sclerites in the thorax (Wiellette, 1999).

Null mutations in Antp result in a transformation of T2 and T3 towards T1 in the embryonic body plan. In addition, Antp mutant embryos develop ectopic head-like sclerites in the dorsal thorax (between T1 and T2), similar in kind but not in position to the ectopic sclerite phenotype seen in spen mutants. To test whether spen and Antp function in an additive or synergistic manner in the repression of head-like sclerites in the thorax, spen^-; Antp^- cuticle phenotypes were examined. Embryos mutant for both spen and Antp have more sclerotic material in dorsal T2 than do Antp mutants alone. In addition, the ectopic head-like sclerites in the ventral thorax of spen^-;Antp^- mutants are more sclerotized and extensive than in spen mutants alone. The sclerotic material in spen^-;Antp^- mutants frequently appears in two distinct bands, one in the center of the segment similar to the position in spen mutants, and at another position in the posterior of T1 and T2. These posterior ectopic sclerites do not develop in T3. The enhanced formation of head-like sclerites in spen^-;Antp^- mutants suggests that spen and Antp function in a common or interacting pathway(s) in subregions of T1 and T2 (Wiellette, 1999).

The synergistic effect of Antp and spen might be due to a regulatory effect of Antp on spen transcription pattern, or to Spen effects on Antp transcript pattern or translation. However, Antp transcript and protein expression patterns are unchanged in spen mutant embryos, and spen transcript expression is unchanged in Antp mutant embryos. Therefore, spen and Antp appear to be acting in parallel, presumably due to direct or indirect regulation of common downstream genes (Wiellette, 1999).

If spen and Antp regulate common targets, then induction of high levels of exogenous Antp expression might result in suppression of the spen mutant phentoype. The ability of excess Antp protein to suppress the spen mutant phenotype was examined. Overexpression of Antp under heat shock promoter control (hsAntp) causes a transformation of head regions to thoracic identity, but leaves T2 and T3 nearly unchanged. When Antp is overexpressed in a spen mutant background, the ectopic head-like sclerites are strongly suppressed. The number of hsAntp; spen^- embryos that exhibit any detectable ectopic sclerites is less than half the expected number compared to spen^- mutant siblings from the same cross, or compared to spen^-; hsAntp embryos that were not subjected to heat shock. In addition, the sclerites which do occasionally appear in heat shocked hsAntp; spen^- embryos are smaller than those in their spen^- siblings. The ability of excess Antp to suppress the spen^- homeotic transformation indicates that the two genes interact to repress ectopic head-like sclerites (Wiellette, 1999).

In the head region, where spen is required for the development of sclerites that also require Dfd, Scr and other head genes, it is also possible that spen might work in parallel to Hox pathways. This is the case for Dfd and Scr, since mutations in these genes have no effect on spen transcript expression pattern, and conversely, spen mutants have no effect on Dfd or Scr transcript or protein expression patterns. Attempts were made to test whether the overexpression of Dfd could rescue the H-piece defect in spen mutant embryos, but the morphology of the head skeleton was so disrupted by heat shock induced ectopic Dfd protein that it was not possible to conclude whether the anterior H-piece was restored (Wiellette, 1999).

Another Drosophila gene involved in distinguishing head from body is teashirt (tsh). Tsh protein is expressed only in the labial segment and trunk region of embryos, where it is required to repress head identity and to promote thoracic and abdominal segment identities. tsh transcription levels in the thorax are maintained by Antp, but a variety of genetic interaction tests have shown that Antp and Tsh have independent functions in repressing head development. Is spen integrated into the Antp;tsh pathways by regulation of the tsh or Antp expression patterns? Experiments show that (1) expression pattern of Tsh protein is unchanged in spen mutant embryos; (2) protein expression patterns of Scr or Antp in spen^-;tsh^- double mutant embryos are unchanged from the pattern seen in tsh mutants alone, and (3) the spen transcript pattern is normal in tsh mutants. Therefore, Spen suppression of head-like sclerites is not exerted by a regulatory effect on the Tsh protein expression pattern, nor by Tsh effects on the spen transcript pattern, nor through combinatorial effects of spen and tsh on Scr or Antp protein abundance (Wiellette, 1999).

The phenotype of tsh ^- , spen^- mutant embryos suggests that the two genes act to promote thoracic development. In tsh mutant embryos, the T1 denticle belt is absent and although the remaining denticle belts appear to have the appropriate segmental identities, the denticles themselves are disorganized and smaller than in wild type. In contrast, tsh^-;spen^- double mutants completely lack denticle belts in the thorax. This may be due to the the death of cells in the denticle field in the thorax of the double mutants, or to the inability of Antp protein, still expressed in the remaining cells, to promote the development of thorax-specific structures (Wiellette, 1999).

As to whether tsh and spen collaborate in repressing head-like sclerites, it was found that the tsh^-, spen^- double mutants still have bits of sclerite in the 'thorax' of the double mutants, so this phenotype is not enhanced. However, the effects of Tsh overexpression on the ectopic head-like sclerites was also examined in spen mutants. In wild-type embryos, overexpression of Tsh protein throughout the embryo results in transformation of head regions toward thoracic identity, as well as poorly differentiated denticle belts, especially in the thorax. In the thorax of spen mutant embryos that also overexpress Tsh, the ectopic ventral head-like sclerites are strongly suppressed. Taken together, these results suggest that spen, tsh and Antp function in a combinatorial manner to repress the development of head-like sclerites and promote the development of thoracic identity (Wiellette, 1999).

split ends mutation enhances E2F overexpression phenotype

The activity of the E2F transcription factor is regulated in part by pRB, the protein product of the retinoblastoma tumor suppressor gene. Studies of tumor cells show that the p16(ink4a)/cdk4/cyclin D/pRB pathway is mutated in most forms of cancer, suggesting that the deregulation of E2F, and hence the cell cycle, is a common event in tumorigenesis. Extragenic mutations that enhance or suppress E2F activity are likely to alter cell-cycle control and may play a role in tumorigenesis. An E2F overexpression phenotype in the Drosophila eye was used to screen for modifiers of E2F activity. Coexpression of dE2F and its heterodimeric partner dDP in the fly eye induces S phases and cell death. Thirty-three enhancer mutations of this phenotype were isolated by EMS and X-ray mutagenesis and by screening a deficiency library collection. The majority of these mutations sorted into six complementation groups, five of which have been identified as alleles of brahma, moira, osa, pointed, and polycephalon (poc: split ends). osa, brm, and mor encode proteins with homology to SWI1, SWI2, and SWI3, respectively, suggesting that the activity of a SWI/SNF chromatin-remodeling complex has an important impact on E2F-dependent phenotypes. Mutations in poc also suppress phenotypes caused by p21(CIP1) expression, indicating an important role for polycephalon in cell-cycle control (Staehling-Hampton, 1999).

split ends as a modifier of cyclin E function

In higher eukaryotes, cyclin E is thought to control the progression from G1 into S phase of the cell cycle by associating as a regulatory subunit with cdk2. To identify genes interacting with cyclin E, a screen was carried out in Drosophila for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of the eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that were identified fall into four complementation groups corresponding to the genes split ends, dacapo, dE2F1, and Cdk2 (Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19) to be addressed. The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, the results presented here demonstrate that the equivalent sites in Cdk2 play no essential role (Lane, 2000).

split ends as a modifier of kinase suppressor of Ras function

kinase suppressor of ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of Ksr is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of Ksr remains unresolved. To further elucidate the role of Ksr and to identify proteins that may be required for Ksr function, a dominant modifier screen was conducted in Drosophila based on a Ksr-dependent phenotype. Overexpression of the Ksr kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with Ksr might modify the Ksr-dependent phenotype. Approximately 185,000 mutagenized progeny were screened for dominant modifiers of the Ksr-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for Ksr in Ras1 signaling. Mutations in genes encoding known components of the Ras pathway were isolated in a screen for the 14-3-3epsilon, Dsor1/mek, rolled/mapk, pointed, yan, and ksr loci. Furthermore, due to the ability of dominant-negative KSR (KDN) to block RAS/MAPK-mediated signaling, mutations in genes expected to function upstream of ksr were also isolated. These included mutations in the Egfr, Star, Sos, and Ras1 loci. In addition, 4 additional complementation groups were identfied. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor (Therrien, 2000).

Since alleles of EK2-9 failed to complement two independent P elements recently shown to disrupt the RRM-motif protein locus split ends (spen), it is concluded that EK2-9 is allelic to spen. This gene encodes at least three large (~5500 amino acids) and closely related protein isoforms. The Spen proteins contain three RRMs at the N terminus and a conserved region of unknown function at the C terminus. The presence of three RRMs suggests that the Spen proteins mediate their effect via a RNA binding-dependent mechanism such as RNA processing or transport. Interestingly, mutant alleles of spen have been isolated in several independent genetic screens in Drosophila. They were initially recovered in a screen for mutations affecting peripheral nervous system development. Subsequently, they were isolated in two related screens that, like the KDN screen, were designed to identify novel components of the Ras1 pathway. Mutations in spen were isolated as dominant enhancers of a Raf-induced [E(Raf) 2A complementation group] and a Yan-induced rough eye phenotype. The fact that three separate screens targeting Ras1 signaling (KSR-, Raf-, and Yan-dependent) recovered mutant alleles of spen suggests that this locus is relevant for Ras1-mediated signal transduction. Examination of the genetic interactions, however, indicates that the role of spen in Ras1 signaling may not be straightforward. For example, the ability of the E(Raf)2A/spen alleles to dominantly enhance an activated Raf-dependent phenotype suggests that spen is a negative regulator of the pathway. However, alleles of spen also enhance Raf^HM7 lethality at 18°, and homozygous mutant clones in the eye are often missing R7 and outer photoreceptor cells, although extra photoreceptor cells are occasionally found. These results are more consistent with a positive role for spen during Ras1 signaling and would agree with the fact that spen mutations have been recovered as enhancers of sE-KDN and gmr-yan^act. Alleles of spen were also recovered as enhancers of a loss-of-function phenotype for the Hox gene Deformed (Dfd). In that context, the genetic analysis of spen suggests that it functions in parallel to Dfd for the specification of head cuticular structures. Finally, mutations in spen have been identified as dominant enhancers of an E2F/Dp-induced rough eye phenotype and as dominant suppressors of p21^CIP1-induced phenotypes. The fact that S-phase entry is stimulated by the overexpression of E2F and Dp proteins in the eye but inhibited by p21^CIP1 overexpression suggests that spen may be involved in the negative regulation of cell cycle progression (Therrien, 2000).

Currently it is unknown how spen activity links Ras1-dependent cell differentiation, Hox-dependent segment specification, and E2F-dependent cell cycle control. Nonetheless, their common requirement for spen function suggests that they are interconnected. In agreement with this idea, other loci have been found in common in the screens mentioned above, as well as in other related screens. One of these loci corresponds to the kismet (kis) gene. In addition to the EK2-4/kis alleles identified in this KDN screen, mutations in kis were recovered as dominant enhancers in the Dfd screen and as dominant suppressors in a Polycomb (Pc) loss-of-function screen. Alleles of kis have also been identified as dominant suppressors of the synthetic lethality generated by the coexpression of activated Sevenless (SevS11) and Ras1^V12 (Therrien, 2000).

Split ends is a tissue/promoter specific regulator of Wingless signaling

Wingless directs many developmental processes in Drosophila by regulating expression of specific target genes through a conserved signaling pathway. Although many nuclear factors have been implicated in mediating Wingless-induced transcription, the mechanism of how Wingless regulates different targets in different tissues remains poorly understood. The split ends gene is shown to be required for Wingless signaling in the eye, wing and leg imaginal discs. Expression of a dominant-negative version of Split ends resulted in more dramatic reductions in Wingless signaling than split ends-null alleles, suggesting that it may have a redundant partner. However, removal of split ends or expression of the dominant-negative has no effect on several Wingless signaling readouts in the embryo. The expression pattern of Split ends cannot explain this tissue-specific requirement, because the protein is predominantly nuclear and present throughout embryogenesis and larval tissues. Consistent with its nuclear location, the Split ends dominant-negative acts downstream of Armadillo stabilization. These data indicate that Split ends is an important positive regulator of Wingless signaling in larval tissues. However, it has no detectable role in the embryonic Wingless pathway, suggesting that it is a tissue or promoter-specific factor (Lin, 2003).

In this study, a total of seven distinct readouts of Wg signaling were examined in imaginal discs. They are: inhibition of interommatidial bristle formation; MF initiation/progression; repression of Wg and DFz2, and activation of Sens expression at the presumptive wing margin; inhibition of dpp expression in the dorsal leg; and reduction of eye size. Wg regulation of six of these readouts is significantly blocked by partial or complete removal of spen and/or the expression of spen^DN. These results provide a strong genetic argument that spen is required for Wg signaling in these tissues (Lin, 2003).

Interpreting spen phenotypes is complicated by the fact that spen has been implicated in several other pathways. Can these functions explain the apparent loss of Wg signaling phenotypes observed? spen has been found to act with Deformed to suppress head identity in the embryonic trunk and spen genetically interacts with cell cycle mutants. It is thought unlikely that these spen functions can account for the phenotypes observed. However, Spen has also been shown to be involved with the Ras and Notch signaling pathways, which do affect the readouts employed for studying Wg signaling. Therefore, it is possible that some of the spen phenotypes documented in this study are due to disruption of these signaling cascades, though it is argued that this is unlikely (Lin, 2003).

spen mutations affect some Ras targets in a way that suggests it acts positively in Ras signaling. This may be the explanation for the non-autonomous derepression of Ac expression adjacent to spen clones in P[sev-wg] eyes, since Dl expression is activated by the EGF/Ras pathway in the eye. Ras signaling plays a positive role in MF progression and elevated Ras signaling can suppress a Wg or Arm induced small eye phenotype. Therefore, a reduction in Ras signaling caused by loss of spen cannot explain the observations. Ras signaling has no effect on wing margin formation and acts downstream of Wg/Dpp crossregulation in the leg, again arguing that the role of Spen in Ras signaling cannot account for the apparent Wg signaling defects observed (Lin, 2003).

Expression of Suppressor of Hairless [Su(H)], a transcription factor required for Notch signaling, is significantly reduced in spen mutant embryos. Can a reduction of Notch signaling explain the results? Notch signaling is required for interommatidial bristle inhibition so this could explain the requirement of spen for Wg-dependent Ac inhibition. However, Notch is absolutely required for Wg expression at the DV stripe in the wing and plays a positive role in MF progression. Thus, reducing Notch activity by loss of spen or spen^DN cannot explain the wider Wg stripe and suppression of the dpp^blk MF defect observed (Lin, 2003).

Though no evidence for elevated Notch signaling in spen mutants has been reported in Drosophila, a recent report has suggested that SHARP, a human Spen homolog, functions as a transcriptional co-repressor for RBP-Jkappa/CBL, the ortholog of Su(H). In addition, the fly homolog of human SMRT, which binds to SHARP, has been shown to act as a negative regulator of Notch signaling. This could mean that loss of spen activity in flies results in higher expression of Notch/Su(H) targets, owing to derepression. Although this could conceivably contribute to the MF and wing phenotypes that were found, such derepression could not account for the suppression of Wg-dependent reduction of eye size and bristle inhibition or the derepression of dpp expression in the leg. In summary, the only explanation consistent with all the spen (or spen^DN) imaginal disc phenotypes is a loss of Wg signaling (Lin, 2003).

In contrast to the data in the imaginal discs, no evidence was found for the involvement of spen in Wg signaling in the embryo, either by removing spen gene activity or expressing spen^DN. Thus, it appears that Spen may be a tissue-specific regulator of Wg signaling. Spen is a predominately nuclear protein expressed ubiquitously in embryos and imaginal discs. It could be that a Spen co-factor is not expressed in embryos, or that Spen is post-translationally modified in a tissue-specific way. Alternatively, the specificity could lie in the promoters of the targets that were tested. This appears to be the case in the wing, where Wg and Sens regulation by Wg signaling is spen dependent, while that of Fz2 is not (Lin, 2003).

The negative results obtained in the embryo cannot be viewed as definitive. Embryos that lack maternal and zygotic spen activity could be normal for Wg signaling because of redundancy. Likewise, even though expression of spen^DN in the imaginal discs causes strong Wg loss of function phenotypes, and causes spen-like phenotypes under mild expression conditions in the embryo, it is possible that adequate amounts of spen^DN were not supplied in embryonic assays (Lin, 2003).

Experiments with loss of function spen alleles indicate that spen is not absolutely required for Wg signaling in the wing and eye. Although reduction of spen activity suppresses a dpp^blk MF defect, which can be explained by a reduction in Wg signaling, complete removal of spen does not cause an ectopic MF. Because removal of Wg signaling is known to induce an ectopic MF, this indicates that sufficient Wg signaling still occurs in the spen clones. In the wing, spen clones affect Wg readouts, but with incomplete penetrance: this again indicates a partial reduction in Wg signaling in the absence of spen (Lin, 2003).

Experiments with spen^DN suggest that the partial loss of Wg signaling in spen mutants may be due to redundancy. Expressing spen^DN causes more severe phenotypes and much higher penetrance in disruption of Sens and expansion of Wg in the wing than complete removal of spen. A likely explanation is that the Spen^DN protein also inhibits the function of another gene that has roles in the Wg pathway redundant to spen (Lin, 2003).

Although many genes exist in the fly genome that encode proteins containing RRMs, only one other besides Spen is predicted to encode a protein with both RRMs and a SPOC domain. This factor has been called short Spen-like protein (SSLP or DmSSp) and is referred to as CG2910 in the annotated genome. No genetic or molecular characterization of SSLP has been reported and its possible redundancy with spen is currently being examined (Lin, 2003).

Where does Spen act in the Wg pathway? Epitasis experiments in the eye indicate that Spen^DN blocks Wg signaling downstream of Arm stabilization. Thus, Spen could act in Arm nuclear import, or in mediating TCF/Arm transcriptional regulation. Consistent with a role in Wg target gene transcription, Spen is predominantly nuclear in imaginal tissues. In addition, the mouse and human homologs of Spen have been implicated as transcription factors (Lin, 2003).

Studies on the vertebrate homologs of Spen have provided functions for the RRM and SPOC domain that these proteins share with Spen. Spen has three predicted RRMs near its N terminus. The role of RRMs in specific RNA binding is well established and the RRM domains in the human Spen homolog SHARP has been shown to bind to the steroid receptor RNA co-activator SRA. By contrast, the RRM domain of the mouse Spen homolog, MINT, has been shown to bind to specific double-stranded DNA, including the proximal promoter of the osteocalcin gene. SHARP also binds to the nuclear receptor co-repressor SMRT and acts as a transcription corepressor by recruiting histone deacetylases (HDACs) through its SPOC domain. A similar co-repressor function for SHARP with the DNA-binding protein RBP-Jkappa/CBL has also been reported. Finally, MINT was also found to interact with Msx2, a known transcriptional repressor. These studies on the vertebrate homologs suggest that Spen may bind DNA or RNA at its N terminus, and may regulate the Wg pathway as a transcription corepressor (Lin, 2003 and references therein).

Why is spen required for only a subset of Wg targets? Based on studies with its vertebrate homologs, could spen only regulate the Wg targets that are transcriptionally repressed by TCF/Arm? Wg-dependent transcriptional inhibition through TCF has been shown for the stripe gene in the embryo and has been suggested for bristle inhibition in the eye. However, no direct targets of Wg signaling in the imaginal discs have been determined and attempts to determine whether stripe repression in the embryo requires spen have been inconclusive. It is interesting to note that two embryonic targets tested which were spen independant, eve and slp1, are both directly activated by TCF/Arm. Identification of spen-dependent direct targets of Wg signaling will be necessary to explore this model (Lin, 2003 and references therein).

Two factors have previously been reported that are tissue/promoter-specific regulators of Wg signaling. tsh has been shown to be required for Wg-mediated inhibition of denticle formation in the ventral embryonic epidermis and lines, which is needed for Wg signaling only in the dorsal epidermis. A third factor, Spen, has been reported, that is only needed for imaginal disc regulation of Wg targets. The existence of these specific factors begs the question: what is the difference between the various Wg targets that requires such specificity (Lin, 2003)?

The product of the split ends gene is required for the maintenance of positional information during Drosophila development

The Drosophila split ends (spen) gene encodes a large nuclear protein containing three RNP-type RNA binding motifs, and a conserved transcriptional co-repressor-interacting domain at the C-terminus. Genetic analyses indicate that spen interacts with pathways that regulate the function of Hox proteins, the response to various signaling cascades and cell cycle control. Although spen mutants affect only a small subset of morphological structures in embryos, it has been difficult to find a common theme in spen mutant structural alterations, or in the interactions of spen with known signaling pathways. By generating clones of spen mutant cells in wing imaginal discs, it has been show that spen function is required for the correct formation and positioning of veins and mechanosensory bristles both on the anterior wing margin and on the notum, and for the maintenance of planar polarity. Wing vein phenotypic alterations are enhanced by mutations in the crinkled (ck) gene, encoding a non-conventional myosin, and correlate with an abnormal spatial expression of Delta, an early marker of vein formation in third instar wing imaginal discs. Positioning defects were also evident in the organization of the embryonic peripheral nervous system, accompanied by abnormal E-Cadherin expression in the epidermis. The data indicate that the role of spen is necessary to maintain the correct positioning of cells within a pre-specified domain throughout development. Its requirement for epithelial planar polarity, its interaction with ck, and the abnormal E-Cadherin expression associated with spen mutations suggest that spen exerts its function by interacting with basic cellular mechanisms required to maintain multicellular organization. This role for spen may explain why mutations in this gene interact with the outcome of multiple signaling pathways (Mace, 2004).

How could Spen instruct cells to maintain a specific position, without affecting their fate directly? A plausible explanation is that it could affect cell adhesion. In fact, in spen mutant embryos, the expression of E-cadherin was up-regulated at sites of high epithelial morphogenetic activity, generating a phenotype similar to the E-cadherin mutant embryos, as it has been observed in other studies. It is plausible that the increase in E-cadherin expression is the result of a wound response to a defect in epithelial integrity, caused by spen mutations. A defect in cell adhesion and/or cytoskeletal rearrangements could also explain specific aspects of the spen embryonic phenotype. The holes that result in abnormal cuticle deposition in the embryonic epidermis are due to a failure of epidermal epithelial integrity. These cells subsequently undergo a wound response at the end of embryogenesis. Additionally, some of the phenotypes resulting from the loss of spen are indeed similar to those seen in mutants for the gene encoding Daschous, a cadherin involved in cell adhesion. However, blistering of the wings, a phenotype that is often found in cell adhesion mutants, was never observed in any of the spen mutant clones (Mace, 2004).

A role for Spen in cell adhesion and/or cytoskeletal rearrangements could also be inferred through its genetic interaction with crinkled (Myosin VIIA), and the planar cell polarity phenotype observed in mutant cells for spen in the wing blade. Myosin VIIA is associated with the cadherin-catenins complex and participates in the creation of a tension force between the actin cytoskeleton and adherens junctions, which is predicted to strengthen cell-cell adhesion. Furthermore, ck acts downstream of Drosophila Rho-associated kinase (Drok), which links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Myosin VIIA mutations have been described in vertebrates, including those causing the Usher syndrome in humans, the shaker-1 mutation in mice, and the mariner mutation in zebrafish. Interestingly, these mutations, among other symptoms, cause splaying and abnormal distribution of sensory hair cells in the inner ear, leading to deafness in mice and humans, and mechanosensory defects in zebrafish. It seems plausible that spen may regulate the expression or function of components affecting the outcome of pathways involved in cytoskeletal rearrangements and epithelial planar polarity and, hence, affect cell positioning. However, a direct requirement for spen function in the Ck or Drok pathways is unlikely, since mutations in these genes result in different phenotypes from those observed in spen mutants (Mace, 2004).

An influential role for spen in mechanisms of intercellular adhesion and/or cytoskeletal rearrangements may also be relevant to understanding its suggestive role in human cancer. The search of public human sequence resources reveals one spen ortholog (SHARP), and three putative Short Spen-like Protein (SSLP) orthologs in the human genome. At least one of these genes (OTT/RBM15) is involved in a recurrent translocation detected in acute megakaryocitic leukemia, and a potentially aberrant transcript for another human SSLP ortholog at 3p21 has been identified in cDNA isolated from human cancer cells. Despite the presence of common domains, the functional relationship between large and small Spen-related polypeptides is still unknown. It is plausible that in Drosophila, SSLP might rescue some of the functions of spen during early embryonic development, as evidenced by the incomplete penetrance of phenotypes seen in spen maternal and zygotic mutant embryos. Complementation at this level has been suggested by others to explain the incomplete penetrance of spen mutations in wing discs, although it should be noted that the region required for Spen to interact with transcription factors such as Msx-2 or nuclear receptors, is apparently missing in SSLP proteins. Therefore, the potential redundancy of Spen and SSLP will have to be determined (Mace, 2004).

This study has shown that the function of the spen gene is essential for all stages of development. The experimental evidence indicates that Spen participates in processes that regulate planar cell polarity and may influence cytoskeletal organization, and its loss results in specific phenotypes that can not be solely explained by defects in a specific signaling pathway. In order to unify this observations with those previously reported by others, it is proposed that the function of Spen is necessary for the maintenance of correct cell positioning during growth, ensuring that structures that are determined early during development are correctly positioned in the adult. Since cells are determined early during development to become part of a specific structure, their position has to be maintained during growth according to a pre-established pattern. If cells were unable to maintain their position, phenotypes similar to those obtained in spen mutant clones would be expected. Structures would be misplaced, and in some cases would be absent if the cells that were predetermined to adopt a specific fate fall within 'forbidden' positions. This mechanistic model could explain why spen interacts genetically with signaling pathways that require and/or specify precise spatial organization during metazoan development (Mace, 2004).

Characterization of the split ends-like gene spenito reveals functional antagonism between SPOC family members during Drosophila eye development

The novel family of SPOC domain proteins is comprised of broadly conserved nuclear factors that fall into two subclasses, termed large and small, based on protein size. Members of the large subgroup, which includes Drosophila SPEN and human SHARP, have been characterized as transcriptional corepressors acting downstream of a variety of essential cell signaling pathways, while those of the small subclass have remained largely unstudied. Since SPEN has been implicated in Drosophila eye development, and the small SPOC protein (Spenito) Nito is also expressed in the developing eye, this context was used to perform a structure/function analysis of Nito and to examine the relationship between the two SPOC family subclasses. The results demonstrate that the phenotypes obtained from overexpressing Nito share striking similarity to those associated with loss of spen. Dosage sensitive genetic interactions further support a model of functional antagonism between Nito and SPEN during Drosophila eye development. These results suggest that large and small SPOC family proteins may have opposing functions in certain developmental contexts (Jemc, 2006).

spen encodes the founding member of a family of proteins characterized by three N-terminal RNA recognition motifs (RRMs) and a novel C-terminal domain, called the SPEN Paralog Ortholog Conserved domain or SPOC domain. SPEN orthologues have been identified in worms, flies, mosquito, mouse, human, and other vertebrates, and more recent studies have identified proteins in plants and yeast carrying the SPOC domain in conjunction with other functional motifs. The RRMs suggest a role for SPOC family proteins in RNA or DNA binding and in the case of SPEN are necessary for nuclear localization, while the SPOC domain of SPEN and its human and mouse orthologs SHARP (SMRT/HDAC1 Associated Repressor Protein) and MINT (Msx2-interacting nuclear target protein) has been implicated in transcriptional regulation and repression (Kuroda, 2003; Oswald, 2002; Shi, 2001; Yang, 2005). SPOC family proteins can be further divided into two subclasses based on their size. In contrast to large SPOC family proteins almost nothing is known about the functions of small SPOC proteins. Thus far, only the human small SPOC family member One Twenty Two (OTT)/ RNA-Binding Motif protein-15 (RBM15) has been studied. Specifically, chromosomal translocations identified in cases of acute megakaryocytic leukemia revealed a fusion with MAL (Megakaryocytic Acute Leukemia)/MKL1 (Megakaryoblastic Leukemia-1) that results in a chimeric protein that includes almost the entire coding region of both genes, with 4 RBM15/OTT at the N-terminus and MAL/MKL1 at the C-terminus (Ma, 2001; Mercher, 2001). Recent evidence suggests that the RBM15-MKL1 fusion may contribute to leukomogenesis through an increased ability to activate serum response factor (SRF) target genes (Jemc, 2006 and references therein).

Sequence conservation defines two distinct SPOC family subclasses: SPOC family proteins fall into two apparent subclasses based on their size. To determine whether such a distinction might be functionally significant, sequence alignments of the conserved Cterminal SPOC motif were performed to compare the level of sequence conservation in the SPOC family in general and subclass members in particular. Analysis revealed only 27% identity and 50% overall similarity between the SPOC domains of SPEN and Nito, the Drosophila representatives of the large and small subfamilies, respectively; however, upon comparison of the SPOC domains of these proteins with those of their respective subclass family members, a higher level of conservation was revealed. Drosophila SPEN and human SHARP exhibit 58% sequence identity and 79% overall sequence similarity, while Drosophila Nito and human RBM15/OTT share 47% sequence identity and 62% overall sequence similarity. Comparable results were obtained by comparing the RRM motifs. These results reveal a higher level of sequence conservation within SPOC family subclasses relative to the family in general, raising the possibility that subclasses may have adopted divergent functions (Jemc, 2006).

Overexpression of nito perturbs adult eye morphology: To better understand the relationship between large and small SPOC proteins, it was determined if spen and nito function synergistically or antagonistically in vivo. Because the large SPOC family member spen is required for Drosophila eye development and the fly eye provides a uniquely powerful system in which to explore functional relationships between signaling molecules, this analyses focused on the eye. RT-PCR confirmed that nito, like spen, is expressed in the developing eye disc (Jemc, 2006).

Because no nito mutants are currently available, an in vivo structure-function analysis was undertaken to investigate nito function during eye development. While the phenotypes resulting from overexpression of a gene must be interpreted with caution, such overexpression models frequently result in sensitized genetic systems that can provide powerful tools for investigating in vivo relationships between signaling molecules. Myc-tagged full-length Nito (Nito-FL), Nito lacking the N-terminus (NitoDN; an exogenous nuclear localization sequence was added to ensure proper nuclear targeting) and Nito lacking the C-terminus (NitoDC) were cloned downstream of a UAS promoter and the transgenes were expressed in flies using eye specific GAL4 drivers. Three different sevenless-Gal4 (sev-Gal4) drivers, which promote expression in photoreceptors R1, R3, R4, R6, R7, the cone cells and the 'mystery' cells, which are poorly understood interommatidial cells that are never recruited to the ommatidia and ultimately apoptose, were utilized in this study: sev^strong couples the sev enhancer to the hsp70 promoter, resulting in the highest levels of expression; sevmedium contains both the sev enhancer and sev promoter, and expresses at an intermediate level; sev^weak contains the same regulator sequences as ^sevmedium, but expresses at lower levels, presumably as a consequence of position effect of the transgene. To avoid unnecessary confusion, these will be referred to collectively as sev-Gal4 (Jemc, 2006).

Sev-Gal4 driven overexpression of Nito-FL and NitoDC yielded dosage dependent adult rough eye phenotypes, while overexpression of NitoDN was indistinguishable from wild type. Western blots confirmed the expression of all transgenes, and immunohistochemistry showed nuclear localization of Nito in all cases, indicating that the lack of a NitoDN phenotype is not due to the absence or mislocalization of protein. While overexpression of Nito-FL and NitoDC both perturb eye development, the resulting phenotypes are distinct. This data is not unexpected given previous results for the large SPOC family protein, SPEN, in which overexpression of SPENDC functions as a dominant negative with respect to spen (Jemc, 2006).

It was therefore speculated that NitoDC functions analogously as a dominant negative relative to nito whereas Nito-FL expression simply augments the pool of full-length Nito. Specifically, it was observed that overexpression of Nito-FL results in roughening of the posterior part of the eye and an overall decrease in eye size, whereas overexpression of NitoDC more uniformly perturbs the external morphology of the eye (Jemc, 2006).

In order to distinguish the Nito-FL and NitoDC rough eye phenotypes at the cellular level, adult eyes were sectioned and examined for defects. In wildtype ommatidia, photoreceptors are arranged in a trapezoidal array with seven of the eight photoreceptors visible in one plane of view. The regular trapezoidal arrangement of photoreceptors is disturbed in both overexpression systems. When Nito-FL is overexpressed, a decrease in the number of photoreceptors per ommatidia, elongated rhabdomeres, as well as a general disorganization of the ommatidia are seen. These observations suggest that the rough eye phenotype is due to a loss of photoreceptors and possible defects in the accessory cells, which normally provide support for the rhabdomeres in the ommatidia. This phenotype is strikingly reminiscent of that seen in sections of spen mutant eye clones, raising the possibility that overexpressed nito may function antagonistically with respect to spen in the developing eye (Jemc, 2006).

Eyes overexpressing NitoDC also appear disorganized compared to wildtype, although in contrast to Nito-FL ommatidia, photoreceptor number is not strongly affected. Rather, the most prevalent defect appears to be ommatidial fusions suggesting that cone and pigment cells, rather than photoreceptors are most affected. Given that the Gal4 driver used for these experiments is expressed primarily in a subset of photoreceptors, the cone cells and interommatidial mystery cells, the accessory cell defects observed upon nito overexpression may be due in part to indirect effects on pigment cells. Thus, Nito-FL ommatidia have defects in photoreceptor number and ommatidial morphology, while NitoDC ommatidia have defects in accessory cells required for the spacing of ommatidia (Jemc, 2006).

To further investigate the defects caused by overexpressing nito, the effects of increasing nito expression in early eye development were examined. First, recruitment of the photoreceptor neurons into ommatidia was examined by looking at expression of the panneural marker ELAV in the larval precursor to the eye, the eye imaginal disc. Consistent with the differences observed in the adult phenotypes, the larval phenotypes associated with sev-Gal4 driven expression of Nito-FL and NitoDC are also distinct. In eye discs overexpressing Nito-FL, initial recruitment of photoreceptors appears normal. However, approximately seven rows posterior to the furrow, there is a decrease in the number of photoreceptors per ommatidia. Thus while Nito-FL expression does not perturb initial photoreceptor recruitment, subsequent development and/or survival are compromised, resulting in the reduced number of photoreceptors observed in the adult eye. The loss of photoreceptors upon overexpression of Nito-FL is also similar to spen mutant clones, which have reduced numbers of photoreceptors in mutant ommatidia in the developing imaginal disc, consistent with the observations made in adult eye sections. In contrast to Nito-FL and spen mutant clones, and consistent with the ommatidial fusions observed in adult eye sections, overexpression of NitoDC causes loss of spacing between ommatidia in the larval eye disc, while recruitment of photoreceptors is not affected (Jemc, 2006).

To examine the possibility that the phenotypes associated with overexpression of Nito-FL and NitoDC were due primarily to cell death, eye discs were stained with the apoptotic marker acridine orange. In the wildtype eye disc very little cell death is observed. In Nito-FL eye discs, a stripe of cell death occurs in the posterior part of the differentiating eye disc, consistent with the loss of photoreceptors observed in the ELAV-probed eye disc and similar to the elevated cell death phenotype observed in spen mutant clones. However coexpression of the apoptotic inhibitor p35 or introduction of the H99 Deficiency that removes the proapoptotic genes hid, reaper and grim, did not suppress the Nito-FL rough eye phenotypes, suggesting that increased apoptotic cell death is unlikely to be the primary factor contributing to the Nito-FL associated eye defects. In discs overexpressing NitoDC, increased cell death is observed more anteriorly relative to that for Nito-FL, consistent with the ommatidial spacing defects observed in the ELAV-probed disc (Jemc, 2006).

The potential for functional antagonism between SPEN and Nito was suggested by the similarity of phenotypes observed in adult eye sections overexpressing Nito-FL and in spen mutant eye clones. To further investigate this potential antagonism, a series of dose-sensitive genetic interactions between spen and nito was performed (Jemc, 2006).

Initially, the effects of reducing spen levels in the Nito-FL overexpression background was examined. If Nito-FL antagonizes SPEN function, as suggested by the phenotypic analysis, further reducing spen should exacerbate the Nito-FL overexpression phenotype. An important requirement for such an experiment is the need for dose-sensitive Nito-FL phenotypes. Two observations suggest Nito-FL provides a dose-sensitive phenotype ideal for studying genetic interactions: (1) expression of independent transgenic lines with the same sev-Gal4 driver results in a range of phenotypes, and (2) expression of a given Nito-FL transgene with sev^weak results in a mild rough eye phenotype whereas expression of the same line at a higher level produces a more severe phenotype. Consistent with the hypothesis of an antagonistic relationship between spen and nito, it was found that heterozygosity for a null spen allele enhanced the rough eye phenotype associated with Nito-FL expression, as demonstrated by an increased number of ommatidia lacking photoreceptors. Next, the consequences of increasing or decreasing nito levels in the background of a dominant negative spen transgene (spenDN), which encodes the C-terminal 936 amino acids of spen), and also produces dose sensitive phenotypes, that were examined. Because both transgenes are capable of perturbing eye development on their own, in order to distinguish between additive and synergistic interactions a Nito-FL transgenic line was used that when expressed with sev^weak exhibits only very mild perturbations of the adult eye (Jemc, 2006).

As expected, given the Nito structure-function analysis, Nito-FL causes an enhancement of the spenDN rough eye phenotype, an increase in necroses in the eye and a complete loss of organization. Thus, overexpression of nito and overexpression of spenDN appear to act in the same direction, suggesting opposing functions for Nito and Spen. Since loss-of-function mutations in nito have not been isolated, a nito transgenic dsRNA construct was generated to investigate the consequences of reducing endogenous nito expression levels with respect to spen function. RT-PCR from Drosophila eye discs confirmed that this construct mediates partial knockdown of nito expression. In vivo, while dsRNA-mediated knockdown of nito expression does not perturb eye morphology on its own, nito-RNAi partially rescues the rough eye phenotype resulting from overexpression of spenDN, again suggesting antagonism between nito and spen. Eye sections show fewer missing ommatidia in nito-RNAi, spenDN adult eyes relative to those overexpressing spen DN alone, as well as fewer missing photoreceptors in ommatidia lacking the full complement of photoreceptors and more normal rhabdomere morphology. Together, these dose-sensitive genetic interactions argue for mutual antagonism between the large SPOC family member spen and the small SPOC family representative nito during Drosophila eye development (Jemc, 2006).

It remains to be determined if the antagonistic relationship between nito and spen is maintained in developmental contexts outside of the eye. Previous work examining the role of SPEN in Wingless signaling suggested the presence of a redundant partner for SPEN, for which Nito would be a good candidate, given their sequence conservation. In situ hybridization for nito and spen suggests they are also both ubiquitously expressed throughout embryonic development, and considering the broad range of embryonic phenotypes attributed to spen mutants, exploration of context specific interactions between spen and nito in the embryo will likely improve understanding of the relationships between these two related proteins. It is predicted that certain developmental events will require synergism between nito and spen, whereas others, as was demonstrate in the eye, will require antagonism (Jemc, 2006).

At the cellular level, spen is implicated as a positive component of Wingless and RTK/RAS signaling, and large SPOC family proteins SHARP and MINT are implicated as negative regulators of Notch signaling (Kuroda, 2003; Oswald, 2002). Given the ability of nito to antagonize spen function in the developing eye, it seems reasonable to speculate that Nito also acts as a downstream regulator/effector of some or all of these pathways. Furthermore, the antagonism between nito and spen may provide a mechanism for differential regulation of output from these pathways. Mechanistically, how might one envision the mutual antagonism between Spen and Nito? Large SPOC proteins have been previously shown to serve as transcriptional corepressors. Thus one attractive possibility is that small SPOC proteins might serve as transcriptional activators. In this model, by virtue of their conserved RRM and SPOC motifs, small and large SPOC proteins might compete for access to common binding partners. The resulting complexes, depending on whether they contain Spen or Nito, would then either repress or activate transcription. In a slight variation of the model, one could propose that SPOC proteins might be able to either repress or activate transcription, and so depending on context, would either act synergistically or antagonistically. Unfortunately, Drosophila cultured cells do not provide an appropriate environment in which to assay the activity of SPOC proteins so it was not possiable to test this model with respect to Spen and Nito. However, using mammalian COS cells, it was observed that while the SPOC motif of SHARP represses transcription, the SPOC motif of RBM15, the human Nito ortholog, strongly activates transcription. Thus, perhaps the antagonistic relationship between Spen and Nito that is reported in this study in the context of Drosophila eye development reflects a conserved antagonistic relationship between large and small SPOC proteins that is manifested at the level of transcriptional output (Jemc, 2006).

In conclusion, an antagonistic relationship has been demonstrated between the large and small SPOC family proteins in the developmental context of the Drosophila eye. The finding that SPOC family proteins function as downstream effectors of a variety of signaling pathways suggests they may act to fine-tune transcriptional output downstream of these cascades. Thus, it will be extremely interesting to determine whether the antagonistic relationship have observed between Nito and Spen in the eye is a general property of small and large SPOC proteins, or if it is unique to Drosophila eye development. Determination of transcriptional targets and cofactors will be required to understand how SPOC family proteins function to regulate and integrate information from these signaling pathways (Jemc, 2006).

Split ends antagonizes the Notch and potentiates the EGFR signaling pathways during Drosophila eye development

The Notch and Epidermal Growth Factor Receptor (EGFR) signaling pathways interact cooperatively and antagonistically to regulate many aspects of Drosophila development, including the eye. How output from these two signaling networks is fine-tuned to achieve the precise balance needed for specific inductive interactions and patterning events remains an open and important question. The gene split ends (spen) functions within or parallel to the EGFR pathway during midline glial cell development in the embryonic central nervous system. This study shows that the cellular defects caused by loss of spen function in the developing eye imaginal disc place spen as both an antagonist of the Notch pathway and a positive contributor to EGFR signaling during retinal cell differentiation. Specifically, loss of spen results in broadened expression of Scabrous, ectopic activation of Notch signaling, and a corresponding reduction in Atonal expression at the morphogenetic furrow. Consistent with Spen's role in antagonizing Notch signaling, reduction of spen levels is sufficient to suppress Notch-dependent phenotypes. At least in part due to loss of Spen-dependent down-regulation of Notch signaling, loss of spen also dampens EGFR signaling as evidenced by reduced activity of MAP kinase (MAPK). This reduced MAPK activity in turn leads to a failure to limit expression of the EGFR pathway antagonist and the ETS-domain transcriptional repressor Yan and to a corresponding loss of cell fate specification in spen mutant ommatidia. It is proposed that Spen plays a role in modulating output from the Notch and EGFR pathways to ensure appropriate patterning during eye development (Doroquez, 2007).

This study demonstrates that loss of spen perturbs the normal balance between the EGFR and Notch pathways as evidenced by the patterning disruptions and aberrant expression of multiple pathway components. These findings raise the question of whether Spen functions primarily in the Notch pathway, primarily in the EGFR pathway, or as a critical component of both. Although definite resolution is difficult given the extensive and intricate feedback regulation within and between these two signaling networks, a model is proposed in which Spen-mediated antagonism of the Notch pathway regulates the signaling flow through the EGFR pathway to achieve proper retinal cell fate specification (Doroquez, 2007).

Loss of spen results in hyperactivation of the Notch pathway as evidenced by elevated levels of both Notch and its transcriptional targets, the E(spl)-bHLHs. Therefore, a normal function of Spen in the developing eye is to limit the activity of Notch. Consistent with this model, heterozygous reduction of spen was shown to be sufficient to suppress the heterozygous Notch wing margin phenotype. However, loss of spen does not lead to the anti-neurogenic phenotypes typically associated with overexpression/overactivation of canonical members of the Notch pathway, suggesting that although Notch signaling output is elevated, the increase is below the threshold needed to achieve such phenotypes. Consistent with this interpretation, recruitment of the initial R8 photoreceptor neuron, a process influenced at multiple stages by Notch signaling, occurs normally in the absence of spen (Doroquez, 2007).

Where might Spen interface with the Notch signaling pathway? The striking increase in Sca expression in spen mutant clones at the MF is consistent with Spen regulating Notch activation by limiting the expression of sca either through transcriptional repression or by destabilizing the transcript. This suggests that in the Drosophila eye Spen may have an upstream role in the Notch pathway in contrast to the downstream role described for Spen mammalian orthologs. In contrast, because of extensive feedback regulation in Notch signaling, it is plausible that Spen interfaces with the network at a more downstream point. For example, ectopic expression of Notch^intra was shown to promote Sca expression, which in turn activates Notch signaling. Additionally, although no such role was detected with respect to yan, it is possible that Spen limits Notch^intra/Su(H)-mediated transactivation at the level of transcriptional repression of other Notch pathway targets, including the E(spl)-bHLHs, as is the case for the mammalian Spen orthologs. This latter mechanism might also be relevant posterior the MF, where Notch signaling remains elevated as judged by increased levels of both Notch and the E(spl)-bHLHs in spen mutant clones, but where Sca is no longer expressed (Doroquez, 2007).

Although pinpointing where Spen interfaces with the Notch signaling pathway remains a challenge, the simplest interpretation of the data is that at the MF, Spen either directly or indirectly regulates Sca expression to restrict Notch pathway output. Posterior to the MF, as discussed below, mutual antagonism between the Notch and EGFR pathways may stabilize the initial signaling imbalance independent of Sca, leading to sustained up-regulation of Notch and down-regulation of EGFR output in spen mutant tissue (Doroquez, 2007).

What might the consequences of a moderate increase in Notch pathway output be? Given the extensive functional antagonism that has been reported between the Notch and EGFR pathways in the eye, a likely outcome is that the increased Notch signaling in spen mutant tissue would dampen EGFR pathway output. Supporting the idea that spen plays a positive role with respect to EGFR signaling, the cell fate specification defects observed in spen mutant clones are highly reminiscent of phenotypes associated with hypomorphic mutants in positive components of the EGFR pathway. Thus, the defective specification of neuronal and non-neuronal cell types and the perturbed R8 spacing adjacent to the MF all suggest reduced, but not ablated, EGFR pathway function in spen clones (Doroquez, 2007).

Lending further support to a model in which elevated Notch signaling in spen clones dampens EGFR pathway output, both Ato and dpERK expression at the MF are reduced. Because previous work has shown that Ato is required for activation of the EGFR pathway at the MF, one possibility is that Spen stabilizes dpERK levels at the MF by antagonizing Notch-mediated lateral inhibition to ensure appropriate Ato expression. Another plausible mechanism for Spen-mediated regulation of dpERK activity would be downstream of or in parallel to Ras. In this scenario, Spen might mediate transcriptional repression of an inhibitor such as a MAPK phosphatase. However, qRT-PCR analysis in imaginal discs predominantly mutant for spen do not indicate a role for Spen in regulating the expression of two characterized Drosophila MAPK phosphatases - dMKP3 and PTP-ER. Thus, validation of such a mechanism will require identification of other MAPK phosphatases or pathway inhibitors that might be regulated by Spen (Doroquez, 2007).

It should be noted that the results of this analysis of spen function in the eye appear contradictory to those from a prior study that suggested spen antagonizes EGFR output and promotes Notch signaling during embryonic neural development (Kuang, 2000). Specifically, elevated EGFR signaling was reported in spen maternal/zygotic null embryos, as evidenced by increased numbers of midline glial cells and loss of Yan expression. However, these results could not be reproduced (F. Chen and I. Rebay, unpublished data reported in Doroquez, 2007). On the contrary, analysis of spen function during midline glial cell development in the embryonic central nervous system was entirely consistent with a role for spen as a positive contributor to EGFR signaling. Thus, at least with respect to EGFR signaling, it is believed that Spen serves an analogous role in multiple developing tissues (Doroquez, 2007).

With respect to Notch signaling, Kuang reported a strong reduction in E(spl)-bHLH expression throughout the embryo but no change in Notch levels, exactly opposite to the current findings in the eye. Additional work will be needed to determine whether and how spen interfaces with the Notch pathway during embryogenesis, and whether distinct or identical mechanisms operate in retinal versus embryonic neural development (Doroquez, 2007).

It is not yet clear whether spen's role in Notch-EGFR interactions posterior to the MF is identical to its role in events occurring at the MF. The failure to down-regulate Yan and the resulting cell fate specification defects show that EGFR signaling posterior to the MF is compromised in spen mutant tissue. Given that Yan up-regulation in spen clones does not result from loss of Spen-mediated transcriptional repression, but rather reflects loss of post-translational control, two models for Spen function seem likely. First, if the inability to detect changes in dpERK protein levels posterior to the MF in situ accurately indicates unaltered dpERK levels, then the ability of dpERK to phosphorylate Yan must be compromised in spen mutants. Alternatively, dpERK levels may be sufficiently reduced to increase Yan stability, but the change may be below the immunohistochemical detection threshold (Doroquez, 2007).

In terms of the signals that impinge on dpERK, whereas Notch signaling and Ato expression are critical for proper dpERK expression in the MF, reiterative EGFR signaling takes over posterior to the MF to maintain dpERK activity. Thus, it is possible that a Spen-dependent, Notch-independent mechanism may regulate EGFR output posterior to the MF. Alternatively, because Notch, E(spl) and Yan expression are all elevated in spen mutant tissue both in and posterior to the MF, Spen-mediated antagonism of Notch signaling may be relevant to EGFR regulation in both contexts. An extension of this idea that results in perhaps the most appealing model is that the initial increase in Notch output at the MF dampens EGFR signaling, which in turn leads to elevated Notch signaling in more posterior regions resulting in reduced EGFR output. In this way, the initial signaling imbalance created by loss of spen at the MF could be maintained over the entire eye disc through mutual antagonism and feedback regulation between the Notch and EGFR pathways (Doroquez, 2007).

In summary, this study analyzed the requirement for spen in regulating the EGFR and Notch pathways during Drosophila eye development, and it is proposed that increased Notch pathway activity upon loss of spen may be sufficient to dampen EGFR signaling, but not to disrupt other downstream effects of Notch signaling. Therefore, because the effects of spen loss appear to be at a threshold below the production of bona fide Notch-related phenotypes, it is suggested that Spen plays a subtle role in the regulation of the Notch pathway or functions redundantly alongside other components. An equally likely hypothesis is that Spen regulates the Notch and EGFR pathways separately and that the phenotypes reported reflect a composite of independent disruptions to both signaling networks (Doroquez, 2007).

Although much of the literature focuses on a primary role for Spen family proteins as co-repressors, recent findings suggest members of this family may also regulate non-coding RNA sequestration, mRNA export, RNA splicing, and proteolysis. Therefore, future identification of the precise molecular mechanisms by which Spen interfaces with the EGFR and Notch pathways may reveal novel modes of interaction between these two critical and conserved signaling networks (Doroquez, 2007).

EVOLUTIONARY HOMOLOGS

Msx2 is a homeodomain transcriptional repressor that exerts tissue-specific actions during craniofacial skeletal and neural development. To identify coregulatory molecules that participate in transcriptional repression by Msx2, a Farwestern expression cloning strategy was applied to identify transcripts encoding proteins that bind Msx2. A lambda gt11 expression library from mouse brain was screened with radiolabeled GST-Msx2 fusion protein encompassing the core suppressor domain of Msx2. A cDNA was isolated that encodes a novel protein fragment that binds radiolabeled Msx2. Homeoprotein binding activity was confirmed by Farwestern analysis of the T7-epitope-tagged recombinant protein fragment, and interactions in vitro require Msx2 residues necessary for transcriptional suppression in vivo. On the basis of biochemical analyses, this novel protein was named MINT, an acronym for Msx2-interacting nuclear target protein. The original clone is part of a 12.6 kb transcript expressed at high levels in testis and at lower levels in calvarial osteoblasts and brain. Multiple clones isolated from a mouse testis library were sequenced to construct a MINT cDNA contig of 11 kb. Starting from an initiator Met, a large nascent polypeptide of 3576 amino acids is predicted, in contiguous open reading frame with the Msx2 interaction domain residues 2070-2394. Protein sequence analysis reveals that MINT has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals. Western blot analysis of fractionated cell extracts reveals that mature approximately 110 kDa (N-terminal) and approximately 250 kDa (C-terminal) MINT protein fragments accumulate in chromatin and nuclear matrix fractions, cosegregating with Msx2 and topoisomerase II. In gel shift assays, the MINT RRM domain selectively binds T- and G-rich DNA sequences; this includes a large G/T-rich inverted repeat element present in the proximal rat osteocalcin (OC) promoter, overlapping three cognates that support OC expression in osteoblasts. MINT and OC mRNAs are reciprocally regulated during differentiation of MC3T3E1 calvarial osteoblasts. Consistent with its proposed role as a nuclear transcriptional factor, transient expression of MINT(1-812) suppresses the FGF/forskolin-activated OC promoter, and does not significantly regulate CMV promoter activity, but markedly upregulates the HSV thymidine kinase promoter in MC3T3E1 cells. These data indicate that the novel nuclear protein MINT binds the homeoprotein Msx2 and coregulates OC during craniofacial development. Msx2 and MINT both target an information-dense, osteoblast-specific regulatory region of the OC proximal promoter: nucleotides -141 to -111. The N-terminal MINT RRM domain represents an authentic dsDNA binding module for this novel vertebrate nuclear matrix protein. Acting as a scaffold protein, MINT potentially exerts both positive and negative regulatory actions by organizing transcriptional complexes in the nuclear matrix (Newberry, 1999).

The recurrent t(1;22)(p13;q13) translocation is exclusively associated with infant acute megakaryoblastic leukemia. The two genes involved in this translocation have been identified. Both genes possess related sequences in the Drosophila genome. The chromosome 22 gene (megakaryocytic acute leukemia, MAL) product is predicted to be involved in chromatin organization, and the chromosome 1 gene (one twenty-two, OTT) product is related to the Drosophila split-end (spen) family of proteins. Drosophila genetic experiments have identified spen as involved in connecting the Raf and Hox pathways. Because almost all of the sequences and all of the identified domains of both OTT and MAL proteins are included in the predicted fusion protein, the OTT-MAL fusion could aberrantly modulate chromatin organization, Hox differentiation pathways, or extracellular signaling (Mercher, 2001).

Notch proteins are the receptors for an evolutionarily highly conserved signalling pathway that regulates numerous cell fate decisions during development. Signal transduction involves the presenilin-dependent intracellular processing of Notch and nuclear translocation of the intracellular domain of Notch, Notch-IC. Notch-IC associates with the DNA-binding protein RBP-Jkappa/CBF-1 to activate transcription of Notch target genes. In the absence of Notch signalling, RBP-Jkappa/CBF-1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. SHARP, a homolog of Drosophila Split ends, is identified as an RBP-Jkappa/CBF-1-interacting corepressor in a yeast two-hybrid screen. In cotransfection experiments, SHARP-mediated repression is sensitive to the HDAC inhibitor TSA and facilitated by SKIP, a highly conserved SMRT and RBP-Jkappa-interacting protein. SHARP represses Hairy/Enhancer of split (HES)-1 promoter activity, inhibits Notch-1-mediated transactivation and rescues Notch-1-induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on these data, a model is proposed in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP-Jkappa to repress transcription of target genes in the absence of activated Notch (Oswald, 2002).

A yeast two-hybrid screen using the conserved carboxyl terminus of the nuclear receptor corepressor SMRT as a bait led to the isolation of a novel human gene termed SHARP (SMRT/HDAC1 Associated Repressor Protein). SHARP is a potent transcriptional repressor whose repression domain (RD) interacts directly with SMRT and at least five members of the NuRD complex including HDAC1 and HDAC2. In addition, SHARP binds to the steroid receptor RNA coactivator SRA via an intrinsic RNA binding domain and suppresses SRA-potentiated steroid receptor transcription activity. Accordingly, SHARP has the capacity to modulate both liganded and nonliganded nuclear receptors. Surprisingly, the expression of SHARP is itself steroid inducible, suggesting a simple feedback mechanism for attenuation of the hormonal response (Shi, 2001).

Spen proteins regulate the expression of key transcriptional effectors in diverse signaling pathways. They are large proteins characterized by N-terminal RNA-binding motifs and a highly conserved C-terminal SPOC domain. The specific biological role of the SPOC domain (Spen paralog and ortholog C-terminal domain), and hence, the common function of Spen proteins, has been unclear to date. The Spen protein, SHARP (SMRT/HDAC1-associated repressor protein), was identified as a component of transcriptional repression complexes in both nuclear receptor and Notch/RBP-J{kappa} signaling pathways. The 1.8 Å crystal structure of the SPOC domain from SHARP has been determined. This structure shows that essentially all of the conserved surface residues map to a positively charged patch. Structure-based mutational analysis indicates that this conserved region is responsible for the interaction between SHARP and the universal transcriptional corepressor SMRT/NCoR (silencing mediator for retinoid and thyroid receptors/nuclear receptor corepressor. This interaction involves a highly conserved acidic motif at the C terminus of SMRT/NCoR. These findings suggest that the conserved function of the SPOC domain is to mediate interaction with SMRT/NCoR corepressors, and that Spen proteins play an essential role in the repression complex (Ariyoshi, 2003).

The structure of the SPOC domain reveals a novel architecture for an independent protein domain. (The ß-barrel domain of Ku forms part of a larger structure.) It appears to be ideally suited to mediate interaction with other proteins through a number of deep grooves and clefts in the surface as well as two nonpolar loops. In addition, the N-terminal region seems to possess an intrinsic propensity to form a ß-sheet with partner proteins. Most significantly, the structure reveals a highly basic patch on the surface, which is absolutely conserved throughout the Spen protein family. It is likely, therefore, that the function of this patch is indicative of the conserved role of the Spen proteins (Ariyoshi, 2003).

Through a variety of interaction and mutagenesis experiments it has been shown that this basic patch mediates the tight and specific interaction of the Spen proteins with the conserved acidic C-terminal LSD peptide from the SMRT/NCoR corepressors. Remarkably, point mutations within the basic patch totally abolish interaction with the LSD peptide. This suggests that although complementary charges play an important role in the interaction, the precise positioning of side chains of the key basic residues is absolutely required for stereospecific recognition of the SMRT/NCoR LSD motif. Whereas the precise details of the interaction remain to be determined, some indication of a possible mode of interaction is seen within the crystal lattice. The N-terminal region of one molecule (Pro 3495-Gln 3500) makes a crystal packing interaction with the ß3 strand of an adjacent molecule (Arg 3548-Arg 3554). The interactions include backbone-backbone hydrogen bonds, as well as both electrostatic and hydrophobic interactions (Ariyoshi, 2003).

The conservation of the SPOC domain in Drosophila and Caenorhabditis elegans suggests that both these species should possess corepressor proteins with LSD motifs. It is clear that the rather divergent Drosophila corepressor SMRTER does have an almost identical LSD motif. The findings of this study suggest that a similar protein must also be present in the worm (Ariyoshi, 2003).

It remains to be seen how other proteins such as HDAC1 may interact with SHARP. It is striking however that the LSD peptide itself serves as a potent transcriptional repressor, suggesting that recruitment of SHARP to a promoter is sufficient to mediate strong repression of basal transcription (Ariyoshi, 2003).

In conclusion, the combination of structural and functional experiments with the SPOC domain of SHARP clearly demonstrate that the conserved function of the SPOC domain is to mediate interaction with corepressors and, therefore, that Spen proteins play an essential role in regulating transcriptional repression (Ariyoshi, 2003).

Msx2-interacting nuclear target protein (MINT) competes with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppresses the transactivation activity of Notch signaling. Although MINT null mutant mice are embryonic lethal, MINT-deficient splenic B cells differentiate about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless (Kuroda, 2003).

Msx2 promotes osteogenic lineage allocation from mesenchymal progenitors but inhibits terminal differentiation demarcated by osteocalcin (OC) gene expression. Msx2 inhibits OC expression by targeting the fibroblast growth factor responsive element (OCFRE), a 42-bp DNA domain in the OC gene bound by the Msx2 interacting nuclear target protein (MINT) and Runx2/Cbfa1. To better understand Msx2 regulation of the OCFRE, functional interactions between MINT and Runx2, a master regulator of osteoblast differentiation, were studied. In MC3T3E1 osteoblasts (with endogenous Runx2 and FGFR2), MINT augments transcription driven by the OCFRE that is further enhanced by FGF2 treatment. OCFRE regulation can be reconstituted in the naive CV1 fibroblast cell background. In CV1 cells, MINT synergizes with Runx2 to enhance OCFRE activity in the presence of activated FGFR2. The RNA recognition motif domain of MINT (which binds the OCFRE) is required. Runx2 structural studies reveal that synergy with MINT uniquely requires Runx2 activation domain 3. In confocal immunofluorescence microscopy, MINT adopts a reticular nuclear matrix distribution that overlaps transcriptionally active osteoblast chromatin, extensively co-localizing with the phosphorylated RNA polymerase II meshwork. MINT only partially co-localizes with Runx2; however, co-localization is enhanced 2.5-fold by FGF2 stimulation. Msx2 abrogates Runx2-MINT OCFRE activation, and MINT-directed RNA interference reduces endogenous OC expression. In chromatin immunoprecipitation assays, Msx2 selectively inhibits Runx2 binding to OC chromatin. Thus, MINT enhances Runx2 activation of multiprotein complexes assembled by the OCFRE. Msx2 targets this complex as a mechanism of transcriptional inhibition. In osteoblasts, MINT may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses (Sierra, 2004).

The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), a protein absolutely required for the production of infectious virions, shares properties with mRNA export factors. By using a yeast two-hybrid screen, the human protein OTT3 has been identified as an EB2-interacting factor. OTT3 is a new member of the Spen (split end) family of proteins (huSHARP, huOTT1, DmSpen, and muMINT), which are characterized by several N-terminal RNA recognition motifs and a highly conserved C-terminal SPOC (Spen Paralog and Ortholog C-terminal) domain that, in the case of SHARP, has been shown to interact with SMRT/NCoR corepressors. OTT3 is ubiquitously expressed as a 120-kDa protein. Transfected OTT3 is a nonshuttling nuclear protein that co-localizes with co-transfected EB2. EB2 interacts with the SPOC domains of both OTT1 and SHARP proteins. Although the OTT3 interaction domain maps within the 40 N-terminal amino acids of EB2, OTT1 and SHARP interact within the C-terminal half of the protein. Furthermore, the capacity of the OTT3 and OTT1 SPOC domains to interact with SMRT and repress transcription is far weaker than that of SHARP. Thus there is no evidence for a role of OTT3 in transcriptional regulation. Most interestingly, however, OTT3 has a role in splicing regulation; OTT3 represses accumulation of the alternatively spliced beta-thalassemia mRNAs, but it has no effect on the beta-globin constitutively spliced mRNA. Thus these results suggested a new function for Spen proteins related to mRNA export and splicing (Hiriart, 2005).

Collagen type II is an extracellular matrix protein important for cartilage and bone formation, and its expression is controlled by multiple cis- and trans-acting elements, including the zinc finger transcription factor alpha A-crystallin-binding protein 1 (CRYBP1). MSX2-interacting nuclear target protein (MINT), a conserved transcriptional repressor, associates with CRYBP1 and negatively regulates the transactivation of the collagen type II gene (Col2a1) enhancer. CRYBP1 was identified as a binding partner of MINT by screening a mouse embryonic cDNA library using the yeast two-hybrid system. The C terminus of MINT interacts with the C terminus of CRYBP1 as determined using the mammalian cell two-hybrid assay, glutathione S-transferase pull-down, and co-immunoprecipitation analyses. Furthermore, MINT and CRYBP1 form a complex on the Col2a1 enhancer, as shown by chromatin immunoprecipitation and gel shift assays. In the presence of CRYBP1, overexpression of MINT or its C-terminal fragment in cells repressed a reporter construct driven by the Col2a1 enhancer elements. This transcription repression is dependent on histone deacetylase, the main co-repressor recruited by MINT. The present study shows that MINT is involved in CRYBP1-mediated Col2a1 gene repression and may play a role in regulation of cartilage development (Yang, 2005).

Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jkappa/CBF1. In the absence of Notch, RBP-Jkappa represses Notch target genes through the recruitment of a corepressor complex. SHARP has been identified as a component of this complex. This study functionally demonstrates that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. The CtIP and CtBP corepressors were identified as novel components of the human RBP-Jkappa/SHARP-corepressor complex; CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. It is proposed that a corepressor complex containing CtIP/CtBP facilitates RBP-Jkappa/SHARP-mediated repression of Notch target genes (Oswald, 2005).

The nuclear matrix protein Msx2-interacting nuclear target protein (MINT) is a transcription factor that regulates the expression of key transcriptional effectors in diverse signaling pathways. To further understand the function and mechanism of the MINT-mediated transcription regulation, the yeast two-hybrid system was employed to screen proteins that interact with the C-terminal fragment of MINT. From a cDNA library of human lymph nodes, a cDNA encoding the ubiquitin-conjugating enzyme UbcH8 was identified. Using different truncated versions of MINT, it was shown that the C-terminal Spen paralog and ortholog C-terminal domain (SPOC) domain, which has been demonstrated to mediate interactions between MINT and a panel of other molecules, might be responsible for interaction between MINT and UbcH8 in yeast, as confirmed by the beta-galactosidase assay. The interaction between MINT and UbcH8 in mammalian cells was further proved by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, and co-immunoprecipitation assay. Using a reporter system, it was found that MINT-mediated transcription suppression was sensitive to MG132, an inhibitor of the proteosome system. These results suggest a novel mechanism of MINT-mediated transcription regulation, and might be helpful for understanding functions of MINT (Li, 2006).

Retroviruses/retroelements provide tools enabling the identification and dissection of basic steps for post-transcriptional regulation of cellular mRNAs. The RNA transport element (RTE) identified in mouse retrotransposons is functionally equivalent to constitutive transport element of Type D retroviruses, yet does not bind directly to the mRNA export receptor NXF1. The RNA-binding motif protein 15 (RBM15) recognizes RTE directly and specifically in vitro and stimulates export and expression of RTE-containing reporter mRNAs in vivo. Tethering of RBM15 to a reporter mRNA showed that RBM15 acts by promoting mRNA export from the nucleus. It was also found that RBM15 binds to NXF1 and the two proteins cooperate in stimulating RTE-mediated mRNA export and expression. Thus, RBM15 is a novel mRNA export factor and is part of the NXF1 pathway. It is proposed that RTE evolved as a high affinity RBM15 ligand to provide a splicing-independent link to NXF1, thereby ensuring efficient nuclear export and expression of retrotransposon transcripts (Lindtner, 2006).

The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs

The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. This study has determined the crystal structure of the region of the human SPEN homolog that contains these RRMs-the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 A resolution. SHARP is a co-regulator of the nuclear receptors. Two of the three RRMs, namely RRM3 and RRM4, were shown to interact via a highly conserved interface. It was shown that the RRM3-RRM4 block is the main platform mediating the stable association with the H12-H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP-RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions (Arieti, 2014).

Rbm15 modulates Notch-induced transcriptional activation and affects myeloid differentiation

RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, the normal functions of RBM15 and MKL must be understood. A role for Rbm15 in myelopoiesis is demonstrated in this study. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJkappa, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJkappa (Ma, 2007).

Drosophila split ends homologue SHARP functions as a positive regulator of Wnt/β-Catenin/T-cell factor signaling in neoplastic transformation.

Wnt ligands have pleiotropic and context-specific roles in embryogenesis and adult tissues. Among other effects, certain Wnts stabilize the beta-catenin protein, leading to the ability of beta-catenin to activate T-cell factor (TCF)-mediated transcription. Mutations resulting in constitutive beta-catenin stabilization underlie development of several human cancers. Genetic studies in Drosophila highlighted the split ends (spen) gene as a positive regulator of Wnt-dependent signaling. This study has assessed the role of SHARP, a human homologue of spen, in Wnt/beta-catenin/TCF function in mammalian cells. SHARP gene and protein expression were found to be elevated in human colon and ovarian endometrioid adenocarcinomas and mouse colon adenomas and carcinomas carrying gene defects leading to beta-catenin dysregulation. When ectopically expressed, the silencing mediator for retinoid and thyroid receptors/histone deacetylase 1-associated repressor protein (SHARP) protein potently enhances beta-catenin/TCF transcription of a model reporter gene and cellular target genes. Inhibition of endogenous SHARP function via RNA inhibitory (RNAi) approaches antagonized beta-catenin/TCF-mediated activation of target genes. The effect of SHARP on beta-catenin/TCF-regulated genes is mediated via a functional interaction between SHARP and TCF. beta-Catenin-dependent neoplastic transformation of RK3E cells is enhanced by ectopic expression of SHARP, and RNAi-mediated inhibition of endogenous SHARP in colon cancer cells inhibits their transformed growth. These findings implicate SHARP as an important positive regulator of Wnt signaling in cancers with beta-catenin dysregulation (Feng, 2007).

Ott1(Rbm15) has pleiotropic roles in hematopoietic development

OTT1(RBM15) was originally described as a 5' translocation partner of the MAL(MKL1) gene in t(1,22)(p13;q13) infant acute mega karyocytic leukemia. OTT1 has no established physiological function, but it shares homology with the spen/Mint/SHARP family of proteins defined by three amino-terminal RNA recognition motifs and a carboxyl-terminal SPOC (Spen paralog and ortholog carboxyl-terminal) domain is believed to act as a transcriptional repressor. To define the role of OTT1 in hematopoiesis and help elucidate the mechanism of t(1,22) acute megakaryocytic leukemia pathogenesis, a conditional allele of Ott1 was generated in mice. Deletion of Ott1 in adult mice causes a loss of peripheral B cells due to a block in pro/pre-B differentiation. There is myeloid and megakaryocytic expansion in spleen and bone marrow, an increase in the Lin(-)Sca-1(+)c-Kit(+) compartment that includes hematopoietic stem cells, and a shift in progenitor fate toward granulocyte differentiation. These data show a requirement for Ott1 in B lymphopoiesis, and inhibitory roles in the myeloid, megakaryocytic, and progenitor compartments. The ability of Ott1 to affect hematopoietic cell fate and expansion in multiple lineages is a novel attribute for a spen family member and delineates Ott1 from other known effectors of hematopoietic development. It is plausible that dysregulation of Ott1-dependent hematopoietic developmental pathways, in particular those affecting the megakaryocyte lineage, may contribute to OTT1-MAL-mediated leukemogenesis (Raffel, 2007).

Ott1 (Rbm15) is essential for placental vascular branching morphogenesis and embryonic development of the heart and spleen

The infant leukemia-associated gene Ott1 (Rbm15) has broad regulatory effects within murine hematopoiesis. However, germ line Ott1 deletion results in fetal demise prior to embryonic day 10.5, indicating additional developmental requirements for Ott1. The spen gene family, to which Ott1 belongs, has a transcriptional activation/repression domain and RNA recognition motifs and has a significant role in the development of the head and thorax in Drosophila. Early Ott1-deficient embryos show growth retardation and incomplete closure of the notochord. Further analysis demonstrated placental defects in the spongiotrophoblast and syncytiotrophoblast layers, resulting in an arrest of vascular branching morphogenesis. The rescue of the placental defect using a conditional allele with a trophoblast-sparing cre transgene allowed embryos to form a normal placenta and survive gestation. This outcome showed that the process of vascular branching morphogenesis in Ott1-deficient animals was regulated by the trophoblast compartment rather than the fetal vasculature. Mice surviving to term manifested hyposplenia and abnormal cardiac development. Analysis of global gene expression of Ott1-deficient embryonic hearts showed an enrichment of hypoxia-related genes and a significant alteration of several candidate genes critical for cardiac development. Thus, Ott1-dependent pathways, in addition to being implicated in leukemogenesis, may also be important for the pathogenesis of placental insufficiency and cardiac malformations (Raffel, 2009).

The spen family protein FPA controls alternative cleavage and polyadenylation of RNA

The spen family protein FPA is required for flowering time control and has been implicated in RNA silencing. The mechanism by which FPA carries out these functions is unknown. This study reports the identification of an activity for FPA in controlling mRNA 3' end formation. FPA functions redundantly with FCA, another RNA binding protein that controls flowering and RNA silencing, to control the expression of alternatively polyadenylated antisense RNAs at the locus encoding the floral repressor FLC. In addition, it was shown that defective 3' end formation at an upstream RNA polymerase II-dependent gene explains the apparent derepression of the AtSN1 retroelement in fpa mutants. Transcript readthrough accounts for the absence of changes in DNA methylation and siRNA abundance at AtSN1 in fpa mutants, and this may explain other examples of epigenetic transitions not associated with chromatin modification (Hornyik, 2010).

REFERENCES

Search PubMed for articles about Drosophila spen

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date revised: 10 August 2018

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