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).

GENE STRUCTURE

The structure of the spen cDNA clones suggests that at least two alternative promoters initiate transcripts. A probe made from the most 5' exons, which initiate the long (spen^L) transcript isoform does not detect maternal transcripts in the 0-2 hour RNA lane of a Northern blot, whereas a probe from the common region does. However, RT-PCR analysis does detect spen^L and spen^S transcripts at all stages, including 0-2 hours, indicating that low levels of the spen^L isoform exist in maternal RNA. Both probes detect transcripts of approximately 20 kb at later stages of embryonic development, although the spen^L isoform is predicted to be approximately 1 kb larger based on the known cDNA structures. A third Spen protein isoform has been characterized by Kuang (2000), and is initiated from an alternative exon that maps upstream of the spen^S 5' exon. The third isoform has a few unique amino acid residues at its N terminus that differ from two Spen protein isoforms reported in this study (Wiellette, 1999).

cDNA clone length - 19444 (Wiellette, 1999)

Bases in 5' UTR - 1005 (Wiellette, 1999)

Exons - 11

Bases in 3' UTR - 1737 (Wiellette, 1999)

PROTEIN STRUCTURE

Amino Acids - 5533 (long isoform)

Structural Domains

The first ATG in the long open reading frame of the spen^L transcript begins a long open reading frame of 5533 codons, which is predicted to make a 597 kDa protein. The first ATG in the spen^S transcript begins an open reading frame of 5476 codons. The SpenS protein isoform is essentially an N-terminal truncation of the SpenL protein with the addition of a Met Arg dipeptide, encoded in S exon 2, substituted for the first 59 aa residues of SpenL. The N terminus of the SpenL isoform may contain unique functions due to its poly-Asn and poly-Gln sequences. Three RNA-binding domains of the RNP type (amino acids 554-806 of the L form) are present in the 5474 aa common region of the predicted SpenL and SpenS proteins. Five bipartite nuclear localization sequences are scattered throughout the middle of the common sequence (positions 1872-1889, 1949-1966, 2135-2152, 2450-2467, 4556-4573 of SpenL). In addition, a region that is highly enriched in Glu (E) and Lys (K) residues is present from positions 1917-2053 of SpenL, and strings of glutamine (Q) residues are found throughout the C-terminal one-third of the Spen proteins (Wiellette, 1999).

Database searches combined with GENESCAN and FGENE predictions of exon structure reveal apparent structural orthologs of Spen in genome sequences from human and C. elegans. These Spen family members are defined by their large size (predicted to be at least 3300 aa for human and 2738 aa for C. elegans), by their possession of three RNP domains that are closely related to those in Spen isoforms, and by a lengthy sequence match to a 165 amino acid motif at the C terminus of the D. melanogaster Spen proteins. The sequence of the first RNP domain of the Spen family also is closely related to the first RNP domain of Nucleolin, a protein involved in ribosome biosynthesis. The second Spen family RNP domain is a good match to the canonical RNP domain, and shares even more identities with an RNP domain in AC binding factor (ACBF), an RNP-motif protein isolated from tobacco. ACBF binds to DNA regulatory sequences of genes in the phenylpropanoid biosynthetic pathways of many plants (Seguin, 1997). The C-terminal motif, which is called the SPOC domain (Spen Paralog and Ortholog C-terminal domain) is very similar among family members (57% identity between D. melanogaster Spen and H. sapiens Spen1), but matches no other sequence motifs with known biochemical function. The entire open reading frames of two EMS-induced spen alleles [spen E(CycE++)D57 and spen E(CycE++)e9 ] were sequenced. Single base substitutions in each of these alter the predicted amino acid sequence at highly conserved positions within the SPOC domain (Wiellette, 1999).

Sequence similarity searches also reveal a separate sub-family of short Spen-like proteins encoded in D. melanogaster and other animal genomes. The short Spen-like proteins have a similar arrangement of Spen-like RNP motifs and SPOC domains as do those in the Spen orthologs, but the short Spen-like proteins are approximately one tenth the size of Spen. All of the known short Spen-like proteins also encode an RGG motif, which is capable of destablizing RNA helices and often found in combination with RNP motifs. The current sequence evidence suggests that a duplication event giving rise to the genes that encode the large and small Spen-like proteins occurred before the divergence of the deuterostome and protostome lineages in metazoan evolution (Wiellette, 1999).

spen encodes a member of the RNA recognition motif (RRM) family of RNA-binding proteins. The RRM sequence is an 80-100-amino-acid stretch of sequence that forms an RNA-binding domain (RBD). This motif is characterized by a bipartite RNP consensus sequence that forms the RNA-binding surface of the domain, as well as several predominantly hydrophobic amino acids dispersed throughout the RRM that are essential for the overall structure of the domain. RRM-containing proteins mediate a variety of post-transcriptional RNA-processing reactions, including mRNA splicing, stability, and transport. In Drosophila, RNA-processing reactions and the RRM-containing proteins that mediate them are critical to multiple developmental processes. For example, in sex determination, sex-specific alternate splicing reactions are regulated by the RRM-containing proteins tra2 and Sxl. Establishment of dorsoventral polarity in the oocyte requires input from the RRM protein Squid to localize the mRNA encoding the Egfr ligand gurken. Another RRM protein, Elav, is expressed in all Drosophila neurons and is required for determination and maintenance of the neuronal fate, although the precise mechanism of function is not clear (Rebay, 2000 and references therein).

BLAST searches using Split ends protein sequence identify proteins that contain related C-terminal sequences in worms, flies and vertebrates. These proteins all contain RNA recognition motifs (RRMs) and are predicted to have relative molecular masses of >300 kDa or <95 kDa. Different genes in these organisms produce the large and shorter Spen-like proteins. The RRMs of the large Spen-like proteins are more related to each other than to the RRM consensus, suggesting that they share similar nucleic acid binding properties. The 360 kDa mouse Spen-like protein, MINT, contains three RRMs that bind to GT-rich dsDNA in vitro (Newberry, 1999). RRMs are found in many proteins involved in RNA processing, but also function as dsDNA binding motifs in several transcription factors. The predicted C-terminal 168 amino acids of the Spen protein are 51% identical and 67% similar in sequence to the corresponding domains of the >300 kDa predicted vertebrate proteins. The <95 kDa Spen-like proteins contain C-terminal sequences that are more distantly related to Spen than those in longer forms. Thus, Spen identifies a new family of RRM-containing proteins that also contain a distinctive C-terminal domain (Kuang, 2000).

spen is allelic to polycephalon (poc), a gene required for head development and an enhancer of mutations in the Deformed homeobox gene (Gellon, 1997). spen is also allelic to En(raf)2A, an enhancer of activated Raf (Dickson, 1996) and to En(yan^ACT)2-7, an enhancer of activated yan (Rebay, 2000). spen also enhances eye phenotypes associated with ectopic expression of the E2F transcription complex (Staehling-Hampton, 1999). Virtually identical sequence results have been obtained for the poc cDNAs (accession no. AF188205) (Wiellette, 1999), with the exception of an additional start site and the internal splice reported in this study. Point mutations in the conserved C-terminal domain have been identified among alleles of spen/poc (Wiellette, 1999). The C-terminal domain is therefore referred to as the SPOC domain (Kuang, 2000 and references therein).

spen has three RRM motifs in tandem within the first 1000 amino acids of the protein. Database searches identify a number of predicted proteins from either genomic sequencing efforts or EST projects that have very similar RRMs. For these proteins, the homology within the RRM region extends beyond the conserved residues that define the motif. Homology between spen and other RRM-containing proteins is less striking and is limited to the residues that comprise the motif. Because the RRM domain is thought to mediate specificity of RNA-binding interaction, it is possible that the spen class of RRM proteins interacts with similar substrates. Further experiments will be required to determine whether spen actually binds RNA, what its in vivo targets are, and what the function of this activity is in the context of RTK signaling events during development (Rebay, 2000).

Two additional structural motifs of note in the spen protein are the C-terminal ~170 amino acids and a region of predicted coiled-coil near the middle of the protein. The coiled-coil domain is likely to mediate protein-protein interactions, either with spen itself or with other proteins. One possibility is that spen interacts directly with Yan via this domain. Alternatively, the interaction between spen and yan could be less direct. Homology searches using the C terminus of the Spen protein detect several proteins, several of which, including a Drosophila protein predicted from the BDGP genomic sequence and a human protein compiled from multiple overlapping ESTs, also have spen-class RRMs. Although the structure of the C terminus of spen is not homologous to any protein domain of known function, the high degree of conservation of this region between fly, worm, and mammalian proteins suggests it is likely to have a conserved function. Apart from the RRM, coiled-coil, and C-terminal domains, the rest of the spen protein is novel, and to date it is uninformative in terms of providing clues as to function. Because the protein is so large (5476 amino acids), structure-function analyses and determination of the molecular lesions associated with the various spen alleles will be required to determine whether there are other important functional domains not detected by sequence homology. Investigation of the function of spen homologues in other species may also be informative (Rebay, 2000).

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).

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).

date revised: 11 September 2001

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