Gene name - knot
Synonyms - collier (col)
Cytological map position - 51C1--51C1
Function - transcription factor
Symbol - kn
FlyBase ID: FBgn0001319
Genetic map position -
Classification - EBF/Olf-1 homolog, HLH protein
Cellular location - nuclear
Segmentation of the Drosophila embryo is based on a cascade of hierarchical gene interactions initiated by maternal morphogens. These interactions define spatially restricted domains of zygotic gene expression within the blastoderm. Although the hierarchy of the segmentation genes that subdivide the trunk is well established, patterning in the head is less well understood. Seven head segments can be assigned on the basis of metameric patterns of segment-polarity gene expression and internal sensory organs. The domains of expression for head gap-like genes broadly overlap; their posterior margins are out of phase by one segment. These observations, taken together with the lack of pair-rule gene expression in the head, have led to the suggestion that head gap genes act in a combinatorial manner, simultaneously determining segmental borders in the head and segmental identities (Crozatier, 1996 and references).
collier (preferentially called knot because the gene was discovered an the basis of a mutant phenotype prior to being name collier) expression at the blastoderm stage is restricted to a single stripe of cells corresponding to part of the intercalary and mandibular segment primordia, possibly parasegment 0. There is a striking similarity between the early stripe of collier expression and the position of a specific mitotic domain at cycle 14, mitotic domain 2 (MD2). Mitotic domains are defined as groups of cells that enter mitosis 14 both synchronously and out of synchrony with other groups of cells (Foe, 1989). The pattern of string (stg) transcription anticipates the pattern of cycle 14 mitoses. At the onset of gastrulation, string and collier are simultaneously expressed in a group of cells that correspond to MD2, suggesting that these cells not only share a mitotic fate, but also share a specific gene expression program. It is thought that col and stg respond to the same patterning information and act in parallel, with col assigning a specific gene-expression program in cells in MD2. stg and col expression in MD2 is concomitant and both require buttonhead (Crozatier, 1996).
Reduction of col activity in early gastrula embryos by antisense RNA expression results in a specific lack of head structures derived from intercalary and mandibular segments. Almost all antisense expressing embryos fail to hatch. However, 80% develop to the point of making a cuticle; in these embryos, the only defects consistently observed are in the head (cephalopharyngeal) skeleton. There is an absence or drastic reduction of the lateral gräten, one of the head skeleton elements. All other skeletal structures appear normal. The lateral gräten are though to originate from the mandibular segment. In antisense expressing embryos, the Engrailed intercalary spot is either reduced or is sometimes missing, whereas the more posterior mandibular Engrailed stripe appears to be unaffected (Crozatier, 1996).
It is suggested that Col may act as a 'second-level regulator' of head patterning. This study, together with the recent characterization of crocodile (a gene required for the formation of structures derived from the intercalary segment, the posterior wall of the pharynx and the ventral arm of the cephalopharyngeal skeleton, indicates that a complex network of transcription factors acts downstream of head gap genes in controlling morphogenesis in the embryonic head. The structural conservation of Col during evolution raises the questions of its conservation of function in head specification and its interactions with other factors conserved between insects and vertebrates (Crozatier, 1996).
Collier is required for formation of a subset of somatic muscles. During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the formation of a single muscle (muscle DA3[A] in the abdominal segments; DA4[T] in the thoracic segments T2 and T3). Col expression is first observed in two promuscular clusters (in segments A1-A7), corresponding to two progenitors and then their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3[A]. This lineage-specific restriction depends on the asymmetric segregation of Numb during the progenitor cell division and involves the repression of col transcription by Notch signaling. In col mutant embryos, the DA3[A] founder cells form but do not maintain col transcription and are unable to fuse with neighbouring myoblasts, leading to a loss-of-muscle DA3[A] phenotype. In wild-type embryos, each of the DA3[A]-recruited myoblasts turns on col transcription, indicating that this conversion, accomplished by the DA3[A] founder cell, induces the naive myoblasts to express founder cell distinctive patterns of gene expression, activating col itself. Muscles DA3[A] and DO5[A] (DA4[T] and DO5[T] respectively) derive from a common progenitor cell, the DA3[A]/DO5[A] progenitor. However, ectopic expression of Col is not sufficient to switch the DO5[A] to a DA3[A] fate. Together these results lead to a proposal that specification of the DA3[A] muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles (Crozatier, 1999a).
The col-expressing promuscular clusters and progenitor cells have a distinctive position, as defined relative to morphological landmarks and ectodermal Engrailed (En) expression. The DA3[A]/DO5[A] progenitor cell lies underneath the anterior epidermal compartment, whereas the DT1[A]/DO4[A] progenitor cell lies on the anterior edge of the posterior compartment, consistent with mapping of the primordium for the somatic mesoderm. Since Wingless (Wg) and Hedgehog (Hh) signaling have been shown to be required for mesoderm segmentation and formation of a subset of muscle founder cells, col expression was analyzed in wg and hh mutant embryos. At stage 10, both mutant embryos show changes in mesodermal col expression: rather than being restricted to specific clusters in the anterior compartment, it appears almost continuous along the anteroposterior axis. Therefore, both wg and hh signalings appear to restrict col transcription to specific clusters. Lack of Wg or Hh activity does not seem, however, to impede specification of the DA3[A]/DO5[A] progenitor, which is singled out in the mutant as well as the wild-type embryos. It was noticed, however, that, while the DA3[A]/DO5[A] progenitor appears to be specified normally, more than one cell is singled out from the DT1[A] /DO4[A] cluster in hh mutant embryos (Crozatier, 1999a).
Following establishment of the promuscular clusters, specification of the progenitors is controlled by lateral inhibition, a cell-cell interaction process mediated by the neurogenic genes Notch (N) and Delta (Dl)). In both N and Dl mutant embryos, promuscular Col expression is initiated normally but fails to become restricted to a single cell per cluster, similar to observations previously made for the expression of lsc. As a consequence, a hyperplasic expression of Col is observed from stage 11. Since it is expressed in promuscular clusters and segregating muscle progenitors, lsc has been proposed to play a role in muscle progenitor selection similar to the role of achaete and scute in neuroblast specification. However, in embryos lacking lsc activity, selection of the Col-expressing progenitors occurs normally at stage 11 and muscle DA3[A] forms as in wild type (Crozatier, 1999a).
A key event in the generation of the muscle diversity is the asymmetric division of progenitor cells. The distinction between sibling muscle founder cells depends on the differential distribution of the membrane-associated protein Numb (Nb), under the control of inscuteable (insc). One proposal for the action of Nb in determining differences in cell fate is that it biases the N-mediated cell-cell interactions by inhibiting Notch activity, so that this interaction becomes, in turn, asymmetric. The formation of muscles DA3[A] and DO5[A] was analyzed in insc, nb and N mutant embryos. In insc mutant embryos, the DA3[A] muscle is duplicated at the expense of DO5[A] in most of the segments. Although not 100% penetrant, the DA3[A] duplication phenotype always correlates with the absence of DO5[A], indicating a transformation of DO5[A] into DA3[A]. The reciprocal phenotype is observed in nb embryos: muscle DA3[A] is missing whereas muscle DO5[A] is duplicated. By analogy with the sensory organ precursor (SOP) lineage, this finding suggests that the DA3[A] founder cell is the cell that inherits Nb. Absence, or duplication, of DA3[A] in nb and insc mutant embryos, respectively, indicates that Nb function is required for specifying the DA3[A] cell fate. This conclusion is supported by the DA3[A] duplication phenotype observed in embryos mutant for sanpodo (spdo), another gene that acts antagonistically to nb in the Notch-mediated determination of alternative cell fates and encodes a tropomodulin-like protein (Crozatier, 1999a).
The question was then raised as to how nb and col functions relate to one another in specifying DA3[A] . col transcription was examined in insc and nb mutant embryos, using the col intronic probe. col transcription is controlled by Notch signaling in the establishment of the DA3[A]/DO5[A] lineage. In wild-type embryos at late stage 12, only one founder cell (DA3[A]) maintains col transcription; in insc mutant embryos, two cells do so. Conversely, no founder cell continues to transcribe col in nb mutant embryos. These data indicate that Nb determines the choice between the DA3[A] and DO5[A] cell fates, by allowing col transcription to persist in the DA3[A] founder cell. In N mutant embryos, a large disruption of the muscle pattern occurs, as a result of the cumulative effects of overproduction of muscle progenitor cells, lack of myoblast fusion, disorganization of the muscle epidermal attachment sites as well as, possibly, lack of a signal to the mesoderm emanating from the epidermis. Despite this cumulative phenotype, N mutant embryos were used to analyse the role of N in establishing the DA3[A] cell fate, taking advantage of the perdurable expression of the col-lacZ transgene. In N embryos, there is a large increase in the number of muscle cells that express high levels of Col and beta-gal at stage 11, resulting from the defective progenitor selection. beta-gal expression persists in these cells up to stage 16, suggesting that they have adopted a DA3[A] fate. All together, and based on the recent finding that Notch is required to maintain progenitor-specific gene expression in one sibling founder cell and repress it in the other, a comparison of the patterns of col expression between wild-type and insc, nb or N mutant embryos indicates that the restriction of col transcription to a single founder cell is under the control of Notch signaling, at two successive levels: Notch activity is first required for restricting col expression to a single cell per cluster during the progenitor selection process. Afterward, Notch signaling is necessary to restrict col transcription to only one of two sibling founder cells and distinguish between the DA3[A] and DO5[A] fates (Crozatier, 1999a).
While col activity is absolutely required for the formation of muscle DA3[A] , it remained uncertain whether it is sufficient to convert the DO5[A] into a DA3[A] muscle. To address this question, Col was ectopically expressed at different time points during embryonic development, using a heat-shock col construct, and the DA3[A] fate was followed with P[col5-lacZ] expression. The A2-A7 pattern of muscles of heat-shock-treated embryos was visualised by double immunostaining for beta-gal and myosin heavy chain at stage 16. Ectopic Col expression induced at 4-5 hours AEL (stage 7-9), i.e., before singling out of the progenitor cell has occurred, does not alter much the final muscle pattern. These data indicate that ectopic col expression is not sufficient by itself to either switch cell fate between DO5[A] and DA3[A] or change the cell fate of other muscle precursors (Crozatier, 1999a).
The vestigial muscle phenotype has not been reported so far, precluding a comparison with the col mutant phenotype. Nevertheless, suggestive evidence that vg might be involved in regulation of col expression is provided by the ectopic col-lacZ expression observed in conditions of heat-shock-induced ubiquitous col expression. It is interesting to note that all three muscles in which col-lacZ is ectopically activated (muscles DA2[A] , VL1[A], VL2[A]) also express vg. All together, these results support the involvment of a combinatorial code of muscle-identity genes expressed in muscle progenitors and controlling the diversification of the somatic muscles. How does Col interact with the myogenic pathway in controlling formation of the DA3[A] muscle, or, put another way, determining the specific targets of Col in this process remains a challenging question. Col belongs to a small family of non-basic HLH transcription factors, the COE proteins, which are highly conserved during evolution. One Xenopus member of this family, XCoe2, is involved in the specification of primary neurons and that Xcoe2 activity is subject to feed-back regulation by lateral inhibition. The present report raises the interesting possibility that, beyond an apparent diversity of function, regulation of Xcoe2 expression during primary neuron formation and col during embryonic muscle formation reflect the existence of an evolutionary conserved pathway linking Notch signaling and col/Xcoe2 function in binary cell decisions in vertebrates and invertebrates (Crozatier, 1999a).
In a complex nervous system, neuronal functional diversity is reflected in the wide variety of dendritic arbor shapes. Different neuronal classes are defined by class-specific transcription factor combinatorial codes. The combination of the transcription factors Knot and Cut is particular to Drosophila class IV dendritic arborization (da) neurons. Knot and Cut control different aspects of the dendrite cytoskeleton, promoting microtubule- and actin-based dendritic arbors, respectively. Knot delineates class IV arbor morphology by simultaneously synergizing with Cut to promote complexity and repressing Cut-mediated promotion of dendritic filopodia/spikes. Knot increases dendritic arbor outgrowth through promoting the expression of Spastin, a microtubule-severing protein disrupted in autosomal dominant hereditary spastic paraplegia (AD-HSP). Knot and Cut may modulate cellular mechanisms that are conserved between Drosophila and vertebrates. Hence, this study gives significant general insight into how multiple transcription factors combine to control class-specific dendritic arbor morphology through controlling different aspects of the cytoskeleton (Jinushi-Nakao, 2007).
To understand the mechanism by which Knot promotes dendrite outgrowth, attempts were made to identify Knot-regulated genes in da neurons. Knot promotes formation of a microtubule-based dendritic arbor cytoskeleton. Therefore, candidate genes for regulation by Knot were chosen based on annotation in the Gene Ontology database indicating their association with microtubule biogenesis and function. The small GTPases rac1, cdc42, and rho, were also analyzed. Ectopic (classes I-III) or endogenous (class IV) Knot activity promotes da neuron dendritic arbor outgrowth. With this in mind, the relative expression of candidate genes was compared between wild-type and ectopic knot-expressing da neurons. To do this the RluA1-Gal4 line was used that drives UAS-mCD8::GFP expression solely in all da and some ES neurons. Purifying Gfp-positive cells from RluA1-Gal4, UAS-mCD8::GFP embryos gave a highly enriched population of da neurons for analysis. Gfp-positive cells were sorted from control (RluA1-Gal4, UAS-mCD8::GFP) and ectopic knot-expressing (RluA1-Gal4, UAS-mCD8::GFP, UAS-kn) embryos by Fluorescence-Activated Cell Sorting (FACS). Total RNA was isolated from the purified cells and the relative expression of candidate genes was compared between these populations by Reverse Transcription Polymerase Chain Reaction (RT-PCR). mRNA expression levels of all candidates were normalized against gapdh, and as a positive control analyzed knot levels were analyzed. Between the wild-type and ectopic knot-expressing cells, knot mRNA expression was upregulated by ratio of 2.6. Of the candidates analyzed, only spastin had an altered expression level; it was strongly upregulated by a ratio of 3.3 (Jinushi-Nakao, 2007).
These RT-PCR findings were confirmed by examining upregulation of Spastin protein via Knot ectopic expression. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) family, all of which have a related protein structure. To avoid cross-reactivity between family members, a Spastin-specific antibody, which was additionally preabsorbed against the other AAA family members, was used. Spastin protein levels were examined in western blots of protein extracts from sorted Gfp-positive cells. Ectopic knot-expressing (RluA1-Gal4, UAS-mCD8::GFP, UAS-kn) da neurons showed a very large upregulation in Spastin protein content as compared with those prepared from wild-type (RluA1-Gal4, UAS-mCD8::GFP) da neurons (Jinushi-Nakao, 2007).
If spastin is a bona fide target of Knot in class IV neurons, then spastin expression may be enriched in class IV da neurons versus other da neuron classes. To examine spastin expression whole-mount embryonic spastin mRNA in situ experiments were carried out. The results confirmed those of previous studies that show that spastin is ubiquitously expressed, with a higher-than-background expression level in nervous system tissues. However, the high ubiquitous background expression level made it impossible to compare levels of spastin in specific da neuron classes. To get around this problem, FACS purified a mixed population of all da neurons (RluA1-Gal4, UAS-mCD8::GFP) and a pure population of class IV neurons (ppk-Gal4, UAS-mCD8::GFP) were examined. spastin expression in these two populations was compared. spastin expression levels were clearly enriched (62% more) in the pure population of class IV neurons as compared with the mixed population of da neurons (Jinushi-Nakao, 2007).
Spastin has microtubule-severing activity in cultured cells and in vitro. It was asked if Spastin is also able to alter microtubule structure in the dendritic arbor of da neurons. To do this, spastin was ectopically expressed in class I ddaE neurons (Gal42-21, UAS-mCD8::GF8, UAS-spastin). Then the entire dendritic arbor was visualized at the wandering third-instar larva stage by staining with an anti-Gfp antibody, and examined the microtubule cytoskeleton was simultaneously by staining the arbor with antibodies to detect Futsch. In wild-type class I neurons, Futsch was present throughout the dendritic arbor. When spastin was expressed ectopically in the class I neuron, a loss of Futsch from the dendritic arbor was observed and disruption of arbor morphology. Therefore, high levels of Spastin disrupt microtubule organization within the arbor, a finding consistent with Spastin's microtubule-severing activity (Jinushi-Nakao, 2007).
Next, whether Spastin activity is required to promote class IV dendritic arbor complexity, as would be expected if it is part of the program controlled by Knot activity, was investigated. To do this class IV neurons were marked with ppk-Gal4, UAS-mCD8::GFP in the background of a null spastin5.75 allele. ppk-Gal4 was used to express an RNAi construct directed against spastin (UAS-spastinRNAi) along with UAS-mCD8::GFP to selectively knock down spastin class IV neurons. The morphology of these neurons was examined at the wandering third-instar larva stage (Jinushi-Nakao, 2007).
Reduction of spastin levels in either heterozygous null background or spastin RNAi-mediated knockdown background lead to large gaps both between neighboring class IV dendritic arbors and within the arbor of an individual neuron. Such gaps were not seen in wild-type control larvae, but were also seen in loss- or reduction-of-function knot mutants. To quantify this effect, dendrite coverage was measured by drawing a 34 × 34 grid of 10 μm × 10 μm squares over the central portion of the neuron. The number of squares that did not contain any portion of a dendrite branch was counted. This analysis provides an approximate measure of the amount of area that is not covered by the dendritic arbor. spastin RNAi knockdown had 17% more uncovered area than wild-type ddaC neurons; spastin5.75/+ mutants had 43%, and kn1/knKN2 mutants had 59% (Jinushi-Nakao, 2007).
Spastin is upregulated by Knot in da neurons and is required for class IV neuron dendritic arbor outgrowth. To confirm that Spastin is part of the program by which Knot mediates dendritic arbor outgrowth, the outcome was investigated of spastin RNAi in either a wild-type or an ectopic knot-expressing class I neuron. UAS-spastinRNAi was crossed to Gal42-21, UAS-mCD8::GFP or UAS-kn; Gal42-21, UAS-mCD8::GFP, and class I ddaE dendritic arbor shape was assayed at wandering third-instar larva stage. The spastinRNAi construct had no effect on either branching or dendrite length when expressed in a wild-type class I neuron. However, the UAS-spastinRNAi construct strongly reduced both branching and total dendrite length (by 18% and 19%, respectively) when expressed in an ectopic knot-expressing class I neuron. Therefore, Spastin activity is an essential part of the program by which ectopic knot expression mediates an increase in dendritic arbor complexity (Jinushi-Nakao, 2007).
This study has shown that Knot and Cut act simultaneously in the class IV neuron to promote dendritic arbor outgrowth and branching. However, the loss-of-function phenotypes for Knot and Cut are different, which demonstrates that each transcription factor works through a dissimilar mechanism. Indeed, ectopic expression experiments show that Knot and Cut regulate different aspects of the cytoskeleton. Knot expressed ectopically in the class I neuron promotes arbor extension that is microtubule-positive. Conversely, ectopic expression of Cut in the class I neuron leads to arbor extension that is F-actin-positive but microtubule deficient. When Cut and Knot are expressed together in the class I neuron, they have a synergistic effect on dendritic arbor area and branching. However, the effect of Cut and Knot coexpression on dendritic arbor total length is additive: both the microtubule-positive and microtubule-negative regions of the arbor are increased. This overall arbor organization mimics that of class IV neurons. The majority of the dendritic arbor of the class IV neuron contains microtubules, but the highest-order branches are microtubule deficient (Jinushi-Nakao, 2007).
When Cut levels are increased and Knot levels are reduced in a class IV neuron, its dendritic arbor takes on characteristics similar to those of class III. This transformation from a class IV to a class III shape is not absolute. Hence, it is likely that other factors are also required to fully control all aspects of class IV dendritic arbor morphology versus those of other da neuron classes. Overall, however, the data suggest that class IV-specific Knot expression demarcates arbor shape via multiple mechanisms. Knot synergizes with Cut in promoting dendrite length, branching, and area. Additionally it represses the ability of Cut to mediate filopodia/spike formation. Finally, Knot induces symmetry in the dendritic arbor of the class IV neuron, as opposed to class I–III neurons, which have asymmetric dendritic arbor shapes (Jinushi-Nakao, 2007).
Suppression of Cut-mediated filopodia/spike formation by Knot does not occur through repression of Cut protein levels and therefore acts either downstream of Cut or in parallel. Interestingly, though, the absolute level of Knot protein is controlled by the level of Cut in the cell. Tuning the level of Knot to the level of Cut protein in each neuron could be a mechanism by which Knot acts to repress only specific aspects of the Cut-driven morphogenesis program (Jinushi-Nakao, 2007).
Knot and Cut also interact very differently with Rac1. A major function of Rac1 is to promote reorganization of the actin cytoskeleton. Filopodia/spikes are rich in F-actin and deficient in microtubules, and indeed Rac1 significantly enhances the ability of Cut to promote filopodia/spike formation. Ectopic coexpression of Knot and Rac1 in class I neurons leads to large increases in the length of the short, thorn-shaped projections that are induced by expression of Rac1 alone. Rac1 has been shown to form focal F-actin in the distal edge of axonal growth cones, which acts as a site of microtubule capture during outgrowth. Perhaps similar processes are occurring in the Rac1-mediated thorn-shaped projections. Knot activity could then promote microtubule invasion and outgrowth at these points of Rac1-mediated F-actin reorganization (Jinushi-Nakao, 2007).
Knot promotes microtubule-mediated dendritic arbor outgrowth by inducing Spastin expression. A large amount of the extra arbor outgrowth induced by ectopic Knot expression is suppressed by reducing Spastin function. Therefore, Spastin is a primary component of the mechanism by which Knot promotes arbor outgrowth (Jinushi-Nakao, 2007).
Spastin acts as a microtubule-severing protein and may function by producing new seeds for microtubule polymerization. Maintenance of a population of dynamic microtubules is important for axonal extension, branching, and growth cone guidance. In vivo, Spastin has been shown to be required for growth of synaptic terminals at the Drosophila neuromuscular junction and for axon outgrowth in zebrafish. This study shows that Spastin activity can also destabilize microtubules in the dendritic arbor, and that Spastin is itself required for class IV da dendritic arbor outgrowth (Jinushi-Nakao, 2007).
The human spastin gene (SPG4) is mutated in over 40% of autosomal dominant hereditary spastic paraplegia (AD-HSP) cases. SPG4 mutation usually causes pure spastic paraplegia of the lower limbs due to degeneration of the corticospinal tract axons. However, in some families SPG4 mutation is associated with additional neurological symptoms that cannot be explained by dysfunction of the corticospinal tract axons alone. This study shows that Spastin is also required for complex dendritic arbor development; hence, defects in dendrite as well as axon development and function may be part of the pathology of some AD-HSP cases (Jinushi-Nakao, 2007).
Accumulating evidence suggests that mechanisms of dendritogenesis are closely conserved between Drosophila and other species. For example, actin binding proteins, rac/rho GTPases, and calcium/calmodulin-dependent protein kinase II (CaMKII) all control dendrite branching and filopodia/spine morphogenesis in Drosophila and in vertebrates. knot (Ebf1, 2, and 3) and cut (Cux1 and 2) homologs are expressed in the developing mouse nervous system and may overlap in some subsets of neurons, e.g., in the spinal cord and cerebellum. In vertebrates, it is possible that these genes may also regulate dendrite morphology. Both human CUX1 and mouse Ebf2 can phenocopy cut and knot, respectively, when ectopically expressed in class I neurons. Interestingly, Ebf2 is involved in the migration and differentiation of Purkinje neurons. However, a specific role for Ebf2 in controlling the highly complex dendritic arbor shape of these neurons remains to be assayed (Jinushi-Nakao, 2007).
This study has elucidated mechanisms of transcription factor-mediated control of dendritogenesis. It was found that Knot and Cut function to control Drosophila class IV da sensory neuron dendritic arbor morphogenesis through different aspects of the cytoskeleton. Further analysis of Knot and Cut targets will provide a powerful entry point into understanding dendritic arbor morphogenetic mechanisms that are potentially conserved between Drosophila and vertebrate species (Jinushi-Nakao, 2007).
Bases in 5' UTR - 512
Bases in 3' UTR - 1268
Two protein regions are particularly well conserved between Col and EBF/Olf-1 (Early B-cell factor). The first one, which is 210 amino-acids long and lies between residues 59 and 269 of Col, shows 86% identity and corresponds to the DNA-binding domain of EBF. The second region (Col residues 297 to 431) shows 89% identity and partially overlaps a region of EBF sufficient for homodimerization in vitro. There is a consensus helix-1-loop-helix-2 motif in this region, which is also conserved in the rodent proteins. A second potential helix-2 reported in EBF/Olf-1 is absent in Col. The HLH dimerization motif is not preceded by a basic region, consistent with the presence of the independent DNA binding domain (between residues 59 and 269). The C-terminal region is rich in alanine, serine and threonine residues and probably represents a transcription activation domain (Crozatier, 1996).
The col gene maps to the chromosomal region 51C1,2. In order to establish its molecular organization, approx. 45 kb of overlapping genomic DNA were isolated covering the col transcription unit and the relevant regions were sequenced. The col transcription unit consists of 12 exons and 11 introns spanning a genomic region of about 30 kb. Introns separate the coding regions for each Col functional domain, defined by biochemical dissection of EBF and sequence conservation during evolution. These are the DNA binding domain (aa 59 to 288), the homodimerization domain (aa 289 to 429), and a putative transactivation domain at its carboxy-terminal end. However, additional introns split the Col DNA binding and homodimerization domains, despite their extensive primary sequence conservation in all COE proteins identified so far, from nematode to vertebrates. Within the homodimerization domain, the helix-loop-helix (HLH) motif is encoded by a single exon, exon 9. Finally, the genomic structure of col indicates that the two predicted Col embryonic protein isoforms, which differ in their carboxy-terminal protein coding region, result from alternative splicing of exon 11 (Crozatier, 1999b).
date revised: 30 May 2008
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