cut: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

Gene name - cut

Synonyms -

Cytological map position - 7B1-2

Function - transcription factor

Keyword(s) - selector gene, ventral midline, peripheral nervous system

Symbol - ct

FlyBase ID:FBgn0004198

Genetic map position - 1-20.0

Classification - homeodomain and cut repeat

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene | UniGene |

Recent literature
Jia, D., Soylemez, M., Calvin, G., Bornmann, R., Bryant, J., Hanna, C., Huang, Y. C. and Deng, W. M. (2015). A large-scale in vivo RNAi screen to identify genes involved in Notch-mediated follicle cell differentiation and cell cycle switches. Sci Rep 5: 12328. PubMed ID: 26205122
During Drosophila oogenesis, follicle cells sequentially undergo three distinct cell-cycle programs: the mitotic cycle, endocycle, and gene amplification. Notch signaling plays a central role in regulating follicle-cell differentiation and cell-cycle switches; its activation is essential for the mitotic cycle/endocycle (M/E) switch. Cut, a linker between Notch signaling and cell-cycle regulators, is specifically downregulated by Notch during the endocycle stage. To determine how signaling pathways coordinate during the M/E switch and to identify novel genes involved in follicle cell differentiation, an in vivo RNAi screen was performed through induced knockdown of gene expression and examination of Cut expression in follicle cells. 2205 RNAi lines were screened, and 33 genes were found regulating Cut expression during the M/E switch. These genes were confirmed with the staining of two other Notch signaling downstream factors, Hindsight and Broad, and validated with multiple independent RNAi lines. Gene ontology software was applied to find enriched biological meaning, and the results were compared with other publications to find conserved genes across tissues. Specifically, earlier endocycle entry in anterior follicle cells was found than those in the posterior, the insulin-PI3K pathway was found to participate in the precise M/E switch, and Nejire was suggested as a cofactor of Notch signaling during oogenesis.

Jia, D., Bryant, J., Jevitt, A., Calvin, G. and Deng, W. M. (2016). The ecdysone and Notch pathways synergistically regulate Cut at the dorsal-ventral boundary in Drosophila wing discs. J Genet Genomics [Epub ahead of print]. PubMed ID: 27117286
Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression. In Drosophila, the wing imaginal disc provides an excellent model for the study of how signaling pathways interact to regulate pattern formation. The determination of the dorsal-ventral (DV) boundary of the wing disc depends on the Notch pathway, which is activated along the DV boundary and induces the expression of the homeobox transcription factor, Cut. This study shows that Broad (Br), a zinc-finger transcription factor, is also involved in regulating Cut expression in the DV boundary region. However, Br expression is not regulated by Notch signaling in wing discs, ecdysone signaling is the upstream signal that induces Br for Cut upregulation. Also, it was found that the ecdysone-Br cascade upregulates cut-lacZ expression, a reporter containing a 2.7 kb cut enhancer region, implying that ecdysone signaling, similar to Notch, regulates cut at the transcriptional level. Collectively, these findings reveal that the Notch and ecdysone signaling pathways synergistically regulate Cut expression for proper DV boundary formation in the wing disc. Additionally, br was shown to promote Delta, a Notch ligand, near the DV boundary to suppress aberrant high Notch activity, indicating further interaction between the two pathways for DV patterning of the wing disc.
de Miguel, C., Linsler, F., Casanova, J. and Franch-Marro, X. (2016). Genetic basis for the evolution of organ morphogenesis. The case of spalt and cut in development of insect trachea. Development [Epub ahead of print]. PubMed ID: 27578790
Changes in body organ morphology have allowed animals to better exploit diverse habitats. As morphogenesis in general and organogenesis in particular are under genetic control, genetic modifications provide the basis for a wide range of morphologies. Knowledge of the genetic basis of phenotypic diversification in evolution has focused mostly on quantitative traits. However, it is not clear how simple genetic changes can account for the coordinated variations that give rise to modified functional organs. This study addressed this issue by analysing the expression and function of regulatory genes in the developing tracheal systems of two insect species. The larval tracheal system of Drosophila can be distinguished from the less derived tracheal system of the beetle Tribolium by two main features. First, the lateral spiracles, which in Tribolium connect the tracheal branches to the exterior in each segment, are not present in Drosophila. Instead, Drosophila has only one pair of strongly derived posterior spiracles. Second, the dorsal trunks, two prominent branches that distribute air from the posterior spiracles and extend longitudinally through the larva, are not present in Tribolium. Both innovations, while considered different structures, are functionally dependent on each other and linked to habitat occupancy. In this regard, buried Drosophila larvae in semi-liquid environments keep their posterior spiracles above the surface and distribute the gas along the body via the dorsal trunks. Conversely, the lateral spiracles of free-living Tribolium larvae provide sufficient airflow to all segments making unnecessary the formation of thick dorsal trunks. This study shows that changes in the domains of spalt and cut expression are associated with the acquisition of each innovation. Moreover, these two genetic modifications are connected both functionally and genetically, thus providing an evolutionary scenario by which a genetic event contributes to the joint evolution of functionally interrelated structures.
Das, R., Bhattacharjee, S., Patel, A. A., Harris, J. M., Bhattacharya, S., Letcher, J. M., Clark, S. G., Nanda, S., Iyer, E. P. R., Ascoli, G. A. and Cox, D. N. (2017). Dendritic cytoskeletal architecture is modulated by combinatorial transcriptional regulation in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 29025914
Studies in Drosophila melanogaster have demonstrated that the conserved transcription factors (TFs) Cut and Knot exert combinatorial control over aspects of dendritic cytoskeleton development, promoting actin- and MT-based arbor morphology, respectively. To investigate transcriptional targets of Cut and/or Knot regulation, systematic neurogenomic studies were conducted, coupled with in vivo genetic screens utilizing multi-fluor cytoskeletal and membrane marker reporters. These analyses identified a host of putative Cut and/or Knot effector molecules and a subset of these putative TF targets converge on modulating dendritic cytoskeletal architecture and are grouped into three major phenotypic categories, based upon neuromorphometric analyses:-- complexity enhancer, complexity shifter, and complexity suppressor. Complexity enhancer genes normally function to promote higher order dendritic growth and branching with variable effects on MT stabilization and F-actin organization, whereas complexity shifter and complexity suppressor genes normally function in regulating proximal-distal branching distribution or in restricting higher order branching complexity, respectively, with spatially restricted impacts on the dendritic cytoskeleton. Collectively, this study implicate novel genes and cellular programs by which TFs distinctly and combinatorially govern dendritogenesis via cytoskeletal modulation.
Xu, K., Liu, X., Wang, Y., Wong, C. and Song, Y. (2018). Temporospatial induction of homeodomain gene cut dictates natural lineage reprogramming. Elife 7. PubMed ID: 29714689
Understanding how cellular identity naturally interconverts with high efficiency and temporospatial precision is crucial for regenerative medicine. This study revealed a natural midgut-to-renal lineage conversion event during Drosophila metamorphosis and identified the evolutionarily-conserved homeodomain protein Cut as a master switch in this process. A steep Wnt/Wingless morphogen gradient intersects with a pulse of steroid hormone ecdysone to induce cut expression in a subset of midgut progenitors and reprogram them into renal progenitors. Molecularly, ecdysone-induced temporal factor Broad physically interacts with cut enhancer-bound Wnt pathway effector TCF/beta-catenin and likely bridges the distant enhancer and promoter region of cut through its self-association. Such long-range enhancer-promoter looping could subsequently trigger timely cut transcription. These results therefore led the autnors to a propose an unexpected poising-and-bridging mechanism whereby spatial and temporal cues intersect, likely via chromatin looping, to turn on a master transcription factor and dictate efficient and precise lineage reprogramming.
Clark, S. G., Graybeal, L. L., Bhattacharjee, S., Thomas, C., Bhattacharya, S. and Cox, D. N. (2018). Basal autophagy is required for promoting dendritic terminal branching in Drosophila sensory neurons. PLoS One 13(11): e0206743. PubMed ID: 30395636
Relatively little is known regarding the developmental role of basal autophagy in directing aspects of dendritic arborization. This study demonstrates that autophagy-related (Atg) genes are positively regulated by the homeodomain transcription factor Cut, and that basal autophagy functions as a downstream effector pathway for Cut-mediated dendritic terminal branching in Drosophila multidendritic (md) sensory neurons. Further, loss of function analyses implicate Atg genes in promoting cell type-specific dendritic arborization and terminal branching, while gain of function studies suggest that excessive autophagy leads to dramatic reductions in dendritic complexity. The Atg1 initiator kinase interacts with the dual leucine zipper kinase (DLK) pathway by negatively regulating the E3 ubiquitin ligase Highwire and positively regulating the MAPKKK Wallenda. Finally, autophagic induction partially rescues dendritic atrophy defects observed in a model of polyglutamine toxicity. Collectively, these studies implicate transcriptional control of basal autophagy in directing dendritic terminal branching and demonstrate the importance of homeostatic control of autophagic levels for dendritic arbor complexity under native or cellular stress conditions.

cut expression is detected in a bewildering variety of organs, including external sensory organs, Malpighian tubules, ovary follicule cells and precursors of adult muscle cells. These organs appear to share no common developmental origin, and this only adds to cut's intrigue and the mystery of its function (Blocklinger, 1993).

Cut contains a unique homeodomain, different from all other homeodomains, and three unusual domains. Taken together they define the Cut repeat. In the mammalian Cut counterpart, one Cut repeat is known to have DNA binding activity independent of the homeodomain (Andres, 1994).

The regulation of cut is as complex as any other gene with tissue specific enhancers. Eight tissue specific enhancers have been identified for cut, including wing margin external sensory organs, Malphighian tubules, posterior spiracles and tracheae. These enhancers are distributed over 80 kb upstream from the structural gene (Jack, 1995). cut expression in different organs has appeared downstream of Krüppel, pox neural and perhaps achaete. However, cut expression persists after these other genes become silent (Jack, 1995).

Somatically expressed cut interacts with Notch to regulate egg chamber formation and to maintain germline cyst integrity during Drosophila oogenesis. Communication between the germline and the soma during Drosophila oogenesis is essential for the formation of egg chambers and to establish polarity in the developing oocyte. Cut expression initiates in somatic follicle cells in region 2b of the germarium, at about the same time that follicle cells interleave and surround germline cysts. It persists in all follicle cells, including the polar follicle cells and the interfollicular stalk cells, until about stage 6. Between stages 6 and 9 of oogenesis, Cut expression ceases in all the follicle cells except the polar follicle cells. At about stage 10, Cut expression resumes, first in the anterodorsal follicle cells, then throughout the layer of columnar follicle cells which surround the oocyte, and continues in these cells until stage 14 (Jackson, 1997).

Genetic manipulations of cut activity results in defective packaging of germline-derived cysts into egg chambers and disintegration of the structural organization of oocyte-nurse cell complexes to generate multinucleate germline-derived cells. Although these egg chambers invariably

contain 16 germline-derived nuclei, the total number of ring canals is frequently decreased to 14 or 13. The distribution of ring canals is also abnormal in affected egg chambers. cut null alleles in combination with wimp result in egg chamber defects. wimp encodes the RNA polymerase II 140 kD subunit. Ectopic cut expression produces compound egg chamber. cut interacts genetically with the Notch gene and with the catalytic subunit of Protein kinase A gene during egg chamber morphogenesis. cut null mutations suppress loss of Notch function during oogenesis. Two different cut null mutations reduce the incidence of compound egg chambers found in mutant Notch ovaries. Since cut expression is restricted to the somatic follicle cells and cut mutant germline clones are phenotypically normal, it is proposed that the defects in the assembly of egg chambers and the changes in germline cell morphology observed in cut mutant egg chambers are the result of altered interactions between follicle cells and germline cells. It is suggested that cut participates in intercellular communications by regulating the expression of molecules that directly participate in this process (Jackson, 1997). Interestingly, Notch and cut also interact, albeit synergistically, during the development of the wing margin; the activation of cut expression along the prospective wing margin has been shown to depend on Notch activity (de Celis, 1996, Micchelli, 1997 and Neumann, 1996).

Depending on the tissue observed, cut mutations cause a variety of transformations and defects. External sensory organs are transformed into chordotonal neurons (Blockinger, 1991). Malpighian tubules become bladder-like structures (Liu, 1992). Multiple dendritic organs are transformed from one subtype to another (Brewster, 1995).

If one generalization is to be made, it must be that cut expression is so broad, it may be found in a subset of cell types for any given tissue. Also relevant is the fact that cut is activated at the precursor stage, and expression persists in the differentiated progeny. It would seem that cut is responsible for subtype specificity in a number of cell lineages, possibly for the maintenance of gene expression in differentiated cells. Additionally amazing is the conservation of this gene's structure and function through 600 million years of evolution.

The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal

The peripheral nervous system of Drosophila offers a powerful system to precisely identify individual cells and dissect their genetic pathways of development. The mode of specification of a subset of larval PNS cells, the multiple dendritic (md) neurons (or type II neurons), is complex and still poorly understood. A morphological categorization of md neurons reveals three subpopulations: md-da neurons are the most abundant subclass, which have extensive dendritic arborizations; md-bd neurons have bipolar dendrites; and md-td neurons extend their dendrites along tracheal branches. Within the dorsal thoracic and abdominal segments, two md neurons, dbd and dda1, apparently require the proneural gene amos but not atonal or Achaete-Scute-Complex (ASC) genes. ASC normally acts via the neural selector gene cut to specify appropriate sensory organ identities. Dbd- and dda1-type differentiation is suppressed by cut in dorsal ASC-dependent md neurons. Thus, cut is not only required to promote an ASC-dependent mode of differentiation, but also represses an ASC- and ato-independent fate that leads to dbd and dda1 differentiation (Brewster, 2001).

The requirement for proneural gene function is arguably the most reliable criterion for distinguishing different sensory cell types. In support of this idea, neurons that require the same proneural gene for their formation have been shown to project to the central nervous system in a similar fashion. ASC is required for the formation of all es neurons, for a large number of md neurons, which are lineage-related to es organs, and for most solo md lineages. In contrast, the ch organs and three ventral ch-related md neurons depend on ato. In the absence of both ASC and ato, the only remaining cells of the PNS are two dorsally located md neurons, suggesting that they are dependent on an unknown proneural gene (Brewster, 2001).

Since all the components of the PNS, including these two md neurons, require the bHLH gene da for their formation, it is likely that the 'missing' proneural gene also codes for a bHLH-containing binding partner for Da. Indeed, a likely candidate gene has recently been identified, which apparently is required for the formation of dbd and a dorsal da neuron (Brewster, 2001).

pdm1 and pdm2, two closely linked genes belonging to the POU family of transcription factors, are co-expressed in two PNS neurons that are potentially coincident with ASC/ato-independent neurons: dbd and a dorsal md-da neuron. In addition, the ligament cells of lch5 also weakly express these genes. To determine if the pdm-expressing cells are ASC-and ato-independent, the pdm1 expression pattern was examined in ASC and ato single and double mutant embryos. In all three mutant configurations pdm1 expression is present in dbd and a dorsal md-da neuron. The latter will be referred to henceforth as dda1 (dorsal da neuron #1). Thus, pdm specifically marks the ASC/ato-independent subclass of PNS neurons (Brewster, 2001).

In order to further characterize ASC/ato-independent md neurons, other markers expressed in these cells were sought. en and the lacZ reporter gene from the E7-3-49 enhancer trap line were identifed. In the PNS, en is expressed in one dorsal md neuron as early as stage 11. The E7-3-49 line confers lacZ expression to several PNS cells, including dbd and 2-4 dorsal da-md neurons. Co-incidence of expression of pdm1 with that of en and E7-3-49 was examined in the PNS. Double-labeling for expression of pdm1 and E7-3-49-lacZ reveals that they overlap in dda1 and dbd. Similarly, pdm and en are co-expressed in dda1 but not in dbd (Brewster, 2001).

The expression pattern of the homeobox neural selector gene cut encompasses all es organs and a large number of md neurons (the majority of which are related to es organs by lineage). cut is clearly not expressed in the readily identifiable dbd neuron. Since dbd and dda1 co-express the markers described above and perhaps are specified by the same proneural gene(s), whether dda1 is indeed negative for cut expression was examined. Embryos double-labeled for cut and E7-3-49-lacZ or pdm show that the pattern of cut expression in the dorsal PNS cluster is complementary to these markers. These results indicate that, unlike the majority of md neurons, dda1 and dbd (along with its sibling glial cell) are specified in a cut-independent fashion (Brewster, 2001).

The observation that dda1 and dbd co-express a number of cell markers and are both ASC/ato-independent raises the possibility that these cells derive from a common precursor. In order to address the lineage relation of these cells, random lacZ-expressing clones previously generated using the flp/FRT method were examined. Of the four embryos in which the dbd neuron and its accompanying glial cell were clearly lacZ-positive: no dorsal da neuron in the position corresponding to dda1 was co-labeled. These data suggests that dda1 and dbd/sibling glial cells are likely to be generated from two independent precursor cells. The dynamic pattern of amos expression in the dorsal cluster region is consistent with this hypothesis (Brewster, 2001).

The expression of ASC and ato in proneural domains is thought to become restricted to and enhanced in single SOPs during a process called lateral inhibition, mediated by the 'neurogenic' genes. A failure to down-regulate proneural gene expression in most cells of a proneural cluster results in a massive overproduction of PNS neurons, as observed in neurogenic mutants. Therefore, the number of dbd and dda1 neurons in neurogenic mutants was examined. en- and pdm-expressing PNS neurons are produced in large excess in mutants of Notch, Delta, mastermind and neuralized, and less so in big brain mutants, similar to the other components of the PNS. These findings suggest that the genetic control mechanism of lateral inhibition is common to all SOPs of the PNS, including the ASC/ato-dependent neurons. The size of ASC proneural clusters that will give rise to thoracic macrochaetes in the adult fly has been estimated at 20-30 cells. Due to the close proximity of sensory cells in the PNS and to the limited number of cell-specific markers, the size of embryonic proneural clusters has been difficult to estimate. Since en is only expressed in dda1, the size of the dda1 proneural cluster was assessed by counting the number of en-expressing dorsal PNS cells in neurogenic mutants. In Notch mutants, 10 En-positive cells are present on average per cluster (with a range of five to 14 cells), consistent with the estimated number of cells in the amos-expressing proneural clusters (Brewster, 2001).

Since en and pdm are specifically expressed in the ASC/ato-independent PNS, the possibility that these two genes may play a role in the formation and/or differentiation of dbd and dda1 was examined. (1) pdm1 expression was examined in embryos that lacked en function and in embryos in which en was overexpressed via a heat shock-driven transgene. In both en loss- and gain-of-function mutants, a normal number of peripheral pdm-expressing cells was found. This suggests that en may not be essential for the formation and correct specification of dda1. It cannot be ruled out, however, that en may have a redundant function in this process. For example, although en4 is a strong loss-of-en-function allele, expression of its neighboring sister gene, invected, which can substitute for en function when over-expressed, may not decline in dda1 in the absence of en function as it does in most other situations. Alternatively, en may be involved in distinguishing dda1 versus the dbd identity (analogous to the role of cut in specifying es versus ch organ fate). This possibility seems unlikely, since the overall morphology of dda1, as determined by pdm expression and Nomarski optics, appears unchanged in en mutants (Brewster, 2001).

(2) en and E7-3-49-driven lacZ expression was examined in embryos that lacked both pdm genes [by using Df(2L)GR4, a genetic deficiency for both genes], and in embryos in which pdm1 or pdm2 were overexpressed using a heat shock vector. In all embryos examined, en and E7-3-49-lacZ expression appeared normal. The characteristic bilateral extensions of dbd were also unchanged in pdm mutants as observed with anti-FasII and 22C10 antibodies. en;Df(2L)GR4 double mutants were generated to determine if dda1 formation is affected when both en and the pdm genes are inactivated. Again, as in the single mutants, double mutant embryos appeared normal at least with respect to dbd formation and morphology. Taken together, en and pdm are unlikely to have a crucial role in the correct specification of the ASC/ato-independent md neurons (Brewster, 2001).

The neural selector gene cut encodes a homeodomain-containing transcription factor and functions as a critical bimodal switch between different cell fates in the PNS. In cut mutants, es organs are transformed into ch-like organs, as assessed by multiple morphological and immunocytochemical criteria. The ASC-dependent md neurons also acquire an alternative fate in cut mutants. For example, E7-3-49-driven lacZ expression is greatly expanded in cut mutants and encompasses nearly all md neurons of the da subtype. In that study, however, it was not clear what type of md fate was in fact assumed by the new E7-3-49-lacZ-expressing cells (Brewster, 2001).

In order to address this question further, the identity of the transformed md-da neurons in cut mutants was examined with markers for ASC/ato-independent neurons. Similar to E7-3-49, the expression of pdm1 is expanded to additional neurons in the dorsal and lateral PNS clusters. The cells expressing pdm1 ectopically are also positive for a marker, the E7-2-36 enhancer trap line, which is specific for all md neurons, suggesting that the extra Pdm1-positive neurons are indeed md neurons. The mechanism for restricting ectopic pdm1 but not E7-3-49- lacZ expression to the dorsal and lateral clusters is not known. Overall, these findings are consistent with the interpretation that in cut mutants many ASC-dependent md neurons are transformed towards an ASC/ato-independent rather than an ato-dependent fate (Brewster, 2001).

In contrast to these findings with E7-3-49-lacZ and pdm1 expression, when en expression was examined in cut mutants, the pattern of en-expressing PNS cells is unaltered, i.e. there is only one En-positive cell per dorsal cluster. Since en is not expressed in dbd but pdm1 is, the possibility that the invariance of en expression reflects the acquisition of a dbd rather than a dda1 cell fate in cut mutants cannot be ruled out. This possibility seems unlikely, however, since the morphology of supernumerary Pdm1-positive cells is unlike that of dbd, and a marker for the dbd-associated glial cell (repo) is not ectopically expressed in cut mutants (Brewster, 2001).

Taken together, it appears that in cut mutants the md neurons that normally depend on ASC in dorsal and lateral clusters are transformed towards an ASC/ato-independent fate, as determined by pdm and E7-3-49-lacZ expression. However, the postulated cell fate change may be incomplete due to the lack of ectopic en expression. This partial phenotype is not surprising, since cut (null) mutants also exhibit variability and incomplete phenotypic penetrance with respect to es organ transformation towards a ch fate. It is thus likely that gene functions other than cut also contribute to the restriction of en and pdm1, similar perhaps to the situation of ato-dependent md neurons, which do not express cut or pdm/en (Brewster, 2001).

Since ASC-dependent md neurons partially exhibit characteristics of the ASC/ato-independent PNS in cut mutants, it was of interest to see if overexpression of cut results in suppression of marker gene expression typical of an ASC/ato-independent fate. It has already been shown that cut overexpression abolishes E7-3-49-driven lacZ expression in the PNS. pdm1 expression was examined in cut overexpression embryos, and a nearly complete loss of Pdm1 protein was observed in dbd and dda1. Moreover, en expression is also suppressed in virtually all dda1 neurons. Thus, en fails to be ectopically expressed in cut mutants, yet ectopic cut expression is apparently sufficient to repress PNS-specific en expression in dda1. These findings lend support to the hypothesis that in ASC-dependent md neurons, cut apparently contributes to the repression of an ASC/ato-independent md fate (Brewster, 2001).


cDNA clone length - 8217 base pairs

Bases in 5' UTR - 210

Exons - four

Bases in 3' UTR - 1388


Amino Acids - 725

Structural Domains

The Cut protein is a homeodomain homolog. Although it is the most divergent of all Drosophila homeodomain proteins, it shows identity at the nine residues that are invariant in all homeodomain proteins. There are three 60 residue repeats showing 55-68% identity to each other which are not homologous to repeats found in other proteins. These are known as the Cut repeats. There are also four stretches of polyglutamate/aspartate and a number of runs rich in single amino acids (Blochlinger, 1988 and Aufiero, 1994).

The CCAAT displacement protein, the homolog of the Drosophila melanogaster Cut protein, contains four DNA-binding domains: three CUT repeats (CR1, CR2, and CR3) and the CUT homeodomain (HD). Using a panel of fusion proteins, it was found that a CUT repeat cannot bind to DNA as a monomer, but that certain combinations of domains exhibit high DNA-binding affinity: CR1+2, CR3HD, CR1HD, and CR2HD. One combination (CR1+2) exhibits strikingly different DNA-binding kinetics and specificities. CR1+2 displays rapid on and off rates and binds preferably to two C(A/G)AT sites, organized as direct or inverted repeats. Accordingly, only CR1+2 is able to bind to the CCAAT sequence, and its affinity is increased by the presence of a C(A/G)AT site at close proximity. A purified CCAAT displacement protein/CUT protein exhibits DNA-binding properties similar to those of CR1+2; and in nuclear extracts, the CCAAT displacement activity also requires the simultaneous presence of a C(A/G)AT site. Moreover, CR1+2, but not CR3HD, is able to displace nuclear factor Y. Thus, the CCAAT displacement activity requires the presence of an additional sequence (CAAT or CGAT) and involves CR1 and CR2, but not the CUT homeodomain (Moon, 2001).

These results reveal that cooperation between various CDP/CUT DNA-binding domains can generate at least two DNA-binding activities with distinctly different binding kinetics and specificities: (1) CR1 and CR2 bind rapidly but transiently to sequences containing two C(A/G)AT sites in either orientation; (2) CR3 and the CUT homeodomain can form a stable complex with the ATCGAT DNA sequence. Moreover, the purified full-length protein binds with higher affinity to oligonucleotides containing two binding sites. Thus, the two bipartite domains CR1+2 and CR3HD can cooperate, albeit weakly, to bind to DNA. Surprisingly, however, the full-length CUT protein binds in vitro with kinetics similar to those of CR1+2, with fast on and off rates. Although a fast on rate was to be expected because of CR1+2, the simultaneous presence of CR3HD should have stabilized the protein on DNA. These results suggest the possibility that CR3HD is not very active in the context of the full-length CDP/CUT protein. This would help explain the lack of extensive cooperativity as noted above. Although such behavior by CDP/CUT in cells could be explained by invoking the phosphorylation of the homeodomain during the G1 phase of the cell cycle, the same explanation cannot hold in the case of the purified full-length protein that was dephosphorylated in vitro prior to DNA binding. The possibility is considered that CR1+2, in a manner analogous to high mobility group proteins, may impart a conformational change to DNA that would cause the quick release of CR3HD from its adjacent binding site. But when tested as individual proteins, CR1+2 and CR3HD are able to bind simultaneously to the N19 probe, which contains binding sites for both proteins. Therefore, CR1+2 does not prevent the stable binding of CR3HD to an adjacent site. The results instead suggest that something else, perhaps some conformational constraint, prevents CR3HD from binding to DNA with high affinity in the context of the purified full-length protein; yet the same protein, when present within nuclear extracts in S phase, can stably bind to DNA. The molecular basis for this discrepancy is not known; however, the results clearly point to a difference in the behavior of the CDP/CUT protein in vitro and in nuclear extracts (Moon, 2001).

cut: Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

date revised: 20 Nov 2001  

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