Gene name - chiffon
Cytological map position - 35F11--36A2
Function - Regulatory subunit of Cdc7-Dbf4 dimer
Keywords - cell cycle
Symbol - chif
FlyBase ID: FBgn0000307
Genetic map position - 2-53
Classification - Dbf4 homolog
Cellular location - presumably nuclear
Chiffon is the Drosophila homolog of yeast Dbf4; its function is associated with the origin recognition complex (ORC), which is a multisubunit protein complex that binds to chromosomal origins of replication and is required for the initiation of cellular DNA replication. In yeast, one of the last steps in activation of the origin of replication appears to be phosphorylation of one or more origin-associated proteins by the Cdc7-Dbf4 protein kinase (Jackson, 1993; Lei, 1997; Owens, 1997). Cdc7 (Drosophila homolog: Cyclin-dependent kinase 7) is a cdk-like serine/threonine protein kinase whose activity is regulated in a cell-cycle-dependent manner by association with its regulatory subunit Dbf4. Dbf4 binds to origin-associated proteins, thus providing a likely mechanism for recruitment of Cdc7 to target(s) at the origin (Dowell, 1994). As the regulatory complex of the Cdc7-Chiffon dimer, Chiffon interacts with unknown regulators and activates the kinase function of its dimerization partner, thus triggering the initiation of cellular DNA replication. A better understanding of Chiffon should lead to an enhanced understanding of the regulation of DNA replication, a central feature of the biology of the cell (Landis, 1999 and references therein).
To meet demand for rapid synthesis of chorion (eggshell) proteins, Drosophila ovary follicle cells amplify the two chromosomal clusters of major chorion genes ~80-fold. Amplification occurs through repeated firing of one or more DNA replication origins located near the center of the gene clusters. Chorion gene amplification is amenable to both molecular and genetic analysis, making it an ideal model system for the study of metazoan chromosomal DNA replication origins and their regulation. Constructs derived from the third chromosome chorion gene cluster are able to amplify when reinserted into the Drosophila genome using P element-mediated transformation. This assay has allowed mapping of a cis-acting control element required for high levels of amplification (termed ACE3 for Amplification Control Element, 3rd chromosome), as well as several stimulatory regions. Two-dimensional gel analysis of DNA replication intermediates originating from the endogenous locus and from transgenic constructs has allowed mapping of the major origin of replication, called Ori-beta, as well as one or two more minor origin regions (Landis, 1999 and references).
Mutations that disrupt genes required in trans for amplification cause female sterility. The sterility is due, at least in part, to underproduction of chorion proteins and synthesis of thin, fragile chorions. Twelve genes have been identified that are required for amplification (Calvi, 1998; Royzman, 1999). The first of these to be cloned (k43), was found to encode the Drosophila homolog of the S. cerevisiae Origin Recognition Complex subunit 2 (ORC2) (Gossen, 1995; Landis, 1997). The ORC is a complex of six proteins (Orc1p-Orc6p) that binds to S. cerevisiae chromosomal DNA replication origins in vivo and in vitro. The phenotypes of mutations in yeast ORC subunit genes demonstrate that ORC is required for origin function, as well as for transcriptional silencing of the yeast mating-type loci. ORC is associated with the origin throughout the cell cycle and activation of the origin appears to result from association of additional proteins with the ORC. The identification of Drosophila k43 as the homolog of S. cerevisiae ORC2 (see Drosophila Origin recognition complex 2) suggests that all or part of the mechanism of origin regulation may be conserved between yeast chromosomal origins and the chorion gene origins. The chiffon gene was originally identified in female sterile mutants, which lay eggs with thin eggshells (Schüpbach, 1991). Experiments have shown that chiffon is required in trans for chorion gene amplification. Cloning and characterization of the Drosophila chiffon gene have revealed that the predicted Chiffon protein contains two domains (designated CDDN for Chiffon, Dbf4, Dfp1 and NimO); these four proteins are related to regulators of DNA replication and cell cycle in lower eukaryotes (Landis, 1999).
Chorion gene amplification occurs through the repeated firing of one or a small number of origins located near the center of each of the chorion gene clusters. Amplification is expected to utilize all or part of the cells general DNA replication machinery. In addition, there must be some mechanism making this process specific to the follicle cells and to only the origins associated with the chorion gene clusters. In S. cerevisiae, ORC is required for origin activity; however, activation of the origin requires binding of additional proteins prior to S phase. Firing of the S. cerevisiae origin in S phase also requires the activity of the Cdc7-Dbf4 protein kinase. Cdc7 is a cdk-like serine/threonine protein kinase whose activity requires binding of the regulatory subunit Dbf4 (Jackson, 1993). Dbf4 binds to proteins associated with the origin, thus providing a likely mechanism for recruitment of Cdc7 to the origin (Dowell, 1994). The Mcm2-7 proteins comprise a family of six highly conserved proteins that associate with the ORC origin complex prior to S phase; they are required for initiation of DNA synthesis. Cdc7-Dbf4 phosphorylates a subset of the Mcm proteins in vivo and in vitro suggesting that this may be part of the mechanism by which Cdc7-Dbf4 activates the origin (Lei, 1997). Cdc7 homologs have now been characterized and cloned from S. pombe, Drosophila, Xenopus laevis, mouse and human. This conservation suggests that regulation of DNA replication origin firing by Cdc7-family protein kinases is conserved through eukaryotic evolution. The cloning of the chiffon gene suggests that function of the regulatory subunit of the Cdc7 protein kinase may also be conserved (Landis, 1999).
The predicted Chiffon protein was found to contain two domains, designated CDDN1 and CDDN2, which are also found in S. cerevisiae Dbf4. The CDDN domains are also found in S. pombe Dfp1 protein, which is the S. pombe homolog of Dbf4 (Brown, 1998), as well as in an S. pombe gene highly related to Dfp1 (called 'similar to rad35/dfp1'; K. Oliver, direct submission to GenBank). The CDDN domains are also found in the predicted translation product of the Aspergillus nimO gene. The nimO gene is required for DNA synthesis and mitotic checkpoint control in Aspergillus (James, 1999). That study points out the homology between nimO protein and Dbf4 in the CDDN1 region, and makes the interesting observation that the structure of the CDDN1 region is consistent with a single Cys2-His2 zinc finger-like motif with a short central loop of 9 bp. Finally, the CDDN domains have also been found in a predicted human protein of unknown function and in several mouse cDNAs of unknown function. The data suggest a family of eukaryotic proteins related to Dbf4 and involved in initiation of DNA replication (Landis, 1999).
The similarity among Chiffon, Dbf4, and Dfp1 suggest a model for Chiffons role in amplification: Chiffon may function in the activation of the chorion gene origins as the regulatory subunit of a kinase involved in origin firing, most likely the Drosophila homolog of Cdc7. S. cerevisiae Dbf4 contacts Cdc7 through the carboxyl terminus, where the CDDN1 domain is located. Thus it is hypothesized that, analogous to Dbf4 function in S. cerevisiae, Chiffon may contact the Drosophila Cdc7 homolog through the conserved CDDN1 domain and recruit it to the ORC via conserved ORC contact sites in the Chiffon amino terminus, perhaps the CDDN2 domain. Chiffon could also be hypothesized to recruit other, as yet unidentified proteins to the origin. Alternative and less direct models for Chiffon function during amplification cannot be ruled out. In addition to the defect in chorion gene amplification, the chiffon null phenotype also includes rough eyes and thin thoracic bristles. While there are several possibilities for how chiffon might be required for normal eye and bristle development, these phenotypes are consistent with a defect in DNA replication and/or S phase control in the cells forming these structures. For example, roughex regulates cyclin levels and entry into S phase, and roughex mutants are viable with rough eyes similar to chiffon nulls (Thomas, 1997). Morula is a regulator of mitotic- and endo-cell cycles; hypomorphic morula mutants have rough eyes and thin thoracic bristles similar to chiffon null mutants (Reed, 1997). Finally, specific hypomorphic mutations in either the dDP or dE2F subunits of the Drosophila cell cycle regulator E2F cause rough eyes, thin thoracic bristles and defective chorion gene amplification nearly identical to chiffon nulls (Royzman, 1997 and 1999). Thus, Drosophila chorion gene amplification, eye development and thoracic bristle development appear to be processes that are particularly sensitive to defects in the cell cycle/DNA replication machinery (Landis, 1999).
There are a number of possibilities for how chiffon might function in S phase regulatory pathways with the above-mentioned and/or other cell cycle and S phase regulators. Identification of proteins that interact with chiffon in vivo will begin to test these models and should facilitate the dissection of the mechanism(s) regulating chorion gene amplification. Identification of proteins that interact with the evolutionarily conserved CDDN domains of Chiffon, using techniques such as yeast two-hybrid system, should prove particularly informative. The chiffon null phenotype demonstrates that Chiffon is required for chorion gene amplification and normal eye and bristle development. Chiffon might therefore be a tissue-specific regulator of DNA replication and/or cell cycle. However, it remains possible that Chiffon might be required in additional tissues, or even in every cell for DNA replication and/or cell cycle control. Because wild-type Chiffon mRNA is maternally supplied to chiffon mutant embryos, wild-type Chiffon protein might persist through embryonic and larval development and mask a more general requirement for chiffon function. Additional experiments, such as analysis of chiffon mutant germline clones will be required to address these questions (Landis, 1999).
chiffon potentially encodes two alternate open reading frames, one 5.085 kb and the other 5.133 kb, encoding proteins of 1695 and 1711 amino acid residues, respectively. The two proteins differ in their 3' amino acid sequences and 3' UTRs
Bases in 5' UTR - 691
Bases in 3' UTR - 859 and 771
The predicted 5.085 kb chiffon ORF sequence was used to search the NCBI databases for related proteins. The predicted Chiffon protein was found to be related to a group of known or predicted proteins: S. cerevisiae Dbf4; S. pombe Dfp1 (which is the S. pombe homolog of S. cerevisiae Dbf4); Aspergillus nidulans NimO protein termed SRAD35 (which stands for 'Similar to RAD35', where RAD35 is another name for Dfp1), and finally the protein predicted by translation of specific human and mouse clones. The protein sequences were aligned. The region of greatest similarity between Chiffon and the other proteins is a 44 amino acid residue domain designated CDDN1 (for chiffon, Dbf4, Dfp1 and NimO). In the CDDN1 domain, Chiffon is 43% identical to Dbf4; 41% identical to Dfp1; 36% identical to NimO, and 32% identical to the human BAC clone translation product. Chiffon is also related to the other proteins in an amino terminal region designated CDDN2, although to a lesser extent than for CDDN1 (Landis, 1999).
date revised: 2 October 99
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