Cell cycle control of chorion gene amplification

Abstract

Over-replication of two clusters of chorion genes in Drosophila ovarian follicle cells is essential for rapid eggshell biosynthesis. The relationship of this amplification to the follicle cell cycles has remained unclear. To investigate the regulation of amplification, we developed a technique to detect amplifying chorion genes in individual follicle cells using BrdU incorporation and FISH. Amplification occurs in two developmental phases. One of the gene clusters begins to amplify periodically during S phases of follicle cell endocycles. Subsequently, after endocycles have ceased, both clusters amplify continuously during the remainder of oogenesis. In contrast to the early phase, late amplification commences synchronously among follicle cells. The pattern of Cyclin E expression mirrors these two phases. We present evidence that Cyclin E is required positively for amplification. We suggest that Cyclin E also acts negatively to inhibit refiring of most origins within a cycle, and that specific factors at chorion origins allow them to escape this negative rereplication control. Our findings suggest that chorion amplification is a model for understanding metazoan replicons and the controls that restrict replication to once per cell cycle.[Key Words: Drosophila; oogenesis; chorion; amplification; replication; cyclin E] Received November 10, 1997; revised version accepted January 15, 1998.To maintain euploid gene balance, DNA sequences must be replicated every cell cycle but not more than once. Recent evidence indicates that cell cycle control of DNA replication is effected by a two-step mechanism (for review, see Diffley 1996). Origins first become competent to replicate by assembling proteins comprising prereplication complexes onto chromatin in G 1 , and then later, during S, those origins initiate replication (Diffley et al. 1994). Replication from or through an origin dissociates functional prereplication complexes. Once destroyed, these complexes cannot reassemble until the subsequent G 1 , thereby precluding refiring of an origin in a single cycle. Several lines of evidence suggest that cyclin dependent kinases (CDKs), in addition to being required positively for cell cycle progression, act negatively and are responsible for blocking reassembly of replication complexes in S, G 2 , and M (Broek et al. 1991;Hayles et al. 1994;Moreno and Nurse 1994;Dahmann et al. 1995;Sauer 1995;Hua et al. 1997;Jallepalli et al. 1997;Tanaka et al. 1997). It is only after passage through mitosis, during a period in G 1 when kinase levels are low, that complexes can reassemble onto chromatin. This two-step mechanism of assembly and firing linked to kinase levels ensures that each region of the genome is replicated only once per cycle.In Drosophila melanogaster, as in many multicellular eukaryotes including humans, certain tissues become polyploid by entering an endocycle characterized by alternating S and G phases without intervening mitoses (for review, see Carminati and Orr-Weaver 1996). As in other cycles, Cyclin E (CycE), with...

Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress

“…The proteolysis of Dup/Cdt1 is now known to be important to prevent rereplication during mitotic cycles of a variety of organisms (Arias and Walter 2007). The proposed importance of Dup regulation for endocycle licensing control is consistent with the observation that MCM proteins periodically associate with chromatin during the Gap phase of each endocycle, and measurements of DNA copy number that showed that most of the genome does not duplicate more than once per endocycle S phase (Lilly and Spradling 1996;Calvi et al 1998;Su and O'Farrell 1998;A. Kolpakas, S. Maqbool, and B. Calvi, unpubl.).…”

“…Egg chambers then move down a structure called the "ovariole" as they mature through stages of oogenesis (stages 1-14) (Spradling 1993). Follicle cells proliferate by a mitotic division cycle up to stage 6, and then switch to the endocycle by stage 7, undergoing three rounds of G/S endoreduplication by stage 10A (Mahowald et al 1979;Lilly and Spradling 1996;Calvi et al 1998;Deng et al 2001;Lopez-Schier and St Johnston 2001). Thereafter, they selectively rereplicate only a few loci, resulting in amplification of gene copy number (Calvi et al 1998).…”

“…Follicle cells proliferate by a mitotic division cycle up to stage 6, and then switch to the endocycle by stage 7, undergoing three rounds of G/S endoreduplication by stage 10A (Mahowald et al 1979;Lilly and Spradling 1996;Calvi et al 1998;Deng et al 2001;Lopez-Schier and St Johnston 2001). Thereafter, they selectively rereplicate only a few loci, resulting in amplification of gene copy number (Calvi et al 1998). Similar to mitotic cycles, MCM proteins periodically associate with chromatin during each endocycle G phase, and Dup protein is proteolyzed at the G/S transition, suggesting that Dup regulation may be important for licensing control of origins in the endocycle (Su and O'Farrell 1998;Whittaker et al 2000;Thomer et al 2004;Hong et al 2007).…”

“…For example, in several tissues in Drosophila, developmental signals induce certain cells to enter a specialized endocycle that is comprised of alternating G and S phases without mitosis, which results in genome polyploidization (Edgar and Orr-Weaver 2001;Lilly and Duronio 2005). Endocycles are controlled by the oscillating activity of the CDK complex, Cyclin E/CDK2, and most of the genome is duplicated exactly once during each endocycle S phase, although some heterochromatic sequences are not duplicated (Gall et al 1971;Sauer et al 1995;Lilly and Spradling 1996;Calvi et al 1998;Parisi et al 2003). This and other evidence suggest that during a single endocycle S phase, origins initiate no more than once (Su and O'Farrell 1998).…”

Sequence requirements for function of theDrosophilachorion gene locus ACE3 replicator and ori-β origin elements

“…Additional conserved DNA replication factor genes required for chorion gene amplification include those encoding Cyclin E (Calvi et al, 1998), E2F (Royzman et al, 1999), DP (Royzman et al, 1999), RBF (Bosco et al, 2001), CDT1 (Whittaker et al, 2000), geminin (Quinn et al, 2001) and MCM6 (Schwed et al, 2002). Antibody to ORC1 or ORC2 reveals that, coincident with the initiation of amplification, ORC moves from a diffuse nuclear distribution into dramatic foci localized at the chorion gene loci (Asano and Wharton, 1999;Calvi et al, 1998;Royzman et al, 1999). The data demonstrate that Drosophila chorion gene amplification uses evolutionarily conserved machinery for initiation; however, some mechanism must exist to uniquely mark the chorion gene loci origins for activation during amplification, when overall genomic replication has ceased.…”