Research Summary and Significance
Every time a cell divides it must fully and accurately duplicate its genomic DNA. Defects in the regulation of DNA replication during the cell division cycle can cause DNA mutations that promote cancer. Despite much progress, we have yet to discover the full repertoire of mechanisms that orchestrate genome duplication and stability. Moreover, it remains unclear what determines where replication starts on chromosomes in multi-cellular animals (metazoa); a DNA consensus for these origins of DNA replication has yet to emerge. A further challenge is to understand how cell cycle regulation is modified during development. Answers to these questions are crucial for understanding what goes awry in the cancer cell. To address these questions, we use the powerful tools available in the model organism Drosophila melanogaster (the fruit fly) integrating imaging methods with genetics, molecular biology, and genomics.
Introduction to the Cell Cycle Regulation of DNA Replication
In recent years, it has become increasingly apparent that defects in DNA replication are common in pre-malignant cells and can cause mutations that promote cancer (Figure 1). In a normal cell, DNA replication starts at thousands of sites (origins) so that that genome is duplicated in a timely fashion. If too few origins initiate, replication forks can collapse into double strand DNA breaks at common fragile sites. Cell cycle checkpoints sense these problems, slow cell division, and mobilize proteins to repair the damage. Checkpoint pathways can also induce permanent cell division arrest (senescence) or programmed cell death (apoptosis). When these checkpoints fail, however, it can cause genome instability and mutations in genes that promote oncogenesis. Moreover, if origins initiate DNA replication more than once per cell cycle, the resulting re-replication can also cause DNA damage and cancer. It is important, therefore, to fully understand how origins are regulated so that DNA replication is coordinated with cell division.
The basic strategy for the cell cycle regulation of origins is conserved in eukaryotes from
yeast to humans. In late mitosis / early G1 a multi-protein pre-Replicative Complex (pre-RC) is assembled onto origins, making them competent for replication, a process known as “licensing” (Figure 2). The assembly of the pre-RC occurs stepwise, the hexameric origin recognition complex (ORC) binds DNA and serves as a scaffold for subsequent association of CDC6 and Cdt1, which in turn are required to load the hexameric mini-chromosome maintenance complex (MCM), the putative replicative helicase. During the subsequent S phase, cyclin dependent kinase 2 (CDK2) and another kinase, CDC7, trigger initiation (pre-RC activation), resulting in the association of other proteins with the origin and establishment of the replication fork.
Re-replication is normally prevented because the pre-RC is disassembled when origins initiate, and reassembly of the pre-RC onto origins is inhibited during S, G2, and early M phase (Figure 3). In addition to their role in origin activation, CDK’s inhibit reassembly of the pre-RC after an origin initiates replication. In addition, multi-cellular eukaryotes have another inhibitor of pre-RC assembly, Geminin, which binds Cdt1 and renders it incapable of loading the MCM complex. It is only after Geminin and cyclins are degraded
at the subsequent metaphase that the pre-RC can reform, thereby restricting origin licensing, and DNA replication, to once per segregation of chromosomes. While this general picture is conserved in eukaryotes, much remains to be learned about the cell cycle regulation of genome duplication and stability.
A Molecular Genetic Model System for Genome Duplication
We have exploited a model system in Drosophila to understand the cell cycle regulation of DNA replication during development (Calvi et al. 1998). This system is based on developmental amplification of eggshell protein (chorion) genes in the Drosophila ovary (Calvi 2006). Amplification is a local increase in gene copy number due to DNA re-replication from only a few genomic sites (Figure 4). It occurs under developmental control late in oogenesis when genomic DNA replication has ceased (Figure 5). The origins that mediate amplification are among the best-defined in multicellular eukaryotes, and a number of molecular assays are available to investigate origin regulation. Moreover, we can visualize the activity of these origins using immunofluorescent microscopy (Figure 6). (Calvi and Spradling 2001, Calvi and Lilly 2002). Chorion amplification is also a forward genetic model for identifying new S phase regulators because defective origin activity results in an easily identified thin-eggshell phenotype. Using these combined methods, we, and others, have shown that an “Amplification Complex” (AC) resembling the pre-RC binds and licenses chorion origins and is regulated by CDK2 and Cdc7 (Figure 7) (Schwed et al. 2002). In the last few years, the study of chorion amplification has revealed new insights into origin structure and cell cycle regulation (Calvi 2006).
Discovery of a New Gene Family Required for S Phase
We used the thin eggshell phenotype for a forward genetic identification of the gene humpty dumpty (hd) (Bandura et al. 2005). hd is conserved in all multi-cellular eukaryotes, with one strong putative ortholog in humans, but the function of this family was previously undefined. Our investigation revealed that hd is required in Drosophila for DNA amplification and genomic replication during development. Both fly and human hd expression peaks at G1/S. The conservation of Hd protein sequence and cell cycle regulated expression suggests that its function in DNA replication is conserved in eukaryotes.
We are currently using live-cell imaging, molecular, genetic,
and genomic approaches to define Hd molecular mechanism
in flies. Antibody labeling and YFP tagging indicate that Hd
protein resides in distinct bodies in the nucleus, nuclear
envelope, and cytoplasm (Figure 8). We have found that Hd
cellular location and function is conserved in human cells
using live cell imaging and siRNA knockdown. In collaboration
with other labs, we have used used Mass Spectroscopy to
identify candidates of an Hd protein complex, and are currently
validating the functional relationship between these candidates
and Hd in vivo. Some of these proteins associate with human
oncoproteins that shuttle between nucleus and cytoplasm.
This suggests that regulated nuclear transport of Hd and these
other proteins may represent a new mechanism to regulate
G1/S phase progression.
Chromatin Regulates Origin Selection During Development
In multicellular eukaryotes, it remains a mystery how certain regions of the genome are selected to be origins. We found that acetylation of nucleosomes is important for the developmental specificity of origins in the Drosophila ovary (Aggarwal and Calvi 2004) (Figure 9). Importantly, our results suggest that epigenetic regulation may be a general mechanism that determines where replication starts on chromosomes (Figure 10). Our results, therefore, have important implications for understanding how perturbed epigenetic regulation contributes to genome instability in human cancers.
To gain further insight into chorion origin structure and regulation, we used cell biological and bioinformatics approaches to examine twelve other fly species whose genomes have been recently sequenced (Calvi et al, 2007; Clark et al. 2007). This study showed that chromatin acetylation and pre-RC recruitment to amplification origins is evolutionarily conserved, and also identified a conserved cis sequence that may recruit a histone acetyl transferase (HAT) to the origin. We are extending these findings using molecular and genomic methods to determine whether chromatin modification and remodeling are general mechanisms that modulate origin activity in different cells in development. The results should have important implications for understanding genome stability in development and cancer.
Preventing Re-replication and DNA Damage During Development
We found that the pre-RC protein Double-parked (Dup), the fly ortholog of
Cdt1, is rapidly degraded at G1/S, and showed that this regulation is critical to
prevent genome re-replication and instability (Thomer et al. 2004) (Figure 11).
We further defined the proteosome-dependent cell cycle mechanisms that
regulate Dup stability (May et al. 2005), which others have shown are
conserved in vertebrates, and relevant to genome instability in cancer cells.
We are currently investigating how Dup mis-regulation conspires with
checkpoint defects in genome instability. We have found that fly cells, like
human cells, respond to re-replication by inducing p53-dependent apoptosis,
an important barrier to oncogenesis in humans. Our novel finding is that cells
in the endocycle (G1/ S cycle) do not trigger apoptosis after re-replication and
genome damage (Mehrotra et al. 2008) (Figure 12). We are using genomic,
genetic, and cell biological approaches to further define endocycle regulation.
The ultimate goal is to understand how genome integrity checkpoints differ
among cells in vivo, which is relevant to why certain human tissues are
susceptible to specific types of cancers.
We are also mapping genomic fragile sites caused by re-replication to gain insight into the mechanism of genome instability. Our preliminary results suggest that heterochromatin may be especially prone to damage during
re-replication (Figure 13). We are pursuing this result to gain mechanistic insight into why certain genomic loci are prone to DNA damage when DNA replication is deregulated in pre-malignant cells.
If you wish to know more details about our research please
contact Dr. Brian Calvi at:
bcalvi @ indiana . edu