Amy Freeland

Reduced Mcm2 expression results in severe stem/progenitor cell deficiency and cancer.

Mcm2 is a component of the DNA replication licensing complex that marks DNA replication origins during G1 of the cell cycle for use in the subsequent S‐phase. It is expressed in stem/progenitor cells in a variety of regenerative tissues in mammals. Here, we have used the Mcm2 gene to develop a transgenic mouse in which somatic stem/progenitor cells can be genetically modified in the adult. In these mice, a tamoxifen‐inducible form of Cre recombinase is integrated 3′ to the Mcm2 coding sequence and expressed via an internal ribosome entry site (IRES). Heterozygous Mcm2IRES‐CreERT2/wild‐type (wt) mice are phenotypically indistinguishable from wild‐type at least through 1 year of age. In bigenic Mcm2IRES‐CreERT2/wt; Z/EG reporter mice, tamoxifen‐dependent enhanced green fluorescence protein expression is inducible in a wide variety of somatic stem cells and their progeny. However, in Mcm2IRES‐CreERT2/IRES‐CreERT2 homozygous embryos or mouse embryonic fibroblasts, Mcm2 is reduced to approximately one‐third of wild‐type levels. Despite the fact that these mice develop normally and are asymptomatic as young adults, life span is greatly reduced, with most surviving to only ∼10–12 weeks of age. They demonstrate severe deficiencies in the proliferative cell compartments of a variety of tissues, including the subventricular zone of the brain, muscle, and intestinal crypts. However, the immediate cause of death in most of these animals is cancer, where the majority develop lymphomas. These studies directly demonstrate that deficiencies in the function of the core DNA replication machinery that are compatible with development and survival nonetheless result in a chronic phenotype leading to stem cell deficiency in multiple tissues and cancer.

DNA Damage Response and Tumorigenesis in Mcm2 Deficient Mice

Minichromosome maintenance proteins (Mcms) are components of the DNA replication licensing complex. In vivo, reduced expression or activity of Mcms has been shown to result in highly penetrant early onset cancers (Shima et al., 2007; Pruitt et al., 2007) and stem cell deficiencies (Pruitt et al., 2007). Here we use mouse embryonic fibroblasts from an Mcm2-deficient strain of mice to show by DNA fiber analysis that origin usage is decreased in Mcm2-deficient cells under conditions of hydroxyurea (HU)-mediated replication stress. DNA damage responses (DDRs) resulting from HU and additional replication-dependent and replication-independent genotoxic agents were also examined and shown to function at wild-type (wt) levels. Further, basal levels of many components of the DDR were expressed at wt levels, showing that there is no acute replicative stress under normal growth conditions. Only very modest, 1.5- to 2-fold increases in the basal levels of γ-H2AX, p21cip1 and 53bp foci were found, consistent with a slight chronic elevation in DDR pathways. The one condition in which a larger difference between wt- and Mcm2-deficient cells was found occurred after ultraviolet irradiation and may reflect the role of Chk1-mediated suppression of dormant origins. In vivo, abrogating p53-mediated DDR in Mcm2-deficient mice results in increased embryonic lethality and accelerated cancer formation in surviving mice. Further, p53 mutation rescues the negative effect of Mcm2 deficiency on the survival of neural stem cells in vitro; however, the enhanced survival correlates with increased genetic damage relative to Mcm2 wt cells carrying the p53 mutation. Together these results show that even relatively minor perturbations to primary or dormant replication origin usage contribute to accelerated genetic damage in vivo. In addition, these studies show that tumor types resulting from Mcm2 deficiency are strongly affected by interaction with both genetic background and p53.

Mcm2 deficiency results in short deletions allowing high resolution identification of genes contributing to lymphoblastic lymphoma

Mini-chromosome maintenance (Mcm) proteins are part of the replication-licensing complex that is loaded onto chromatin during the G1-phase of the cell cycle and required for initiation of DNA replication in the subsequent S-phase. Mcm proteins are typically loaded in excess of the number of locations that are used during S-phase. Nonetheless, partial depletion of Mcm proteins leads to cancers and stem cell deficiencies. Mcm2 deficient mice, on a 129Sv genetic background, display a high rate of thymic lymphoblastic lymphoma. Here array comparative genomic hybridization is used to characterize the genetic damage accruing in these tumors. The predominant events are deletions averaging less than 0.5 Mbp, considerably shorter than observed in prior studies using alternative mouse lymphoma models or human tumors. Such deletions facilitate identification of specific genes and pathways responsible for the tumors. Mutations in many genes that have been implicated in human lymphomas are recapitulated in this mouse model. These features, and the fact that the mutation underlying the accelerated genetic damage does not target a specific gene or pathway a priori, are valuable features of this mouse model for identification of tumor suppressor genes. Genes affected in all tumors include Pten, Tcfe2a, Mbd3 and Setd1b. Notch1 and additional genes are affected in subsets of tumors. The high frequency of relatively short deletions is consistent with elevated recombination between nearby stalled replication forks in Mcm2-deficient mice.

Effect of minichromosome maintenance protein 2 deficiency on the locations of DNA replication origins

Minichromosome maintenance (MCM) proteins are loaded onto chromatin during G1-phase and define potential locations of DNA replication initiation. MCM protein deficiency results in genome instability and high rates of cancer in mouse models. Here we develop a method of nascent strand capture and release and show that MCM2 deficiency reduces DNA replication initiation in gene-rich regions of the genome. DNA structural properties are shown to correlate with sequence motifs associated with replication origins and with locations that are preferentially affected by MCM2 deficiency. Reduced nascent strand density correlates with sites of recurrent focal CNVs in tumors arising in MCM2-deficient mice, consistent with a direct relationship between sites of reduced DNA replication initiation and genetic damage. Between 10% and 90% of human tumors, depending on type, carry heterozygous loss or mutation of one or more MCM2-7 genes, which is expected to compromise DNA replication origin licensing and result in elevated rates of genome damage at a subset of gene-rich locations.

Cell Cycle Heterogeneity in the Small Intestinal Crypt and Maintenance of Genome Integrity

Stem cell quiescence has been hypothesized to suppress the rate at which genetic mutations accumulate within tissues by reducing the number of divisions a cell undergoes. However, recent studies have suggested that stem cells in the small intestine are rapidly dividing. This observation raises the issue of whether replication related errors are an important contributor to the accumulation of genetic damage and, if so, how genomic integrity is maintained within the small intestine. Here, reporter‐marked small intestinal epithelial cells, resulting from mini‐chromosome maintenance protein 2 (Mcm2) gene driven Cre‐mediated recombination, are shown to be retained at the +1 position within the crypt and to contribute to the intestinal epithelia over long periods. Additionally, we show that the rate of cycling of +1 position Mcm2‐expressing stem cells is heterogeneous with cycling times ranging between 1 and 4 days. Further, this heterogeneity depends on the p53 signaling pathway and could provide the basis for retention and expansion, through niche succession and crypt fission, of genetically intact stem cells. This somatic selection process would require active cellular replication. STEM CELLS 2010;28:1250–1259

Cdkn1b overexpression in adult mice alters the balance between genome and tissue ageing

Insufficient cell proliferation has been suggested as a potential cause of age related tissue dysgenesis in mammals. However, genetic manipulation of cell cycle regulators in the germ lines of mice results in changes in animal size but not progeroid phenotypes. Here we increase levels of the cyclin dependent kinase inhibitor Cdkn1b (p27kip1) in adult mice through doxycycline inducible expression and show this results in reduced cell proliferation in multiple tissues. The mice undergo changes resembling aging even in the absence of an elevated DNA damage response or evidence of senescent cells suggesting an altered balance between genetic and tissue aging. In contrast, suppressing cell proliferation by doxycycline treatment of neonates retards growth, but the onset of degenerative changes is delayed during the period of reduced body mass. These results support the hypothesis that many of the most recognizable features of mammalian aging can result from an imbalance between cell production and the mass of tissue that must be maintained.

Stem/Progenitor Cell‐Specific Enhanced Green Fluorescent Protein Expression Driven by the Endogenous Mcm2 Promoter

Previous studies have demonstrated expression of the minichromosome maintenance protein Mcm2 in cells that remain competent to divide, including stem/progenitor cells of the subventricular zone (SVZ) within the brain. Here, a transgenic mouse line in which the Mcm2 gene drives expression of enhanced green fluorescent protein (EGFP) was constructed by insertion of an internal ribosomal entry site (IRES)‐EGFP cassette into the last exon of the gene, 3′ to the stop codon. In these mice, expression of EGFP is observed in the SVZ and several other tissues with high proliferative activity, including the spleen, intestine, hair follicles, and bone marrow. These observations suggest that EGFP fluorescence in this mouse line provides an index of the proliferative capacity of different tissues. Immunohistological analysis demonstrates a direct concordance between expression of EGFP and Mcm2, consistent with a transcriptional level downregulation of Mcm2 expression in postmitotic cells. To test the utility of EGFP expression for recovery of live cells retaining the capacity to divide, EGFP‐expressing and ‐nonexpressing cells from bone marrow and brain were isolated from an adult Mcm2IRES‐EGFP mouse by fluorescence‐activated cell sorting and assayed for clonal growth. The EGFP‐positive fraction contained the entire clonogenic population of the bone marrow and greater than 90% of neurosphere‐forming cells from the brain. Brain‐derived clonogenic cells were shown to remain competent to differentiate towards all three neural lineages. These studies demonstrate that the Mcm2IRES‐EGFP transgenic line constructed here can be used for recovery of proliferation competent cells from different tissue types.

DNA replication stress restricts ribosomal DNA copy number

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number.

A Signature of Genomic Instability Resulting from Deficient Replication Licensing

Insufficient licensing of DNA replication origins has been shown to result in genome instability, stem cell deficiency, and cancers. However, it is unclear whether the DNA damage resulting from deficient replication licensing occurs generally or if specific sites are preferentially affected. To map locations of ongoing DNA damage in vivo, the DNAs present in red blood cell micronuclei were sequenced. Many micronuclei are the product of DNA breaks that leave acentromeric remnants that failed to segregate during mitosis and should reflect the locations of breaks. To validate the approach we show that micronuclear sequences identify known common fragile sites under conditions that induce breaks at these locations (hydroxyurea). In MCM2 deficient mice a different set of preferred breakage sites is identified that includes the tumor suppressor gene Tcf3, which is known to contribute to T-lymphocytic leukemias that arise in these mice, and the 45S rRNA gene repeats.