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Title: Ribosomal DNA copy number loss and sequence variation in cancer}
Article:
Abstract:
Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.
Introduction:
Repetitive regions in the human genome include ribosomal DNA( rDNA), telomeres, and centromeres, and these regions have profoundly important functions for genome stability[1, 2, 3]. While challenging to study, genetic variability of these regions is ripe for selection. The rDNA encodes the functional RNAs of the ribosome, the most conserved and utilized genes in the genome. Three of the four RNAs are transcribed initially as a single transcript by RNA polymerase I. The repeats encoding the RNAs are present on five different regions on human acrocentric chromosomes, with each region composed of several copies of the gene, referred to as 45S. 45S is processed into three separate RNAs( 28S, 5. 8S, and 18S). The fourth RNA is encoded by the 5S genes, located on human chromosome 1 and transcribed by RNA polymerase III. The number of 5S copies correlates with 45S[4]. The loci that encode the rRNAs are transcribed at incredibly high rates, with rRNA often constituting as much as 80% of the RNA in actively proliferating cells, to meet the demand for ribosome production. Even so, only about 50% of the repeats are transcribed. The number of repeats is variable in human genomes[5], with one recent computational study estimating between 14–410 copies of 45S[4]. The variability in number combined with five different chromosomal locations makes the 45S pattern unique in each human genome. The human 45S genes are highly recombinogenic, with an estimated 10% recombination in a single generation[6]. The sequence of the 45S repeat within a species is highly conserved due presumably to high rates of recombination which allow for concerted evolution[7]. While repeats exist mainly in a head to tail tandem arrangement, palindromic arrangements have also been observed, especially in syndromes with increased genomic instability[8]. One can imagine that one way for a cell to achieve high rates of protein synthesis would be to expand the number of rDNA copies, especially given that ribosomal RNA can be limiting for ribosome biogenesis[9]. In fact, increases in copy number occur in different developmental and disease contexts. Expansions occur in frog oocytes[10] and the macronucleus of tetrahymena[11] as part of the normal course of development, presumably to help achieve high rates of ribosome biogenesis and protein synthesis. Southern blotting experiments demonstrate that recombination at the rDNA is common in adult lung and colorectal cancer[12], suggesting the possibility that the rDNA may be affected in the cancer genome. However, copy number of the rDNA in mammalian development and disease has not been examined. The TOR( target of rapamycin) kinase detects the nutritional environment and promotes ribosome biogenesis when amino acids are plentiful. The PTEN( phosphate and tensin homolog) phosphatase is a negative regulator of mTOR activity and acts as a tumor suppressor. Somewhat paradoxically, overfeeding flies, which overstimulates mTOR, has been associated with contraction of the rDNA copy number in the germline in Drosophila, transmitted transgenerationally[13]. TOR signaling was required for the amplification from low copy number back to normal copy number in budding yeast[14]. Together these results suggest there may be a connection between TOR signaling and the rDNA copy number, but this has not been explored in the mammalian genome. Furthermore, if the copy number or sequence changes in response to the environment, then rDNA may act as both a sensor and adaptor under stress conditions. In addition to its role in ribosome biogenesis, the rDNA has many extra-ribosome functions, such as regulating gene expression, chromosome organization, and titrating chromatin factors[15, 16, 17, 18]. The nucleolus, which contains the ribosomal DNA, is a hub for many signaling pathways and can act as a stress sensor. Chromosomal domains from most human chromosomes are associated with the nucleolus[19, 20], highlighting its role as an organizer. Dramatically reducing the copy number in yeast does not cause reduction in rRNA levels, but causes all repeats to become active( sometimes termed “compensation”) and the yeast are hypersensitive to DNA damage[21]. In contrast, low copy compromises protein synthesis and development in the bobbed mutants in Drosophila[22]. Furthermore, copy number of the rDNA titrates position effect variegation in flies[15, 18, 23]. Therefore, modulation of copy number has the potential to regulate signaling, genome stability, chromosome organization, and gene expression without necessarily affecting ribosome biogenesis. Recent technological developments enable accurate and high throughput measurements of the copy number of the rDNA. Copy number can be estimated from whole-genome sequence( WGS) and droplet digital PCR( ddPCR). The plethora of cancer genome sequencing projects allows comparison between the copy number in the tumor genome versus the normal genome from a single individual. We used these new technologies to analyze how rDNA copy number changes in different tissues and in cancer. Surprisingly, rDNA copy number is reduced in cancer genomes with mTOR activation, providing evidence that copy number can be altered in a natural system. We also discovered co-occurring increased copy number of additional genes, and evidence for single nucleotide variation in the repeats. Using cancer stem cells derived from a mouse model for leukemia driven by loss of Pten, we find that copy loss does not compromise proliferation, rRNA production, or protein synthesis, but cells are hypersensitive to DNA damage. These data suggest that the ribosomal DNA can act as a sensor and adaptor to cancer-associated stress. Copy number may be a biomarker with predictive potential.
Discussion:
We have used both computational approaches and ddPCR to analyze the copy number and sequence of the rDNA in mammalian cells. We find tissue to tissue variation in copy number in a single mouse is relatively low, as is individual to individual variation within an inbred strain. However, in an outbred mouse strain, the level of variation is higher, more resembling the situation in human genomes. We report for the first time that some cancer genomes, and in particular genomes associated with high mTOR activity, tend to have fewer copies than the matched normal genomes, a finding replicated in mouse cancer stem cells, and consistent with a previous report demonstrating transgenerational loss of rDNA copies in D. melanogaster with overactive TOR[13]. The low copy cancer genomes show concerted copy number changes in additional genes, suggesting a structural signature for these genomes. rDNA sequence variation can also occur in cancer genomes. Low copy in the Pten-/- HSCs is associated with sensitivity to DNA damage, extending previous reports that PTEN guards against genome instability. Our results suggest that copy number and sequence can change in the mammalian genome in cancer, and that loss of copies may have both costs and benefits. Future studies further analyzing the mechanisms and functional consequences of genomic alterations at the rDNA in mammals are warranted. Given our findings, it seems possible that ribosomal DNA could exhibit changes in other contexts. Loss of rDNA repeats may affect genome function and chromosomal processes via the release of protein factors. The rDNA locus houses many pluripotency factors in mammalian cells, including Oct4[46]. The rDNA also contains binding sites for Myc[47], a key transcription factor for proliferation that can also affect DNA replication[48], and CTCF[49], a key chromosome organization protein. The rDNA chromatin contains many different histone modifications[49], which could sequester chromatin readers and writers. Finally, nucleolar associated domains are enriched in regions displaying heterochromatin signatures in Arabidopsis[50] and rDNA copy number titrates position effect variegation, a heterochromatin based silencing phenomenon in Drosophila[23]. Losing repeats has the potential to liberate factors for re-distribution to the rest of the genome which could affect chromosome organization, gene expression, and replication. Interestingly, PTEN has been shown to directly control the function of the DNA replication factor MCM2 during DNA replication stress[51]. Replication stress occurs at the rDNA and has been reported as a potent driver of functional decline in HSCs[52]. In a mouse model for cancer driven by deficiency in Mcm2, genomic deletions can occur[53]. Spontaneous DSBs also occur at higher levels upon loss of PTEN[39]. Together these findings suggest that one possible reason that rDNA repeats are lost in the absence of PTEN is that DSBs and replication stress are handled without MCM2 function, and repair events are required. The repair events that result in deletions may be selected for, since the rDNA is difficult to replicate and loss of repeats might facilitate a successful cell cycle. We suggest that rDNA may both sense and adapt to genomic stress, with PTEN-mTOR normally guarding against copy loss. Extrachromosomal expansions of rDNA have been reported in frog oocytes[10]. This expansion is thought to facilitate the production of rRNA for ribosome biogenesis and the translational requirements in these cells. Based on this finding we predicted that cancer cells might expand the repeats due to similar requirements. Instead, we found recurrent evidence for contractions. We note that there is a key difference between the oocyte and the cancer cell—rounds of DNA replication. We speculate that the requirement to replicate the rDNA in cancer may select for the loss of repeats while the lack of replication in the oocyte may allow the expansion to be tolerated. The timing of the loss of the repeats relative to the development of cancer cannot be determined from the cancer genome projects. However, in the mouse HSCs loss occurs prior to the development of leukemia and aneuploidy, at the stage of a cancer stem cell. Further characterization of the selective forces and timing of loss will be interesting questions for the future. Linking the rDNA to both genome stability and ribosome biogenesis may enable it to act as a critical molecular sensor. Changes in translation are associated with cancer[54]. Myc and PI3K-AKT-mTOR are major oncogenic signaling pathways that promote reprogramming of translation. Studies to date have focused on mRNA regulatory elements, tRNA function and codon usage bias, and adaptations to stress that affect translation, but not ribosomal DNA copy number as a factor under the control of TOR signaling. Protein synthesis is quite tightly regulated in HSCs and defects that arise via loss of Pten or silencing of rDNA repeats cause functional decline and aging[44, 52]. Our study suggests that rDNA copy number and sequence can be altered in human cancers. HSCs with fewer copies are more sensitive to DNA damage, but are not compromised for rRNA production, proliferation, or protein synthesis. We speculate that the extra inactive copies may normally serve in part to counteract rDNA instability. Together, our data and others show that multiple mechanisms regulate protein synthesis and genome stability to control aging and prevent leukemogenesis in HSCs, and that these processes may be linked using the rDNA as a sensor. In summary, the rDNA copy number and sequence can change in cancer, with high mTOR activity associated with contractions and DNA damage sensitivity. With this recognition comes the possibility to target these loci. Cancer stem cells with low copy number may be more sensitive to DNA damaging agents. We speculate that this single copy number measurement could be used as a proxy detector for the variety of mutations that can occur in the PI3K-AKT-mTOR pathway in cancer and a predictor of whether DNA damaging drugs would selectively target the cancer stem cells. These findings may be applicable to cancer diagnosis and therapeutic choice.
The ribosomal DNA encodes the RNAs needed to make ribosomes for protein synthesis and cellular proliferation . However , ribosomal DNA has been excluded from most mammalian genome-wide studies due to challenges associated with its analysis . We find that both the sequence and copy number of the ribosomal DNA can change in human cancer genomes . mTOR is a kinase that senses the nutritional environment and is often over-active in cancer . Given mutational evidence for mTOR activation in the human cancer genomes with loss of ribosomal DNA copies , we analyzed ribosomal DNA in hematopoietic stem cells derived from mice under conditions of mTOR activation . Like the human cancer genomes , the ribosomal DNA copy number contracts in mTOR activated hematopoietic stem cells relative to normal stem cells . Loss is associated with high rates of cellular proliferation , rRNA production , and protein synthesis , but compromised survival in the presence of DNA damage . Contractions are a recurrent feature in cancer genomes with overactive mTOR and may predict sensitivity to DNA damaging chemotherapeutics . Ribosomal DNA may be altered in other disease contexts .