Migration of mitochondrial DNA in the nuclear genome of colorectal adenocarcinoma
- Vinodh Srinivasainagendra1,
- Michael W. Sandel†1, 7,
- Bhupendra Singh†2,
- Aishwarya Sundaresan1,
- Ved P. Mooga2,
- Prachi Bajpai2,
- Hemant K. Tiwari1Email author and
- Keshav K. Singh3, 4, 5, 6Email author
© The Author(s). 2017
Received: 3 November 2016
Accepted: 9 March 2017
Published: 29 March 2017
Colorectal adenocarcinomas are characterized by abnormal mitochondrial DNA (mtDNA) copy number and genomic instability, but a molecular interaction between mitochondrial and nuclear genome remains unknown. Here we report the discovery of increased copies of nuclear mtDNA (NUMT) in colorectal adenocarcinomas, which supports link between mtDNA and genomic instability in the nucleus. We name this phenomenon of nuclear occurrence of mitochondrial component as numtogenesis. We provide a description of NUMT abundance and distribution in tumor versus matched blood-derived normal genomes.
Whole-genome sequence data were obtained for colon adenocarcinoma and rectum adenocarcinoma patients participating in The Cancer Genome Atlas, via the Cancer Genomics Hub, using the GeneTorrent file acquisition tool. Data were analyzed to determine NUMT proportion and distribution on a genome-wide scale. A NUMT suppressor gene was identified by comparing numtogenesis in other organisms.
Our study reveals that colorectal adenocarcinoma genomes, on average, contains up to 4.2-fold more somatic NUMTs than matched normal genomes. Women colorectal tumors contained more NUMT than men. NUMT abundance in tumor predicted parallel abundance in blood. NUMT abundance positively correlated with GC content and gene density. Increased numtogenesis was observed with higher mortality. We identified YME1L1, a human homolog of yeast YME1 (yeast mitochondrial DNA escape 1) to be frequently mutated in colorectal tumors. YME1L1 was also mutated in tumors derived from other tissues. We show that inactivation of YME1L1 results in increased transfer of mtDNA in the nuclear genome.
Our study demonstrates increased somatic transfer of mtDNA in colorectal tumors. Our study also reveals sex-based differences in frequency of NUMT occurrence and that NUMT in blood reflects NUMT in tumors, suggesting NUMT may be used as a biomarker for tumorigenesis. We identify YME1L1 as the first NUMT suppressor gene in human and demonstrate that inactivation of YME1L1 induces migration of mtDNA to the nuclear genome. Our study reveals that numtogenesis plays an important role in the development of cancer.
KeywordsCancer Tumor Colorectal cancer Mitochondria Mitochondrial DNA YME1L1 NUMT Numtogenesis mtDNA transfer Genetic instability
Natural transfer of mitochondrial DNA (mtDNA) into the nuclear genomes of eukaryotic cells is a well-established and evolutionarily ongoing process. The nuclear copies of mtDNA are described as NUMTs (nuclear mtDNA sequences) . Frequently, intact mitochondria containing mtDNA, mitochondrial RNA (mtRNA), and mitochondrial proteins are also reported to localize into the nucleus [2–9]. We have named this phenomenon of occurrence of nuclear mitochondria as numtogenesis. We define numtogenesis as the occurrence of any mitochondrial components into the nucleus or nuclear genome. Numtogenesis is reported in at least 85 sequenced eukaryotic genomes . These include human, plant, yeast, fruit fly, Plasmodium, Caenorhabditis, and other species [1, 10, 11]. In human, NUMT insertions are estimated to occur at a rate of ~5 × 10−6 per germ cell per generation .
Evolutionary studies suggest that the origin and insertion of germline NUMTs are distributed non-randomly in humans and other mammals [13–15]. Germline NUMTs tend not to originate from the mtDNA displacement loop (“d-loop”), and they tend to be located in damage-prone regions of the nuclear genome, such as open chromatin and fragile sites [13–15]. These studies implicate NUMTs in double-strand break repair . The mechanism(s) of NUMT accumulation is not well understood. It is suggested that mitochondria migrate towards the nucleus and accumulate near the nuclear membrane [16–18]. The most parsimonious mechanism explaining NUMT accumulation involves de novo transposition from the mitochondrion to the nucleus; however, NUMTs are also known to accumulate via segmental duplication (sometimes within repetitive elements), and possibly RNA retro-transposition [19–21]. The human genome contains between 755 and 1105 germline NUMTs, with mtDNA identities ranging from 64–100% [12, 22]. Germline NUMTs with the lowest similarity to mtDNA have been evolutionarily conserved for tens of millions of years, while the most recent insertions occurred after certain Homo sapiens populations migrated to Eurasia [22–26]. Human germline NUMTs are relatively well described but little is known about the somatic NUMT and its role in human pathology .
We and others have demonstrated that mito-nuclear interactions play a key role in tumorigenesis [28–35], but a role of mtDNA integration within the nuclear genome remains relatively unexplored. In this study, we analyzed the prevalence of NUMT in colorectal cancer (CRC) because a number of mitochondrial associations are relatively well characterized in CRC. There is a reported relationship between CRC risk and mtDNA copy number [36–38], and there are associations between germline mtDNA variants and CRC risk and mortality [39, 40]. Similarly, colorectal adenocarcinomas tend to have aberrant mtDNA copy number and somatic variant frequencies compared to matched blood-derived normal genomes [41–44]. We differentiated two classes of NUMTs with distinct characteristics, those that are inherited in the germline and those that are somatic NUMTs acquired during tumorigenesis. We present the first quantitative analysis on the abundance of somatic NUMTs in human colorectal adenocarcinoma genomes relative to matched blood-derived normal samples. Further, we compare the distributions of somatic NUMTs and germline NUMTs, describe sex-based differences in numtogenesis, and demonstrate that NUMT abundance in blood reflects NUMT abundance in tumor. In addition, we identify YME1L1 as the first “NUMT suppressor” gene in humans whose inactivation leads to increased numtogenesis.
Whole-genome sequence data were obtained for colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) patients participating in The Cancer Genome Atlas (TCGA), via the Cancer Genomics Hub (CGHub) , using the GeneTorrent file acquisition tool. In order to appraise alternative protocols for transactions on big-data endpoints, we evaluated GTFuse, an innovate software that offers faster access to DNA sequence data without the need for staging the data locally. Although data bandwidth hungry, GTFuse still enabled us to rapidly prototype our research on selected regions of the genome, thereby indicating signals of NUMT deposition in the nuclear genome. A symbiotic combination of GeneTorrent and GTFuse was used to choreograph the downstream data analysis pipeline. Our data harvester robots adopt a high-throughput analysis model by spreading GTFuse across a computational cluster fabric available locally on-campus At the time of manuscript generation, although the GTFuse web-link within AnnaiSystems webportal was unavailable, an alternate web URL https://annaisystems.zendesk.com/hc/en-us was found through web search means.
TCGA sequence data for individuals with matched colorectal tumor and blood-derived normal samples were harvested from CGHub. Downloaded data went through an intense quality control (QC) pipeline, which involved the use of (a) clinical information downloaded from TCGA Data Matrix, (b) short-reads alignment statistics derived from the Binary AlignMent (BAM) sequence data downloaded from CGHub, and (c) tagging and elimination of duplicate reads in the alignment data (BAM) using a popular next-generation sequencing tool, Picard 2.5. All sequence data used in this study were generated at Harvard Medical School using Illumina paired-end read technologies GAII and HiSeq, and reads were mapped to the hg18 human reference assembly. Since paired-end read technology offers better mapping coverage, improved directional sequence accuracy, and reliable mapping of reads to a reference genome, we used sequencing datasets generated using Illumina’s paired-end read technology as opposed to single-end read technology. Paired-end reading, with its increased coverage across several genomic bases, also improves the ability to identify relative positions of the two ends of a single read, especially when each read-end maps to different genomes, nuclear and mitochondrial.
A two-pronged analytic approach is taken to determine (a) NUMT proportion and distribution on a genome-wide scale and (b) hot spots in the nuclear and mitochondrial genomes experiencing more than blood-derived healthy (normal) NUMT abundance.
The primary obstacle to quantifying non-homologous recombinant elements from high-throughput sequence data is the reliable detection of reads that map to the sequence breakpoint. Although conceptually intuitive, the mapping and alignment of breakpoint reads is complicated by the large number of NUMT inserts that are not represented in the reference genome sequence. Alternatively, de novo genome assembly is computationally intensive, and any low-stringency mapping algorithm that would accurately identify insertion sites would also return many false positive hits. Fortunately, paired-end read technology offers a convenient and reliable method of detecting genome structural rearrangements, effectively bypassing an intensive search for non-homologous breakpoints. In the mapping step, paired-end coordinates are recorded for each read, which we filtered using simple text edit scripts. We used utilities of the SAMtools framework to quantify all paired-end reads that mapped to different chromosomes (mismatches). Further NUMTs were filtered from the mismatch output files, where one paired end mapped to mtDNA and the other to a nuclear coordinate. Since breakpoints were not precisely identified, we used the paired-end coordinate as a proxy for nuclear insertion site because the average distance between properly mapped reads was short (ca. 150 bp).
Let the R i /represent the rescaled proportional fold-change in relative NUMT abundance between tumor and matched blood-derived normal samples. R i /was calculated for the nuclear genome using five nested data partitions, where i represents whole genomes (gen), chromosomes (chr), chromosome arms (arm), cytobands (cyt), and sliding windows (win). For nuclear genome-scale comparisons, normalization to mapped reads (M ij ) did not include sex chromosomes due to sequence representation bias associated with differences in chromosome length. For small-partition comparisons (e.g., sub-band), samples were pooled before normalization to avoid bias associated with zero denominators. For the mitochondrial genome, R i /was calculated for three nested data partitions, where i represents genome (mtg), replication strand (mst), and gene (mgn).
The relationship between R arm /and mapped read count was evaluated with linear regression to assess whether NUMT transposition is coincident with aneuploidy. The relationships between R win /were evaluated between GC content to assess whether transcriptional activity predisposes the nuclear genome to integration of non-homologous DNA. All statistical analyses were conducted in the R statistical environment (http://www.R-project.org/). The NUMT proportions and abundance in blood-derived normal and primary tumor sites are quantified in Additional file 1: Table S1.
YME1L1 gene knockout and NUMT analyses in isolated nuclear DNA
Using the CRISPR-Cas9 method, we knocked out the human YME1L1 gene in human breast epithelial MCF-7 cells as described earlier . To avoid any mtDNA contamination in NUMT analyses, we isolated nuclear fractions free of mitochondrial contamination. Briefly, YME1L1 knockout and wild-type MCF-7 cells were lysed using lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA) containing 10% IGEPAL detergent for 10 min at room temperature and centrifugation at 15,000xg for 3 minutes was carried out to pellet the intact nuclei. To completely avoid cytoplasmic fraction contamination in the nuclear pellet, the lysis step was repeated one more time. The purity of the nuclear fraction was ascertained by performing western blotting for mitochondrial encoded cytchrome oxidase II (COXII) protein. Nuclear DNA was prepared from the nuclear pellet using the NAOH boiling method . Mitochondrial DNA content in the nuclear fraction was analyzed by real-time PCR by absolute quantification using primers for COXII (mtDNA-encoded gene) and Beta-2 microglobulin (B2M; nuclear DNA-encoded gene). B2M served as an internal control.
Yeast transformation and genetic selection
Yeast expression vectors (pYX113, pPT31-yYme1, and pYX113-hYme1L) were transformed using the lithium acetate, single-stranded DNA, polyethylene glycol method [48–50]. Following transformation, yeast harboring the desired vector was selected using synthetic drop-out (SD) medium (0.67% [w/v] nitrogen base without amino acids, 0.07% [v/v] drop-out amino acid mix (-His/-Trp/-Ura), 0.02% [w/v] L-histidine and excluding the amino acid that is a selectable marker, 2% [w/v] dextrose, and 1.5% agar for agar plates). Single cell colonies from plates lacking uracil were cultured in glucose medium and 1 × 104 and 5 × 107 or 5 × 108 cells were plated in triplicate onto YPD and SD media lacking tryptophan.
Sacharomyces cerevisiae strains used in the study
MATa ura3-52 lys2 leu2-3, 112 trp1-Δ1 yme1-1
ρ + (TRP1)
Adenocarcinoma genomes contain increased NUMTs compared to healthy genomes
NUMT abundance in blood correlates with NUMT abundance in tumor
Colorectal tumors in women harbor more NUMTs
NUMT abundance is associated with patient survival
NUMT abundance positively correlates with GC content and gene density
Overall, among the groups that had a fold change of 2 or more in NUMT abundance, although the low GC-content cytobands gpos50 and gpos100 were at the top of the NUMT abundance list for chromosomal cytoband windows, GC-rich “gneg” regions were prominent among cytobands exhibiting more than a 4.2-fold change in NUMT abundance. This pattern observed for the outlier data above 4.2 fold-change of NUMT abundance within GC-rich regions of gneg clearly shows a strong correlation between elevated NUMT abundance and GC content. Based on Fig. 7, as indicated by data points above the third quartile level, gneg regions are accountable for the greater than normal NUMT abundance, indicating a positive correlation between gene density and NUMT abundance. A previous study has shown that gpos100 sub-bands are also enriched with LINEs , and other researchers have demonstrated secondary amplification via LINE transposition to be an important mechanism of NUMT accumulation [11, 20]. Our results corroborate the empirical evidence. Clearly, a great deal of new research is needed to elucidate the causes and consequences of NUMT transposition. We hope that our discovery will serve as an impetus for such inquiry.
Mitochondrial fragile sites associated with NUMTs
YME1L1 inactivation leads to increased numtogenesis
In the yeast Saccharomyces cerevisiae, YME1 is reported to be an important suppressor of mtDNA migration to the nucleus . Interestingly, the YME1L1 gene encodes the human homologue of yeast mitochondrial AAA (ATPases associated with diverse cellular activities) metalloprotease, Yme1p. YME1L is a functional homologue of Yme1p, with conserved roles in mitochondrial assembly, integrity, and DNA metabolism ; however, its function in suppressing NUMTs is not known.
We expanded our analysis of Yme1L1 in TCGA database. This analysis revealed a high incidence of Yme1L1 mutations in CRC (Fig. 9c). The mutations in Yme1L1 include missense and synonymous substitutions and inframe and frameshift deletions (Fig. 9c). Most of the mutations in Yme1L1 in human colorectal cancer fall into two categories: missense substitutions (~68%) and synonymous substitutions (20%). The relative distribution of various mutations is summarized as a pie chart in Fig. 9c. We also analyzed Yme1L1 mutations in other human cancer types and observed a high mutation frequency in all the tested cancer types (Fig. 9d).
We determined whether inactivation of the human YME1L1 gene increases NUMT formation. For this, we created a YME1L1 knockout in a human cell line using YME1L1 gene-specific CRISPRs. We prepared nuclear fractions free of mitochondrial contamination and quantified the amount of mtDNA present in the nuclear fraction of these cells. We observed a strikingly increased amount of mtDNA in the nuclear fraction of YME1L1 knockout cells compared to wild-type cells (Fig. 9e). These results identify YME1L1 as the first NUMT suppressor gene in humans and suggest that inactivation of YME1L1 leads to increased numtogenesis.
Human homologue of YME1 suppresses migration of mtDNA to the nucleus
We asked whether the phylogenetically conserved role of human YME1L1 can rescue the migration of mtDNA in a yeast strain in which YME1 is disrupted. We utilized the Yme1-1 yeast strain, which harbors a mutation which leads to inactivation of the YME1 gene . In this strain, the auxtotrophic endogenous nuclear TRP1 gene is deleted and inserted into the mitochondrial genome. Since the required transcription machinery for TRP1 is only present in the nucleus, the mitochondrially inserted TRP1 gene is only functional when it migrates to the nucleus, permitting analysis of mtDNA migration to the nucleus [57, 58].
To determine whether human Yme1L1 is expressed in the Yme1-1 strain, western blotting was performed. Indeed, hYme1L1 was expressed in the Yme1-1 hYme1L1 strain (Fig. 9f). When the Yme1-1 vector was plated under tryptophan selection, it showed a significantly large number of colonies (Fig. 9g). These data support a previous observation that migration of mtDNA to the nucleus is high in Yme1-1 cells . Yme1-1 cells expressing yeast Yme1 (yYme1) display only a few (<50) tryptophan-positive colonies, suggesting that accumulation of mtDNA fragments in the nucleus in the Yme1-1 strain was prevented to a greater degree by re-introducing yYme1 (Fig. 9g). The same number of Yme1-1 cells harboring hYme1L1 also produced significantly low number of tryptophan-positive colonies compared to Yme1-1 cells (Fig. 9g). However, the number of typtophan-positive colonies in this case was a little higher (>100) compared to yYme1-expressing cells. This suggests that hYme1L1 can partially rescue the phenotype of mtDNA escape in Yme1-1. We conclude that hYme1L1 suppresses migration of mtDNA to the nucleus.
Numtogenesis, a natural phenomenon leading to migration of mitochondria, mitochondrial proteins, mtRNA, or mtDNA into the nucleus, is an ongoing cellular process reported in eukaryotic cells [2–9]. Although the occurrence of NUMT and the phenomenon of numtogenesis have been reported, its role in cellular and organismal function and in human health and disease remains relatively unexplored. Our study revealed that somatic NUMTs are frequently found in colorectal cancer. Consistent with our finding, two previous studies have associated NUMTs with carcinogenesis; one that found NUMTs containing LINEs in rat and mouse tumors  and a study of a cervical carcinoma cell line .
We provide evidence for increased NUMT insertions in the nuclear genomes of colorectal adenocarcinomas relative to matched control samples. NUMT occurrence was influenced by pathological cancer stage and sex between tumor and control groups. Germ line NUMT insertions leading to diseases have been identified . These diseases include severe plasma factor VII deficiency and bleeding diathesis , mucolipodosis IV , Usher syndrome , and a rare Pallister-Hall syndrome . These NUMT insertions were found in coding genes. NUMT insertions in these genes can reduce cellular fitness, leading to cellular dysfunction-induced cell death, which may underlie these human diseases . Conceivably, somatic NUMT insertion in tumor suppressor gene(s) may disrupt pathways which can contribute to tumorigenesis. Similarly, NUMT may activate oncogene(s) involved in tumor development. Indeed, integration of mtDNA fragments in the MYC locus in HeLa cells  and in the nuclear genome of mouse embryonic fibroblasts  has been identified. It appears that the integration of mtDNA in the nuclear genome of mouse embryonic fibroblasts led to the malignant transformation .
Increased somatic NUMT insertion in the nuclear genome of tumors may be associated with mitochondrial dysfunction. Mitochondrial dysfunction is a consistent feature of a variety of tumors and is described to be a hallmark of cancer [67–72]. We have previously demonstrated that mitochondrial dysfunction induces genomic instability in the nucleus [30, 31, 33]. However, genetic instability associated with mitochondrial dysfunction has been described to be point mutations or chromosomal aneuploidy [73–75]. The nuclear genome instability was induced due to increased oxidative stress caused by the changes in the nucleotide pool . Hadler et al.  proposed “a unitary hypothesis for carcinogenesis”, speculating that a breakdown of mito-nuclear symbiosis leads to development of cancer; they suggested release of mtDNA due to damage to the mitochondrial membranes. Using a sliding window approach, we determined NUMT insertion in tumor genomes. When we searched for the origin of these nuclear NUMT landing sites among colorectal cancer samples, we found three potential fragile sites within the mitochondrial genes ND1, COX1, and COX3, whose association with colorectal cancer has already been well investigated [77, 78]. Interestingly, in most females NUMTs originated from the same mitochondrial regions while in males NUMTs originated from different regions of mtDNA.
It is conceivable that a direct physical association or fusion between the mitochondrial and nuclear membranes and encapsulation of mitochondria in the nucleus [83, 84] may contribute to numtogenesis. Observations supporting encapsulation of mitochondria in the nucleus have been reported [2–5, 7–9]. The nuclear envelope breaks down during mitosis, leading to disruption of the physical barrier seperating the nucleoplasm and cytoplasm . This stage of the cell cycle can provide an opportunity for mitochondria to enter into the nucleus. Furthermore, cancer cells often exhibit a ruptured nuclear envelope . Decreased expression of lamins, important constituents of the nuclear membrane, contributing to nuclear rupture in cancer cells, has also been reported . Lamins in the nuclear membrane bind to chromatin and hold chromosomes in place and thus reduce chromosome breakage . Indeed, patients with laminopathy resulting from reduced lamin expression contain mitochondria in the nucleus. It is likely that loss of lamin expression in cancer cells helps migration of mitochondria into the nucleus, resulting in eventual integration of mtDNA into the nuclear genome. This phenomenon might be a survival mechanism for cancer cells .
It is unclear how numtogenesis alters the nuclear genome functions. Tsuji and coauthors  hypothesized that the underrepresentation of d-loop NUMTs in the germline may be due to protein binding sites located in this region, which may interrupt mtDNA fragmentation and immigration to the nucleus. We propose an alternative mechanism which involves structural alteration of the chromosome. Under this model, the inserted mitochondrial d-loop insertion functions as a telomeric t-loop, whereby it stabilizes a double-strand break and truncates the chromosome arm, resulting in aneuploidy. This mechanism involves transfer of the mtDNA displacement loop (d-loop) to the nuclear genome, where it could promote aneusomy by functioning as a telomeric “t-loop” structure, which typically caps the linear DNA molecule with a triple-stranded loop. The insertion of a mitochondrial d-loop could therefore directly interfere with the secondary structure of open chromatin and histone binding sites, leading to genome instability and/or dysregulation of gene expression. The second putative mechanism involves a mismatch between nucleotide composition between the mtDNA origin and nuclear DNA insertion site, which again draws from comparisons with work of Tsuji and coauthors . Previous work has shown that non-homologous recombination and transposable element insertion can lead to genome instability by modifying local methylation patterns and altering molecular thermodynamics, which could lead to dysregulation of the cell cycle and aneuploidy. These two putative mechanisms are not mutually exclusive and we anticipate that novel mechanisms will be revealed through further investigation.
Our study reveals that numtogenesis plays an important role in the development of cancer and that NUMTs may serve as a biomarker for tumorigenesis. This study also identifies YME1L1 as the first NUMT suppressor gene in human and demonstrate that inactivation of YME1L1 induces migration of mtDNA to the nuclear genome. Exploration of mtDNA migration into the cancer genome should provide impetus for further studies to identify the mechanism(s) underlying numtogenesis.
Cancer Genomics Hub
nuclear mtDNA sequence
The Cancer Genome Atlas
We thank Dr. Andreas Ivessa for generously providing us with PTY62 (Yme1-1) yeast strains, Dr. Thomas Fox for the yeast expression construct pPT31-yYme1, Dr. Thomas Langer for pYX113-hYme1L1, and Dr. Diana Stojanovski for pRS414-yYme1 constructs.
This study was supported by grants from the Veterans Administration 1I01BX001716 and a NCTN–LAPS Program Translational Research Award to KKS and T32HL072757 (PI: HKT) to MWS.
Availability of data and materials
TCGA data sets are publicly available to researchers upon individual institutional IRB approval and approval from dbgap.
KKS and HKT conceived the project and designed the experiments. VS, MWS, AS, VPM, and BS performed the experiments and analyzed the data. PB analyzed YME1L1 mutations in tumors. VS, MWS, HKT, BS, and KKS wrote the manuscript. All authors read and approved the final manuscript.
MWS and BS contributed equally to this study. MWS’s current affiliation is the Department of Biological and Environmental Sciences, School of Natural Sciences and Mathematics, University of West Alabama, Livingston, Alabama.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Datasets utilized in part of this research fall under dbGaP’s “Protected/Controlled Data Access System”, which warrants a multi-step protocol involving the IRB team of UAB for gaining complete access to the requested research data. The data access process was initiated by Dr. Hemant Tiwari through his dbGaP eRA Commons account by submitting a research proposal description. Upon successful submission and approval to use identified TCGA Sequencing data, the Genomic Data Commons (GDC) data portal was used to access authorized sequencing datasets. GDC’s data download and storage protocol was followed carefully to maintain integrity of the downloaded data and ensure a secure sandbox was staged to store and analyze the datasets. The research data part of dbGaP’s study ID ‘phs000178.v9.p8’ (referenced and approved by TCGA as Project ID 4538) was downloaded and analyzed as part of our research effort. Furthermore, we de-identified the sample IDs by replacing the original Barcode-based TCGA ID convention with our internal numeric ID format. No individual consent was acquired as the study utilized the TCGA database.
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