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Child leukemias are mistakes in DNA rearrangement
A portion of the KMT2A gene is swapped out with DNA from a "partner" gene located on a separate chromosome. The resulting recombination causes an abnormal genetic rearrangement called a translocation which leads to leukemia, or cancer of the blood cells. Patients with these types of leukemias tend to have poor outcomes.
A joint effort by University of Pennsylvania and Children's Hospital of Philadelphia (CHOP) researchers has applied an innovative new genome sequencing technique to catalog sites of DNA cleavage by the enzyme topoisomerase II (2) — called TOP II — as a step toward better understanding leukemic cancers in children.
The work was led by Brian D. Gregory, an associate professor in Penn's Department of Biology in the School of Arts & Sciences, along with Xiang Yu, postdoctoral researcher in Gregory's lab, and Carolyn A. Felix, the Joshua Kahan Endowed Chair in Pediatric Leukemia Research and attending physician at CHOP and professor of pediatrics in Penn's Perelman School of Medicine, and James W. Davenport, a research associate in the Felix lab. They reported their findings in the journal Genome Research.
"This tool opens new possibilities to better understand and eventually be able to manipulate TOPII cutting in order to prevent DNA rearrangements that give rise to leukemias." explains Carolyn A. Felix PhD, a professor of pediatrics in Pediatric Oncology at the Children's Hospital of Philadelphia, Perelman School of Medicine, in the University of Pennsylvania.
Translocations that lead to infant leukemias and treatment related secondary leukemias, involve the TOP II enzyme.
Certain chemotherapeutic agents are TOP II poisons and, while they can effectively kill cancer cells, sometimes they lead to abnormal DNA rejoining causing translocations — the hallmark of treatment related secondary leukemias. Felix and colleagues had previously reported that babies exposed in utero to TOP II poisons, sometimes found in foods in a mom's diet, are at an increased risk of developing infant leukemia.
Felix's lab has identified several specific leukemia-causing translocations between the KMT2A gene and partner genes. But, he wanted a more efficient way to identify all points in the genome that are cleaved by TOP II in order to confirm its role in DNA damage repaired incorrectly.
That's where Gregory's group came in. Over several years they had developed a technique to perform genome-wide sequencing of locations where an enzyme makes a covalent bond to DNA, which is what TOP II does while snipping strands of DNA.
"We designed a way to pull down the DNA bound to TOP II, then break that bond so only the DNA covalently attached to TOP II is free to be sequenced to single base-pair precision," Gregory said. "This enabled us to map, for the first time, topoisomerase II cleavage on a genome-wide scale."
The team performed the analysis on a human leukemia cell line derived from a patient with leukemia, obtaining all of the cleavage sites, then repeated the technique on the same cells treated with either chemotherapy drugs or other TOP II poisons found in food or the environment.
Examining the patterns they amassed, one of the key findings was that the cleavage events clustered in certain areas of the genome. They found hundreds of thousands of these clusters, mostly in gene introns, the non-coding portion of genic DNA, or in long non-coding RNAs, which play important roles in regulating gene expression. The clusters also tended to occur toward what is known as the 3-prime end of a gene, or the tail-end that is the last to be synthesized or transcribed.
"We think that TOP II cleaves in this region perhaps to decrease torsion at the end of the DNA [strand] and permit elongation of the transcript," Yu surmises. "It lets stress out of DNA that is highly expressed [read over and over]."
The chromosome on which KMT2A is located has a higher density of cleavage clusters than other regions, and cleavage clusters are more prevalent in KMT2A's known partner genes and in other genes that are translocated in leukemia.
"But even more surprising is that genes involved in translocations are in many forms of cancer, not just leukemia, and are more likely to show TOP II cleavage."
Several of the findings in the cells treated with chemotherapy agents or dietary or environmental TOP II poisons matched with patterns already identified in regions of genes involved in translocations in treatment-related leukemias and in infant leukemias, supporting the importance of disturbances in TOP II cleavage in causing these diseases.
Ciprofloxacin is an antibiotic used to treat a number of bacterial infections including bone and joint infections, intra abdominal infections, certain types of infectious diarrhea, respiratory tract infections, skin infections, typhoid fever, and urinary tract infections, among others. For some infections it is used in addition to other antibiotics. It can be taken by mouth or used intravenously and is a type II topoisomerase.
The researchers compared their findings with functional genomic information from the National Human Genome Research Institute's ENCODE project to show that TOP IIA cleavage clusters occurred in areas of the human genome that tended to be less variant, or conserved, across the human population.
"TOP IIA cleavage regions seem to be quite conserved in humans," Yu adds.
The researchers hope to move their work from the arena of basic science into findings that will benefit patients.
"One of our future questions is: Why do these TOP II poisons both kill cancer cells and also lead to the formation of leukemia-causing translocations in normal blood cells?" Gregory said. "Perhaps it is because of different patterns of TOP II cleavage in the normal and cancer cell populations."
Felix said that the findings open possibilities for new clinical approaches.
"The better we identify where cleavage occurs, the better we can understand how the drugs act and how the translocations happen," she said. "We could use that knowledge to design smarter drugs to target the TOP II enzyme that don't have such a high risk of causing translocations or drugs to protect sequences in the genome from unwanted cutting."
Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treatment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution. We find high TOP2A cleavage levels in genes involved in translocations in TOP2 poison–related leukemia. In addition, we find that a large proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. Comparisons to ENCODE data reveal distinct TOP2A CCR clusters that overlap with marks of transcription, open chromatin, and enhancers. Our findings implicate TOP2A cleavage as a broad DNA damage mechanism in oncogenic translocations as well as a functional role of TOP2A cleavage in regulating transcription elongation and gene activation.
Authors: Xiang Yu1, James W. Davenport, Karen A. Urtishak, Marie L. Carillo, Sager J. Gosai, Christos P. Kolaris, Jo Ann W. By, Eric F. Rappaport, Neil Osheroff, Brian D. Gregory and Carolyn A. Felix
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
Support for the study came from the National Insitutes of Health (grants CA776683, CA80175, GM033944 and ES013508-06, CHOP's Department of Pediatrics Academic Enrichment Program and Penn's Center of Excellence in Environmental Toxicology.
Felix is named as an inventor on an unlicensed patent, Methods and Kits for Analysis of Chromosomal Rearrangements Associated with Leukemia. Felix and Gregory are also named inventors on a patent application, Compositions and Methods for the Detection of DNA Cleavage Complexes.
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Chromosomal rearrangement is continuous in a developing body as well as in
repair of injuries. Understanding errors or Translocations in DNA cleavage
will help us protect, avoid or perhpas even "fix" mistakes in the process.
Image credit: Institute of Organic Chemistry and Biochemistry,
Czech Academy of Sciences