Life begins and ends in low oxygen. Mammalian embryos are submerged in a hypoxic environment before the cardiovascular system and placenta develop. In this low oxygen state, embryonic stem cells hum with activity. They proliferate, activate developmental genes, and transcribe DNA in an intricate dance that choreographs the first buds of existence.1,2 When this early mass of pluripotent stem cells, the blastocyst, burrows into the lining of the uterus, access to higher oxygen levels through the maternal blood supply triggers stem cells to differentiate into cells that form the various tissues and organs.3,4 The genes that initiate and wind the clock of life eventually go dormant as key developmental milestones are reached.
The will to live
That’s life. But what about death? Less than a decade ago, researchers debunked the long-held assumption that gene expression—a hallmark of life—ceases at the time of death. While most gene activity is extinguished after an organism dies, certain zombie genes are reawakened, sometimes days later. Some of these are the very same genes that are active during development, then repressed throughout an organism’s lifetime. Death also activates other genes involved in mechanisms such as cell stress responses, inflammation, immunity, and cancer.5,6 Why and how their resurrection occurs remains a mystery.
Cells don’t want to die.
-Gulnaz Javan, Alabama State University
Cell death is a natural and essential part of the biological life cycle. During the dance of development, cell death choreographs tissue maturation and corrects developmental errors.7 Cell death also plays an important role in the body’s response to cancer by mitigating genetic mutations and uncontrolled cell proliferation.8 Despite this, when an organism dies, cells rage against the process. “Cells don’t want to die,” said Gulnaz Javan, a forensic scientist at Alabama State University. Survival is programmed into their molecular makeup.
While the moment of clinical death is absolute, some cells defy this moment. As postmortem time marches on, cells that remain stable in low-nutrient and oxygen conditions, such as stem cells, survive.9 During this period of cellular afterlife, these cells release molecular distress calls in an ongoing display of death resistance. “Cells within tissues struggle to survive by changing their transcriptional programs to cause upregulation of developmental pathways,” Javan said.
Unwinding the clock
Javan coined the term thanatotranscriptome, which derives from the Greek word for death, thanatos, to describe postmortem gene expression. Javan and her colleagues examined gene expression in postmortem human liver tissue and found a substantial increase in expression of a gene that promotes cell survival known as X-linked inhibitor of apoptosis protein (XIAP).10 They also found increased expression of XIAP and other prosurvival genes such as BAG1 and BCL2 in human prostate autopsy tissue.11
After death, all hell breaks loose and just starts unwinding.”
-Peter Noble, University of Alabama at Birmingham
Peter Noble, adjunct professor of microbiology at the University of Alabama at Birmingham, and his team examined gene transcription in zebrafish and mice in the days after death and made an unexpected discovery.5 “There was about one to two percent of the total transcriptome that was active,” Noble said. This included one thousand and sixty-three genes to be exact, some of which became active up to two days after death. Other researchers also discovered increased gene transcription after death in human tissues, including brain, blood, and skin samples.12-16
Noble and his team categorized these zombie genes into functional categories, including those that play a role in development, cancer, stress responses, inflammation, immunity, cell death, nutrient transport, and epigenetic processes. One of the key developmental genes activated was hypoxia inducible factor (HIF), which is part of a group of transcription factors that respond to low oxygen levels by regulating other oxygen sensitive genes that are active in early embryonic development and certain physiological and disease states.17 HIF transcription factors regulate the expression of hundreds of genes through various molecular signaling pathways that have far reaching roles in cell proliferation, growth, metabolism, and survival.
Noble described the zombie gene phenomenon as a genetic unraveling of the developmental clock. “After death, all hell breaks loose and just starts unwinding,” he said. The usual genetic and epigenetic brakes that silence developmental genes throughout an organism’s lifetime are released.
When we started this work, people thought we were nuts. They thought ‘who wants to study death?’
-Peter Noble, University of Alabama at Birmingham
Beyond the veil
As researchers unraveled the secrets encoded in zombie genes, they also discovered their far-reaching scientific relevance. “When we started this work, people thought we were nuts. They thought ‘who wants to study death?’” Noble said. “But it turns out that there are many practical reasons for doing so.” For example, zombie gene expression is being explored as a forensic tool to predict the postmortem interval—the time between death and the start of a criminal investigation—based on their precise and time-sensitive expression.18
Thanatotranscriptome research also informs cancer and organ transplant science. “When you transplant an organ, you're taking it from a dead donor. In some cases, there's an increase in cancer gene expression,” Noble said. This is important, given that the incidence of cancer in organ transplant recipients is significantly higher than the general population. “The common theme is that there is an immunological problem, but when you transfer a kidney or liver to a donor, the cancer genes have already been turned on in the dead person, and it's being transferred to the recipient,” Noble explained.
Javan intends to further study postmortem decomposition of the prostate and liver, which are among the organs that remain intact the longest after death, to inform organ transplant research. “My team is assessing mRNA transcript abundance in postmortem prostate and liver tissues to obtain the list of candidate genes that can be used in the development of test kits to be used by organ transplantologists,” Javan said. Such biomarkers can improve the match between organ donors and recipients and reduce the rate of transplant rejection.
When you transplant an organ, you're taking it from a dead donor. In some cases, there's an increase in cancer gene expression.
-Peter Noble, University of Alabama at Birmingham
Despite advances in understanding the thanatotranscriptome, the cellular afterlife remains shrouded in mystery. “When I was in college, I wondered what happens after we die. Does every cell in our body die at the same time or is there life that goes on?” Javan said. As scientists continue to unearth the answer to this question, zombie genes may hold the molecular keys to understanding far-reaching processes in the human body. The conserved molecular pathways that shape early embryonic development and resurrect postmortem gene activity suggest a continuum along the thin strand that binds cellular life from the cradle to the grave. “We really don't know what happens when an organism dies,” Noble said. How long certain genes remain active and whether they might stay dormant for protracted periods in cells that survive below the threshold of oxygen and nutrient availability that currently defines the needs of a living cell remains to be seen. In the meantime, scientists continue to unravel the truth one gene at a time in their quest to uncover what lies beyond the veil.
- Fathollahipour S, et al. Oxygen regulation in development: lessons from embryogenesis towards tissue engineering. Cells Tissues Organs. 2018;205(5-6):350-371.
- Michiels C. Physiological and pathological responses to hypoxia. Am J Pathol. 2004;164(6):1875-1882.
- Larsen’s Human Embryology—5th Edition. Available online: https://www.elsevier-elibrary.com/product/larsens-human-embryology82193 (accessed on 30 October 2023).
- Podkalicka P, et al. Hypoxia as a driving force of pluripotent stem cell reprogramming and differentiation to endothelial cells. Biomolecules. 2020;10(12):1614.
- Pozhitkov AE, et al. Tracing the dynamics of gene transcripts after organismal death. Open Biol. 2017;7(1):160267.
- Scott L, et al. Life and death: A systematic comparison of antemortem and postmortem gene expression. Gene. 2020;731:144349.
- Arya R, White K. Cell death in development: Signaling pathways and core mechanisms. Semin Cell Dev Biol. 2015;39:12-19.
- Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature. 2001;411(6835):342-348.
- Cieśla J, Tomsia M. Cadaveric stem cells: their research potential and limitations. Front Genet. 2021;12:798161.
- Javan GT, et al. The apoptotic thanatotranscriptome associated with the liver of cadavers. Forensic Sci Med Pathol. 2015;11(4):509-516.
- Tolbert M, et al. The thanatotranscriptome: Gene expression of male reproductive organs after death. Gene. 2018;675:191-196.
- Dachet F, et al. Selective time-dependent changes in activity and cell-specific gene expression in human postmortem brain. Sci Rep. 2021;11(1):6078.
- Antiga LG, et al. Cell survival and DNA damage repair are promoted in the human blood thanatotranscriptome shortly after death. Sci Rep. 2021;11(1):16585.
- Ferreira PG, et al. The effects of death and post-mortem cold ischemia on human tissue transcriptomes. Nat Commun. 2018;9(1):490.
- Zhu Y, et al. Systematic analysis of gene expression patterns associated with postmortem interval in human tissues. Sci Rep. 2017;7(1):5435.
- Abouhashem AS, et al. The prolonged terminal phase of human life induces survival response in the skin transcriptome. Preprint. bioRxiv. 2023;2023.05.15.540715.
- Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. 2012;148(3):399-408.
- Hunter MC, et al. Accurate predictions of postmortem interval using linear regression analyses of gene meter expression data. Forensic Sci Int. 2017;275:90-101.