Gene expression is the complex process of producing proteins from a specific gene sequence encoded in DNA. The DNA in the nucleus of a cell is constantly surrounded by the machinery of gene expression. That machinery will attempt to start the process of transcription, the first step of gene expression, for any sequence it bumps into. Recall that every part of a cell is a chemical soup of molecules moving at incredible speeds, interacting with everything that they can possibly interact with, as fast as possible, countless times every second. Control over which genes are expressed at any given time is a matter of the structure of DNA, which sequences are exposed and which are spooled around histone molecules so that they are hidden from transcriptional machinery. DNA structure is shaped by epigenetic processes, which include the addition and removal of decorations such as methyl groups to specific locations on DNA that alter its shape, modifications to the histone molecules that DNA wraps itself around, and so forth.
DNA becomes damaged constantly - again, the cell nucleus is a soup of fast-moving molecules with countless collisions and reactions taking place in every moment. DNA is protected by highly efficient repair machinery, and near every incident is fixed, even dramatic damage such as complete breakage of both strands of the double helix of DNA. A new and potentially important area of research is focused on the potential for DNA double strand break repair to produce lasting changes to DNA structure and epigenetic regulation of gene expression. This may allow researchers to explain why similar detrimental epigenetic changes occur across all cells with advancing age, driven by stochastic DNA damage that is different in every cell and largely fails to harm any sequence that is actually used by a damaged cell. Importantly, given a sufficient understanding of exactly why long-term effects result from DNA double strand break repair, researchers can focus on developing therapies to prevent this outcome.
One obvious form of therapy already known to fix these issues is partial reprogramming, exposing cells to Yamanaka factors for a period of time. But perhaps there are other approaches that do not present the same challenges that partial reprogramming presents when it comes to fixing an entire body's worth of cells. Delivery is hard, and different cell types need different degrees of Yamanaka factor exposure. If it turns out that depletion of just a few factors involved in DNA repair is the cause of epigenetic change resulting from DNA double strand break repair, perhaps restoring those targets to youthful levels will be an easier goal to achieve. But these are early days yet, and a great deal more time and funding is needed for a deeper investigation of DNA double strand breaks and their potential contribution to degenerative aging.
Repair of DNA double-strand breaks leaves heritable impairment to genome function
Eukaryotic genomes are subjected to hierarchical folding that is required to accommodate DNA wrapped around the histone scaffold (collectively called chromatin) within the three-dimensional (3D) nuclear space. Evolution harnessed the 3D arrangement of nuclear chromatin to facilitate interactions among genomic segments such as promoters and enhancers, whose proximity influences gene expression and who thus have an important role in cell fate decisions such as orderly execution of developmental programs, adaptation to a new environment, or transmission of cell identity across successive generations of dividing cells. Although beneficial in these and other physiological contexts, the 3D arrangement of the nuclear genome also enables a distinct vulnerability to environmental or metabolic assaults that can modify chromatin folding and thus derail cellular functions.
A prominent example of such stress assaults is the DNA double-strand break (DSB). Besides disrupting DNA integrity, DSBs are intrinsically coupled to massive chromatin alterations that include changes in 3D arrangement and gene silencing across megabase distances from the primary DNA lesions. Although the DSB-induced chromatin response is initially beneficial to attract genome caretakers and generate structural scaffolds for timely and efficient DNA repair, its fate after restoring the integrity of DNA sequence is unknown. This seems to be a formidable gap in understanding genome maintenance that poses important questions: Do cells restore DSB-induced chromatin folding and the associated gene expression after completion of DNA repair? If yes, is the restoration of postrepair chromatin complete and back to the predamage level? If not, do the lingering chromatin alterations cause physiological impairments that can be inherited by successive cell generations?
To answer these questions, we directed Cas9-induced DSBs to genomic loci harboring topologically sensitive protein-coding genes, as well as regulatory RNA species, to interrogate long-term consequences of DNA breakage on chromatin topology and gene activity. By combining quantitative imaging of large cell populations, DNA and RNA fluorescence in situ hybridization (FISH), and Region Capture Micro-C as readouts, we found that DSB-induced chromatin alterations do not recover to predamage level but persist as lasting changes in 3D arrangement and impaired gene expression throughout large chromatin neighborhoods that encounter, and subsequently repair, a single DSB. We show that such impairments persist through several rounds of successive cell divisions and can trigger concrete pathophysiological consequences. We term this phenomenon as chromatin fatigue and propose that it represents a hitherto unknown dimension of heritable responses to DNA breakage, with a potential to permanently alter physiology of cells that encounter DSBs through environmental or metabolic stress - but also lineages engineered for various experimental or therapeutic purposes by nuclease-based genome editing.
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