By Rebecca Delker, PhD
The blueprint of an organism is its genome, the most fundamental code necessary for life. The carefully ordered – and structured – composition of As, Ts, Cs and Gs provides the manual that each cell uses to carry out its diverse function. As such, unintended alterations to this code often produce devastating consequences, manifesting themselves in disease phenotypes. From mutations to insertions and deletions, changes in the sequence of nucleotides alter the cell’s interpretation of the genome, like changing the order of words in a sentence. However, arguably one of the most threatening alterations is the double-strand break (DSB), a fracture in the backbone of the helical structure, splitting a linear piece of DNA in two, as if cut by molecular scissors. While the cell has a complex set of machinery designed to repair the damage, this process can be erroneous generating deletions, or even worse, translocations – permanently reordering the pages of the manual and ultimately transforming the cell. Given the central role translocations can play in oncogenic transformation, DSBs have understandably received a bad rap; but, as can be expected, not all is black and white and it’s worth asking whether there is an upside to DSBs.
One such commendable pursuit of the DSB serves to expand the capabilities of our genome. While it is true that the genome is the most basic code necessary for life, many of the processes within a cell actually require changes to the code. These can occur at all levels of the Central Dogma – modifications of proteins, RNA, and even DNA. B- and T-lymphocytes, cells that provide a good amount of heft to our immune system, are notable for their DNA editing skills. Tasked with protecting an organism from billions of potential pathogens, B- and T-cells must generate receptors specific for each unique attack. Rather than encoding each of these receptors in the genome – an impossibility due to size restrictions – B- and T-lymphocytes use DSBs to cut and paste small gene fragments to build a myriad of different receptor genes, each with a unique sequence and specificity (reviewed here). For immune cells, and for the survival of the organism, these DSBs are essential. Although tightly controlled, DNA rearrangements in immune cells are mechanistically similar to the sinister DSB-induced translocations that promote cancer formation; however, rather than causing disease, they help prevent it.
New research published this summer points to exciting, and even more unusual uses of DSBs in the regulation of gene expression. In a quest to understand the molecular effects of DSBs that are causally linked to a variety of neurological disorders, Ram Madabhushi, Li-Huei Tsai and colleagues instead discovered a necessary role for DSBs in the response of neurons to external stimulus. To adapt to the environment and generate long-term memories, changes in the “morphology and connectivity of neural circuits” occur in response to neuron-activation. This synaptic plasticity relies on a rapid increase in gene expression of a select set of early-response genes responsible for initiating the cascade of cellular changes needed for synaptogenic processes. In their paper published in Cell this summer, the authors reveal that the formation of DSBs in the promoter of early-response genes induces gene expression in response to neuron stimulation.
By treating neuronal cells with etoposide, an inhibitor of type-II topoisomerase enzymes (TopoII) that causes DSB formation, the researchers expected to find that DSBs interfere with transcription. In fact, most genes found to be differentially expressed in cells treated with the drug showed a decrease in expression; however, a small subset of genes, including the early-response genes, actually increased. Through a series of in vivo and ex vivo experiments, the researchers showed that even in the absence of drug treatment, DSB formation in the promoters of early-response genes is critical for gene expression – beautifully weaving a connection between neuronal activation, DSB formation and the rapid initiation of transcription in this subset of genes.
The serendipitous discovery of the positive effect of etoposide on gene expression lead the researchers to focus in on the role of topoisomerases, the guardians of DNA torsion, in DSB formation. As a helical structure composed of intertwined strands, nuclear processes like replication and transcription cause the over- or under-twisting of the DNA helix, leading the DNA molecule to twist around itself to relieve the torsional stress and form a supercoiled structure. Topoisomerases return DNA to its relaxed state by generating breaks in the DNA backbone – single-strand breaks by type I enzymes and DSBs by type II – untwisting the DNA and religating the ends. While etoposide can artificially force sustained DSBs, physiological TopoII-induced breaks are typically too transient to allow recognition by DNA repair proteins. The finding that TopoIIb-induced DSBs at the promoters of neuronal early-response genes are persistent and recognized by DNA repair machinery suggests a non-traditional role for TopoII enzymes, and DSBs, in transcription initiation and regulation.
In fact, the contribution of TopoII and DSBs in the regulation of neuronal genes may not be so niche. Another study published recently found a similar relationship between transcriptional activation and Topo-mediated DSB formation. Using the primordial cells of the germline in C. elegans as a model system, Melina Butuči, W. Matthew Michael and colleagues found that the abrupt increase in transcription as embryonic cells switch from a dependence on maternally provided RNA and protein to activation of its own genome induced widespread DSB formation. Amazingly, TOP-2, the C. elegans ortholog of TopoII is required for break formation; but, in contrast to neuronal activation, these DSBs occur in response to transcription rather than as a causative agent.
These recent studies build upon a growing recognition of a potentially intimate relationship between DSBs, torsion and transcription. DNA repair proteins, as well as topoisomerase enzymes have been shown to physically interact with transcription factors and gene regulatory elements; topoisomerase I and II facilitate the transcription of long genes; and, as in neuronal cells, studies of hormone-induced gene expression in cell culture reveal an activation mechanism by which TopoIIb induces DSBs selectively in the promoters of hormone-sensitive genes. Thus, DSBs may constitute a much broader mechanism for the regulation of gene-specific transcription than previously thought.
Given the grave danger associated with creating breaks in the genome, it is curious that the use of DSBs evolved to be an integral component of the regulation of transcription – an inescapable and ubiquitously employed process; however, as we expand our understanding of transcription to include the contribution of the higher-order structure of DNA, the utility of this particular evolutionary oddity comes into focus. Genomic DNA is not naked, but rather wrapped around histone proteins and packaged in the 3D space of the nucleus such that genomic interactions influence gene expression. Changes in the torsion and supercoiling of DNA have been associated with histone exchange, as well as changes in the affinity of DNA-binding proteins for DNA. In addition, the necessity of topoisomerase for the transcription of long genes occurs early as RNA polymerase transitions from initiation to elongation, suggesting that the role of TopoI and II is not to relieve transcription-induced torsion, but rather to resolve an inhibitory, likely 3D, genomic structure that is specific to genes of longer length. A similar mechanism may be involved at the neuronal early-response genes. In these cells, genomic sites of TopoIIb-binding and DSB-formation significantly overlap binding sites of CTCF – a crucial protein involved in genomic looping and higher-order chromatin structure – and, again, DNA breaks may function to collapse a structure constraining gene activation. Whatever the exact mechanisms at play here, these results inspire further inquiry into the relationship between DSBs, genome topology and transcription.
A cell’s unique interpretation of the genome via distinct gene expression programs is what generates cell diversity in multicellular organisms. Immune cells, like B- and T-lymphocytes, are different from neurons, which are different from skin cells, despite working from the same genomic manual. In B- and T-cells, DSBs are essential to piece together DNA fragments in a choose-your-own-adventure fashion to produce a reorganization of the manual necessary for cell function. And, as is emphasized in this growing body of research, DSBs function along with a variety of other molecular mechanisms to highlight, underline, dog-ear, and otherwise mark-up the genome in a cell-specific manner to facilitate the activation and repression of the correct genes at the correct time. Here, DSBs may not reorder the manual, but, nevertheless, play an equally important role in promoting proper cell function.