Final Report Summary - DIVAC (Locus specificity of immunoglobulin gene diversification)
Enhancers serve to restrict potentially dangerous hypermutation to antibody genes
How B lymphocytes are able to direct mutations to their antibody genes to produce millions of different antibodies has fascinated biologists for decades. Our study published in the open access journal PLOS Biology showed that this process of programed, spatially targeted genome mutation (somatic hypermutation) is controlled by nearby transcription regulatory sequences called enhancers. Enhancers are usually known to control gene transcription, and these antibody enhancers are now shown to also act in marking the antibody genes as sites of hypermutation. This work illustrates how undesirable off-target effects of hypermutation can be spatially restrained.
Studies in the late eighties by the laboratories of two prominent immunologists, Ursula Storb and Michael Neuberger, provided some early evidence that the hypermutation process was targeted by sequences neighbouring antibody genes. However, the phenomenon could not be nailed down, because the experimental set-up was laborious and hampered by background noise. Further confounding this analysis was the fact these sequences correspond to enhancers, and enhancers regulate transcription, which happens to be another requirement for hypermutation. To overcome these hurdles, we developed a novel, highly sensitive, and carefully controlled assay with which we finally provide convincing evidence that enhancers play an important role in somatic hypermutation. The new results vindicate earlier pioneering work, and thus resolve the often confusing scientific literature on this topic.
The experimental advantages of a chicken B cell line termed DT40 turned out to be the key to success. This is a little ironic as the DT40 model system has often been criticized for being artificial and of limited relevance for mice and man. However, human enhancers to antibody genes actually increased hypermutation in DT40 cells even more than equivalent sequences from chicken antibody genes. The result demonstrates once more the power of simple experimental models as well as the clear conservation of the targeting mechanism from chicken to humans.
While the study provides an unambiguous resolution of a long-standing question, more work on the precise molecular mechanism is required. Follow-up studies should focus on the interaction of enhancers, gene transcriptional machinery and AID (Activation-Induced Deaminase), the enzyme that initiates hypermutation. With respect to human disease it will be equally important to understand why the mutation targeting mechanism is not fool proof and why AID increases the background mutation rate throughout the genome of B lymphocytes leading to leukemia and lymphomas. It might be possible to use gene targeting to produce lethal mutations in precancerous AID expressing cells.
DIVAC sequences stimulates AID mediated DNA repeat recombination
In another study published in the open access journal elife we demonstrate that the AID enzyme can also cause sections of repeat DNA to be deleted from the genome in a DIVAC dependent fashion. We constructed pieces of DNA that include, in order, a gene that makes cells glow red, a gene that makes cells resistant to an antibiotic, and a gene that could make cells glow green. However, the very start of the ‘green’ gene was missing, which meant that it was switched off. Stretches of DNA were repeated in front of the ‘red’ and ‘green’ genes in some of the ‘constructs’. After inserting this DNA into cells from chickens or mice, most cells glowed red, but some started to glow green instead. The appearance of green cells was strongly stimulated, if DIVAC sequences were included in the constructs. Green cells were killed by the antibiotic; and were only seen when cells carried the constructs with the repeating DNA. Cells that lacked the AID enzyme only glowed red, regardless of which DNA construct they carried.
We then show that AID causes the DNA constructs to align and re-arrange at the repeated sequences. As such, when the cells divide and their DNA is separated and packaged into newly formed cells, the DNA between the repeating sequences can be deleted. Thus, cells started to glow green because the ‘on’ switch at the start of the red gene ended up at the start of the green gene when the region in between was deleted. This also explains why green cells always died when exposed to the antibiotic, because this deletion removed the resistance gene too. This suggests that when a cell attempts to correct the errors caused by the AID enzyme changing the letters in the DNA, it actually can trigger the exchange and deletion of repeated sequences. Future work is now needed to understand how this new role for the AID enzyme is regulated, and whether this role beneficial or harmful to the immune cells.
This research contributes to our understanding how B cell are transformed by the step wise accumulation of mutations and may lead to new approaches how B cell lymphomas can be treated and prevented. There are also important implications for our understanding of autoimmune diseases and the suppression of organ transplant rejection. The incidence of all these diseases are increasing in the member states of the European community.
How B lymphocytes are able to direct mutations to their antibody genes to produce millions of different antibodies has fascinated biologists for decades. Our study published in the open access journal PLOS Biology showed that this process of programed, spatially targeted genome mutation (somatic hypermutation) is controlled by nearby transcription regulatory sequences called enhancers. Enhancers are usually known to control gene transcription, and these antibody enhancers are now shown to also act in marking the antibody genes as sites of hypermutation. This work illustrates how undesirable off-target effects of hypermutation can be spatially restrained.
Studies in the late eighties by the laboratories of two prominent immunologists, Ursula Storb and Michael Neuberger, provided some early evidence that the hypermutation process was targeted by sequences neighbouring antibody genes. However, the phenomenon could not be nailed down, because the experimental set-up was laborious and hampered by background noise. Further confounding this analysis was the fact these sequences correspond to enhancers, and enhancers regulate transcription, which happens to be another requirement for hypermutation. To overcome these hurdles, we developed a novel, highly sensitive, and carefully controlled assay with which we finally provide convincing evidence that enhancers play an important role in somatic hypermutation. The new results vindicate earlier pioneering work, and thus resolve the often confusing scientific literature on this topic.
The experimental advantages of a chicken B cell line termed DT40 turned out to be the key to success. This is a little ironic as the DT40 model system has often been criticized for being artificial and of limited relevance for mice and man. However, human enhancers to antibody genes actually increased hypermutation in DT40 cells even more than equivalent sequences from chicken antibody genes. The result demonstrates once more the power of simple experimental models as well as the clear conservation of the targeting mechanism from chicken to humans.
While the study provides an unambiguous resolution of a long-standing question, more work on the precise molecular mechanism is required. Follow-up studies should focus on the interaction of enhancers, gene transcriptional machinery and AID (Activation-Induced Deaminase), the enzyme that initiates hypermutation. With respect to human disease it will be equally important to understand why the mutation targeting mechanism is not fool proof and why AID increases the background mutation rate throughout the genome of B lymphocytes leading to leukemia and lymphomas. It might be possible to use gene targeting to produce lethal mutations in precancerous AID expressing cells.
DIVAC sequences stimulates AID mediated DNA repeat recombination
In another study published in the open access journal elife we demonstrate that the AID enzyme can also cause sections of repeat DNA to be deleted from the genome in a DIVAC dependent fashion. We constructed pieces of DNA that include, in order, a gene that makes cells glow red, a gene that makes cells resistant to an antibiotic, and a gene that could make cells glow green. However, the very start of the ‘green’ gene was missing, which meant that it was switched off. Stretches of DNA were repeated in front of the ‘red’ and ‘green’ genes in some of the ‘constructs’. After inserting this DNA into cells from chickens or mice, most cells glowed red, but some started to glow green instead. The appearance of green cells was strongly stimulated, if DIVAC sequences were included in the constructs. Green cells were killed by the antibiotic; and were only seen when cells carried the constructs with the repeating DNA. Cells that lacked the AID enzyme only glowed red, regardless of which DNA construct they carried.
We then show that AID causes the DNA constructs to align and re-arrange at the repeated sequences. As such, when the cells divide and their DNA is separated and packaged into newly formed cells, the DNA between the repeating sequences can be deleted. Thus, cells started to glow green because the ‘on’ switch at the start of the red gene ended up at the start of the green gene when the region in between was deleted. This also explains why green cells always died when exposed to the antibiotic, because this deletion removed the resistance gene too. This suggests that when a cell attempts to correct the errors caused by the AID enzyme changing the letters in the DNA, it actually can trigger the exchange and deletion of repeated sequences. Future work is now needed to understand how this new role for the AID enzyme is regulated, and whether this role beneficial or harmful to the immune cells.
This research contributes to our understanding how B cell are transformed by the step wise accumulation of mutations and may lead to new approaches how B cell lymphomas can be treated and prevented. There are also important implications for our understanding of autoimmune diseases and the suppression of organ transplant rejection. The incidence of all these diseases are increasing in the member states of the European community.