Role of Spt5 in regulating AID during antibody maturation

Background: Antibody diversification lies at the heart of the humoral immune response by generating the vast antibody repertoire required to combat the plethora of pathogenic challenges we face daily. This process occurs at the immunoglobulin (Ig) gene loci and involves the interplay of targeted mutation, DNA repair and recombination. The enzyme that initiates the diversification reactions is Activation Induced Deaminase (AID), which generates mutation in single-stranded DNA in a transcription-dependent manner. AID can also target non-Ig genes which can result in genome instability leading to B cell cancers, like Burkitts lymphoma and diffuse large cell lymphoma. Transcription is essential for AID activity and our group has previously shown that the transcription stalling factor, Spt5, is required for AID recruitment and class switch recombination, one mechanism of antibody maturation. To address the role of Spt5 in these events in further detail, a conditional knockout mouse has been generated. Preliminary studies with these mice reveal that Spt5 loss results in AID recruitment defects. However, further analysis showed that the communication between Ig enhancers and promoters was defective in these mice. Since these interactions are important for the diversification reactions, these finding suggest, for the first time, a role for Spt5 in regulating enhancer-promoter interactions. These findings are particularly exciting because of very recent studies demonstrating that AID is preferentially targeted to enhancer-promoter clusters located within topologically associated domains (TADs), thus strongly implicating enhancers in AID recruitment. Aims: Based on these studies and our preliminary data, I hypothesize that Spt5 recruits AID, at least in part, by regulating enhancer-promoter interactions. Using the Spt5 knockout cells and a diverse array of techniques, I aim to investigate: (1) whether this role of Spt5 is specific to Ig genes or is a genome-wide feature of Spt5 function, and (2) probe the mechanism by which Spt5 executes this function. Major goal: I want to understand the role of Spt5 in antibody diversification and AID targeting. Significance: This proposal aims to advance our understanding of how AID is recruited to its targets within B cells during antibody diversification reactions. Given the pivotal importance of AID in humoral immunity and in the etiology of major B cell malignancies, my research will further our understanding of humoral immunity and could aid in the evaluation of B cell lymphomagenesis.

Understanding the immune system better! To fight diseases, antibodies must bind antigens on pathogens with the best fit. For this, antibody genes, like Igh, are mutated. Two processes contribute to antibody maturation: Class switch recombination and somatic hypermutation. Both depend on transcription of Igh from DNA to RNA by the enzyme RNA polymerase II, Pol II, and its co-regulator protein Spt5. As a result of my research, we refine the existing model of how antibody class switching succeeds by unmasking the mechanism by which transcription at multiple sites in the Igh gene, causes the gene to fold correctly. Additionally, we show for the first time that features of active transcription and the precise position of mutations during somatic hypermutation are not causally linked. Rather, we propose that the surrounding DNA sequence and termination of transcription are key to the necessary mutations. These findings reveal new mechanisms inside the cells of the immune system and help explain antibody maturation in greater detail. Now for the details: Spt5 is required for transcription by Pol II, which starts at several sites in the antibody gene. These are regulatory sequences, enhancers and promoters, which must come close to each other by folding the DNA into loops. If Spt5 is missing, no transcription and no folding occurs. Excitingly, we have uncovered two mechanisms how Spt5 regulates loop formation: First, Spt5 activates transcription at enhancers, which initiates loop formation to switch promoters; second, transcription from switch promoters creates a local barrier and ensures that a DNA loop ends there. As a result, a double loop forms, bringing three DNA regions of the antibody gene close, including two switch regions. This close proximity is important because during recombination, the DNA of the two switch regions is broken and must be correctly reconnected to eventually return a useful antibody after the class switching. In the second part, we revealed that different features of transcription do not determine the exact positions of the mutations. The double strand is opened when Pol II moves along the DNA: it starts, pauses, and moves in both directions on the Igh gene. These features of transcription free up single-stranded DNA, and it has been assumed until now that this regulates AID, the enzyme that sets the mutations. We tested several sequences, but specific starts or pauses of Pol II or some other feature could not explain the observed mutation patterns. As a last possibility, we propose that the surrounding DNA influences the patterns. This and our new hypothesis that AID can access single-stranded DNA in the short time window after Pol II stops transcription and falls off and before the DNA closes again, are important questions for the future. ursula.schoeberl@imp.ac.at