Mechanism of R‐Loop formation at Immunoglobulin Class Switch sequences

Transcription‐induced R‐loop structures, where the transcript hybridizes to the template DNA strand and the nontemplate exists as a single‐stranded entity, are formed at class switch sequences lying upstream of immunoglobulin heavy chain genes, and are targeted by the class switch recombination (CSR) machinery to facilitate a DNA rearrangement resulting in heavy chain isotype switching from μ to γ, α or ε in B lymphocytes. To characterize the formation and stabilization requirements for R‐loop formation at these sequences, we performed in vitro experiments with mouse Sγ3 as our model. We find that R‐looping efficiency decreases with decreasing number of switch repeats; and more drastically with decreasing number of Gs in a cluster from GGGG to GGG, with no R‐loops found if the cluster size is GG. G‐clustering on the nontemplate strand is the major determinant of R‐looping, but has to be present in context of high G‐density to be more stable; although a highly G‐dense but cluster disrupted region can independently support R‐loops at a much lower frequency. We show that a “thread‐back” mechanism operates during R‐loop formation, where the transcript comes back and hybridizes with the template DNA after exiting from exit channel in the RNA polymerase, as opposed to an “extended‐hybrid” formation where the transcript is laid on the DNA. We investigated the role of G‐quartets in R‐looping and our data suggests that R‐loops are formed and stabilized independently of any supportive influence from G‐quartets. We have also observed that inherent negative superhelicity or unwinding of DNA enhances R‐looping downstream of a promoter, even without switch sequences. Putative R‐loop motifs have been identified and are currently being tested for R‐loop forming abilities.