Somatic hypermutation patterns in immunoglobulin variable regions are established independently of the local transcriptional landscape

Reviewer #1 (Public review):

Summary:

In this study, the authors take a closer look at whether AID-mediated SHM occurs at stalled RNA polII complexes. Through experimental and bioinformatic overlaps, authors observe that AID target sites really do not overlap with RNA polII stalling, convergent transcription, or H3K27Ac marks. Rather, AID target sites just exist around transcription start sites. The authors thus bring up an important argument, that RNA poll II stalling is not the driving mechanism for AID targeting. This is important since research groups work with the assumption that transcription stalling drives AID access to single-strand DNA. The authors are also clarifying their previous studies, where they suggested that stalled Spt5-associated RNA polII recruits AID DNA deamination activity.

Comments:

Transcription start sites (TSS) of promoter genes. Most AID mutations occur at the first 500 pbs to 1 kb from the TSS of promoters or enhancers, but not in the rest of the transcription module or gene body. To this end, existing literature (including work done by the author(s)) has suggested that transcription stalling or pausing of elongating RNA polymerase and/or chromatin modifications such as H3K27Ac (markers of promoters and enhancers) have something to do with helping AID see single-strand DNA substrates for SHM. These conclusions, initially being drawn from AID's functional interaction with Spt5 and RNA exosome -two factors involved in the resolution of stalled RNA polII - and further supported through co-relative data of AID SHM sites overlapping S2-P RNA polII. As with genomics data, these observations were drawn through the bioinformatic window of overlap by the respective authors of the previously published studies.

In this study, the authors take a closer look at these overlaps and observe that AID target sites really do not overlap with RNA polII stalling, convergent transcription, or H3K27Ac marks. Rather, AID target sites just exist around transcription start sites that accumulate promoter-proximal terminated transcripts. The authors thus bring up an important argument, that RNA poll II stalling is not the driving mechanism for AID targeting. This is important since research groups work with the assumption that transcription stalling drives AID access to single-strand DNA.

The authors are clarifying the models and literature that they themselves had set earlier, and are doing this with quite detailed analyses, with some well-done experiments. I feel they need to be heard. The experiments are well done, and the text is well written. Since the study is associative (versus being directly mechanistic) due to constant use of bioinformatics overlaps of SHM genomics data with ChIP data, some concerns will remain (and have been outlined by the authors), but that will be future work.

Reviewer #2 (Public review):

Summary:

In this manuscript, Pavri and colleagues examine in-depth how the local transcriptional landscape affects somatic hypermutation (SHM) of variable region genes. They use the human Burkitt lymphoma Ramos cell line as a model system to examine AID-induced SHM.

The authors delete Emu and demonstrate that the epigenetic marks at the Ig loci do not correlate with their mutability. They define algorithms to map the V gene promoters and their mutational load in Ramos cells overexpressing AID and failed to find a correlation between mutation frequency and nascent transcription or transcription strength or between mutation frequency and polII stalling. Additionally, the authors show that convergent transcription may not be a major player for SHM. The authors additionally knock-in two other human V genes into the endogenous Vh gene in Ramos cells, and again failed to observe any significant correlation between PolII stalling and SHM. The authors also observe a similar lack of correlation between SHM (at the B-18 gene) and nascent transcription features in germinal center B cells. Overall, the authors conclude that mutation patterns in V genes are not linked to transcriptional features but are rather hard-wired into the sequence. The authors propose that premature transcription termination might have a role in promoting AID recruitment and activity at Ig genes.

Strengths:

The mechanisms that allow AID recruitment to Ig genes during SHM are very poorly understood. Many mechanisms have been proposed, with most invoking transcriptional features, including stalling, convergent transcription, etc. This work, demonstrating the lack of correlation with the proposed models, is of much importance to the field. The experiments are well done, and even though the results are generally "negative", they are highly relevant to our current understanding of SHM.

Weaknesses:

The authors propose premature transcription termination as a possible mechanism to determine V gene mutability, but the study does not experimentally address such possibilities.

Comments:

(1) It would be important for the authors to compare their results in Figure S1 at the B1-8 locus with those reported several years ago by Schatz and colleagues (Odegard et al, Immunity, 2005) and discuss if the results are different from what the authors report here. This is important as the first two figures essentially corroborate previous results that the Emu enhancer is important for transcription through the V genes.

(2) The authors mention that AID recruitment is facilitated by Ig enhancers. Is endogenous AID recruited to the V genes in the absence of Emu in the Ramos cells?

(3) The authors should explain how their results are different from those reported by the Schatz lab in their recent study (Wu et al, Mol Cell, 2025), demonstrating that ELOF1-mediated transcriptional pausing might promote SHM.

Reviewer #3 (Public review):

Summary:

The manuscript by Schoeberlet et al. aims to elucidate the relationship between somatic transcription and nascent transcription. Using PRO-seq data across V regions and 275 non-immunoglobulin targets, the authors show that there is no statistically significant correlation with SHM hotspots and localized Pol II enrichment within V regions. They further confirm this conclusion by comparing SHM levels with reduced transcription and reduced activating epigenetic marks. They have revised the model for SHM regulation to emphasize transcription-independent targeting.

Comments:

(1) The sum of the mutation class percentages in Figure 3G should be one hundred percent.

(2) A quantitative bar of transcription and mutation levels could be added to make it clear across these V regions.

(3) The authors propose that transcriptional termination may contribute to the boundaries of the SHM (e.g., the ~2 kb from the V promoters). If this is the case, the slowing of Pol II velocity prior to termination would theoretically provide more opportunities for AID to access ssDNA, which should lead to higher mutation rates in regions upstream of termination sites (3-4 kb from TSSs). However, the observed SHM peaks in the V(D)J region, and declines exponentially within 1-2 kb downstream, which seems contradictory. The related statement could be revised.

(4) Recent ELOF1 stories published by the Schatz and Meng labs should be discussed. ELOF1 could be listed in the model in Figure 7.