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A High-Resolution Landscape of Mutations in the BCL6 Super-Enhancer in Normal Human B Cells

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A High-Resolution Landscape of Mutations in the BCL6 Super-Enhancer in Normal Human B Cells A high-resolution landscape of mutations in the BCL6 super-enhancer in normal human B cells Jiang-Cheng Shena, Ashwini S. Kamath-Loeba, Brendan F. Kohrna, Keith R. Loeba,b, Bradley D. Prestona, and Lawrence A. Loeba,c,1 aDepartment of Pathology, University of Washington, Seattle, WA 98195; bClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and cDepartment of Biochemistry, University of Washington, Seattle, WA 98195 Edited by James E. Cleaver, University of California San Francisco Medical Center, San Francisco, CA, and approved October 28, 2019 (received for review August 14, 2019) The super-enhancers (SEs) of lineage-specific genes in B cells are clustered mutations (8). The first of these targets to be identified off-target sites of somatic hypermutation. However, the inability was the promoter/SE region of the BCL6 gene (8, 9). The BCL6 to detect sufficient numbers of mutations in normal human B cells gene encodes a transcription corepressor protein, whose ex- has precluded the generation of a high-resolution mutational pression is tightly coordinated with entry and exit from germinal landscape of SEs. Here we captured and sequenced 12 B cell SEs at centers (10, 11). Germinal center B cells have high levels of the single-nucleotide resolution from 10 healthy individuals across BCL6 protein, which regulates the expression of many genes diverse ethnicities. We detected a total of approximately 9,000 involved in B cell differentiation. On the other hand, antibody- subclonal mutations (allele frequencies <0.1%); of these, approx- secreting plasma cells and memory B cells that exit the germinal imately 8,000 are present in the BCL6 SE alone. Within the BCL6 SE, center turn off BCL6 expression to facilitate the switch between we identified 3 regions of clustered mutations in which the muta- differentiation and stable cell state maintenance. It has been tion frequency is ∼7 × 10−4. Mutational spectra show a predomi- hypothesized that mutations within the BCL6 SE dysregulate nance of C > T/G > A and A > G/T > C substitutions, consistent BCL6 expression to promote lymphomagenesis (12, 13). Indeed, with the activities of activation-induced-cytidine deaminase (AID) Sanger sequencing of PCR amplicons from diffuse large B cell and the A-T mutator, DNA polymerase η, respectively, in mutagen- lymphomas has identified clonal mutations in the BCL6 SE locus esis in normal B cells. Analyses of mutational signatures further (12). Furthermore, reporter assays in cultured cells showed that corroborate the participation of these factors in this process. Sin- some of these mutations interfere with the binding of tran- MEDICAL SCIENCES gle base substitution signatures SBS85, SBS37, and SBS39 were scriptional repressor factors to up-regulate BCL6 expression found in the BCL6 SE. While SBS85 is a denoted signature of AID (14). More recently, next-generation whole-genome sequencing in lymphoid cells, the etiologies of SBS37 and SBS39 are unknown. studies of diffuse large B cell lymphomas reported the presence Our analysis suggests the contribution of error-prone DNA poly- of mutation clusters within the BCL6 SE locus (7); however, only merases to the latter signatures. The high-resolution mutation approximately 30 clonal mutations were observed in a total of 10 landscape has enabled accurate profiling of subclonal mutations lymphoma samples, an inadequate number for characterizing the in B cell SEs in normal individuals. By virtue of the fact that sub- types and distributions of mutations. clonal SE mutations are clonally expanded in B cell lymphomas, our A high-resolution landscape of SE mutations in B cells from studies also offer the potential for early detection of neoplastic healthy individuals will aid in unravelling the process of aSHM alterations. and enable early detection of preexisting SE mutations that might signal lymphomagenesis. We reasoned that DNA mutated duplex sequencing | somatic hypermutation | BCL6 | super-enhancer | mutational signatures Significance n response to antigen stimulation, naïve B cells congregate in We used duplex sequencing to detect low-frequency mutations Igerminal centers, where they undergo multiple rounds of cell in the BCL6 super-enhancer locus in normal human B cells. The division and antigenic selection ultimately maturing into landscape of preexisting mutations is remarkably conserved antibody-producing plasma cells and memory B cells (1–3). In across different ethnicities and reveals clustered mutational the germinal center, somatic hypermutation (SHM) occurs, tar- hotspots that correlate with reported sites of clonal mutations geting the variable domain of the Ig receptor and resulting in and translocation breakpoints in human B cell lymphomas. This nonfunctional, low-affinity, or high-affinity antibodies. This high-resolution genomic landscape revealed by duplex se- process involves the creation of multiple single-base substitutions quencing offers accurate and thorough profiling of low- in Ig genes by the mutagenic SHM machinery (4). SHM includes frequency preexisting mutations in normal individuals along 2 stages of mutagenesis: cytidine deamination by activation- with the potential for early detection of neoplastic alterations. induced cytidine deaminase (AID) to generate a G:U mispair and subsequent error-prone repair synthesis by an error-prone Author contributions: J.-C.S., B.D.P., and L.A.L. designed research; J.-C.S. and A.S.K.-L. performed research; B.F.K. and K.R.L. contributed new reagents/analytic tools; J.-C.S., DNA polymerase. A.S.K.-L., B.F.K., K.R.L., B.D.P., and L.A.L. analyzed data; and J.-C.S., A.S.K.-L., and L.A.L. Although SHM is a tightly regulated process, it can act aber- wrote the paper. rantly, even in normal cells, to somatically mutate other (off- Competing interest statement: L.A.L. is a founder and equity holder at TwinStrand target) sites. Aberrant SHM (aSHM) sites are frequently found Biosciences. in noncoding DNA regions containing cis-regulatory elements, This article is a PNAS Direct Submission. such as intragenic super-enhancers (SEs)—binding sites for Published under the PNAS license. – multiple factors that increase transcription (5 7). These regions Data deposition: The data in this paper have been uploaded to NCBI BioProject (accession are proximal to the transcription start site (TSS) of actively no. PRJNA574179). transcribed genes and are in an “open” chromatin conformation 1To whom correspondence may be addressed. Email: [email protected]. (p300- and H3K27ac-positive). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. aSHM targets were delineated by low-resolution single-cell– 1073/pnas.1914163116/-/DCSupplemental. based polymerase chain reaction (PCR) sequencing of sites with www.pnas.org/cgi/doi/10.1073/pnas.1914163116 PNAS Latest Articles | 1of7 Downloaded by guest on September 26, 2021 by aSHM is preserved in circulating memory B cells such that 1.0 x10-03 deep sequencing of purified B cells would reveal details of the mutational landscape. We used duplex sequencing (DS) (15, 16) for this purpose. DS provides a mutational landscape of genomic DNA at single-nucleotide resolution to reveal mutational pat- 1.0 x10-04 terns and potential underlying mechanisms. The accuracy of DS stems from copying both strands of single DNA molecules; mutations are defined based on complementarity and presence in both strands at the same position. As a result, DS is approx- 1.0 x10-05 imately 1,000-fold more accurate than routine next-generation sequencing and allows the identification of rare mutations— those present at frequencies as low as 1 base substitution in 7 10 nucleotides sequenced. Frequency Mutation 1.0 x10-06 Using targeted capture, we purified and sequenced 12 SE loci + in genomic DNA samples of human CD19 B cells isolated from 10 healthy individuals of diverse ethnic backgrounds. The mu- tational landscape shows clustered mutations, most elevated in 1.0 x10-07 the SE of BCL6 and to a lesser extent in the SEs of PAX5 and ∼ MYC BCL6 CD83 PIM1 PAX5 EBF1 CD83. A total of 9,000 base substitutions were detected in the H2AFXRHOH AICDACD79BCD79A 10 samples; ∼8,000 of these were found in the BCL6 SE. The POU2AF IgHV3-23 mutational profiles of the BCL6 SE are remarkably similar in Fig. 1. Mutation frequencies in various TSS-SE gene loci and in the Ig gene individuals across various ethnic groups, suggesting that they are IgHV3-23 in B cells from 10 healthy individuals. Twelve aSHM targets as well not the result of random stochastic events but instead may as the Ig gene IgHV3-23 were captured from CD19+ B cells purified from 10 function in normal developmental processes. Mutational spectra independent blood donors and sequenced by DS. The mutation frequency— and signatures at the BCL6 SE locus further suggest that mu- the total number of mutations divided by total number of nucleotides se- tations result from the activities of AID and of the error-prone quenced—was calculated for each sample and plotted as shown. Note that − η the mutation frequency at the BCL6 SE (2.2 × 10 4) is as high as that of DNA polymerase, Pol . − IgHV3-23 (1.3 × 10 4). Data are presented as mean ± SEM (n = 10). SEs with − − − Results mutation frequencies >1.0 × 10 4, between 2.5 × 10 6 and 1.0 × 10 4, and <2.5 × 10−6 are highlighted in red, blue, and orange, respectively. High Frequency of Mutations in the BCL6 SE. We sequenced 12 + aSHM targets (SI Appendix, Table S1) in CD19 B cells from 10 healthy blood donors of different ethnicities (SI Appendix, Table SE locus (Fig. 2 A and B). We found that close to 90% of the ∼ ′ S2). The target gene segments are located 1kb3 of the TSS ∼8,000 mutations are located in a 600-bp region that corre- – and are reported SEs of the associated genes (5 7).
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