Integrity of immunoglobulin variable regions is supported by GANP during AID-induced somatic hypermutation in germinal center B cells

Eid, Mohammed Mansour Abbas; Shimoda, Mayuko; Singh, Shailendra Kumar; Almofty, Sarah Ameen; Pham, Phuong; Goodman, Myron F.; Maeda, Kengo; Sakaguchi, Nobuo

doi:10.1093/intimm/dxx032

Cited by 3 publications

(3 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, it has been reported that this "mutation-hot" region is also a hotbed for DNA breaks leading to genomic translocations, frequently fusing BCL6 with the Ig heavy chain genes (20,47). In addition to chromatin conformation, AID access to nuclear DNA and AID-induced C > T mutations are also regulated by proteins such as GANP, which facilitates translocation of AID from the cytoplasm into the nucleus and targets it to sites of SHM (48).…”

Section: Discussionmentioning

confidence: 99%

A high-resolution landscape of mutations in the BCL6 super-enhancer in normal human B cells

Shen

Kamath-Loeb

Kohrn

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The super-enhancers (SEs) of lineage-specific genes in B cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B cells has precluded the generation of a high-resolution mutational landscape of SEs. Here we captured and sequenced 12 B cell SEs at single-nucleotide resolution from 10 healthy individuals across diverse ethnicities. We detected a total of approximately 9,000 subclonal mutations (allele frequencies <0.1%); of these, approximately 8,000 are present in the BCL6 SE alone. Within the BCL6 SE, we identified 3 regions of clustered mutations in which the mutation frequency is ∼7 × 10−4. Mutational spectra show a predominance of C > T/G > A and A > G/T > C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA polymerase η, respectively, in mutagenesis in normal B cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signatures SBS85, SBS37, and SBS39 were found in the BCL6 SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations.

show abstract

Section: Discussionmentioning

confidence: 99%

A high-resolution landscape of mutations in the BCL6 super-enhancer in normal human B cells

Shen

Kamath-Loeb

Kohrn

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…While the order of AID and Pol η mutagenesis during SHM varies slightly from that during aSHM, it is evident that a balance of these two mutagenic activities is required for fine-tuning antibody diversity. This argument is further supported by the finding that GANP (germinal center-associated nuclear protein) plays a critical role in modulating AID and error-prone polymerase activities in germinal center B-cells to facilitate the production of high-affinity antibodies (44). The cold region is devoid of H3K27ac, suggesting that the nucleosome is in a closed conformation and lacks regulatory function.…”

Section: Discussionmentioning

confidence: 92%

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

Shen¹,

Kamath-Loeb²,

Kohrn³

et al. 2019

Preprint

View full text Add to dashboard Cite

The super-enhancers (SE) of lineage-specific genes in B-cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B-cells has precluded the generation of a high-resolution mutational landscape of SEs. Here, we captured and sequenced 12 B-cell SEs at single-nucleotide resolution from ten healthy individuals across diverse ethnicities. We detected a total of ~9000 subclonal mutations (allele frequencies <0.1%); of these, ~8000 are present in the BCL6 SE alone. Within the BCL6 SE, we identified three regions of clustered mutations where the mutation frequency is ~7X10 -4 . Mutational spectra show a predominance of C>T/G>A and A>G/T>C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA Polymerase η, respectively, in mutagenesis in normal B-cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signature SBS85, SBS37, and SBS39 were found in the BCL6 SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are still unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B-cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B-cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations. SignificanceWe used Duplex Sequencing to detect low-frequency mutations in the BCL6 superenhancer locus in normal human B-cells. The landscape of pre-existing mutations is remarkably conserved across different ethnicities and reveals clustered mutational hotspots that correlate with reported sites of clonal mutations and translocation breakpoints in human B-cell lymphomas. This high-resolution genomic landscape revealed by Duplex Sequencing offers accurate and thorough profiling of low frequency, pre-existing mutations in normal individuals, and the potential for early detection of neoplastic alterations.

show abstract

“…We then selected for further analysis 6 hits present in the different clusters that were not already known to regulate Dazl: BEND3, MCM3AP, KDM5C, KMT2D, SPOP, and ZBTB14 (Figure 3A-C, S3A). Among the 3 members of TREX-2 mRNA export complex, we chose to pursue MCM3AP since it is a scaffolding protein, important for the stability of the complex (Wickramasinghe et al, 2014) and it is also known to have additional nuclear functions (Eid et al, 2017;Evangelista et al, 2018).…”

Section: Screen Validation Rules Out Differentiation Artifacts and Global Effects On Known Dazl Regulatorsmentioning

confidence: 99%

A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

Gupta

Yakhou

Albert

et al. 2021

Preprint

View full text Add to dashboard Cite

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements, and higher-order chromatin structures. Expectedly, these chromatin marks are indispensable for mammalian development and alterations often lead to diseases such as cancer. Molecularly, epigenetic mechanisms rely on factors to establish patterns, interpret them into a transcriptional output, and maintain them across cell divisions. A global picture of these phenomena has started to emerge over the years, yet many of the molecular actors remain to be discovered. In this context, we have developed a reporter system sensitive to epigenetic perturbations to report on repressive pathways based on Dazl, which is normally repressed in mouse ES cells. We used this system for a genome-wide CRISPR knock-out screen, which yielded expected hits (DNMT1, UHRF1, MGA), as well as novel candidates. We prioritized the candidates by secondary screens, and led further experiments on 6 of them: ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D. Our results show that all 6 candidates regulate the expression of germline genes. In addition, we find that removal of ZBTB14, KDM5C, SPOP and MCM3AP led to similar transcriptional responses, including a reactivation of the 2-cell like cell (2CLC) signature. Therefore, our genetic screen has identified new regulators of key cellular states.

show abstract

Integrity of immunoglobulin variable regions is supported by GANP during AID-induced somatic hypermutation in germinal center B cells

Abstract: GANP maintains antibody structural integrity during SHM

Cited by 3 publications

References 52 publications

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

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

A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

Contact Info

Product

Resources

About