Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. Recurrent loss-of-function mutations in TET proteins are frequent in human diffuse large B cell lymphoma (DLBCL). Here, we investigate the role of TET proteins in B cell homeostasis and development of B cell lymphomas with features of DLBCL. We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B cell homeostasis and results in spontaneous development of germinal center (GC)-derived B cell lymphomas with increased G-quadruplexes and R-loops. At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double-strand breaks (DSBs) at immunoglobulin switch regions. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of GC B cells, diminished the accumulation of G-quadruplexes and R-loops and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss of function might predispose to the development of B cell malignancies.
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The code used to process the next-generation sequencing datasets has been deposited in the GitHub repository at https://github.com/dsamanie7/Tet2-Tet3_DKO_CD19_cre.
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We thank H. Yuita and I. Lopez-Moyado for generating the ERT2-cre Tet1fl/fl Tet2fl/fl Tet3fl/fl, ERT2-cre TKO mice, D. Kitamura at the Tokyo University of Science for sharing the 40LB cells, U. Basu and B. Laffleur at Columbia University for help with the HTGTS protocol, our collaborators at Cambridge Epigenetix (UK) for providing the 5hmC mapping kits, the LJI Flow Cytometry Core team (C. Kim, D. Hinz, C. Dillingham, M Haynes and S. Ellis) for help with cell sorting and the LJI next-generation sequencing core members (J. Day, S. Alarcon, H. Dose, K. Tanaguay and A. Hernandez) for help with sequencing. The BD FACSAria II is supported by the NIH (NIH S10OD016262, NIH S10RR027366), and our research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract number DE-AC02-05CH11231. The NovaSeq 6000 and the HiSeq 2500 were acquired through the Shared Instrumentation Grant (SIG) Program (S10) (NovaSeq 6000 S10OD025052 and HiSeq 2500 S10OD016262). K.S. acknowledges support from NIH grant DP2-NS105576. V.S. was supported by a Leukemia and Lymphoma Society Postdoctoral Fellowship (grant ID 5463-18) and currently by a K99/R00 award from the National Cancer Institute (grant ID CA248835). D.S.-C. and E.G.-A. were supported by University of California Institute for Mexico and the United States and El Consejo Nacional de Ciencia y Tecnología (UCMEXUS/CONACYT) pre-doctoral fellowship. This work is supported by the NIH grants R35 CA210043, R01 AI109842 and AI128589 to A.R. and K99/R00 CA248835, research funds from LLS grant 5463-18 and the Tullie and Rickey families SPARK award from LJI to V.S.
The authors declare no competing interests.
Peer review information Nature Immunology thanks Jayanta Chaudhuri and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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. a) H&E staining in spleens from 8 week-old Dfl and CD19 DKO mice The data is representative of 2 independent experiments. b) Kaplan-Meir curves displaying lymphoma-free survival of CD19 DKO (red) and control Dfl (blue) and CD19 Cre (green) mice. Y-axis, percent mice without lymphoma, defined by ≥ 3-fold increase in spleen weight and ≥ 2-fold increase in cellularity. c) – f) Quantification of c) cell numbers, d) spleen weights, e) B cell numbers, f) percent of B cells in spleens of 9 week-old CD19 DKO (red) and control Dfl (blue) and, CD19 Cre (green) mice from at least 5 biological replicates and 4 independent experiments. g) Percent of B cells in spleens of CD19 DKO (red) and control Dfl (blue) and CD19 Cre (green) mice at different ages from 3 independent experiments. h) Quantification of cell numbers of CD4+ T, CD8+ T and CD4+ Tfh cells from spleens of 9 week-old CD19 DKO (red) and control Dfl (blue) mice from 3 biological replicates and 2 independent experiments. i) Percent of activated CD4+ and, CD8+ T cells, and CD4 Tfh cells from spleens of 9 week-old CD19 DKO (red) and control Dfl (blue) mice from 3 biological replicates and 2 independent experiments. j) and k) Flow cytometry plots of j) Activated CD62Llow (X-axis) and CD44+ (Y-axis) CD4+ and CD8+ T cells and k) CD4+ Tfh cells PD1+ (Y-axis) CXCR5+ (X-axis) from spleen of 9 week-old CD19 DKO (red) and control Dfl (blue) mice. Numbers represent frequency of gated populations. l) Scheme of adoptive transfer experiment. B cells from Dfl and CD19 DKO CD45.2 mice were transferred retro-orbitally into sub-lethally irradiated CD45.1 immunocompetent host mice. m) Kaplan-Meir curves displaying overall survival of CD45.1 host mice transplanted with B cells from Dfl or CD19 DKO mice. X-axis denotes time in weeks after transplantation. n) Enlarged spleens in CD45.1 host mice transplanted with CD19 DKO compared to Dfl B cells 8 weeks after adoptive transfer. o) Representative flow cytometry data from the spleens of CD45.1 host mice 8 weeks after transplantation with Dfl and CD19 DKO B cells. Frequencies of the CD45.1+ and CD45.2+ cell populations are shown. Statistical significance is calculated using the log-rank test for b) and m), one-way ANOVA for c)-f) and two-way ANOVA for g)-i). Error bars represent mean +/− standard deviation, ** p value ≤0.01 *** p value ≤0.001, **** p value ≤0.0001.
. a), b) Gene set enrichment analysis (GSEA) plots showing enrichment for a GC B cell transcriptional signature in the transcriptional profile of CD19 DKO compared to Dfl B cells, using gene sets from a) GC versus follicular B cells and b) early GC versus late GC B cells. Y-axis denotes enrichment score. NES, Normalized enrichment score, FDR, False discovery rate. c) Representative flow cytometry data gated on splenic B cells from 8 week-old Dfl, CD19Cre (YFP+) and CD19 DKO (YFP+) mice. Numbers in the rectangles represent frequencies of GC B cells, identified as EFNB1+ (Y-axis) and IgDlow (X-axis). d) Representative flow cytometry data showing Ig isotype expression, gated on splenic B cells from 8 week-old Dfl, CD19Cre (YFP+) and CD19 DKO (YFP+) mice. Top, IgG1; middle, IgG2b; bottom, IgG3 X-axis shows expression of the default IgD isotype. Numbers represent frequencies of gated cell populations. e) Immunoblot showing AID expression in Dfl and CD19 DKO B cells (2 replicate experiments). Actin is used as a loading control. f) Relative fold-change in expression of μ and γ1 germline, Tet3, Irf4 and Myc transcripts measured by qRT-PCR in Dfl and CD19 DKO B cells from 2 biological replicates. g) Histogram (left panel) and bar-graph (right panel) showing staining with BCL6 antibody compared to isotype IgG controls in B cell from 8 week-old CD19Cre, Dfl and CD19 DKO mice from 3 independent experiments. h) and i) Bar plots displaying the proportions of (h) IgVH and (i) Igκ clonotypes (rearranged variable gene segments) from Dfl (blue) and CD19 DKO (red) B cells, identified from BCR repertoire analysis of RNA-Seq data. Y-axis represents the proportion of each clonotype. Each individual IgVH and Igκ clonotype is displayed using a different color in the bar plots. Numbers at the bottom represent the number of clonotypes identified in two independent replicates of Dfl (blue) and CD19 DKO (red) B cells. j) Representative flow cytometry data gated on Peyer’s patch B cells from 8 week-old Dfl and CD19 DKO mice. Numbers represent frequency of GC B cells, identified as FAS+ (Y-axis) and CD38− (X-axis). k) Quantification of GC B cell frequency in Peyer’s patches of Dfl (blue) and CD19 DKO (red) mice from 3 independent experiments. Statistical significance is calculated using two-tailed student t-test in f), k) and one-way ANOVA in g). Error bars represent mean +/− standard deviation in f), g) and k). ** p value ≤0.01.
Extended Data Fig. 3 TET deficiency is associated with increased levels of G-quadruplexes and R-loops.
. a) Flow cytometric detection of G-quadruplexes with BG4-Ig antibody or isotype IgG controls in primary B cells stimulated with 25 µg/ml LPS for 48 hours and treated with 10 µM pyridostatin (PDS, G4 ligand) for 24 hours. Numbers represent median fluorescence intensity. b) Quantification of median fluorescence intensity (MFI) of BG4-Ig signal from primary B cells treated with (red) or without (blue) PDS. Lines connect paired samples treated with or without PDS from 3 independent experiments. c) Fluorescence emission spectrum of NMM in the presence of a G4-forming oligonucleotide (oligo) from the human c-Kit gene promoter or a control oligo in which guanines in G4-forming regions (G-tracts) were mutated. d) Fluorescence enhancement over background (no oligos) for NMM at 610 nm in presence of known G4-forming oligos (Kit1, Kit2, Spb1) from the c-Kit gene locus or the telomeric repeat (Telo) or their respective mutated versions. e) G-quadruplex levels assessed by NMM or DMSO vehicle control (Veh) staining in untreated CH12 B cells or cells treated with 5 µM pyridostatin (PDS, G4 ligand) for 24 hours. Numbers represent median fluorescence intensity from 3 independent experiments. f) Flow cytometric detection of R-loops using V5-epitope-tagged recombinant RNASE H1 (rRNASE H1) in CH12 cells with or without RNASE H enzyme digestion during staining. Numbers represent median fluorescence intensity. g) Quantification of median fluorescence intensity (MFI) of R-loops (rRNASE H1) signal from CH12 cells with (red) or without (blue) RNASE H enzyme digestion. Lines connect paired samples with or without RNASE H digestion from 3 independent experiments. h) - m) Representative images (h, j, l) and quantification of mean fluorescence signal (i, k, m) of CD19cre and CD19 DKO YFP+ B cells stained with DAPI or propidium iodide and CD19, BG4-Ig (h, i), NMM (j, k) and rRNASE H1 (l, m) or respective controls using the AMNIS imagestream. Data are from two independent experiments. Statistical significance is calculated using paired student t-test in b) and g), two-tailed student t-test i), k) and m). Error bars represent mean +/− standard deviation, ** p value ≤0.01, **** p value ≤0.0001.
Extended Data Fig. 4 TET deficiency in multiple primary cell types is associated with increased DNA G-quadruplex structures.
. a) Representative flow cytometry data gated on splenic B cells from 8 week-old Cγ1Cre, Dfl and Cγ1 DKO mice 12 days after immunization with SRBCs. GC B cells are identified as FAS+ (Y-axis) and CD38− (X-axis). Numbers represent frequency of GC B cells. b), c) Quantification of (b) GC B cell frequencies and (c) absolute numbers of splenocytes from 8 week-old Cγ1Cre, Dfl and Cγ1 DKO mice 12 days after immunization with SRBCs from 3 independent experiments. d) Experimental design. ERT2Cre DKO or control Dfl mice were injected for 5 consecutive days with tamoxifen to induce Cre expression and TET deletion, then rested for 2 days. Splenic B cells were activated for 72 hours in vitro with LPS and IL-4 in the presence of 4-hydroxytamoxifen (4-OHT). e) G-quadruplex levels in naïve (left panel) and activated (right panel) B cells from tamoxifen-treated ERT2Cre DKO (YFP+) or control Dfl mice. Numbers represent median fluorescence intensity. f) Quantification of median fluorescence intensity (MFI) of NMM signal from naïve and activated B cells from ERT2Cre DKO (YFP+) or control Dfl mice from 3 independent experiments. g), i) G-quadruplex levels assessed by NMM or DMSO vehicle staining (Con) in (g) transferred CD45.2+ myeloid cells from ERT2Cre TKO (YFP+) or control Tfl mice, and (i) transferred CD45.2+ T cells from CD4Cre DKO (YFP+) or control Dfl mice. h), j) Quantification of median fluorescence intensity (MFI) of NMM signal in (h) transferred CD45.2+ myeloid cells from ERT2Cre TKO (YFP+) or control Tfl mice from 2 biological replicates and j) transferred CD45.2+ T cells from CD4Cre DKO (YFP + ) or control Dfl mice from 3 biological replicates. Statistical significance is calculated using one-way ANOVA in c), two-way f) and two-tailed student t-test in h) and j). Error bars represent mean +/− standard deviation, * p value ≤0.01, ** p value ≤0.005.
Extended Data Fig. 5 Increased apoptosis and DNA DSBs in TET-deficient B lymphoma cells depleted of enzymes that resolve G-quadruplexes and R-loops.
. a) Experimental design. Primary B cells from Dfl and CD19 DKO B cells were nucleofected with Cas9 RNPs loaded with sgRNAs against Rnase H1 or the known G4-binding helicases Atrx, Blm and Fancd2, then stimulated with 10 µg/ml LPS for 48 hours before assessing the frequency of apoptotic cells by flow cytometry for cleaved Caspase 3. b) Representative immunoblots showing decreased protein levels of ATRX, BLM, FANCD2 and RNASE H1 in CD19 DKO B cells nucleofected 48 hours earlier with the corresponding or CD4 Cas9 RNPs (Ctrl). The data is representative of at least 2 independent experiments. c) Representative flow cytometry plots quantifying percent apoptotic cells in Dfl and CD19 DKO B cells nucleofected with Cas9 RNPs. Y-axis, staining for cleaved Caspase 3; X-axis, forward scatter (FSC). d) Quantification of apoptosis, measured as percent of cells showing staining for cleaved Caspase 3, in cells nucleofected with Cas9 RNPs to Atrx, Blm, Fancd2, Rnase H from 3 biological replicates. e) Quantification of G-quadruplexes as NMM median fluorescence intensity (MFI) in Dfl and CD19 DKO B cells 48 hours after nucleofection with Cas9 RNPs. The signal is normalized to the NMM MFI of the same biological sample nucleofected with CD4 Cas9 RNPs (Ctrl) from 3 biological replicates. f) Quantification of DNA DSBs, assessed by γH2AX median fluorescence intensity (MFI) in Dfl and CD19 DKO B cells 48 hours after nucleofection with the indicated Cas9 RNPs from 3 biological replicates. The signal is normalized to the γH2AX MFI of the same biological sample nucleofected with control Cas9 RNP loaded with sgRNA against CD4 (Ctrl). g) Experimental design. Dfl and CD19 DKO B cells were treated for 2 days with the G-quadruplex stabilizing compound pyridostatin (PDS) prior to activation for 48 hours with LPS. h) Quantification of apoptosis, measured as percent of cells showing staining for cleaved caspase 3, in Dfl and CD19 DKO B cells cultured without (untreated) or with 10 µM PDS from 5 biological replicates and 3 independent experiments. i) Representative flow-cytometry plots quantifying percent apoptotic cells in Dfl and CD19 DKO B cells with or without PDS treatment. Y-axis, staining for cleaved caspase 3; X-axis, forward scatter (FSC). Statistical significance is calculated using two-way ANOVA. Error bars represent mean +/− standard error, * p value ≤0.05, ** p value ≤0.01, *** p value ≤0.0005 **** p value <0.0001.
Extended Data Fig. 6 TET deficiency is associated with genome-wide accumulation of G-quadruplexes and R-loops.
. a) Genome annotations of regions enriched for G-quadruplexes (G4) and R-loops (right bar) compared to their representation in the mouse genome (mm10) (left bar). b) Relative representation of different classes of motifs predicted to form G-quadruplexes (pG4) in control regions selected randomly from the genome (left bar) and regions enriched for G-quadruplexes and R-loops (right bar). c) Heat maps showing enrichment (RPM) for G-quadruplexes and R-loops in CD19 DKO and control B cells. The signal is plotted in a +/− 2 kb window from the center of the regions ordered based on decreasing intensity from top to bottom in the entire 4 kb window. R-loop signal is plotted after background subtraction of MNase-alone control. d) Profile histograms showing the signals for G-quadruplexes (G4) (RPM, reads per million), R-loops (RPM), WGBS (percent of 5mC + 5hmC/unmodified C) and 5hmC (RPM). The 9722 regions enriched for both G-quadruplexes and R-loops are divided into two categories – 6212 regions overlapping promoters (left panels) and 3510 regions not at promoters (right panels). Dashed grey lines indicate the center of the region and the 1 kb boundaries located on either side of the center. Blue and red lines show data from Dfl and CD19 DKO B cells, respectively. Asterisks represent statistical significance calculated by comparing the signals between Dfl and CD19 DKO B cells, either within the G-quadruplex and R-loop forming regions, the region to +/−1kb window or +/−1kb to 2 kb window for respective datasets. e) Profile histograms showing the 5hmC signal in Dfl (blue) and CD19 DKO (red) B cells in 23,467 regions identified as enriched for 5hmC signal. f) Violin plots quantifying enrichment (RPKM) of 5hmC signal in Dfl B cells in the +/−1 kb from G-quadruplex and R-loop forming regions at promoters, non-promoter regions and control regions randomly located in euchromatin (Hi-C A genomic compartment) from 2 biological replicates. g) Pie chart showing the differentially methylated regions (DMRs) in CD19 DKO compared to control Dfl B cells. Of a total of 6948 DMRs identified by WGBS, 1014 (15%) showed reduced DNA methylation (hypomethylation) and 5934 (85%) showed increased DNA methylation (hypermethylation). h) Box and whisker plots quantifying percent of 5mC + 5hmC/unmodified C (from WGBS) at and near the G4 and R-loop forming regions overlapping promoters and regions not overlapping promoters in Dfl (blue) and CD19 DKO (red) B cells from 2 biological replicates. The signal is plotted in three windows; window 1, within the G4 and R-loop regions; window 2, from region to +/− 1 kb on either side and; window 3, +/-1kb to +/−2kb on either side. i) Percent of 5mC + 5hmC/unmodified C (from WGBS) in random genomic regions of Dfl (blue) and CD19 DKO (red) B cells. j) Heatmaps of enrichment (RPM) for G-quadruplexes (left) and R-loops (right) in Dfl and CD19 DKO B cells, ordered in descending order of gene expression. k) MA plot (left) showing differentially expressed genes (DEGs) in CD19 DKO B cells. Red dots, upregulated DEGs; blue dots, downregulated DEGs; black dots, DEGs with G-quadruplexes and R-loops at their promoters (+/-1kb of TSS); yellow dots, non-DEGs with G-quadruplexes and R-loops at their promoters; grey dots, non-DEGs without G-quadruplexes and R-loops. The pie-chart (right) shows the percent of DEGs with (green) and without (brown) G-quadruplexes and R-loops at their promoters. Asterisks indicate statistical significance. Statistical significance is calculated using Kruskal-Wallis test and the ad hoc Dunn’s test in d), f) and h), Chi-square test in j). Boxes in box and whisker plots represent median (center) with 25th to 75 th percentile and whiskers represent maxima/minima. **** p value ≤0.0001 and ***** p value <0.000001.
. a)-c) Genome browser tracks showing the distribution of G-quadruplexes (G4), R-loops, RNA-Seq, WGBS, and 5hmC datasets for Dfl (blue tracks) and CD19 DKO (red tracks) B cells. Grey boxes indicate regions of interest. The blue arrows at the bottom show the location of the TSS and the direction of transcription. d) Circos plots to visually depict all translocations identified by HTGTS in Dfl and CD19 DKO replicates. Colored lines connect the Sµ bait with the translocation partner regions. Color scale represents the number of translocation partner regions identified in a 10 kb window. Translocations from two Dfl and CD19 DKO replicates are represented separately. e) Relative representation of different classes of motifs predicted to form G-quadruplexes (pG4) in control genomic regions selected randomly (right bar) and +/−300 bp from the center of translocation partner junctions identified from translocations in Dfl and CD19 DKO B cells (right bar). The numbers (n) of Dfl and CD19 DKO hits, and control regions are included in the plots. f) Density of AID motifs (WRCY/RGYW) in +/−300bp from the center of translocation partner junctions identified from translocations in Dfl and CD19 DKO B cells compared to control random regions in euchromatin (Hi-C A compartment) from 2 biological replicates. Statistical significance is calculated using the Wilcoxon signed rank test f), ***** p value <0.000001.
. a) Diagrammatic representation of the strategy used to confirm G-quadruplex binding. Nuclear lysates of activated B cells were incubated with biotin-conjugated single stranded G4- or non-G4-forming control oligonucleotides (Oligos) captured using streptavidin beads. b) Immunoblots showing flag-tagged BG4 (positive control), ATRX, BLM and DNMT1 proteins. Left lane, 1/10th input from nuclear lysates (1/10th Input); middle lane, proteins pulled down with G4 forming oligonucleotides; and right lane, proteins pulled down with non-G4 control oligonucleotides. The data is representative of at least 2 independent experiments. c) - d) Quantification of c) cell numbers, d) spleen weights, of 10 week-old Tfl (grey), CD19 Dnmt1 KO (purple), CD19 DKO (red) and CD19 TKO (brown) mice from 5 independent experiments. e) Enlarged spleen of 75-week-old CD19 TKO mice compared with Tfl control mice. f) Flow cytometric detection of G-quadruplexes with BG4-Ig antibody or isotype IgG controls in B cells from Tfl (YFP−, grey), CD19 Dnmt1 KO (YFP+, purple), CD19 DKO (YFP+, red) and CD19 TKO (YFP+, brown) mice. g) – h) Quantification of median fluorescence intensity (MFI) of g) BG4-Ig signal, h) γH2AX signal from Tfl (YFP−), CD19 Dnmt1 KO (YFP+), CD19 DKO (YFP+) and CD19 TKO (YFP+) B cells from 4 independent experiments. i), k) Flow cytometric detection of i) G-quadruplexes with BG4-Ig antibody or isotype IgG controls and k) R-loops using V5-epitope-tagged recombinant RNASE H1 (rRNASE H1) or IgG controls in GC B cells (Fas+) from Tfl (YFP−, grey), CD19 DKO (YFP+, red) and CD19 TKO (YFP+, brown) mice. j), l) Quantification of median fluorescence intensity (MFI) of j) BG4-Ig signal and l) R-loops (rRNASE H1) signal in GC B cells (Fas+) from Tfl (YFP−, grey), CD19 DKO (YFP+, red) and CD19 TKO (YFP+, brown) mice from 3 independent experiments. m) Model proposing functional interplay between TET and DNMT activities to limit GC B cell expansion. TET deficiency in B cells leads to increased G4 and R-loop structures and is associated with altered gene expression, DNA damage and development of B cell lymphoma. Statistical significance is calculated using one-way ANOVA in c), d), g), h), j) and l). Error bars represent mean +/− standard deviation, * p value ≤0.05, ** p value ≤0.01, *** p value ≤0.0005 **** p value <0.0001.
. a) Sequential gating strategy used for the flow cytometry analysis. The respective gate names are mentioned in the corresponding figures. b)-e) scanned immunoblots for Extended Data Fig. 2 (b), 5 (c) and 8 (d).
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Shukla, V., Samaniego-Castruita, D., Dong, Z. et al. TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures. Nat Immunol 23, 99–108 (2022). https://doi.org/10.1038/s41590-021-01087-w
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