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Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation

Nature Immunology volume 23, pages 1763–1776 (2022)Cite this article

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Abstract

The nuclear corepressors NCOR1 and NCOR2 interact with transcription factors involved in B cell development and potentially link these factors to alterations in chromatin structure and gene expression. Herein, we demonstrate that Ncor1/2 deletion limits B cell differentiation via impaired recombination, attenuates pre-BCR signaling and enhances STAT5-dependent transcription. Furthermore, NCOR1/2-deficient B cells exhibited derepression of EZH2-repressed gene modules, including the p53 pathway. These alterations resulted in aberrant Rag1 and Rag2 expression and accessibility. Whole-genome sequencing of Ncor1/2 DKO B cells identified increased number of structural variants with cryptic recombination signal sequences. Finally, deletion of Ncor1 alleles in mice facilitated leukemic transformation, whereas human leukemias with less NCOR1 correlated with worse survival. NCOR1/2 mutations in human leukemia correlated with increased RAG expression and number of structural variants. These studies illuminate how the corepressors NCOR1/2 regulate B cell differentiation and provide insights into how NCOR1/2 mutations may promote B cell transformation.

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Fig. 1: B cell development requires nuclear corepressors NCOR1 and NCOR2 and is sensitive to their gene dosage.
Fig. 2: Nuclear corepressors regulate both proliferation and differentiation during B cell development.
Fig. 3: Absence of nuclear corepressors results in defective pre-BCR signaling and in turn promotes STAT5-mediated transcription.
Fig. 4: scATAC-seq reveals the epigenetic landscape regulated by nuclear corepressors.
Fig. 5: Nuclear corepressors segregate RAG expression from proliferating cells and protect the genome integrity.
Fig. 6: NCOR1 deficiency promotes leukemic transformation with phenotypic heterogeneity.
Fig. 7: Human B-ALL patients with nuclear corepressor mutations harbor structural variants with high RAG expression.

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Data availability

The data supporting this study are available within the paper and supplementary information file. A reporting summary is also available. Mouse genome mm10 was used as reference sequence (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/). scRNA-seq and scATAC-seq data were deposited at Gene Expression Omnibus with the accession code GSE208656. Whole-genome sequencing data were deposited at SRA with the accession code PRJNA860179. STAT5 ChIP-seq peak data were obtained from GSE86878, and H3K27ac ChIP-seq peak data were obtained from http://www.cistrome.org via project GSM1463433. IMMGEN RNA-seq count data were from GSE124829. Human B-ALL NCOR1 and NCOR2 mutation and RNA-seq data were obtained from St Jude ProteinPaint (https://pecan.stjude.cloud/proteinpaint). Source data are provided with this paper.

Code availability

Only publicly available software tools were used for analysis (for example, CellRanger, Seurat, Signac, whole-genome sequencing and GSEA). No custom software was used or developed in this study, but analysis scripts will be provided upon request.

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Acknowledgements

We thank G. Hubbard, A. Rost and N. Keller for technical assistance and mouse husbandry; E. Stanley, J. Daniels and K. Beckman and the University of Minnesota Genomics Center for 10x Genomics single-cell capture and sequencing; J. Motl, R. Arora and P. Champoux for cell sorting and Flow Cytometry Core Facility maintenance at University of Minnesota (5P01AI035296); and S. Dehm and K. Schwertfeger (University of Minnesota) for comments on the manuscript. We thank E. Olson (UT-Southwestern), W. Ellmeier (Medical University of Vienna) and J. Auwerx (École Polytechnique Fédérale, Lausanne, Switzerland) for providing the Ncor1FL/FL mice. The Minnesota Supercomputing Institute at the University of Minnesota provided bioinformatic support and resources that contributed to the research results reported within this paper. We are thankful for support from the German Gene Trap Consortium, which assisted in the generation of the Ncor2FL/FL mice. This work was supported by an individual predoctoral F30 fellowship from the National Institutes of Health (NIH) (F30CA232399) and a T32 training grant (T32 GM008244) to R.D.L.; St Jude Children’s Research Hospital Cancer Center Core Grant CA021765 and NIH grant R35CA197695 to C.G.M.; and NIH grants R01AI124512, R01AI147540 and R01CA232317 to M.A.F. M.A.F. was supported by the Virginia and David Utz endowed chair in fundamental immunobiology.

Author information

Authors and Affiliations

  1. Department of Laboratory Medicine and Pathology, Center for Immunology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA

    Robin D. Lee, Lynn M. Heltemes-Harris & Michael A. Farrar

  2. Supercomputing Institute for Advanced Computational Research, University of Minnesota, Minneapolis, MN, USA

    Todd P. Knutson, Sarah A. Munro & Jeffrey T. Miller

  3. Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Charles G. Mullighan

  4. Institute for Genomic Medicine, University of California, San Diego, San Diego, CA, USA

    Kristen Jepsen

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Contributions

Conceptualization, R.D.L. and M.A.F.; Methodology, R.D.L. and M.A.F.; Formal Analysis, R.D.L., S.A.M., T.P.K., J.M., L.H.H. and M.A.F.; Investigation, R.D.L. and L.H.H.; Resources, C.G.M., K.J. and M.A.F.; Data Curation, S.A.M., T.P.K. and L.S.; Writing – Original Draft, R.D.L. and M.A.F.; Writing – Review & Editing, R.D.L, S.A.M., T.P.K., C.G.M., K.J. and M.A.F.; Visualization, R.D.L., L.H.H., S.A.M., T.P.K. and J.M.; Supervision, M.A.F.; Project Administration, M.A.F.; Funding Acquisition, R.D.L. and M.A.F.

Corresponding author

Correspondence to Michael A. Farrar.

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Competing interests

C.G.M. provides consultation and advise for Illumina (compensated), Faze Medicines (compensated) and Beam Therapeutics (compensated) and receives research funding from Pfizer and Abbvie. The remaining authors declare no competing interests.

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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Editor recognition statement (if applicable to your journal) 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. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Ncor2 conditional knockout mouse model and flow cytometry gating scheme.

a. Schematic for generating Ncor2 flox mice using the flip-excision system (FlEx). A retrovirus vector containing a strong splice acceptor site followed by a lacZ reporter gene was flanked by FRT and loxP sites. This gene trap was targeted in the intronic region between exon 1 and 2. Mice harboring the gene trap were bred to FLPe constitutive expressing mice, inverting the gene trap and allowing for normal splicing of Ncor2. Subsequent breeding with Cd79a-Cre mice then allows for conditional deletion of Ncor2 in developing B cells. b. Representative NCOR1 protein expression in CD19 + B220 + bone marrow B cells with isotype controls (left) and quantification of NCOR1-positive B cells (right). Each genotype represents n = 3, from three independent experiments. Center of measure indicates mean and error bars indicate standard deviation. A one-way ANOVA was used to calculate statistical significance. c. Representative NCOR1 protein expression in CD19 + B220 + splenic B cells with isotype controls (left) and quantification of NCOR1-positive B cells (right). Each genotype represents n = 3, from three independent experiments. Center of measure indicates mean and error bars indicate standard deviation. A one-way ANOVA was used to calculate statistical significance. d. Quantification of Ncor1 and Ncor2 expression via qPCR in wildtype and NCOR1/2 double-knockout B cells from the bone marrow and the spleen. Each genotype represents n = 3, from two independent experiments. Center of measure indicates mean and error bars indicate standard deviation. A two-tailed t-test was performed to calculate statistical significance. e. Whole-genome sequencing read depth coverage across the Ncor1 gene for wildtype and NCOR1/2 DKO B cells. f. Whole-genome sequencing mapping of reads across the Ncor2 gene trap for wildtype (Cre- littermate) and NCOR1/2 DKO B cells; left and right panels indicate 5’ and 3’ inversion breakpoints, respectively. g. Lymphocytes are gated using FSC-A, SSC-A, and then subsequently on SSC-A and SSC-W for singlets. Dead cells are excluded using the GhostRed780 Live/dead dye and gated on B220 and CD43. CD43+ and CD43- cells are gated on to assess Hardy fractions A-C and D-F, respectively.

Source data

Extended Data Fig. 2 Single-cell RNA-sequencing multiplet removal and cluster identification.

a. Multiplet identification using HTOdemux at a resolution of k = 22 (left). Multiplet and singlet classification based on genotype is demonstrated on the right. b. Feature plots of temporally regulated genes during B cell development. c. Feature plot of CITE-Seq antibodies, B220, CD19, CD25, CD43, CD93, and IgM (top) and violin plot of the CITE-seq antibodies (bottom) are shown. These gene expression profiles and CITE-seq antibody expression patterns were used to characterize different subsets of B cell development.

Extended Data Fig. 3 Overlap between scRNA-seq and scATACseq differentially upregulated expression and accessibility genes.

a. Cluster comparison between scRNAseq and scATACseq genes that are differentially upregulated in expression and accessibility in the NCOR1/2-knockout (DKO) B cells.

Extended Data Fig. 4 Nuclear corepressors regulate the p53 pathway and repress BIM expression.

a. Landscape in silico analysis (LISA)-based prediction of transcriptional regulators of significantly upregulated genes in either WT (left) or NCOR1/2 DKO (right) of cycling pro B cells (top) or pre-BCR-dependent cells (bottom). Number in parentheses indicates rank. A two-tailed wilcoxon rank-sum test was used to calculated statistical significance. b. GSEA of p53 pathway and senescence pathway in NCOR1/2 DKO cells in both cycling pro B (top) and pre-BCR-dependent cells (bottom). c. Changes in expression of p53 pathway genes, Cdkn1a, Btg2, and Trp53inp1, between WT, NCOR1 KO and NCOR1/2 DKO B cell clusters. d. Pro-apoptosis factor, BIM expression of WT and NCOR1/2 DKO in pre-pro B, pro B, pro B/pre B, small pre B, immature B and mature B cells. Plot represents n = 3 of each genotype from two independent experiments. Error bar indicates standard deviation. A one-way ANOVA was used to calculate statistical significance. e. BIM (Bcl2l11) gene expression and accessibility differences in wildtype, NCOR1 KO and NCOR1/2 DKO B cells. Asterisk indicates comparisons with at least a log2 fold change of 0.25 that have a p-value <0.05 when compared to wildtype. RNA expression of Bcl2l11 (left) represents WT (n = 7454 cells), NCOR1 KO (n = 7806 cells), NCOR1/2 DKO (n = 8637 cells) from two biological replicates for each genotype from a single capture. Accessibility of Bcl2l11 (right) represents WT (n = 8552 cells), NCOR1 KO (n = 6008 cells), and NCOR1/2 DKO (n = 8574 cells) from a single biological sample derived from three separate captures. A two-tailed Wilcoxon rank-sum test was used to calculate statistical significance. All measure of centers indicate mean.

Source data

Extended Data Fig. 5 KLF2 and its target genes are aberrantly expressed in NCOR1/2-deficient B cells.

a. RNA expression of Klf2 and its target genes Cd69 and Sell (CD62L; L-selectin) in wildtype (WT), NCOR1-knockout (KO) and NCOR1/2-knockout (DKO) B cells. Asterisk (*; P < 0.05) indicates statistically significant expression changes compared to wildtype cells for Klf2 in PreBCRi-II (DKO; P = 4.24E-15), preBCRi-III (DKO; P = 1.43E-9), and immature B (DKO; P = 1.34 E-8), for Cd69 in PreBCRi-II (DKO; P = 1.86E-39), PreBCRi-III (DKO; P = 2.74E-8), Kappa Pre B (DKO; P = 1.47E-37), Lambda Pre B-I (DKO; P = 1.69E-12), Lambda Pre B-III (DKO; P = 9.14E-14), and Immature B (DKO; P = 1.89E-7). b. Gene activity of Klf2 and its target genes Cd69 and Sell (CD62L; L-selectin)in WT, KO and DKO B cells. Asterisk (*; P < 0.05) indicates statistically significant accessibility changes compared to wildtype cells for Klf2 in Pro B VDJ (KO; P = 1.23E-9), Cycling Pre B (KO; P = 2.94E-36, DKO; P = 4.97E-24), Kappa/Lambda Pre B (KO; P = 5.31E-33), Immature B (KO; P = 1.22E-14, DKO; P = 6.40E-11), mature B (KO; P = 4.94E-13), plasma cells (KO; P = 4.94E-13, DKO; P = 5.39E-7) and for Cd69 in Cycling Pro B (KO; P = 1.74E-6, DKO; P = 2.12E-12), Pro B VDJ (KO; P = 4.20E-13, DKO; P = 2.47E-30), Cycling Pre B (KO; P = 1.29E-27, DKO; P = 6.87E-84), Kappa/Lambda Pre B(KO; P = 1.96E-57, DKO; P = 6.92E-46), Immature B (KO; P = 3.72E-27, DKO; P = 3.13E-22), mature B (KO; P = 1.68E-20, DKO;P = 5.39E-7), plasma cells (KO; P = 1.68E-20, DKO; P = 5.39E-7) and for Sell in Kappa/Lambda Pre B (DKO; P = 4.27E-7), and immature B (DKO;P = 1.47E-16). c. CD62L expression of different B cell development stages in wildtype (WT) and NCOR1/2-knockout (DKO) B cells. Top represents representative flow cytometry plots for CD62L expression and bottom represents summarized frequency of CD62L + cells for prepro B (B220 + CD43 + CD24−CD19-, n = 4), proB/preB (B220 ((eB (eB (for 220 ((e, n = 4), small pre B (B220 + CD43 + CD19 + IgM-, n gM4), immature B (B220lowCD43immgM M4, n n4), and mature B cells (B220highCD43andgM M4, n n4) from three independent experiments. A two-tailed t-test was used to calculate statistical significance. Center of measure indicates mean and error bars indicate standard deviation.

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Extended Data Fig. 6 Transcriptional and accessibility changes in genes associated with Rag-mediated genomic changes in human B-ALLs.

a. Transcriptional changes between WT, NCOR1 KO, and NCOR1/2 DKO derived from scRNA-seq. b. Accessibility changes between WT, NCOR1 KO, and NCOR1/2 DKO derived from scATAC-seq. Asterisk (*) indicates comparisons with at least a log2 fold change of 0.25 that have a p-value <0.05 when compared to wildtype. RNA expression (left) data represents WT (n = 7454 cells), NCOR1 KO (n = 7806 cells), NCOR1/2 DKO (n = 8637 cells) from two biological replicates for each genotype from a single capture. ATAC accessibility data (right) represents WT (n = 8552 cells), NCOR1 KO (n = 6008 cells), and NCOR1/2 DKO (n = 8574 cells) from a single biological sample derived from three separate captures. A two-tailed Wilcoxon rank-sum test was used to calculate statistical significance.

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Supplementary Tables 1–5

Supplementary Table 1. Pairwise comparison of differentially expressed genes among WT, NCOR1 KO and NCOR1/2 DKO. A two-tailed Wilcoxon rank-sum test was performed to calculate statistical significance. Cluster labels correspond to the following cell type as following: cluster 0: κ pre-B, cluster 1: Pro B VDJ, cluster 2: PreBCRi-I, cluster 3 – PreBCRi II, cluster 4: pre-pro-B, cluster 5: Immature B, cluster 6: Cycling Pro B, cluster 7: Mature B, cluster 8: PreBCRi-III, cluster 9: Pre B-II, cluster 10: λ Pre B-III, cluster 11: PreBCR-d, cluster 12: High mitochondrial B, cluster 13: λ Pre B-I, cluster 14: Committed Pro B, cluster 15: Plasma Cells, cluster 16: Cycling Immature B. Supplementary Table 2. Pairwise comparison of differential transcription factor motifs present among WT, NCOR1 KO and NCOR1/2 DKO. Transcription factor motifs were derived from the JASPAR database. A two-tailed Wilcoxon rank-sum test was performed to calculate statistical significance. Supplementary Table 3. Pairwise comparison of differentially accessible genes among WT, NCOR1 KO and NCOR1/2 DKO. Accessibility of the gene was defined as peaks found within the gene body and peaks present within the upstream 2,000-bp promoter. A two-tailed Wilcoxon rank-sum test was performed to calculate statistical significance. Supplementary Table 4. Structural variants found in WT and NCOR DKO B cells. Structural variants including BND (break end), DEL (deletion), INS (insertion), TRA (translocation) and INV (inversion) and their genomic position and affected genes are listed. Structural variant calling was performed using Smoove. Supplementary Table 5. Human B-ALL NCOR1 mutation type and the presence of intrachromosomal or interchromosomal structural variants. Types of NCOR1 mutations and structural variants that are present are broken down into intrachromosomal and interchromosomal. Interchromosomal translocations that are B-ALL subtype defining are present. Structural variant information was derived from StJude ProteinPaint

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Lee, R.D., Knutson, T.P., Munro, S.A. et al. Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation. Nat Immunol 23, 1763–1776 (2022). https://doi.org/10.1038/s41590-022-01343-7

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