Co-repressor CBFA2T2 regulates pluripotency and germline development

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Abstract

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs)1,2, precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own ‘latent’ form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2, 3, 4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell- and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2−/− mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional ‘passenger’ role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

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Figure 1: PRDM14 and the co-repressor protein CBFA2T2 interact and bind to chromatin interdependently.
Figure 2: PRDM14 and CBFA2T2 regulate pluripotency.
Figure 3: Cbfa2t2−/− mice are defective in their germ line.
Figure 4: Mechanism of CBFA2T2–PRDM14 complex chromatin binding and direct regulation of PGC epigenetic reprogramming.

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Primary accessions

Gene Expression Omnibus

Data deposits

Sequencing data have been deposited in the Gene Expression Omnibus under accession number GSE71676.

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Acknowledgements

We thank P. Andrews and X.-J. Sun for providing plasmids. We are grateful to L. Vales, M. E. Torres-Padilla and L. Bu for critical comments, H. Zheng for MS analysis, and D. Hernandez, C. Leek, M. Yamaji, A. Paradkar and M. Alu for excellent technical assistance. The work was supported by the Howard Hughes Medical Institute (HHMI) and National Institutes of Health (NIH; RO1GM064844-12) (D.R.). B.A.G. acknowledges funding from NIH grant R01GM110174. T.T. was supported by the HHMI, NIH, Starr Foundation, and Tri-Institutional Stem Cell Initiative. M.Y. was a recipient of a Japan Society for the Promotion of Science (JSPS) Research Fellowship.

Author information

S.T. designed and performed majority of experiments; V.N. analysed ChIP-seq and RNA-seq data; S.E.V., S.T. and M.S. performed iPSC reprogramming experiments; X.W. and B.A.G. quantified histone modifications. S.Y.K. did CRISPR zygotic injection. S.T., S.Y.K., M.Y. and L.A.R. characterized mice. M.Y., S.T. and T.T. designed and performed PGC experiments. S.T., V.N., M.Y. and D.R. wrote the manuscript.

Correspondence to Danny Reinberg.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Biochemical interaction between PRDM14 and CBFA2T2.

a, Mass spectrometry peptide counts from Flag affinity purification from NCCIT control cells and stable lines expressing Flag–HA–PRDM14 (PRDM14-F), and Flag–HA–CBFA2T2 (CBFA2T2-F). b, Characterization of in-house human PRDM14 antibody. Western blot performed using 30 μg of NCCIT and KH2 mES cell lysate. Human PRDM14 antibody is specific and does not cross-react with mouse PRDM14. c, Immunoprecipitation (IP) using antibodies against the indicated endogenous proteins in mES cells. d, Western blot of Superose 6 column fractionation of Flag-purified CBFA2T2 complex in NCCIT cells stably expressing Flag–HA–CBFA2T2. e, ChIP analysis using the indicated antibodies in 293T-REx harbouring a UAS-TK-Luciferase transgene. Fold enrichment represents the ratio of enrichment by ChIP-qPCR upon induction of GAL4–PRDM14 expression via doxycycline addition. Positions of the primer set are indicated by small arrows in the schematic. qPCR source data are included in the Supplementary Information. Source data

Extended Data Figure 2 PRDM14 and CBFA2T2 exhibit an overlapping and interdependent distribution on chromatin in NCCIT cells.

a, Heat map depicting PRDM14, CBFA2T2, RING1B and SUZ12 read density across a 5-kb window centred about the PRDM14 (top) or RING1B (bottom) SERs identified in NCCIT cells. b, Representative genome browser tracks depicting SERs at the indicated genomic loci. c, Gene Ontology (GO) analysis of PRDM14 and CBFA2T2 common target genes. d, Western blot analysis of PRDM14 and CBFA2T2 protein levels in knockdown (KD) experiments (Fig. 1d, e).

Extended Data Figure 3 Characterization of knockout ES cell mutants and quantification of human iPS cell reprogramming efficiency.

a, b, Strategy for generating Prdm14- and Cbfa2t2-knockout (KO) mES cells via CRISPR–Cas9 genome editing. Sequencing chromatograms confirming homozygous disruption of the locus are depicted. c, Cbfa2t2- and Prdm14-knockout ES cells require 2i to maintain growth. ES cell lines generated under FBS plus LIF plus 2i conditions were continuously cultured in FBS plus LIF plus 2i (top, middle), or switched to FBS plus LIF (bottom). Eight days after 2i withdrawal (FBS plus LIF), well-formed ES cell colonies were undetectable; instead, mutant ES cells appeared to be differentiated. Scale bar, 100 μm. d, Proliferation rates of wild-type (WT) and mutant knockout ES cells as described in c. Data were obtained from three biological replicates. Please note error bars shown in the plots. Owing to the logarithmic scale used here, some error bars are very small and might be invisible. e, RNA-seq MA plot (log ratio (M) versus mean average (A)) in the indicated ES cells. Data are representative of three biological replicate experiments for each line. Mean abundance is plotted on the x axis and enrichment (both in log2 scale) is plotted on the y axis. Genes depicted in red are differentially expressed with a FDR < 0.0001. f, Heat map showing relative expression of all differentially expressed genes as described in Fig. 2c. The only difference is now the heat map is centred on CBFA2T2 differentially expressed genes, rather than PRDM14 differentially expressed genes. g, Scheme of human fibroblast reprogramming to iPS cells. Fibroblasts were transduced with lentiviruses expressing polycistronic OCT4/KLF4/SOX2/c-MYC (OKSM) and either PRDM14 or CBCFA2T2. Three weeks later, bright-field images of successfully reprogrammed colonies (left) and live TRA-1-81 staining (right) were recorded. Scale bar, 500 μm. h, Quantification of human iPS cell reprogramming efficiency based on TRA-1-81 staining with secondary antibody conjugated with horseradish peroxidase (HRP) and substrate DAB. Error bars are based on four biological replicates of each condition. The source data are included in the Supplementary Information. Source data

Extended Data Figure 4 Cbfa2t2−/− mouse genotypes and sperm defects.

a, One representative Cbfa2t2−/− mouse genotype wherein a 7-bp fragment is deleted. b, Testes of multiple wild-type (n = 4) and Cbfa2t2−/− (n = 4) male mice at 8 weeks old were dissected and weighed. c, Number of sperm in the epididymis of Cbfa2t2+/+ (n = 4) and Cbfa2t2−/− (n = 4) mice is shown with standard error of the mean. P value was determined by Student’s t-test. d, Near loss of gonocytes in Cbfa2t2-knockout mutant P0 testes by DDX4 (MVH) staining. Visualization of MVH-positive (red) gonocytes in Cbfa2t2+/− (top) or Cbfa2t2−/− (bottom) testis at P0 stage. The merged images with Hoechst (left; white) are shown on the right. Scale bars, 100 mm. e, Numbers of AP-2γ-positive PGCs in Cbfa2t2+/+ (black), Cbfa2t2+/− (grey) and Cbfa2t2−/−(red) embryos at the indicated embryonic stages. LS, late-streak stage; EB, MB, and LB, early-, mid-, and late-bud stage; EHF, early-head fold stage; 2 st., 2 somites stage. Student’s t-test: *P = 0.03, **P = 0.003. f, Numbers of AP-2γ-positive PGCs in Cbfa2t2+/+ (black), Cbfa2t2+/− (grey) and Cbfa2t2−/−(red) embryos at the indicated embryonic stages. 0B, zero-bud stage; LHF, late-head fold stage. g, Left, expression of SOX2 (red) in AP-2γ-positive (green) PGCs in Cbfa2t2+/+ (top) or Cbfa2t2−/− (bottom) embryo at mid-bud stage, E7.25, shown as z-projection images of posterial confocal sections. Arrow indicates a minor PGC with relatively normal activation of SOX2. Scale bar, 50 mm. Right, percentage of SOX2-positive (red) cells in AP-2γ-positive (green) PGCs in the indicated genotypes of Cbfa2t2 at E7.0–7.25 (zero- to mid-bud stage) are shown with statistical significance (Student’s t-test: *P = 0.0006, **P = 0.0001; Cbfa2t2+/+, n = 7; Cbfa2t2+/−, n = 5; Cbfa2t2−/−, n = 5).

Extended Data Figure 5 Cbfa2t2-m7 mutant characterization and the related mechanism.

a, Gene Ontology (GO) analysis of PRDM14 ChIP-seq target genes. PRDM14 target genes are enriched in histone methyltransferase activities by DAVID functional annotation tool analysis. b, Cbfa2t2-m7 mutant genotyping. The mutant 7 amino acids are in red and corresponding wild-type (WT) residues are highlighted in blue in the displayed protein sequences. c, Bright-field images of wild-type and Cbfa2t2-m7 mES cells. Scale bar, 100 μm. d, Western blot analysis of PRDM14, CBFA2T2 and OCT4 protein levels in Prdm14-knockout (KO), Cbfa2t2-knockout or m7 mutant ES cells under feeder-free FBS plus LIF plus 2i condition. Nonspecific bands are denoted with an asterisk. e, ChIP-qPCR using antibodies against PRDM14, CBFA2T2 or OCT4 at selected OCT4 target genes. Occupancy is compared between wild-type, Cbfa2t2-knockout and Cbfa2t2-m7 mES cells. ChIP-qPCR primer sequences are included in Supplementary Table 3. f, RT–qPCR quantification of Ehmt1 mRNA levels in wild-type and mutant lines. P values are 0.004 (**) and 0.0142 (*).The source data are included in the Supplementary Information. g, Mass spectrometry quantification of histone H3K9 modifications in wild-type and mutant lines. P values are 0.00956, 0.04248 (*). The source data are included in the Supplementary Information. h, i, Additional immunofluorescence analysis of H3K9me2 (red) of AP-2γ-positive (green; arrowheads) PGCs in Cbfa2t2+/− and Cbfa2t2−/− embryos at E8.75 as described in Fig. 4i. Source data

Supplementary information

Supplementary Table 1

This table contains PRDM14 and CBFA2T2 target genes identified by ChIP-seq in NCCIT cell. (XLSX 155 kb)

Supplementary Table 2

This table contains differentially expressed genes in Prdm14 and Cbfa2t2 Knockout lines. (XLSX 361 kb)

Supplementary Table 3

This table contains qPCR and PCR primers used in ChIP-qPCR and off-target verification. (XLSX 43 kb)

Supplementary Table 4

This table contains PRDM14 and CBFA2T2 target genes identified by ChIP-seq in mouse ESC. (XLSX 182 kb)

Supplementary Information

This file contains Supplementary Figure 1, the uncropped western blot images. (PDF 367 kb)

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Tu, S., Narendra, V., Yamaji, M. et al. Co-repressor CBFA2T2 regulates pluripotency and germline development. Nature 534, 387–390 (2016) doi:10.1038/nature18004

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