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A PAX5–OCT4–PRDM1 developmental switch specifies human primordial germ cells

Nature Cell Biology volume 20, pages 655–665 (2018)Cite this article

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Abstract

Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analysed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined the distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrated that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, an epistasis analysis indicated that PAX5 acts upstream of OCT4 and PRDM1. The PAX5–OCT4–PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programmes during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.

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Fig. 1: Global redistribution of OCT4 binding in PGCs compared with ESCs.
Fig. 2: Co-occurrence of OCT4 with PAX5 and PRDM1 in PGCs.
Fig. 3: Overexpression PAX5 and PRDM1 enhances the germ cell potential of ESCs.
Fig. 4: Knockout of PAX5 or PRDM1 reduces the germ cell potential of ESCs.
Fig. 5: PAX5 acts upstream of OCT4.
Fig. 6: PAX5 and OCT4 act upstream of PRDM1.
Fig. 7: Role of PAX5 and PRDM1 in hPGC specification in vitro.

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Acknowledgements

This work was supported by P50 HD 068158 to R.A.R.P. (Project I).

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Authors and Affiliations

  1. Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT, USA

    Fang Fang, Benjamin Angulo, Ninuo Xia, Jun Cui & Renee A. Reijo Pera

  2. Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA

    Fang Fang, Benjamin Angulo, Ninuo Xia, Jun Cui & Renee A. Reijo Pera

  3. Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, School of Medicine, Magee Women’s Research Institute, Pittsburgh, PA, USA

    Meena Sukhwani & Kyle E. Orwig

  4. Genomic Medicine Division, Hematology Branch, NHLBI/NIH, Rockville, MD, USA

    Zhengyuan Wang

  5. Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA

    Charles C. Carey, Aurélien Mazurie, Royce Wilkinson & Blake Wiedenheft

  6. Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    Naoko Irie & M. Azim Surani

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  1. Fang Fang
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Contributions

The study was conceived and designed by F.F. and R.A.R.P. F.F. performed most experiments (including ChIP–seq, immunohistochemistry, RNA-seq, protein pull-down assays, luciferase reporter assays, gene expression profiling) and analysed the data. N.X. performed bioinformatics analyses for ChIP–seq and luciferase reporter assays, flow cytometry and gene expression analysis for the xenotransplantation experiments. B.A. generated the PAX5-knockout hESC lines and performed part of the immunohistochemistry in xenotransplantation samples. Z.W. performed the initial bioinformatics analysis for ChIP–seq data. M.S. and K.E.O. conducted the xenotransplantation. C.C.C and A.M. performed RNA-seq analysis. J.C. constructed the H1-DDX4 reporter. R.W. and B.W. designed and constructed the PAX5-knockout plasmids. M.A.S. and N.I. provided the PRDM1-knockout hESC line and protocol. The manuscript was written by F.F. and R.A.R.P. with input from the other authors.

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Correspondence to Fang Fang.

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Integrated supplementary information

Supplementary Figure 1 Staining of OCT4-positive cells in human fetal testis and comparison between mixed ChIP-Seq and Standard ChIP-Seq.

(a) Cross-section of human fetal testis (22 weeks) with immunostaining of OCT4. Scale bar represents 50 µm. (b) Immunostaining of OCT4 and cKIT in human fetal testis. Arrows indicate co-staining cells. Scale bar represents 50 µm. (c) Immunostaining of OCT4 and DDX4 proteins in human fetal testis. Arrows indicate cells that only express OCT4. Scale bar represents 50 µm. Immunostaining experiments in (a-c) were independently repeated a minimum of three times with similar results. (d) Schematic strategy for comparing mixed ChIP with conventional ChIP. (e) ChIP-qPCR for detection of peaks at OCT4 locus. ChIP-qPCR were independently repeated a minimum of three times with similar results. (f) Scatterplot comparing OCT4 ChIP-seq data generated in pure ESCs and 1% ESCs mixed with fibroblast cells. Correlation was computed using whole genome data within 10kb of transcription start site (TSS) of RefSeq genes. Sample size n=2 and Pearson’s correlation coefficient was used for the correlation analysis. (g) Venn diagram showing overlapping genes bound by OCT4 generated by ChIP-seq data in pure ESCs and 1% ESCs mixed with fibroblast cells. (h) Venn diagram showing overlapping genes bound by OCT4 generated by ChIP-seq data derived from two biological replicates.

Supplementary Figure 2 PAX5 and PRDM1 expression and binding in hPGCs.

(a) Cross-section of human fetal testis (22 weeks) with immunostaining for OCT4, PAX5, PRDM1, SOX2 and IgG control. Scale bars represent 50 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (b) Expression level of transcription factors in hESCs and human fetal testis. Data are represented as mean ± SD of n=3 independent replicates. (c) Heatmap visualization of PAX5 and PRDM1 ChIP-seq data, depicting all binding events centered on the peak region within a 5kb window around the peak. (d) GST-pull down assay to assess protein interactions between PRDM1 and PAX5. Western blot images are representative of three independent experiments with similar results. Unprocessed scans of western blot analysis are available in Supplementary Fig. 8. Source data for b are in Supplementary Table 2.

Supplementary Figure 3 Overexpression of PAX5 and PRDM1 enhances germ cell potential of hESCs during in vitro differentiation.

(a) RT-qPCR analysis of expression level of PAX5 in H1 ESCs, PAX5 KO and cells overexpressing PAX5. Data are represented as mean ± SD of three replicates. (b) Bright field view of cells PAX5 OE and PRDM1 OE cells. Scale bars represent 50 µm. Experiments were independently repeated a minimum of three times with similar results. (c) Immunostaining of OCT4, PAX5 and PRDM1 in PAX5 and PRDM1 overexpression hESCs. OE represents overexpression. Scale bars represent 25 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (d) RT-qPCR analysis of control, PAX5 OE and PRDM1 OE H1 hESCs before and after BMPs-induced differentiation. Data are represented as mean ± SD of n=3 independent replicates. (e) Genome browser representation of ChIP-seq tracks for PAX5 at the SYCP3 and SYCP1 loci. ChIP-seq were independently repeated twice with similar results. (f) RT-qPCR analysis of control and PAX5&PRDM1 double OE H1 hESCs after BMPs-induced differentiation. Data are represented as mean ± SD of n=3 independent replicates. Source data for a, d and f are in Supplementary Table 2.

Supplementary Figure 4 Overexpression of PAX5 and PRDM1 enhances germ cell differentiation of hESCs in vivo in xenotransplantation.

(a) Immunostaining of GFP and DAPI in untransplanted mouse testis. Scale bars represent 100 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (b) Immunostaining analysis of testis xenografts derived from PAX5&PRDM1 double OE H1 hESCs. All images are merged from DDX4 (red), GFP (green) and DAPI-stained nuclei. Scale bars represent 50 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (c) Percentage of tubules positive for GFP+/ DDX4+ cells was calculated across multiple cross-sections (relative to total number of tubules). Data are represented as mean ± SD of n=3 independent replicates. (d) For each positive tubule, the ratio of GFP+/DDX4+ cells per tubule was determined. Data are represented as mean ± SD of n=3 independent replicates. Source data for c and d are in Supplementary Table 2.

Supplementary Figure 5 Construction of PAX5 knockout hESC line with CRISPR.

(a) Targeting strategy of PAX5 knockout in hESC with the designated guide RNA (gRNA) and the resulting deleted sequences. (b) Sequences of wide type PAX5 and PAX5 KO line that show homologous recombination and deletions are shown. Grey box indicates CRISPR recognition site and black bars indicate deleted sequences. (c) Immunofluorescence of PAX5 on wild-type (WT) and PAX5 KO after BMPs-induced differentiation. Scale bars represent 50 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (d) Western blot of PAX5 and ACTIN on wild type (WT) and PAX5 knockout (KO) cells. Western blot images are representative of three independent experiments. Unprocessed scans of Western blot analysis are available in Supplementary Fig. 8.

Supplementary Figure 6 Gene expression of in vitro derived hPGCs and sorting of hPGCs derived from mouse seminiferous tubules.

(a) PAX5 expression level from previously published RNA-seq data19,31,38. Data are represented as mean of three technical replicates. (c) Expression level of germ cell genes from RNA-seq data19,38. Data are represented as mean of three technical replicates. (d) RT-qPCR analysis of OCT4 expression in control and PAX5 KO differentiated cells in vitro. “protocol” refers to the in vitro human germ cell differentiation protocols developed in the specific paper19,38. Data are represented as mean ± SD of n=3 independent replicates. P-value was calculated by two-tailed Student’s t-test and "ns" means not significant. Source data for a-c are in Supplementary Table 2.

Supplementary Figure 7 Identification and mutation of PAX5 binding motifs in OCT4 and PRDM1 enhancer.

(a, e) scanning of OCT4 (a) and PRDM1 (e) enhancer regions to look for key regulatory elements (RE). OCT4 or PRDM1 enhancer region is shown on the top. Red bars represent putative RE. Luciferase activity is shown on the bottom. Red box indicates key RE that has the strongest enhancer activity. Activity is presented relative to the full-length enhancer construct and minimal promoter construct (MP). MP: minimal promoter; RE: regulatory element; Full-length: Full-length enhancer. Data are represented as mean ± SD of n=3 independent replicates. (b, f) Sequence of OCT4 RE4 (b) or PRDM1 RE1 (f). Grey boxes indicate putative binding site for PAX5. PBS: putative binding site. (c, g) The effects of deleting the specific PBS regions in RE4 for OCT4 enhancer (c) and in RE1 for PRDM1 enhancer (g) on luciferase reporter activity. Activity is presented relative to the wild-type construct (RE4 or RE1) and minimal promoter construct (MP). Red box indicates PBS whose deletion abolished the induction of luciferase activity by PAX5 OE. Data are represented as mean ± SD of n=3 independent replicates. (d, h) The effects of mutating PBS2 for OCT4 enhancer (d) or PBS2 for PRDM1 enhancer (h) on luciferase reporter activity. Sequence of wide-type PBS and mutated PBS is shown on the top. Luciferase activity is presented relative to the wild-type construct (RE4 or RE1) and minimal promoter construct (MP). Data are represented as mean ± SD of n=3 independent replicates. Source data for a, c, d, e, g and h are in Supplementary Table 2.

Supplementary Figure 8 Unprocessed gel blots.

Of note, for some immunoblotting assays membranes were cut into several pieces to incubate with different antibodies, and therefore the raw images of these membranes are small in size.

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and Supplementary Table legends

Reporting Summary

Supplementary Table 1

Primer sequences used by category for qRT-PCR analysis

Supplementary Table 2

Statistics source data for Figure 1–7 and Supplementary Figures 1–7

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Fang, F., Angulo, B., Xia, N. et al. A PAX5–OCT4–PRDM1 developmental switch specifies human primordial germ cells. Nat Cell Biol 20, 655–665 (2018). https://doi.org/10.1038/s41556-018-0094-3

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