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ZFP462 safeguards neural lineage specification by targeting G9A/GLP-mediated heterochromatin to silence enhancers

Nature Cell Biology volume 25, pages 42–55 (2023)Cite this article

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Abstract

ZNF462 haploinsufficiency is linked to Weiss–Kruszka syndrome, a genetic disorder characterized by neurodevelopmental defects, including autism. Though conserved in vertebrates and essential for embryonic development, the molecular functions of ZNF462 remain unclear. We identified its murine homologue ZFP462 in a screen for mediators of epigenetic gene silencing. Here we show that ZFP462 safeguards neural lineage specification of mouse embryonic stem cells (ESCs) by targeting the H3K9-specific histone methyltransferase complex G9A/GLP to silence meso-endodermal genes. ZFP462 binds to transposable elements that are potential enhancers harbouring pluripotency and meso-endoderm transcription factor binding sites. Recruiting G9A/GLP, ZFP462 seeds heterochromatin, restricting transcription factor binding. Loss of ZFP462 in ESCs results in increased chromatin accessibility at target sites and ectopic expression of meso-endodermal genes. Taken together, ZFP462 confers lineage and locus specificity to the broadly expressed epigenetic regulator G9A/GLP. Our results suggest that aberrant activation of lineage non-specific genes in the neuronal lineage underlies ZNF462-associated neurodevelopmental pathology.

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Fig. 1: CRISPR screen identifies heterochromatin regulators required for heritable Oct3/4 gene silencing.
Fig. 2: ZFP462 elicits silencing function through interaction with G9A/GLP and HP1γ.
Fig. 3: Depletion of Zfp462 leads to aberrant expression of lineage-specifying genes.
Fig. 4: Zfp462 mutant cells show abnormal cell fate specification during neuronal differentiation.
Fig. 5: ZFP462 establishes H3K9me2 containing heterochromatin by recruiting GLP and WIZ proteins.
Fig. 6: ZFP462-targeted heterochromatin restricts DNA accessibility and TF binding.
Fig. 7: ZFP462 represses meso-endodermal enhancers in ESCs.

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Data Availability

High-throughput sequencing data produced in this study are deposited at the Gene Expression Omnibus (GEO) database under super series accession number GSE175369 (RNA-seq: GSE176321, QuantSeq: GSE176319, ATAC-seq: GSE176322 and ChIP–seq: GSE177058). G9a/GLP DKO ESC ATAC-seq and RNA-seq data are obtained from GSE138102 (ref. 52). REST and ADNP ChIP–seq data are obtained from GSE27148 (ref. 95) and GSE97945 (ref. 51), respectively. Meso-endoderm cell ATAC-seq data are obtained from GSE116262 (ref. 66). Processed single-cell RNA-seq data of mouse embryo stage E4.5 to E7.0 are obtained from ftp://ftp.ebi.ac.uk/pub/databases/scnmt_gastrulation43. Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD037238. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We are grateful to all members of the Bell and Brennecke laboratories for support, feedback and discussions. HP1γ and GLP Avi-tagged cell lines are kind gifts from M. Bühler and J. Betschinger labs at FMI, Switzerland. We thank N. Urban for providing mouse NSCs. We thank C. Buecker, F. Mohn and K. Jensen for sharing unpublished date, experimental advice and helpful discussions. We thank F. Berger, L. Cochella and J. Schnabl for feedback on the manuscript. We thank the Vienna Biocenter Core Facility Next Generation Sequencing. The GMI/IMBA/IMP Scientific Service units, especially the BioOptics facility and the Mass Spectrometry unit, provided outstanding support. DNA methylation analysis by LC–MS/MS was performed by the Metabolomics Facility at Vienna BioCenter Core Facilities (VBCF), funded by the City of Vienna through the Vienna Business Agency. We thank Life Science Editors for editorial assistance. We apologize to colleagues whose work could not be cited due to space limitations. Funding: O.B., J.B., U.E. and S.M. were supported by the Austrian Academy of Sciences. O.B. was supported by the New Frontiers Group of the Austrian Academy of Sciences (NFG-05), the Human Frontiers Science Programme Career Development Award (CDA00036/2014-C), the National Institute of Mental Health (R01MH122565) and the Norris Comprehensive Cancer Center of USC and its NCI Award (P30CA014089). R.Y. was supported by EMBO Long-Term Fellowship (ALTF 256-2015). D.S. acknowledges support from the Novartis Research Foundation and the European Research Council under the European Union’s (EU) Horizon 2020 research and innovation program grant agreement (ReadMe-667951). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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  1. Luca Michetti

    Present address: Università Vita-Salute San Raffaele, Milan, Italy

  2. These authors contributed equally: Ramesh Yelagandula, Karin Stecher.

Authors and Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria

    Ramesh Yelagandula, Karin Stecher, Luca Michetti, Georg Michlits, Pablo Hofbauer, Gintautas Vainorius, Sasha Mendjan, Ulrich Elling, Julius Brennecke & Oliver Bell

  2. Department of Biochemistry and Molecular Medicine and Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA

    Ramesh Yelagandula & Oliver Bell

  3. Vienna BioCenter PhD Program, Vienna, Austria

    Karin Stecher

  4. Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria

    Maria Novatchkova, Jingkui Wang & Carina Pribitzer

  5. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    Luke Isbel & Dirk Schübeler

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Contributions

R.Y., K.S. and O.B. initiated and designed the study. R.Y and K.S. generated cell lines, performed CRISPR–Cas9 genetic screen and differentiation assays. R.Y. performed all molecular biology experiments and revisions. S.M. and P.H. provided experimental advice and P.H. carried out immunohistochemistry. R.Y. and M.N analysed transcriptome and epigenome data. J.W. and G.M. analysed CRISPR–Cas9 genetic screen data. U.E., G.M and G.V. provided the CRISPR sgRNA library and helped with the screen design. L.M. and C.P. participated in experiments. L.I. and D.S. shared reagents and experimental expertise. J.B. co-supervised part of the project in his laboratory. O.B. supervised all aspects of the project. The manuscript was prepared by R.Y. and O.B. D.S. and J.B. edited the manuscript. All authors discussed results and commented on the manuscript.

Corresponding authors

Correspondence to Ramesh Yelagandula or Oliver Bell.

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Extended data

Extended Data Fig. 1 Characterization of WT and mutant CiA Oct4 dual reporter cells.

a) Western blot confirms expression of TetR-FLAG and TetR-FLAG-HP1 proteins in WT and Dnmt1 knockout (KO) CiA Oct4 dual reporter ESCs. LMNB1 is used as protein loading control. b) FACS gating strategy used to analyse the GFP fluorescence in cells. c) Flow cytometry histograms show GFP expression in CiA Oct4 dual reporter cells after TetR-FLAG recruitment and after TetR-FLAG release following Dox addition for four days. d) ChIP-qPCR shows relative enrichment of HP1γ upstream of the TetO site before tethering, in the presence of TetR-FLAG-HP1 and after Dox-dependent release of TetR-FLAG-HP1 for four days. n = 2 independent biological replicates. e) Bar plot shows fraction of cytosine methylation (5mC) in WT and Dnmt1 KO CiA Oct4 dual reporter ESCs measured by LC-MS. n = 3 independent biological replicates. Data are presented as mean values + /− SD. f) Western blot shows expression of TetR-FLAG-HP1 and ZFP462 in WT and two independent Zfp462 KO CiA Oct4 dual reporter ESC lines. LMNB1 is used as loading control.

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Extended Data Fig. 2 ZFP462 is conserved across vertebrates and acts as transcriptional repressor.

a) Phylogenetic tree of ZFP462 protein orthologues in vertebrate species. Bootstrap values are shown on branches. b) Western blot shows expression ZFP462 fusions with TetR-FLAG in CiA Oct4 dual reporter ESCs. Bottom image shows long exposure. Hashtag indicates cleaved TetR-FLAG protein from TetR-FLAG-ZFP462-FL (full-length). TetR-FLAG fusions initially express mCherry for selection which is cleaved via P2A signal. Asterisk marks P2A uncleaved protein product. c) Representative flow cytometry histograms show GFP expression in CiA Oct4 dual reporter ESCs expressing TetR-FLAG fusions with full-length or truncated ZFP462 proteins. Each histogram is average profile of 100,000 analysed cells.

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Extended Data Fig. 3 Zfp462 expression analysis and impact of Zfp462 deletion on ESC morphology and gene expression.

a) UMAP plots visualize lineage assignments of cells in mouse embryonic developmental stages (embryonic day (E) E4.5 cells, E5.5 cells and E6.5-7 cells) as previously described (Wolf Reik et.al) (top). UMAP plots of Zfp462 RNA expression at corresponding developmental stages (bottom). b) Sanger sequence chromatograms of heterozygous Zfp462 mutants. Heterozygous non-sense mutations are highlighted in grey. c) Alkaline phosphatase staining of Zfp462 +/Y1195* and Zfp462 −/− cl.2 ESCs (scale bar = 100 μm). d) Correlation plot shows principal component analysis (PCA) of replicate RNA-seq experiments from WT, two Zfp462 +/− and two Zfp462 −/− ESC lines. e) Volcano plot shows differential gene expression of Zfp462 −/− cl.2 ESCs compared to WT ESCs. (n = three replicates). f) Bar plots show gene ontology (GO) terms enriched in the four clusters of heatmap in Fig. 3e.

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Extended Data Fig. 4 Lineage-specifying genes are deregulated during neuronal differentiation.

a) Alkaline phosphatase staining of WT and Zfp462 mutant ESCs cultured in 2i/LIF/Serum medium (scale bar = 180 μm). b) Representative bright field images show Zfp462 +/Y1195* and Zfp462 −/− cl.2 at corresponding stages of neural differentiation. Scale bar = 100μm. c) Western blot analysis shows levels of ZFP462, G9A and H3K9me2 in WT and Zfp462 −/− ESCs during neural differentiation. LMNB1 is used as loading control. d) Line plot shows RT-qPCR analysis of Zfp462 RNA expression during neural differentiation (n = two replicates). Expression level is shown relative to ESCs (2i/S/L). e) Heatmaps show differential expression of selected marker genes specific for endodermal (EN), mesodermal (ME) and neural lineages (EC) in heterozygous and homozygous Zfp462 mutant cells during neural differentiation.

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Extended Data Fig. 5 ChIP-seq profiles of ZFP462, REST, ADNP, HP1γ and H3K9me2 in WT and Zfp462 KO ESCs.

a) Heatmap shows Spearman correlation between input controls and two independent ZFP462 ChIP-seq experiments. b) Heatmap shows Spearman correlation between input controls and two independent ChIP-seq experiments of GLP and HP1γ in WT and Zfp462 KO ESCs. c) Western blot shows ZFP462 expression in WT and Zfp462 KO Avi-FLAG-tagged GLP and HP1γ ESCs. d) Heatmap shows Spearman correlation between input controls and two independent ChIP-seq experiments of WIZ and H3K9me2 in WT and Zfp462 KO ESCs. e) Heatmaps of ZFP462, REST, ADNP, HP1γ and H3K9me2 ChIP-seq signals at ZFP462, REST and ADNP peaks in WT and Zfp462 KO ESCs. Blue-to-red scaled heatmap represents ChIP-seq enrichment ratios between Zfp462 KO versus WT (KO/WT). Each heatmap represents a 10 kb window centred on peak midpoints, sorted by ZFP462, REST and ADNP signal in their respective clusters. Below are scale bars (n = average distribution of two replicates).

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Extended Data Fig. 6 Rescue of Zfp462 KO ESCs with ZFP462 full-length and ZFP462 C-terminal truncation.

a) Heatmap shows Spearman correlation between three independent ATAC-seq experiments in WT and Zfp462 KO ESCs. b) Scatter plot shows pairwise correlation of gene expression changes between Zfp462 KO vs WT and G9a/Glp dKO vs WT. coefficient of determination (R²): black - all genes, red – differentially regulated genes in Zfp462 KO vs WT (padj. ≤ 0.05, LFC ≥ 1). c) Cartoon depicts CRISPR strategy to knock-in coding sequences for ZFP462 full-length and ZFP462 NT+Mid proteins at the Zfp462 gene locus. Insertions were targeted in-frame with exon 3. d) Bar plot shows RT-qPCR analysis of Zfp462 RNA transcript levels in WT, KO and rescue ESCs. n = 2 independent biological replicates. e) Western blot analysis shows ZFP462 protein levels in WT, Zfp462 KO and rescue ESCs. LMNB1 is used as loading control. f) Alkaline phosphatase staining of WT and Zfp462 KO and rescue ESCs (scale bar = 100μm). g) Heatmaps of ATAC-seq signal ratios at ZFP462 and REST peaks of Zfp462 KO vs WT (KO/WT) and Zfp462 rescue ESCs (ZFP462 FL/WT) or (ZFP462 NT+Mid/WT). Each row represents a 10 kb window centred on peak midpoints, sorted by KO/WT enrichment ratio (n = average distribution of two ATAC-seq replicates). h) Line plots show RT-qPCR analysis of lineage marker expression during neural differentiation (n = two replicates) in WT, Zfp462 KO and rescue ESCs. Expression levels are shown relative to ESCs (2i/S/L).

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Extended Data Fig. 7 Correlation between genomic distribution of ZFP462 and pluripotency transcription factors.

a) Heatmaps show ATAC-seq and ChIP-seq signal of ZFP462, H3K9me2, OCT4, SOX2, NANOG, ESRRB and NR5A2 at peaks of ZFP462, OSN (shared OCT4-SOX2-NANOG peaks not overlapping with ZFP462 peaks) and REST. Red-to-blue scaled heatmaps represent signal ratios between Zfp462 KO versus WT (KO/WT). Each row represents a 10 kb window centred on peak midpoints, sorted by H3K9me2 KO/WT ChIP signal loss. (n = average distribution of two ChIP-seq replicates / ATAC-seq three replicates). b) Genomic screenshot shows DNA accessibility (ATAC-seq) and ChIP-seq signals of H3K27ac and ZFP462 at the Oct3/4 locus in WT ESCs. ChIP-seq signals of OCT4, SOX2 and NANOG in WT (black line) and Zfp462 KO ESCs (green fill) are superimposed. ATAC-seq and ChIP-seq profiles are normalized to library size. c) Bar plot shows percentage of ZFP462-bound TE families overlapping with ChromHMM-annotated enhancers in ESCs. ZFP462-bound TEs contribute a total of 35.37% of ChromHMM-annotated enhancers in ESCs. d) HOMER analysis of known DNA sequence motifs enriched at ZFP462 peaks overlapping ChromHMM-annotated ME/EN-specific enhancers. Top ranked DNA sequence motifs and respective significance values are shown in the table. e) Box plots shows enrichment of NR5A2 and ESRRB ChIP signal in WT and Zfp462 KO ESCs at ZFP462 peaks overlapping with Mesoderm (ME)/Endoderm (EN)- and Ectoderm (EC)-specific enhancers. n = 553 (EN/ME), n = 314 (EC). Shown are median (horizontal line), 25th to 75th percentiles (boxes), and 90% (whiskers). Outliers are not shown. f) Bar plot shows frequency distribution of significantly up- and down-regulated genes located proximal to ZFP462 peaks annotated as ME/EN-specific enhancers (KO/WT, LFC ≥ 1 and padj. ≤ 0.05). g) Western blot shows ZFP462 protein expression in NSCs isolated from mouse brain and NPCs differentiated from WT ESCs. LMNB1 is used as loading control.

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Supplementary information

Reporting Summary

Peer Review File

Supplementary Table

List of genes enriched in genetic screen. Proteins enriched in IP–MS experiments. Information about oligos used in this study.

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Yelagandula, R., Stecher, K., Novatchkova, M. et al. ZFP462 safeguards neural lineage specification by targeting G9A/GLP-mediated heterochromatin to silence enhancers. Nat Cell Biol 25, 42–55 (2023). https://doi.org/10.1038/s41556-022-01051-2

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