Abstract
Pericentric heterochromatin is a critical component of chromosomes marked by histone H3 K9 (H3K9) methylation1,2,3. However, what recruits H3K9-specific histone methyltransferases to pericentric regions in vertebrates remains unclear4, as does why pericentric regions in different species share the same H3K9 methylation mark despite lacking highly conserved DNA sequences2,5. Here we show that zinc-finger proteins ZNF512 and ZNF512B specifically localize at pericentric regions through direct DNA binding. Notably, both ZNF512 and ZNF512B are sufficient to initiate de novo heterochromatin formation at ectopically targeted repetitive regions and pericentric regions, as they directly recruit SUV39H1 and SUV39H2 (SUV39H) to catalyse H3K9 methylation. SUV39H2 makes a greater contribution to H3K9 trimethylation, whereas SUV39H1 seems to contribute more to silencing, probably owing to its preferential association with HP1 proteins. ZNF512 and ZNF512B from different species can specifically target pericentric regions of other vertebrates, because the atypical long linker residues between the zinc-fingers of ZNF512 and ZNF512B offer flexibility in recognition of non-consecutively organized three-nucleotide triplets targeted by each zinc-finger. This study addresses two long-standing questions: how constitutive heterochromatin is initiated and how seemingly variable pericentric sequences are targeted by the same set of conserved machinery in vertebrates.
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Data availability
The raw sequencing data reported in this paper have been deposited to the Genome Sequence Archive of the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA011890) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa. The processed data have been deposited in BIG OMIX under accession number OMIX004553 and are publicly accessible at https://ngdc.cncb.ac.cn/omix. Accession codes of the public data in ENCODE used in this study are as follows: ZNF512 ChIP–seq in human K562 cells, ENCSR696URR; ZNF512B ChIP–seq in human MCF-7 cells, ENCSR761LRR. The expression atlas data for Pax3, Pax9, Foxd3, Zfp512 and Zfp512b used in Extended Data Fig. 1 were downloaded from https://www.ebi.ac.uk/gxa/home. We used mouse genome version mm10 and human genome versions hg38 and CHM13 in this study. Source data for Figs. 1b, 2d, 3b,c,e,f and Extended Data Figs. 2b, 4b and 8d are provided with this paper. The gel for Extended Data Fig. 3d and uncropped film scans for Fig. 4e,f and Extended Data Figs. 3c and 8j are presented in Supplementary Fig. 1.
Code availability
The software used to analyse these data is listed in the Methods and is all publicly available.
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Acknowledgements
We thank G. Li and L.-L. Du for helpful discussion; F. Suo, Q. Zhang and H. Zhang for assistance with protein structure prediction using AlphaFold; J. Jia and S. Meng for assistance with FACS experiments; and J. Wang and M. Zhang from the Laboratory of Proteomics of the Institute of Biophysics for assistance with mass spectrometry. This work was supported by grants from the China Natural Science Foundation (32288102, 32130021 and 92153302), the National Key R&D Programme of China (2018YFE0203302), the Chinese Academy of Sciences (XDB39010100 and JZHKYPT-2021-05), and the New Cornerstone Science Laboratory. Z.Z. is supported by the Youth Innovation Promotion Association (2017133) of the Chinese Academy of Sciences.
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B.Z. designed and supervised the project. R.M. designed and performed most of the experiments. Y.Z. performed most of the bioinformatics analysis. J.Z. participated in performing experiments during revision. Y.F. performed experiments for Extended Data Fig. 3c. H.-T.W. helped to prepare Extended Data Figs. 6b, 7 and 9b. P.Z., Z.L. and Z.Z. assisted with data analysis. R.M. and B.Z. wrote the manuscript, and Y.Z. participated in writing the methods and figure legends. All the authors read and commented on the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 The spatiotemporal expression pattern and binding sites of pericentric sequence-specific DNA binding proteins.
(a) The expression atlas of Pax3, Pax9, Foxd3, Znf512, and Znf512b in 51 experiments. TPM: transcript per million. (b) upper: major satellite consensus sequence in mouse pericentric regions and three types of satellite repeats in human pericentric regions. The binding sites of PAX3 and PAX9 in major satellite consensus sequence were highlighted in orange, and the binding site of FOXD3 in major satellite consensus sequence was highlighted in blue. TTC triples in mouse major satellite consensus sequence and human satellite repeats were highlighted in bold. lower: binding motifs of PAX3, PAX9 and FOXD3.
Extended Data Fig. 2 Workflow and the overlap analysis to characterize the protein composition of pericentric heterochromatin.
(a) Schematic diagram of the approach to map specific genomic site-associated proteome based on proximity labeling. (b) Overlap between proteins identified at pericentric heterochromatin by dCas9-APEX2 and candidates identified by PICh. A full list of pericentric heterochromatin associated proteins was included in Source Data for Extended Data Fig. 2.
Extended Data Fig. 3 ZNF512 and ZNF512B can interact with SUV39H1 and SUV39H2 in vivo and in vitro.
(a) upper: A schematic diagram showing the LacO-LacI targeting system for testing whether a factor can establish H3K9me3 modified heterochromatin de novo. lower: A schematic diagram showing the LacO-LacI targeting system for detecting protein-protein interaction. (b) Validation of the LacO-LacI reporter system for testing whether a factor can establish H3K9me3 modified heterochromatin de novo. A representative cross-section immunofluorescence image showing the relative localization of LacI-mCherry fused SUV39H1 or SUV39H2, H3K9me3, and DAPI in CHO A03-1 cells. Condensed chromatin was stained by DAPI. Scale bar, 5 μm. (c) Co-IP and western blot analyzing the interaction between ZNF512 or ZNF512B and SUV39H1 or SUV39H2 in vivo. The mobilities of protein markers were indicated on the left of each panel. The asterisk refers to the degradation band of proteins. (d) MBP-Flag-ZNF512 or MBP-Flag-ZNF512B and MBP-HA-SUV39H1 or MBP-HA-SUV39H2 or MBP-HA-GFP-APEX2 were pulled down by HA beads. Bound proteins were eluted by HA peptide, separated on SDS-PAGE gels, and stained with Coomassie Brilliant Blue. The mobilities of protein markers were indicated on the right of each panel. For panels b, c, and d, representative data from two independent biological repeats are shown.
Extended Data Fig. 4 ZNF512 and ZNF512B do not appear to be essential for maintaining the molecular signature of pericentric heterochromatin.
(a) Transcriptome browser tracks representing the knockout of Znf512, Znf512b, Suv39h1 and Suv39h2 in wild type cell, Znf512 single KO cell, Znf512b single KO cell, Znf512 and Znf512b double knockout cell, and QKO cell. (b) RT-qPCR showing the relative expression level of major satellite RNA in wild-type and different KO cells. The expression level was normalized to Gapdh. Bars indicate the mean; dots indicate individual replicate. (c) A representative cross-section image showing the localization of H3K9me3 in wild type, Znf512 single KO, Znf512b single KO, Znf512 and Znf512b double KO cells. Scale bar, 2 μm. For panel b, data are from two independent biological replicates. For panel c, representative data from four independent biological repeats are shown.
Extended Data Fig. 5 The localization of H3K9me3 and mutated EGFP-SUV39H in QKO cells and the localization of endogenous SUV39H2 in H1 TKO cells.
(a) A representative cross-section image showing the localization of H3K9me3 in Znf512, Znf512b, Suv39h1 and Suv39h2 quadruple KO (QKO) cells. Scale bar, 5 μm. (b) The localization of EGFP-SUV39H1, EGFP-SUV39H2 wild type or mutated versions and H3K9me3 were analyzed in Znf512, Znf512b, Suv39h1, Suv39h2 quadruple KO (QKO) cell line. SUV39H1-W64A/Y67A, the aromatic cage mutant, was abbreviated as SUV39H1-2A, and SUV39H2-W139A/W142 A, was abbreviated as SUV39H2-2A. SUV39H1-H324K and SUV39H2-H398K were catalytically dead mutants. Scale bar, 5 μm. (c) A representative cross-section image showing the localization of endogenous SUV39H2 and DAPI in wild-type and different KO cells. Scale bar, 5 μm. For panel a, b, and c, representative data from two independent biological repeats are shown.
Extended Data Fig. 6 ZNF512 and ZNF512B utilize their zinc fingers to target the pericentric sequences.
(a) Schematic diagram of ZNF512 and ZNF512B protein sequences. Zinc finger domains and linkers’ lengths were labeled. (b) Multi-sequence alignments of zinc finger domains in mouse ZNF512 and ZNF512B. The zinc fingerprint residues at positions −1, 3, 6 of each C2H2 zinc finger were highlighted. (c) The binding motifs of mouse ZNF512 and ZNF512B were predicted by a polynomial SVM-based algorithm. (d) Representative cross-section fluorescence images showing the relative localization of EGFP-ZNF512 with one or two zinc fingerprints mutated and DAPI in Znf512, Znf512b, Suv39h1, Suv39h2 quadruple KO (QKO) cells. Zinc fingerprint residues mutated versions, in which one zinc finger of ZNF512 mutated to AAA, were abbreviated as ZF-3A. Zinc fingerprint residues mutated versions, which two zinc fingers of ZNF512 mutated to AAA, were abbreviated as ZF-6A. Scale bar, 5 μm. (e) Representative cross-section fluorescence images showing the relative localization of EGFP-ZNF512B with one or two zinc fingerprints mutated and DAPI in Znf512, Znf512b, Suv39h1, Suv39h2 quadruple KO (QKO) cells. Zinc fingerprint residues mutated versions, which one zinc finger of ZNF512B mutated to AAA, were abbreviated as ZF-3A. Zinc fingerprint residues mutated versions, which two zinc fingers of ZNF512B mutated to AAA, were abbreviated as ZF-6A. Scale bar, 5 μm. Data are representative of three (d) or one (e) independent biological experiments.
Extended Data Fig. 7 Evolution of the zinc finger domains and the length of linkers in ZNF512 and ZNF512B.
Phylogenetic tree showing the evolutionary relationship of zinc finger domains of ZNF512 and ZNF512B in humans, mice, chicken, mainland tiger snake, western clawed frog, zebrafish, sea lamprey, and Florida lancelet. Zinc fingerprints were highlighted in red, and predicted binding motifs were highlighted in blue. The length of linkers in ZNF512 and ZNF512B was also evolutionarily conserved among vertebrates.
Extended Data Fig. 8 The long linkers play an important role in ZNF512 and ZNF512B for targeting the pericentric sequences.
(a) Bar plots showing the ratio of the percentage of reads mapped on mouse major satellite repeats between biotin ChIPed samples and the corresponding input samples in indicated cell lines. (b) Bar plot showing the fold enrichment of TTC triplets in the consensus sequence of mouse major satellite repeats. (c) Box plots showing the fold enrichment of TTC triplets in human pericentric satellite III/II/I. Centre line, median; box, 25th and 75th percentiles; whiskers, 1.5 × the interquartile range (IQR). Numbers of satellites: n = 189 (satellite III), n = 61 (satellite II), n = 88 (satellite I). (d) Bar plots showing the ratio of the percentage of reads mapped on human pericentric satellite repeats between ZNF512 or ZNF512B ChIPed samples and the corresponding control samples in indicated cell lines. Bars indicate the mean; dots indicate individual replicate. (e, f) Predicted structures of wild-type or linkers-swapped ZNF512 or ZNF512B proteins using AlphaFold. Zinc finger domains were highlighted in red color. The fourth zinc finger domain of ZNF512B was colored green. (g) Representative cross-section fluorescence images showing the relative localization of EGFP-ZNF512 or EGFP-ZNF512B linker-swapped versions in QKO cells. Scale bar, 5 μm. (h) After overexpressing Flag-tagged APEX2, ZNF512 or ZNF512B wild type or linker-swapped version in Znf512,Znf512b,Suv39h2 triple KO (H2 TKO) cells, H3K9me3 modification at pericentric regions was visualized. Scale bar, 5 μm. (i) After overexpressing mCherry-tagged APEX2, ZNF512 or ZNF512B wild type or linker-swapped version in H2 TKO cells, endogenous SUV39H1 at pericentric regions was visualized. Scale bar, 5 μm. (j) DNA pull-down with biotin-major satellite probe or no probe followed by western blot analysis for MBP tag using purified recombinant MBP-Flag-tagged ZNF512 or ZNF512B. The mobilities of protein markers were indicated on the left of the panel. Data are representative of two (h, i and j) or one (g) independent biological experiments.
Extended Data Fig. 9 H3K9me3 ChIP-seq signals around LINEs.
(a) Profile (top) and heatmap (bottom) showing H3K9me3 signals in WT and Suv39h1 and Suv39h2 double knockout mESCs around LINEs (n = 6,996). The difference in H3K9me3 between Suv39h1 and Suv39h2 double knockout and WT mESCs was also shown. The x-axis represented the distance from the start or end of LINE in kilobases (Kb). Heatmaps were sorted by H3K9me3 signal. The average signals of two replicates were shown. (b) Percentage of ZNF512-bound full-length LINEs per subfamily in mouse ESCs arranged from the youngest to the oldest subfamily. LINE ages obtained from previously published studies. (Myr) Million years.
Extended Data Fig. 10 Reproducibility of replicates in this study.
(a) Overall similarity of ZNF512 signals within ZNF512 peaks in WT mouse ES cells (n = 3,218). Pearson correlation coefficients were indicated. (b) Overall similarity of H3K9me3 signals within H3K9me3 peaks in WT mouse ES cells (n = 29,982). Pearson correlation coefficients were indicated.
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Ma, R., Zhang, Y., Zhang, J. et al. Targeting pericentric non-consecutive motifs for heterochromatin initiation. Nature 631, 678–685 (2024). https://doi.org/10.1038/s41586-024-07640-5
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DOI: https://doi.org/10.1038/s41586-024-07640-5