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. 2023 Oct 27;51(19):10309-10325.
doi: 10.1093/nar/gkad731.

Unraveling the role of ZNF506 as a human PBS-pro-targeting protein for ERVP repression

Qian Wu  1 , Lu Fang  1 , Yixuan Wang  2 , Peng Yang  1
Affiliations

Unraveling the role of ZNF506 as a human PBS-pro-targeting protein for ERVP repression

Qian Wu et al. Nucleic Acids Res. .

Abstract

Krüppel-associated box zinc finger proteins (KZFPs) function as a defense mechanism to maintain the genome stability of higher vertebrates by regulating the transcriptional activities of transposable elements (TEs). While previous studies have characterized ZFP809 as responsible for binding and repressing ERVs containing a proline tRNA primer-binding site (PBS-Pro) in mice, comparable KZFPs have not been identified in humans yet. Here, we identified ZNF506 as a PBS-Pro-binding protein in humans, which functions as a transcriptional repressor of PBS-Pro-utilizing retroviruses by recruiting heterochromatic modifications. Although they have similar functions, the low protein similarities between ZNF506, ZFP809 and KZFPs of other species suggest their independent evolution against the invasion of PBS-Pro-utilizing retroviruses into their respective ancestor genomes after species divergence. We also explored the link between ZNF506 and leukemia. Our findings suggest that ZNF506 is a unique human KZFP that can bind to PBS-Pro, highlighting the diverse evolution of KZFPs in defending against retroviral invasions. Additionally, our study provides insights into the potential role of ZNF506 in leukemia, contributing to the expanding knowledge of KZFPs' crucial function in disease and genome stability.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Screening KZFPs targeting PBS-Pro in humans. (A) Pipeline for screening KZFPs targeting PBS-Pro in humans. The ChIP-seq data of KZFPs mainly come from the Cistrome Data Browser, with most of the raw data derived from the study conducted by Imbeault et al. (16) (GEO: GSE78099). (B) The top five KZFPs with overlapping ChIP-seq peaks in PBS-Pro sites, along with their corresponding counts. PBS-Pro sequences were chosen based on RetroTector scores, with records scoring <100 excluded. (C) DNA-binding motifs of the other four KZFPs as presented in (B).
Figure 2.
Figure 2.
Genome-wide mapping of ZNF506-binding sites shows enrichment at ERVP subfamilies. (A) Strong ZNF506–GFP ChIP-seq peaks overlapped with repeat regions and the human repeat genome as determined by genomic overlap with RepeatMasker annotations (top). Plot showing ZNF506 strong peak numbers and scores at ERV regions (bottom). ERVP subfamilies are marked in purple. Bubbles are sized according to average scores of ChIP-seq peaks. The cell line used is 293T. (B) ChIP-seq motifs of ZNF506 compared with the PBS-Pro consensus sequences. Consensus ZNF506 target motifs were analyzed from the high confidence peaks (peak score > 500). HA-tagged ZNF506 ChIP-seq data were from Imbeault et al. (16) (SRR accession: SRR5197160). ZFP809 ChIP-seq data were from Wolf et al. (28) (SRA accession: SRR1188176). (C) Relative luciferase activity of 293T cells overexpressing ZNF506 or an empty vector (EV) and SV40 promoter-driven reporter containing the PBS-Pro sequence with the indicated mutations highlighted in red (left). t-test: error bars indicate the standard deviation (SD); *P < 0.05, **P < 0.01, ns P > 0.05; n = 3. (D) ITC binding assay of PBS-Pro double-stranded oligonucleotides titrated with ZNF506-ZF1–8 protein. (E) Violin plot showing ZNF506–GFP signals at ERVP subfamilies with or without PBS-Pro sites. Means are marked in red, and quartiles are represented by dashed lines. (F) Venn diagram showing the overlap among strong ZNF506, ZNF253, ZNF682, ZNF93, ZNF90 and ZNF486 ChIP-seq peaks at repeat regions, with the ChIP-seq motif indicated.
Figure 3.
Figure 3.
ZNF506 recruits KAP1 and SETDB1 to promote the formation of H3K9me3 modifications. (A) Heatmap showing ZNF506, KAP1 and H3K9me3 signals at ERV-associated PBS-Pro sites and at strong non-repetitive ZNF506 ChIP-seq peak regions. The 20 kb regions are displayed with the PBS-Pro sequences or with non-repetitive peaks positioned at the center of the peak regions. OE, overexpression. (B) ChIP-seq signals of ZNF506, ZNF765, KAP1, H3K9me3, H3K27ac, ATAC-seq and RNA-seq at ERVP subfamilies containing PBS-Pro sequences. (C) Co-immunoprecipitation and western blotting for the FLAG-ZNF506 KRAB domain with Myc-KAP1 and HA-SETDB1. (D) Heatmap showing H3K9me3 signals at ERVP subfamilies with PBS-Pro sites before and after SETDB1 knockdown (KD) in a melanoma cell line. (E) Profiles showing H3K9me3 signals at different distances from ZNF506 strong peaks in ZNF506 OE, ZNF506 WT and ZNF506 KO 293T cells.
Figure 4.
Figure 4.
ZNF506 restricts PBS-Pro-utilizing pseudoviral infectivity. (A and B) FACS analysis (A) and statistical plot (B) showing the pMX-RFP viral infection rate in 293T cells stably overexpressing GFP-tagged ZNF506 or ZNF417, or WT cells. t-test: error bars indicate the SD; ****P < 0.0001, n = 6. (C and D) FACS analysis (C) and statistical plot (D) showing the pBABE-CMV-RFP viral infection rate in 293T cells stably overexpressing GFP-tagged ZNF506 or ZNF417, or WT cells. t-test: error bars indicate the SD; ***P < 0.001, n = 6. (E) Western blot showing expression of RFP in 293T cells co-transfected with the pMX-RFP vector and ZFP809, ZNF506 vectors, or ZFP961 as a control. (F) Western blot showing expression of RFP in 293T cells co-transfected with the pMX-RFP vector and different amounts of ZNF506 plasmids. (G) ChIP assays with antibodies against Pol II, H3K9me3 and H3K27ac and an immunoglobulin G (IgG) control in 293T cells overexpressing GFP-tagged ZNF506, or ZNF417 as a control, followed by qPCR using primers specific for the pMX LTR. t-test: error bars indicate the SD; ***P < 0.001, ****P < 0.0001, ns P > 0.05; n = 3 or 6. OE, overexpression. (H) Histogram showing viral DNA copy numbers in 293T cells overexpressing GFP-tagged ZNF506, or ZNF417 as a control. t-test: error bars indicate the SD; **P < 0.01, ns P > 0.05; n = 3. RT, reverse transcription.
Figure 5.
Figure 5.
ZNF506 evolves independently in humans against ERVP. (A) Histogram showing the timing of genome insertion of PBS-Pro-containing ERV subfamilies during primate evolution. Myr, million years. (B) Violin plot showing dN/dS ratios of ZNF506 orthologs in primates. (C) Phylogenetic tree showing the evolutionary relationship of KZFPs in humans and mice according to similarities of the KRAB domains. Larger images show details on ancient and homologous KZFPs between mice and humans (green block), KZFPs with high similarities to ZNF506 (yellow block) and KZFPs with high similarities to ZFP809 (blue block). Mouse KZFPs are in blue, and human KZFPs are in red. (D) Alignment of DNA-binding motifs of ZNF506 and ZFP809 between the predicted motif and ChIP motif. The correspondence between ZFs and bases is represented by blue trapezoids. The overlap between the predicted motif and the ChIP motif is indicated by a black rectangle. The fingerprint of each zinc finger is shown below. The ChIP-seq motif of ZFP809 matched the positive strand of the predicted result, while ZNF506 matched the reverse complementary strand of the predicted result. Their ZFs that are critical for binding are also different. High confidence peaks (peak score > 500 or fold change > 50) are used to extract the ChIP-seq motif for either ZNF506 or ZFP809. (E) Domain structure of ZNF506 (top). Multi-sequence alignments of DNA-contacting residues in the ZF array of selected ZNF506 orthologs (bottom). The four key amino acids shown per ZF are defined at positions −1, 2, 3 and 6 relative to the first histidine of each C2H2 ZF.
Figure 6.
Figure 6.
ZNF506 and other KZFPs may play a role in leukemia. (A) Top 10 KZFPs significantly up-regulated in AML compared with normal tissue. The KZFPs are ranked by the significance of up-regulation (*P < 0.05, 173 samples from AML patients and 70 samples from healthy individuals), and ZNF506 ranks second among them. (B) (a) Changes in various subfamilies of repeats in AML, with ZNF506-bound ERVs marked as red dots (GEO database accession: GSE175701). (b) Changes in the expression of each ERV in AML cells after SETDB1 KO (GEO database accession: GSE103409). (C) Expression levels of ZNF506-bound ERVs before and after ZNF506 KO in NB4 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3. (D) FACS analysis showing the viral infection rate of pMX-IRES-GFP in WT and ZNF506 KO NB4 cells. (E) Disease-free survival of high and low KZFP expression groups. Compared with the high expression group, patients in the low expression group of KZFPs had better disease-free survival. (F) Schematic diagram illustrating that in AML patients, abnormal KZFP expression disrupts the balance of repeat element expression. As an illustration, the inhibition of ERVP by ZNF506 is utilized as a paradigmatic case.
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