An evolutionary arms race between KRAB zinc-finger genes ZNF91/93 and SVA/L1 retrotransposons

Abstract

Throughout evolution primate genomes have been modified by waves of retrotransposon insertions. For each wave, the host eventually finds a way to repress retrotransposon transcription and prevent further insertions. In mouse embryonic stem cells, transcriptional silencing of retrotransposons requires KAP1 (also known as TRIM28) and its repressive complex, which can be recruited to target sites by KRAB zinc-finger (KZNF) proteins such as murine-specific ZFP809 which binds to integrated murine leukaemia virus DNA elements and recruits KAP1 to repress them. KZNF genes are one of the fastest growing gene families in primates and this expansion is hypothesized to enable primates to respond to newly emerged retrotransposons. However, the identity of KZNF genes battling retrotransposons currently active in the human genome, such as SINE-VNTR-Alu (SVA) and long interspersed nuclear element 1 (L1), is unknown. Here we show that two primate-specific KZNF genes rapidly evolved to repress these two distinct retrotransposon families shortly after they began to spread in our ancestral genome. ZNF91 underwent a series of structural changes 8-12 million years ago that enabled it to repress SVA elements. ZNF93 evolved earlier to repress the primate L1 lineage until ∼12.5 million years ago when the L1PA3-subfamily of retrotransposons escaped ZNF93's restriction through the removal of the ZNF93-binding site. Our data support a model where KZNF gene expansion limits the activity of newly emerged retrotransposon classes, and this is followed by mutations in these retrotransposons to evade repression, a cycle of events that could explain the rapid expansion of lineage-specific KZNF genes.

Extended Data Figure S1. KAP1 associates with recently emerged transposable elements

a, Immunoblot incubated with anti-KAP1 antibody loaded with 1% input and eluates of KAP1-ChIP or IgG-ChIP derived from human ESC lysates. b, Diagram showing numbers of KAP1 peaks identified in two independent biological replicates and common peaks. c, Distribution of 9174 KAP1-ChIP-seq peaks over various DNA elements. d, Distribution of retrotransposon classes among KAP1-ChIP peaks from hESCs (left) or genome wide (right) e, KAP1 and H3K4me3 ChIP-seq and RNA-seq coverage tracks for a representative region on human chromosome 11 in hESCs (white-, grey-shaded) and TC11-mESCs (yellow-shaded). Blue arrows: de-repressed retrotransposons; black arrows: re-activated transcription; Red vertical shading: reactivated SVAs; orange shading: reactivated LTR12C. Blue and tan in RNA-seq tracks indicate positive and negative strand transcripts, respectively. Note that while the majority of SVAs display aberrant H3K4me3 signal, for unclear reasons not all SVAs display aberrant transcription in TC11-mESCs. sup: supernatant; Rep: biological replicate; TSS: transcription start site.

Extended Data Figure S2. Mouse KAP1 associates with mouse-specific retrotransposons in mouse ESCs

a, Distribution of KAP1-ChIP-Seq reads from mouse ESCs (left) and the mouse genome (right) for retrotransposon families as defined by repeatmasker (RepeatMasker Open-3.0. http://www.repeatmasker.org. 1996–2010; Smit AFA, Hubley R, Green P.). b, UCSC Browser image displaying ChIP-seq tracks for input (grey shading) and KAP1 (red shading) as well as gene annotation and repeat element tracks for a region on mouse chromosome 1. Blue shading: KAP1-positive active mouse L1-subtypes Purple shading: KAP1-positive active IAP retrotransposons. TEs: transposable elements; IAP: Intracisternal A-particle.

Extended Data Figure S3. Selection of primate-specific KRAB ZNFs genes with high expression in hESCs

a, Schematic of primate-specific KRAB zinc finger genes subdivided in different clades based on previous analysis. KZNFs in (b) are in red. b, DESeq-calculated basemean expression levels for the 17 highest expressed KRAB zinc finger genes in hESCs (dark blue) and macaque ESCs (light blue), subdivided by clades.

Extended Data Figure S4. The SVA VNTR domain is necessary and sufficient for ZNF91-mediated repression of luciferase activity

a–c, Schematic of SV40-Luciferase constructs used (left) and relative luciferase activity after transfection of the indicated constructs in mESCs (right). a, SVA and SINE-R are strong enhancers. (n = 6 biological replicates) b, Deletion analysis reveals the VNTR of SVA is required for ZNF91-mediated reporter regulation. Luciferase activity in the presence of ZNF91 expressed as a ratio of that observed for EV with the same reporter. Biological replicates: ‘no VNTR’ (n=9); ‘partial VNTR’ (n=3); ‘ no hex/Alu’ (n=2); ‘no hex’ (n=2); ‘full length SVA’ (n=15); ‘SINE-R’ (n=3). EV is set to 100% for comparison. c, 1.5 VNTR repeats is sufficient to confer ZNF91-mediated regulation on an OCT4Enh-SV40-luciferase reporter. Biological replicates: n=3. Student’s T-Test, two tailed; equal variance; Error bars: SEM. **p <0.01

Extended Data Figure S5. SVA is specifically repressed in vivo by ZNF91

a, b Normalized DESEQ basemean values for H3K4me3 ChIP-seq (a) and RNA-seq (b) for retrotransposon classes that showed a significant change in ZNF91-transfected TC11-mESCs relative to EV. SVAs were the only transposable elements that showed a significant decrease (**= p-adj<0.01) in H3K4me3 and RNA-seq values. c, UCSC browser images for a representative SVA element, promoter and L1PA4 element, showing H3K4me3 ChIP-seq signal for hESC (grey) TC11-mESCs transfected with EV (yellow), pools of primate specific KRAB zinc fingers (green), and ZNF91 (red).

Extended Data Figure S6. Evolutionary history of ZNF91

a, The phylogenetic tree used in multiple sequence alignment and ancestral reconstruction of ZNF91 (http://compbio.soe.ucsc.edu/arms-race-znfs-retrotransposons/znf91/znf91msa.html). “hu 1.1”, “ch 1.1” and “go 1.1” represent human, chimpanzee and gorilla domain 6, respectively, “hu 1.2”, “ch 1.2”, “go 1.2” represent human, chimpanzee and gorilla domains 7-12, respectively, and “hu 2”, “ch 2” and “go 2” represent ZNF91 sequence from start to domain 5, a breakpoint, and from domain 13 to the end (see Methods). Ancestors are labeled with first letters of leaf species below them, e.g. HCG is human-chimp-gorilla ancestor. b, Immunoblot incubated with anti-HA antibody on lysates of HEK293FT cells transfected with HA-tagged human, great ape, hominine and macaque ZNF91 proteins or lysates transfected with EV and pCAG-GFP. *= reconstructed ancestral protein. c, ZNF91 domain deletion analysis showing relative luciferase activities on the SVA_D-SV40 luciferase reporter after transfection of EV or ZNF91 deletion constructs in mESCs. Error bars: stdev. Numbers in parenthesis indicate zinc fingers present in the ZNF91 deletion construct. Student’s T-Test, two tailed; equal variance; *P< 0.05; **P<0.01. Biological replicates: EV (n = 42): ZNF91 (1-11) (n = 4); ZNF91 (1-24) (n = 7); ZNF91 (1-30) (n =4 ); ZNF91 (1,2; 23-36) (n = 3). EV= empy vector.

Extended Data Figure S7. L1PA4 elements are repressed by primate-specific ZNF93

a, Relative luciferase activity on a L1PA4- and a OCT4enhancer-SV40-luciferase reporter after transfection of 14 KZNFs in mESCs. Significance measured relative to EV. Student’s T-Test, two tailed; equal variance; Error bars: SEM; *=p<0.05; **=p<0.01; (biological replicates: n=3 ). b, Immunoblot showing ChIP with antibody ab104878 predominantly reacts with a protein of ~70 kDA (left panel) and co-immunoprecipitates KAP1 (right panel). HC: heavy chain of IgG. c, Immunoblot demonstrating that ChIP with ab104878 detects overexpressed ZNF93 in mouse 46c ESCs as a ~70 kDA protein. d, Repeat Browser (see Methods) displaying ChIP-seq coverage tracks for ab104878 (ZNF93) (yellow shading) and KAP1 (blue shading) for a selection of KAP1-bound retrotransposons. e, ChIP-qPCR for amplicons in L1PA4 and LTR12C elements on chromosome 11 in mouse TC11 mESCs after transfection with EV or ZNF93 and ChIP with ab104878. ChIP enrichment is plotted as percentage of input. n=3 biological replicates; error bars, SEM; Student’s T-Test, two tailed; equal variance, *= p<0.05

Extended Data Figure S8. Reconstruction of the evolutionary history of ZNF93

a, Schematic based on the multiple sequence alignment of ZNF93 orthologs (http://compbio.soe.ucsc.edu/arms-race-znfs-retrotransposons/znf93/znf93msa.html). Red shaded area: deletion of zinc fingers. Green shaded area: gain of zinc fingers. Green stripes: gained zinc fingers; Dark blue stripes: zinc fingers that changed contact residues in the lineage to humans; light blue stripes: changes in other lineages. Brown stripes: zinc fingers with different binding residues between macaque and gibbon, with gibbon sharing the great ape conformation: for these zinc fingers, it is unknown (‘?’) whether the change happened in monkey or in the LCA of gibbon and great apes after divergence of old world monkey (see methods). *= reconstructed ancestral protein. b, Relative OCT4enhancer-SV40p-luciferase activity for reporters with the indicated L1PA4 derived sequences after co-transfection of EV or various ZNF93 constructs. Error bars: SEM; **=p<0.01.

Extended Data Figure S9. Schematic of L1Hs retrotranspostion assay

a, Schematic of constructs tested indicating the site of 129^L1PA4 transplant into L1Hs and concept of L1-GFP assay in which GFP expression marks cells where a transfected L1-episome has retrotransposed into a HEK293 cell’s chromosomes.

Extended Data Figure S10. Evolutionary history of L1PA3-6030, L1PA3-6160 and the VNTR size in SVA

a, Phylogenetic tree, rooted on L1PA4, generated using the Minimum Evolution method for fifty 3′-end sequences of L1PA3-6030 and L1PA3-6160, and three 3′-end sequences for L1PA2 and L1PA4. b, Bargraphs showing the number of SVA-A through SVA-F insertions in each great ape genome. c, Distribution of VNTR size for untruncated SVA elements in the human genome plotted for each SVA-subfamily. Number of untruncated elements identified for each subtype is indicated.

Figure 1. SVAs and L1PAs are de-repressed in a non-primate cellular environment

a, KAP1, H3K4me3 ChIP-seq and RNA-seq coverage tracks for a selection of KAP1-bound primate-specific retrotransposons de-repressed in TC11-mESCs (yellow) relative to human ESCs (grey). H3K4me3 signal on promoters is similar in hESCs and TC11-mESCs. b, Percentages of SVA, L1Hs and L1PA elements on human chromosome 11 positive for KAP1, H3K4me3 (MACS ChIP-seq analysis) and arbitrary levels of transcription (see Methods) in hESC and TC11-mESCs. Total elements of each type on human chromosome 11 in parentheses.

Figure 2. SVA elements are repressed by primate-specific ZNF91

a, Relative luciferase activity of a SVA_D-SV40-luciferase reporter after co-transfection of KZNFs in mESCs. b, KAP1 and H3K4me3 ChIP-seq coverage tracks for a selection of loci in hESCs and TC11-mESCs transfected with EV or ZNF91. Pie charts: percentages of H3K4me3-positive SVAs on human chromosome 11. c, Median fold expression change (ZNF91/EV), for genes with (blue circles) or without (gray crosses) an SVA within the indicated genomic distance among the 994 expressed human chromosome 11 genes. d, ZNF91 structural evolution. Green stripes: duplicated zinc fingers; Blue stripes: zinc fingers that changed contact residues in the lineage to humans (dark blue) or in other lineages (light blue). Green arrows: segmental duplications. *= reconstructed ancestral protein. e, Relative SVA_D-SV40-luciferase activity in the presence of various ZNF91 proteins. Error bars: SEM; ** p<0.01

Figure 3. L1PA elements are repressed by primate-specific ZNF93

a, Green peaks represent genome-wide ab104878-ChIP-seq peak-summits mapped to L1PA consensus sequences. Black horizontal bars: alignment to L1PA4, red lines: divergent positions. b, The 129 bp deletion and predicted 51 bp ZNF93 binding motif (grey bar) relative to L1PA4. c, Relative activity of OCT4-enhancer-luciferase reporters after co-transfection of EV or ZNF93. d, Consensus central sequence of ab104878-ChIP-seq summits for L1PA4, aligned with the predicted recognition motif of ZNF93 zinc fingers 8-13. e, Relative activity for OCT4-enhancer-luciferase reporters after co-transfection of EV, ZNF93SerF or ZNF93. f, Number of GFP-positive cells derived from retrotransposition events of L1Hs, L1Hs+129 and L1Hs+129-scrambled constructs in HEK cells (n=7). g, Same as (f) but showing the ratio of retrotransposition events after co-transfection with ZNF93 compared to EV. Error bars: SEM. *= p<0.05; **= p<0.01

Figure 4. Dynamic patterns of co-evolution between ZNFs and target retrotransposons

Schematic showing the evolution of L1PA and SVA retrotransposons parallel to the structural evolution of ZNF93 and ZNF91 along an evolutionary timescale. Red zinc fingers: deletion; blue zinc fingers: change in contact residues; green zinc fingers: duplication. Coloring of ZNF91/ZNF93 horizontal bars: Zinc finger changes (changes in DNA-contacting residues, zinc finger deletions and duplications)/myr during the time interval indicated. Coloring of TE horizontal bars: basepair substitutions/deletions/insertions per site/myr (L1PA), or percentage increase in VNTR size/myr (SVA). myr = million years; owm = old world monkey