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

a, Immunoblot incubated with anti-KAP1 antibody loaded with 1% input and eluates of KAP1-ChIP or IgG-ChIP derived from hESC lysates. b, Diagram showing numbers of KAP1 peaks identified in two independent biological replicates and common peaks. c, Distribution of 9,174 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- or grey-shaded) and TC11-mESCs (yellow-shaded). Blue arrows, derepressed 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. Rep, biological replicate; sup, supernatant; TSS, transcription start site.

a, Distribution of KAP1-ChIP-Seq reads from mESCs (left) and the mouse genome (right) for retrotransposon families as defined by RepeatMasker (http://www.repeatmasker.org/). 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-subtypes⁴⁵; purple shading, KAP1-positive active intracisternal A-particle (IAP) retrotransposons. LINES, long interspersed nuclear elements; LTR, long terminal repeat; MMERVK10C, mouse endogenous retrovirus subtype K10C; RMER, medium reiteration frequency repetitive sequence; SINES, short interspersed nuclear elements; TEs, transposable elements.

a, Schematic of primate-specific KRAB zinc-finger genes subdivided in different clades based on previous analysis⁷. KZNFs shown in b are highlighted in red. b, DESeq-calculated gene expression levels for the 17 highest expressed KRAB zinc-finger genes in hESCs (dark blue) and macaque ESCs (light blue), subdivided by clades.

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 empty vector 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. Empty vector is set to 100% for comparison. c, 1.5 VNTR repeats are sufficient to confer ZNF91-mediated regulation on an OCT4Enh–SV40–luciferase-reporter. n = 3 biological replicates. **P < 0.01; error bars are s.e.m.

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 empty vector. SVAs were the only transposable elements that showed a significant decrease in H3K4me3 and RNA-seq values. **Benjamini–Hochberg adjusted-P < 0.01. c, UCSC browser images for a representative SVA element, promoter and L1PA4 element, showing H3K4me3 ChIP-seq signal for hESCs (grey), TC11-mESCs transfected with empty vector (yellow), pools of primate-specific KRAB zinc-fingers (green) and ZNF91 (red). TSSC4: tumor-suppressing subtransferable candidate 4.

a, The phylogenetic tree used in multiple sequence alignment and ancestral reconstruction of ZNF91 (Supplementary Information File 3). ‘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 the ZNF91 sequence from start to domain 5, a breakpoint, and from domain 13 to the end (see Methods). Ancestors are labelled with first letters of leaf species below them, for example, HCG is a 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 an empty vector and pCAG–GFP. Asterisks denote reconstructed ancestral proteins. c, ZNF91 domain deletion analysis showing relative luciferase activities on the SVA-D–SV40 luciferase reporter after transfection of empty vector or ZNF91 deletion constructs in mESCs. Error bars are standard deviation. Numbers in parenthesis indicate zinc-fingers present in the ZNF91 deletion construct. *P < 0.05; **P < 0.01. Biological replicates: empty vector, n = 42; ZNF91 (1–11), n = 4; ZNF91 (1–24), n = 7; ZNF91 (1–30), n = 4; ZNF91 (1, 2, 23–36), n = 3.

a, Relative luciferase activity on a L1PA4– and a OCT4-enhancer–SV40–luciferase-reporter after transfection of 14 KZNFs in mESCs. Significance measured relative to empty vector. n = 3 biological replicates; *P < 0.05; **P < 0.01; error bars are s.e.m. b, Immunoblot showing that 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 46c mESCs 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 TC11-mESCs after transfection with an empty vector or ZNF93 and ChIP with ab104878. ChIP enrichment is plotted as percentage of input. n = 3 biological replicates; *P < 0.05; error bars are s.e.m.

a, Schematic based on the multiple sequence alignment of ZNF93 orthologues (Supplementary Information File 4). 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 macaques and gibbons, with gibbons sharing the great ape conformation. For this last group of zinc-fingers, it is unknown (represented with a ? symbol) whether the change happened in monkeys or in the LCA of gibbons and great apes after the divergence of Old-World monkeys (see Methods). Asterisks denote reconstructed ancestral proteins. b, Relative OCT4-enhancer–SV40p–luciferase activity for reporters with the indicated L1PA4-derived sequences after co-transfection of an empty vector or various ZNF93 constructs. **P < 0.01; error bars are s.e.m.

a, Schematic of constructs tested indicating the site of 129^L1PA4 transplant into L1Hs and concept of L1–GFP assay²⁴ in which GFP expression marks cells where a transfected L1 episome has retrotransposed into a HEK293 cell’s chromosomes. ORF, open reading frame; CMV, cytomegalovirus promoter; SD, splice donor; SA, splice acceptor; PvuII, restriction enzyme site.

a, Phylogenetic tree, rooted on L1PA4, generated using the Minimum Evolution method⁴² for fifty 3′-end sequences of L1PA3-6030 and L1PA3-6160, and three 3′-end sequences for L1PA2 and L1PA4. b, Bar graphs 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. The number of untruncated elements identified for each subtype is indicated.