Genome surveillance by HUSH-mediated silencing of intronless mobile elements

All life forms defend their genome against DNA invasion. Eukaryotic cells recognize incoming DNA and limit its transcription through repressive chromatin modifications. The human silencing hub (HUSH) complex transcriptionally represses long interspersed element-1 retrotransposons (L1s) and retroviruses through histone H3 lysine 9 trimethylation (H3K9me3)1–3. How HUSH recognizes and initiates silencing of these invading genetic elements is unknown. Here we show that HUSH is able to recognize and transcriptionally repress a broad range of long, intronless transgenes. Intron insertion into HUSH-repressed transgenes counteracts repression, even in the absence of intron splicing. HUSH binds transcripts from the target locus, prior to and independent of H3K9me3 deposition, and target transcription is essential for both initiation and propagation of HUSH-mediated H3K9me3. Genomic data reveal how HUSH binds and represses a subset of endogenous intronless genes generated through retrotransposition of cellular mRNAs. Thus intronless cDNA—the hallmark of reverse transcription—provides a versatile way to distinguish invading retroelements from host genes and enables HUSH to protect the genome from ‘non-self’ DNA, despite there being no previous exposure to the invading element. Our findings reveal the existence of a transcription-dependent genome-surveillance system and explain how it provides immediate protection against newly acquired elements while avoiding inappropriate repression of host genes. The human silencing hub (HUSH) complex uses introns to distinguish intronless foreign DNA from intron-containing host DNA and modifies chromatin to silence transcription of retrotransposons and retroviruses.

All life forms defend their genome against DNA invasion. Eukaryotic cells recognize incoming DNA and limit its transcription through repressive chromatin modifications. The human silencing hub (HUSH) complex transcriptionally represses long interspersed element-1 retrotransposons (L1s) and retroviruses through histone H3 lysine 9 trimethylation (H3K9me3) 1-3 . How HUSH recognizes and initiates silencing of these invading genetic elements is unknown. Here we show that HUSH is able to recognize and transcriptionally repress a broad range of long, intronless transgenes. Intron insertion into HUSH-repressed transgenes counteracts repression, even in the absence of intron splicing. HUSH binds transcripts from the target locus, prior to and independent of H3K9me3 deposition, and target transcription is essential for both initiation and propagation of HUSH-mediated H3K9me3. Genomic data reveal how HUSH binds and represses a subset of endogenous intronless genes generated through retrotransposition of cellular mRNAs. Thus intronless cDNA-the hallmark of reverse transcription-provides a versatile way to distinguish invading retroelements from host genes and enables HUSH to protect the genome from 'non-self' DNA, despite there being no previous exposure to the invading element. Our findings reveal the existence of a transcription-dependent genome-surveillance system and explain how it provides immediate protection against newly acquired elements while avoiding inappropriate repression of host genes.
The mammalian genome is under constant threat from invasion by mobile genetic elements including transposons and viruses. Controlling this activity is fundamental to genome integrity. These defence strategies often use repressive chromatin to silence target gene expression and major chromatin-silencing factors in mammalian cells include: (1) small RNA guides complementary to nascent transcripts and (2) sequence-specific DNA-binding proteins 4 . PIWI-interacting RNAs (piRNAs) guide PIWI proteins to transposon transcripts and promote repressive chromatin at germline transposon loci 5 . piRNAs are derived from piRNA clusters, genomic loci enriched in transposon-derived sequences 6,7 . The piRNA pathway therefore relies on the memory of transposon invasions to provide adaptive, sequence-based immunity. The large KRAB-containing zinc-finger protein (KRAB-ZFP) family of sequence-specific DNA-binding proteins recruit TRIM28 and the SETDB1 methyltransferase to deposit H3K9me3 heterochromatin at target loci 7,8 . piRNA and KRAB-ZFP pathways are mostly active in the germ line and pluripotent stem cells, whereas the HUSH complex silences mobile elements in pluripotent stem cells and differentiated cells. HUSH represses evolutionary young L1 retrotransposons 2,3 , the only active autonomous mobile transposons in humans, as well as integrated lentiviruses 1 and unintegrated murine retroviral DNA via NP220 9 . The importance of HUSH in controlling lentiviral infection is emphasised by the finding that complex primate lentiviruses encode accessory proteins (Vpr and Vpx) that degrade HUSH 10-12 .
To silence mobile elements, the HUSH complex of TASOR, MPP8 and periphilin, recruits two effectors: MORC2-an ATP-dependent chromatin remodeller-enables chromatin compaction 13,14 , and SETDB1 deposits H3K9me3 1 . The chromodomain of MPP8 binds to H3K9me3-modified chromatin anchoring HUSH at the target locus. However, how HUSH recognizes its targets to initiate H3K9me3 deposition is unknown.

Intronless transgenes are HUSH-repressed
Since HUSH-repressed L1s are found in diverse genomic integration sites 2,3,15 , the signal for HUSH recognition must be intrinsic to the L1. To confirm that the L1 sequence confers HUSH repression independent of its integration site, we expressed a lentiviral fluorescent reporter encoding the L1 open reading frame (ORF) and a P2A-iRFP cassette. L1 expression was monitored by flow cytometry with iRFP fluorescence reflecting L1 mRNA abundance (Extended Data Fig. 1a). Inactivation of the ORF2 endonuclease 16 (D205A mutation) reduces retrotransposition; the reporter thus monitors expression from initial L1 integrations (Extended Data Fig. 1c, d). Lentiviral L1 reporter (L1 lenti ) expression is repressed within the entire wild-type population (Fig. 1a), and disrupting HUSH by knockout of HUSH subunits or by TASOR degradation by lentiviral Vpx 10-12 restores L1 lenti expression, whether the reporter is integrated before or after HUSH disruption (Fig. 1a,Extended Data Fig. 1b,. As L1 lenti is expressed from most integration sites following Nature | Vol 601 | 20 January 2022 | 441 HUSH depletion (Fig. 1a,Extended Data Fig. 1e,g), HUSH-mediated L1 silencing is independent of integration site. Lentiviruses predominantly integrate in transcribed gene bodies 17 , whereas the piggyBac transposase directly integrates at randomly distributed TTAA sites 18 . L1 reporter expression from an inducible, piggyBac transposon vector (L1 pb ) confirmed HUSH-dependent repression from most integration sites (Fig. 1b, Extended Data Fig. 1h) and HUSH-mediated H3K9me3 deposition that led to decreased RNA Poll II occupancy and reporter mRNA levels (Fig. 1c). The signal for HUSH repression is therefore intrinsic to L1 and independent of the mechanism and site of genome integration.
HUSH restriction of L1 retrotransposition depends on the native nucleotide sequence of the L1 ORF 2 . By testing the HUSH sensitivity of reporters bearing single L1 ORFs 19 (ORF1 or ORF2), we found that the ORF2 sequence alone is responsible for HUSH-mediated repression of L1 (Fig. 1d, left, Extended Data Fig. 1i). However, replacing the 4-kb ORF2 with 4 tandem repeats of the 1-kb ORF1 also caused HUSH repression (Fig. 1d, right, Extended Data Fig. 1j), suggesting that HUSH repression is not unique to ORF2.
We therefore tested the HUSH sensitivity of lentiviral transgenes with different DNA sequences (Fig. 1e,. To exclude effects on mRNA translation, we inserted DNA sequences lacking an ATG start codon, with a single-nucleotide frameshift at the 3′-untranslated region (3′ UTR) of the GFP reporter (Fig. 1e). Diverse, integrated transgenes containing cDNA sequences from a wide range of human genes were all HUSH-repressed (Fig. 1e), as were transgenes entirely 'foreign' to the human genome, for example, the bacterial Cas9 nuclease (Fig. 1e, Extended Data Fig. 2d). HUSH therefore silences sequence-diverse self and foreign mobile genetic elements, the latter being important as it excludes the possibility of 'genetic memory'. HUSH-mediated transgene repression was maintained over multiple cell divisions, was independent of the number of transgene integrations and showed a significant correlation with the length of inserted DNA (Extended Data Fig. 2e-g). While the L1 ORF1 (1kb) reporter is HUSH-insensitive, tandem repeats of ORF1 gradually acquire HUSH repression as their size increases (Fig. 1d, Extended Data Fig. 3h). Transgene length therefore contributes to HUSH susceptibility, with short (up to 1 kb) transgenes most likely to escape HUSH-mediated repression (for example, L1 ORF1, iRFP or a fragment of Xist long noncoding RNA (lncRNA)) ( Fig. 1d, Extended Data Fig. 2h). However, lentiviral reporters encoding short 1-kb fragments of ORF2 (or 3-kb ORF2 deletion mutants) remained HUSH-repressed (Extended Data Fig. 2i-k), indicating a role for nucleotide composition in HUSH targeting.
We found no correlation between HUSH-mediated repression and adenine and thymine (AT) sequence content (Extended Data Fig. 3a), and decreasing the overall AT content of ORF2 did not alleviate HUSH-dependent silencing (Extended Data Fig. 3b, e). However, HUSH-mediated repression strongly correlates with the A nucleotide content of the sense strand (Extended Data Fig. 3c), with ORF2 showing a strong A (41%) versus T (20%) bias in the sense strand 20 . Indeed, a reverse-complement ORF2 reporter is completely HUSH-resistant, despite expressing a full-length transcript (Extended Data Fig. 3d-g). The HUSH complex therefore represses a broad range of invading DNAs, with transgene length and high A content in the sense strand acting as key determinants of HUSH targeting.
HUSH targets endogenous, full-length, young L1s that are often enriched within transcriptionally permissive euchromatin, suggesting a role for transcription in HUSH targeting 2,3,15 . To directly test whether transcription is required to initiate HUSH-mediated silencing, we transduced HeLa cells with either the standard, spleen focus forming virus (SFFV) promoter-driven L1 lenti reporter or an otherwise identical promoterless reporter. HUSH-dependent H3K9me3 accumulated over the transcriptionally active L1 reporter, but was significantly reduced in the absence of a promoter (Fig. 2b,. Deletion of the promoter region from TAF7, an endogenous HUSH target gene also reduced transcription (Extended Data Fig. 4i, right) and locus-specific H3K9me3 deposition (Extended Data Fig. 4i, left, j) confirming that transcription is required to both initiate and maintain H3K9me3 over HUSH-sensitive loci. Furthermore, silencing cannot be conferred solely by the DNA sequence, as the sequences of HUSH-sensitive and HUSH-insensitive transgenes are identical.
A transcriptional requirement in HUSH-mediated silencing suggests that HUSH binds reporter RNA. Native RNA immunoprecipitation (RIP) showed that periphilin specifically binds RNA from a HUSH-sensitive reporter but not from a HUSH-resistant reporter (Fig. 2c   Article Fig. 4k,l). Notably, these results in SETDB1-deficient cells indicate that HUSH must bind reporter RNA prior to and independent of H3K9me3 deposition (Extended Data Fig. 4m). Transcription is therefore required for transgene repression, and periphilin binding to transgene RNA is likely to contribute to its recognition by HUSH.
To gain a global view of RNAs bound by endogenous periphilin (Extended Data Fig. 5a, b), we performed UV-cross-linked RIP and genome-wide analysis. Periphilin binding showed a significant overlap with genomic repeats, with specific enrichment over L1 elements (Fig. 2d, e, Extended Data Fig. 5c, d, f). There was no significant overlap between periphilin peaks and other repeat classes, with only transcripts of the Tigger DNA transposon family showing significant binding (Extended Data Fig. 5d, f). Periphilin preferentially bound transcripts from full-length, evolutionary young L1s (Extended Data Fig. 5e), reflecting the selective, genome-wide, HUSH-mediated H3K9me3 deposition over these L1 elements 2,3 , as well as from other HUSH-targeted loci (Extended Data Fig. 5f, right). Periphilin recognition of nascent RNA therefore specifies target loci for HUSH repression.

Introns protect against HUSH repression
We next investigated why transcribed cDNA sequences, but not their endogenous genomic loci (Fig. 1e, Extended Data Fig. 6a), are HUSH-repressed. A key difference is that coding regions of neither cDNAs nor L1s are separated by long intragenic non-coding DNA regions (that is, introns) prompting us to investigate whether HUSH sensitivity was intron-dependent.
We compared HUSH repression of: (1) an intronless reporter in which iRFP is followed by non-coding ORF2 (iRFP-ORF2) and (2) an otherwise identical reporter with the second intron of human β-globin (HBB IVS2) inserted within the iRFP (Fig. 3a). Intron insertion abrogates HUSH-mediated repression (Fig. 3a, Extended Data Fig. 6b), and HUSH-mediated repression was also abolished by intron insertion at the 5′ or 3′ end of ORF2 (Fig. 3c, Extended Data Fig. 7c, d). Insertion of an antisense GFP 'stuffer' sequence had no effect (Fig. 3c,Extended Data Fig. 7a). This loss of HUSH repression was associated with decreased periphilin binding (Extended Data Fig. 7g-h) and decreased H3K9me3 deposition (Fig. 3b,Extended Data Fig. 7b). Intron-mediated HUSH protection was also observed for: (1) ORF2 reporters of different architecture expressed from an inducible, piggyBac transposon vector, (2) Cas9 reporters expressed from the piggyBac transposon vector, and (3) lentiviral reporters (Extended Data Fig. 6c-e), and was lost following Cre-lox-mediated deletion of an intron sequence from the integrated transgene, implying that the intron is required continuously to maintain protection (Extended Data Fig. 6f).
Four additional human introns (EEF1A1, NXF1, SMC5 and ACTB) cloned into the iRFP-ORF2 reporter also provided protection from HUSH-mediated repression (Fig. 3d, f, Extended Data Fig. 7e), an effect not seen with a small artificial intron (chimeric β-globin-IgG), or reporters with similar-length control 'stuffer' sequences. The reduction in HUSH sensitivity correlated with the length of intron (Fig. 3f, Extended Data Fig. 7f). The SMC5 intron, despite being poorly spliced, prevented HUSH-mediated repression more effectively than fully spliced HBB and EEF1A1 introns (Fig. 3d, f, Extended Data Fig. 7e), suggesting that intron excision by the splicing machinery may not be required for protection against HUSH repression. To investigate whether splicing is required for intron-mediated protection, we generated a series of HBB IVS2 5′ and 3′ splice-site mutants (Extended Data Fig. 8a)   Fig. 2 | HUSH binds target RNA and initiates silencing before DNA integration. a, HUSH-mediated repression of non-integrated reporters. Left, HUSH-mediated repression of integrated and non-integrated GFP reporter lentiviruses with no insert (empty) or with synthetic ORF2 measured by flow cytometry 24 h after transduction and calculated as the ratio of reporter expression in wild-type and TASOR knockdown (KD). Data are mean of n = 3 biological replicates ± s.d.; two-sided ***P = 0.002, **P = 0.008 versus corresponding no-insert sample, unpaired t-test with Welch's correction. Right, flow cytometry histograms showing expression from GFP lentiviral plasmids containing different untranslated sequences transfected into wild-type or TASOR KD 293T cells. gMFI, geometric mean fluorescence intensity. b, Top, genome browser track depicting input and H3K9me3 chromatin immunoprecipitation with sequencing (ChIP-seq) signal over the unique fragment of the SFFV-driven or promoterless L1 reporter integrated into wild-type and TASOR KO Hela cells. Bottom, ChIP-qPCR quantifying H3K9me3 and total histone H3 levels at a SFFV-driven or promoterless L1 lentiviral reporter integrated into wild-type and TASOR KO HeLa cells. Data are mean of n = 3 biological replicates (independent polyclonal integrations of the reporters) ± s.d.; ***P = 0.0006, **P = 0.002, *P = 0.003 versus wild-type promoter, paired two-tailed t-test.  (mix) is a polyclonal cell pool after SETDB1 CRISPR-Cas9. Significant enrichment is defined as a fold change score above 1 with empirical Benjamini-Hochberg adjusted one-sided P-values (q); ***q = 0.0002 e, Genome browser tracks depicting periphilin and control RIP-seq signal over intronic L1 elements in wild-type and SETDB1 KO (mix) cells. intron ( Fig. 3e, f). Mutant intron no. 1 has a 5′ splice-site deletion critical for early spliceosome assembly at the transcript 22 , suggesting that intron-mediated HUSH protection is independent of assembly of the core spliceosome at the transgene RNA. HBB IVS2 splice mutants with either a 3′ splice-site mutation or deletion of the last 60 nucleotides (including the branch-point site that pairs with the 5′ splice site to form a splicing intermediate) not only counteracted HUSH, but provided more effective protection from HUSH-mediated repression than the wild type intron (Fig. 3e, f, Extended Data Fig. 8a, b). Therefore, even in the absence of splicing, introns protect transgenes against HUSH-mediated repression, whereas effectively spliced stuffer sequences flanked by a 5′ splice site, a branch point and a 3′ splice site, did not counteract HUSH (Fig. 3f, Extended Data Fig. 8c). Thus it is the intron itself rather than the splicing process that protects against HUSH-mediated repression.

HUSH targets endogenous intronless loci
Our data suggest that HUSH provides a genome-surveillance system to repress diverse transcribed, intronless invading DNAs, and predict that genomic loci from similar invading DNAs are bound and silenced by HUSH. Such loci include retrogenes and processed pseudogenes, created when reverse-transcribed cellular mRNA integrates into the genome, as part of a retrotransposition event 23 . We detected HUSH binding and HUSH-mediated H3K9me3 at the loci of transcribed processed pseudogenes and retrogenes, but not on their intron-containing, transcribed parent genes (Fig. 4a, Extended Data Fig. 9a-c, e). Many HUSH-repressed pseudogenes and retrogenes are positioned within transcriptionally active genes, similar to HUSH-regulated L1s. The MAB21L2 retrogene-a non-transcribed paralogue of the HUSH-repressed MAB21L1 retrogene-is not HUSH-repressed, confirming the critical requirement for transcription in HUSH-mediated repression (Extended Data Fig. 9d). Similarly, periphilin bound only retrotranscribed and not intron-containing parent genes (Extended Data Fig. 10a-c). Genomic analysis revealed that 20% of transcribed, non-L1-overlapping pseudogenes and 17% of intronless genes showed at least twofold enrichment of the periphilin RIP signal (Fig. 4b, Extended Data Fig. 10d). There was no enrichment of periphilin binding over intron-containing genes (Extended Data Fig. 10d), with the 5% of genes with bound periphilin predominantly containing HUSH-repressed long (over 2 kb) exons or zinc-finger family (ZNF) members as seen for HUSH-dependent H3K9me3 1,15 (Fig. 4b,Extended Data Figs. 9f,10e). HUSH repression of processed pseudogenes and retrogenes-all bona fide endogenous mobile elements-emphasises the physiological role of HUSH in defending the genome against invading retroelements.

Discussion
Our study reveals how the HUSH epigenetic repressor complex provides a versatile defence system against genome invasion. Without previous exposure to its targets, HUSH is able to recognize and transcriptionally repress a broad range of sequence-diverse, intronless

Article
DNAs, whereas intron-containing DNAs are resistant to HUSH-mediated repression. The defining feature of HUSH targets is therefore the presence of long, intronless transcription units, an intrinsic feature of retroelements, including L1 retrotransposons. Non-reverse-transcribed, intronless invading DNAs are also targeted for repression, including transfected cDNA plasmids. HUSH is therefore 'programmed' to control the spread of integrating, RNA-derived mobile elements within the host genome, representing a universal, cell-autonomous genome-surveillance system (Fig. 4c). The HUSH-mediated repression of endogenous L1s 2,3,15 is a consequence of this programming rather than a recognition of unique L1 sequences. Genomic evidence for HUSH repression of sequence-diverse, retrotransposition-derived, endogenous genes supports this conclusion and validates our findings with reporter genes. HUSH specificity for target length and A-rich bias in the sense strand may reflect retroviral reliance on 'structurally poor' A-rich RNA sequences to support viral cDNA synthesis during reverse transcription 24 and may therefore allow a more selective targeting of reverse-transcribed elements. Moreover, HUSH silencing of transgenes, including most cDNAs larger than about 1.5 kb, explains why many cDNAs remain difficult to express, a practical problem in both gene therapy and in ectopic gene expression in cultured cells.
The dependence of HUSH-mediated repression on transcription is reminiscent of transcription-coupled heterochromatin formation in Schizosaccharomyces pombe 25 , where, as with HUSH, transcription is required for both the initiation and propagation of H3K9me3. -The association of periphilin with its target RNAs even in the absence of H3K9me3 deposition provides support for a critical role of RNA in HUSH-mediated repression. Binding of periphilin to nascent RNA provides specificity for target recognition by recruiting and stabilizing HUSH at target loci independent of the MPP8 chromodomain, and enables HUSH to respond to increased transcription if H3K9me3 levels decline, such as during cell division. Similar to S. pombe, transcription-induced recruitment of HUSH to replicated chromatin may ensure inheritance of the repressed state following DNA replication 26,27 . This requirement for active transcription explains preferential targeting of full length L1s in euchromatic environments by HUSH, and conversely, why HUSH ignores older, degenerate L1s that have lost transcriptional activity 2,3,15 .
Importantly, intron-mediated protection from HUSH-mediated silencing does not require efficient intron splicing or spliceosome recruitment. Given the complex network of RNA-binding proteins involved in exon-intron definition and splicing 28 , intronic sequences may counteract HUSH by recruiting proteins other than core splicing factors that compete with periphilin for transcript binding. Alternatively, HUSH may be sensitive to nucleosome distribution, with the increased occupancy over exons versus introns 29,30 correlating with reduced elongation rates 29-31 . Slow elongation through long exons may trigger HUSH recruitment, which is counteracted by the decreased nucleosome density and increased elongation in cellular introns, consistent with HUSH-mediated H3K9me3 deposition over long exons of endogenous genes. Shorter introns are much less likely to affect nucleosome positioning (with each nucleosomes occupying 147 nt) than longer introns, consistent with the limited or absent HUSH protection afforded by the short ACTB and very short artificial intron. The well-recognized ability of introns to enhance gene expression (intron-mediated enhancement) can, at least in part, be explained by the capacity of introns to protect transgenes from HUSH-mediated silencing 32 .
To distinguish self from non-self, the host immune system recognizes conserved molecular patterns that are maintained in invading pathogens but are absent from the host. Most mammalian genes are organized such that exons comprise small islands within a sea of intronic sequences, whereas the cDNA products of reverse transcription are RNA-derived and intronless. Long, intronless cDNA, the product of reverse transcription, is therefore the molecular pattern recognized by HUSH, which provides a means to distinguish invading retroelements from host genes. Thus, HUSH comprises a component of the innate immune system. To avoid HUSH recognition, retroelements would need to maintain long, non-coding intron sequences, but are constrained by selective pressure for a compact genome. Bypassing the restriction imposed by HUSH therefore poses a major challenge. Whereas retroviral transcripts are often spliced, the intervening sequences are coding  sequences and very different from the classical long non-coding introns of cellular genes. Consequently, primate lentiviruses evade HUSH by encoding accessory proteins that degrade HUSH 10-12 , whereas endogenous retroelements are unable to evade HUSH activity. The innate immune response provides immediate defence but does not confer long-lasting immunity. HUSH-selective targeting of evolutionary young L1s 2,3 suggests a limited ability to provide long-lasting repression over evolutionary timescales. By contrast, DNA sequence-specific KRAB-ZFPs are less agile in repressing young retroelements, as it takes several million years to evolve a KRAB-ZFP with high affinity for a new DNA sequence 33,34 . By rapidly repressing transcription of novel retroelements without the need for genetic memory, HUSH buffers any potentially deleterious effects on cellular fitness. This gives the host a time window to establish sequence-specific adaptive repression to effectively restrict these retroelements and may facilitate their domestication.

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-021-04228-1. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Plasmids
A list and details of all plasmids used in the study are in Supplementary  Table 1.

Cell culture
HeLa cells were obtained from ECACC and HEK 293T and Jurkat cells were from ATCC. Cell morphology was assessed for authentication. All cell lines were grown in IMDM plus 10% FCS and penicillin/streptomycin (100 U ml −1 ). Cell cultures were routinely tested and found to be negative for mycoplasma infection (MycoAlert, Lonza).

CRISPR-Cas9 mediated gene disruption
HeLa or HEK 293T cells were transfected with a pool of sgRNAs cloned into a Cas9-containing plasmid (pSpCas9(BB)-2A-Puro) using TransIT HeLa Monster or TransIT 293T (Mirus) according to the manufacturer's protocol. Transfected cells were enriched with 24 h of puromycin selection (2 µg ml −1 ) starting 24 h after transfection. Hela TASOR KD, HEK 293T TASOR KD and SETDB1 KO (mix) cell lines were maintained as mixed KO populations. HUSH, SETDB1 and MORC2 KO HeLa cells were generated as described 1,13 and are polyclonal KO populations derived from a HeLa clone harbouring a repressed GFP reporter (pHRSIN-p SFFV -GFP-WPRE-P GK -Zeo R ) integrated at pericentromeric site on chromosome 7: 57848728 (hg19). Parental HeLa cells are GFP − and HUSH, SETDB1 and MORC2 KO cells are GFP + because of de-repression of the GFP reporter.

Lentiviral production and transduction
Lentivirus was produced by transfecting HEK 293T cells with the lentiviral vector plus the packaging plasmids pCMVΔR8.91 and pMD2.G using TransIT-293 transfection reagent (Mirus). The viral supernatant was collected 48 h later, cell debris was removed with a 0.45-µm filter and target cells transduced by spin infection at 1,800 rpm for 60 min. Transduced HeLa cells were selected with the following drug concentrations: puromycin, 2 µg ml −1 ; hygromycin, 100 µg ml −1 ; and blasticidin, 5 µg ml −1 . For experiments with non-integrated virus, cells were transduced in the presence of 1 µM raltegravir.
For the 'one-pot' establishment assay, WT HeLa cells were initially transduced with lentiviral vector encoding mCherry (pHRSIN-p SFFV -mCherry-WPRE) at a multiplicity of infection (MOI) <1 and mCherry + cells were purified by fluorescence-activated cell sorting (FACS), resulting in 98% pure mCherry + populations (Supplementary Figure 2). mCherry + WT and mCherry − TASOR KD cells were mixed at a 1:1 ratio and transduced with the lentiviral GFP reporters by spin infection. Reporter expression was typically analysed 2, 4 and 6 days after transduction by flow cytometry. Gating strategy is depicted in Extended Data Fig. 2c, Supplementary  Fig. 2. Reciprocal mixing (mCherry + TASOR KD and mCherry − WT) was used to validate results.

PiggyBac-mediated integration of reporter constructs
HeLa or HEK 293T cells were co-transfected with pB-transposon plasmid and piggyBac transposase-expression plasmid at 5:1 or 2.5:1 ratio using TransIT-HeLa Monster or TransIT-293T (Mirus). Transfected cells were selected with blasticidin (5 µg ml −1 ) for at least 3 days starting from 2 days after transfection. For flow cytometry assays, two cell lines were mixed at a 1:1 ratio prior to transfection. For assays with GFP reporters, WT mCherry + cells were mixed with TASOR KD mCherry − HeLa cells. For assays with iRFP reporters, WT GFP − HeLa cells were mixed with TASOR KO GFP + HeLa cells, which both harbour additional a HUSH-sensitive GFP reporter at chr7:57848728 (hg19). See Supplementary Figure 2 for gating strategies in flow cytometry analyses. Reporter expression was typically analysed 7 and 12 days after transfection and was induced by plating cells in media with doxycycline (1 µg ml −1 ) 24 h prior to flow cytometry analysis or ChIP-qPCR.

Immunoblotting
Cells were lysed in 100 mM Tris pH 7.4 with 1% SDS followed by boiling and vortexing to shear genomic DNA. Lysates were then boiled in SDS sample buffer, separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were probed with the indicated antibodies and reactive bands visualised with ECL, Supersignal West Pico or West Dura (Thermo Scientific).

CRISPR-Cas9 mediated knock-in of HA tag
For C-terminal periphilin tagging, the HA sequence was inserted upstream of the stop codon at the PPHLN1 endogenous locus via CRISPR homology-directed repair. For N-terminal TASOR tagging, HA was inserted downstream of the TASOR start codon. Single-stranded donor oligonucleotides (ssODN) were used as donor templates and purchased from IDT. HEK 293T cells were transfected with single guide RNA (sgRNA) plasmid (pSpCas9(BB)-2A-Puro) and single-stranded donor template. Transfected cells were enriched by puromycin selection and single-cell cloned. Clonal populations were screened for the presence of HA tag by intracellular flow cytometry staining using anti-HA antibody. The genetic modifications were validated by PCR on genomic DNA followed by sequencing. sgRNA and ssODN sequences listed in Supplementary Table 1.

CRISPR-Cas9 mediated deletion of TAF7 promoter
Prior to the modification of the TAF7 locus, HeLa cells were transduced with lentivirus encoding codon-optimized C-terminally HA-tagged TAF7 (TAF7 (opt) -HA) and blasticidin resistance as a selection marker. TAF7 is an essential gene 36 and stable expression of exogenous TAF7 (opt) was used to compensate for the loss of expression from the endogenous TAF7 locus due to promoter deletion. Sequence was codon-optimized so that exogenous TAF7 (opt) was not detected in RT-qPCR or ChIP-PCR.
Two sgRNAs targeting the TAF7 promoter region were cloned into pSpCas9(BB)-2A-Puro (PX459,V2.0): one targeting within the first 80 nucleotides of the TAF7 5′ UTR and a second approximately 850 nt upstream of the transcription start site. Two sgRNA plasmids were mixed at a 1:1 ratio and transfected into HeLa TAF7 (opt) -HA-expressing cells. Twenty-four hours later, cells were treated with puromycin (2 µg ml −1 ) for 24 h and single-cell cloned 5 days after transfection. The genetic deletion effects were validated by PCR on genomic DNA and loss of TAF7 expression measured by RT-qPCR. Sequences of primers and sgRNAs are detailed in Supplementary Table 1.

Chromatin immunoprecipitation
Cells were cross-linked in 1% formaldehyde for 10 min, quenched in 0.125 M glycine for 5 min and lysed in cell lysis buffer (1 mM HEPES, 85 mM KCl and 0.5% NP-40). Nuclei were pelleted by centrifugation and then lysed in nuclear lysis buffer (5 mM Tris, 10 mM EDTA and 1% SDS) for 10 min. The chromatin was sheared with a Bioruptor (Diagenode Pico) to obtain a mean fragment size of <300 bp. Insoluble material was removed by centrifugation. The chromatin solution was diluted to a final SDS concentration of 0.1% and precleared with Pierce Protein G magnetic beads (Thermo Fisher) and then immunoprecipitated overnight with 5 µg primary antibody and Protein G-magnetic beads. Beads were washed twice with low-salt buffer (20 mM Tris pH 8.1, 2 mM EDTA, 50 Mm NaCl, 1% Triton X-100, 0.1% SDS), once with high-salt buffer (20 mM Tris pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% TritonX-100,0.1% SDS), once with LiCl buffer (10 mM Tris pH 8.1, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate) and twice with TE. Protein-DNA complexes were eluted in 150 mM NaHCO 3 and 1% SDS at 65 o C. Cross-links were reversed by overnight incubation at 65 °C with 0.3 M NaCl and RNase A. Proteinase K was then added, the samples were incubated for 2 h at 45 °C, and then the DNA was purified with a spin column (Qiagen PCR Purification Kit). Quantification by qPCR was performed on a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) using SYBR green PCR mastermix (Thermo Fisher Scientific). qPCR primer sequences are detailed in Supplementary Table 1. For ChIP-seq, immunoprecipitated DNA was subjected to library preparation (NEBNext Ultra II DNA Library Prep Kit, Illumina). Libraries were purified, quantified, multiplexed (with NEBNext Multiplex Oligos for Illumina kit, E7335S) and sequenced with 2× 50-bp pair-end reads on Illumina Novaseq platform (Genomics Core, Cancer Research UK Cambridge Institute).

Native RIP-qPCR
Reporter expression was induced by doxycycline (1 µg ml −1 ) for 24 h prior to the experiment. Cells were lysed in HLB-N buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl 2 and 0.5% NP-40), incubated on ice for 5 min and lysate was underlaid with 1/4 volume of HLB + NS (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl 2 , 0.5% NP-40 and 10% (wt/vol) sucrose). Nuclei were pelleted by centrifugation (420g, 5 min) and then lysed in RIP buffer (25 mM Tris pH 7.4, 150 mM KCl, 5 mM EDTA, 0.5 mM DTT, 0.5% NP-40 and 100 U ml −1 SUPERase-IN). The nuclear fraction was sonicated (Diagenode Pico) and insoluble material was removed by centrifugation (8,000g, 10 min). The nuclear fraction was immunoprecipitated with Pierce anti-HA magnetic beads (Thermo Fisher) for 2 h at 4 °C. Beads were washed four times with RIP buffer and RNA was extracted from beads (and input samples) using TRIzol and standard phenol-chloroform extraction. The aqueous phase containing the RNA was loaded onto RNeasy mini columns (QIAGEN) with 2 volumes of 100% ethanol and RNA was purified according to the manufacturer's protocol. RNA was on-column DNase I treated and reverse transcribed using random hexamers and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Quantification by qPCR was performed on QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) using SYBR green PCR mastermix (Thermo Fisher Scientific). qPCR primers sequences are detailed in Supplementary Table 1.

Northern blot
Sample preparation, agarose gel separation and transfer to the membrane were all performed using a NorthernMax Kit (Invitrogen) according to the manufacturer's recommendation. In brief, 1-10 µg of sample RNA or 2 µg Millennium Markers (Invitrogen) were suspended in formaldehyde Article loading dye and loaded onto a 6-mm-thick 1% Agarose-LE gel and run at 5 V cm −1 (150 V, 110 min) in 1× MOPS running buffer. The samples were transferred to a BrightStar-Plus positively charged nylon membrane (Invitrogen) over 120 min, via the described downward transfer apparatus stacked on paper towels. Following transfer, the membrane was UV (254 nm) cross-linked using 120 mJ energy (Stratagene, Stratalinker 1800) and photographed under UV to record the marker positions (Invitrogen, iBright CL1000 Imaging System). Following a 30 min, 68 °C, prehybridization in ULTRAhyb ultrasensitive hybridization buffer, the membrane was incubated overnight at 68 °C with 100 pM digoxigenin-labelled RNA probes, directed against iRFP (nucleotides 4-300) and ACTB (nucleotides 69-618 of mRNA, NM_001101). Membrane was washed with 1× low stringency wash solution (room temperature) and 2× NorthernMax high stringency wash buffer (68 °C), prior to blocking at room temperature with 1× casein blocking buffer (Sigma-Aldrich). The membrane was incubated for 60 min with 50 mU ml −1 anti-digoxigenin-POD (poly), Fab fragments (Roche) in 1× blocking buffer, followed by 4 washes in 1× PBS + 0.1% Tween 20 and visualised using a SuperSignal West chemiluminescent substrate (Thermo Fisher) and the Invitrogen, iBright CL1000 Imaging System. Primers used to generate PCR amplicons against the indicated regions of each gene are listed in Supplementary Table 1. The amplicons were used in a T7 polymerase reaction substituting the NTPs for DIG RNA labelling mix (Roche), to generate antisense digoxigenin labelled RNA probes. The reaction was digested with TURBO DNase (Invitrogen) for 15 min at 37 °C, before purification using an RNeasy MinElute cleanup kit (Qiagen).

RT-qPCR
Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen) with on-column DNase I treatment according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using an equimolar mixture of random hexamers and oligo (dT) 16 primers by SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). RNA quantification was performed using the ΔΔC t method and normalized against ACTB or GAPDH transcript levels. Primer sequences are detailed in Supplementary Table 1.

Analysis of splicing
Efficiency of splicing of the reporter transcripts were determined by semi-quantitative PCR using intron-flanking primers (see Supplementary Table 1) detecting both unspliced and spliced reverse-transcribed mRNA. cDNA was prepared as for RT-PCR. Corresponding plasmids served as DNA controls.

Statistics and reproducibility
Statistical details, including the statistical test used, type (one-or two-sided), adjustments for multiple comparison and sample sizes (n), are reported in the figures and figure legends. The following figure panels show representative data from at least two independent experiments that showed similar results: Fig. 3e, Extended Data Figs. 1b,e,i,2a,g,k,3a,c,d,4d,l,m,5b,f,6b,f,7g. The following figure panels show representative data from at least three independent biological replicates that showed similar results: Figs. 1d,2a,right,3a,d,Extended Data Figs. 1f,2d,h,4b,c,k,6c,7c,e,h,8b,c. The following figure panels show representative data from at least four independent biological replicates that showed similar results: Fig. 1a, b, e, Fig. 2e, Extended Data Figs. 2d, 3b, f, 10a, b, e. The experiments in Fig. 1d and Extended Data Fig. 1c were performed once, but where internally controlled for both positive and negative results. The Northern blot experiments in Extended Data Figs. 1h, j, 3g, 4f, 7d were performed once, but were internally controlled for both positive and negative results. The ChIP-seq experiments in Fig. 2b (top) and Extended Data Fig. 4h, j were performed once, but the results were independently validated by two independent ChIP-qPCR experiments.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability
Gels and blots source images are provided in Supplementary Fig. 1. Next-generation sequencing data have been deposited at the Gene Expression Omnibus (GEO) with accession number GSE181113. The publicly available data 2 are available at GEO under accession number GSE95374 (ChIP-seq and RNA-sequencing data on the HUSH complex). The version of the human reference genome used in this study is GRCh38 (GENCODE v35, https://www.gencodegenes.org/ human/). Repeats were obtained from RepeatMasker (v UCSC hg38) and L1Base 48,49 . Source data are provided with this paper.

Code availability
For details about the bioinformatics data analyses, check the GitHub page for this study at http://github.com/semacu/hush. Periphilin-HA HEK293Ts and control HEK293Ts. p97/VCP as a loading. c,d, Enrichment of periphilin RIP-seq peaks at different repetitive elements in c, WT cells and d, WT (left) and SETDB1 KO (mix) (right). Significant enrichment is defined as a fold change score greater than one with Benjamini-Hochberg empirical adjusted one-sided p-value calculated using simulations and genomic association testing 47 , ***q < 0.001 (for exact p-values see source data). e, Fraction of full length, non-full length L1s and L1s from different families overlapping with periphilin RIPseq peaks. Full length L1s definitions are based on L1Base 48,49 . Blue heatmap indicates age of L1 families predicted from the phylogenetic analysis 50 . Periphilin-bound L1Hs may be underestimated in comparison to L1PA2-L1PA3 due to lower mappability of L1Hs as this is the least sequence-divergent L1 family. f, Genome browser tracks showing periphilin RIP signal over intronic L1s, Tigger DNA transposon and 3'UTR of ZNF37A. Fig. 6 | Introns protect different reporters from HUSH and are continuously required to prevent repression. a, Quantification of H3K9me3 and RNAseq signal over endogenous genes in WT and TASOR KO K562 cells from a publicly available dataset 2 . None of these endogenous genes are HUSH-repressed, unlike lentiviral reporters containing cDNA sequences of these genes. b, Northern blot analysis of mRNAs produced from intronless reporter or reporter with HBB IVS2 cloned within the iRFP gene. ACTB is a loading control. c, Flow cytometry histograms showing expression from GFP and GFP-ORF2 intronless or intron-containing lentiviral reporters in WT and TASOR KD HeLa cells 72h post transduction (bottom). Schematic of the construct (top). To prevent intron splicing during transcription in the virusproducing cells, the reporter cassette driven by the SFFV promoter was cloned in reverse orientation with respect to lentiviral transcription. The polyadenylation signal (pA) in reverse orientation provides a signal for termination of transcription from the reporter cassette in transduced cells. ORF2 is untranslated and intron (HBB IVS2) is cloned 5' of ORF2. SA-splice acceptor, SD-splice donor. d, HUSH-mediated repression of integrated intronless or intron-containing ORF2 piggyBac reporters measured by flow cytometry (histograms in centre panel) and calculated as the ratio of reporter expression in TASOR KO and WT HeLa (right). Expression from the reporter is driven by a dox-responsive CMV promoter. Reporters contain either human beta globin coding sequence or genomic sequence (containing 2 introns) followed by P2A-iRFP and ORF2 sequences (schematics on the left). A HUSHresistant reporter without ORF2 is the negative control. n biological replicates (independent polyclonal integrations of the reporters) ± SD; ***p ≤ 0.0001, one-way ANOVA post-hoc pairwise comparisons vs -introns with Bonferroni correction. e, HUSH-mediated repression of integrated intronless or introncontaining Cas9 piggyBac reporters measured by flow cytometry (histograms in centre panel) and calculated as the ratio of reporter expression in TASOR KO and WT HeLa (right). Expression from the reporter is driven by a doxresponsive CMV promoter. Reporters contain either HBB coding sequence or genomic sequence (containing 2 introns) followed by P2A-iRFP and Cas9 sequences (schematics on left panel). A HUSH-resistant reporter without Cas9 is the negative control. n biological replicates (independent polyclonal integrations of the reporters) ± SD; ***p ≤ 0.0001, one-way ANOVA post-hoc pairwise comparisons vs -introns with Bonferroni correction. f, HUSHmediated repression of reporter with intron removed by Cre-loxP recombination following the reporter integration (upper schematic). Flow cytometry histograms of expression from iRFP-ORF2 reporters driven by EF1a promoter: (i) intronless or (ii) reporter-bearing intron (HBB IVS2) flanked by loxP sites in the absence or presence of Cre expression (left). Gel image (right) confirms intron deletion.

Corresponding author(s): Paul J Lehner
Last updated by author(s): Oct 3, 2021 Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see our Editorial Policies and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All data supporting the findings of this study are available within the Article files. Gels and blots source images are provided in Supplementary Figure 1. In addition,

April 2020
the following figures have associated source data: Fig. 2d, Extended Data 2g, 3a, 3c, 3h, 5c, 5d, 5e, 9e, 10c. Next generation sequencing data have been deposited at the Gene Expression Omnibus with accession number: GSE181113. The accession number for the publicly available data from Liu et. al 2018 is GSE95374 (ChIP sequencing and RNA sequencing data).

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.