Fig 2: Transcribed processed pseudogenes and protein coding retrogenes, but not their parent genes, are bound and silenced by HUSH.Genome browser tracks showing HUSH-dependent H3K9me3, HUSH/MORC2-occupancy and RNA-seq in WT and HUSH KO K562 cells at: a, additional, representative loci of retrogenes; b, FNBP1P1 pseudogene (left) and its parent gene FNBP1 (right); c, UTP14C retrogene (left) and its parent gene UTP14A (right) d, at the locus of MAB21L2, a non-transcribed paralog of HUSH-repressed MAB21L1 retrogene, Data from 2. e, HUSH-repressed genes obtained from the dataset in ref. 2: 378 genes were obtained when there was at least 30% reduction of H3K9me3 signal in all 3 knockout cell lines: TASOR KO, MPP8 KO and MORC2 KO (log2 FC H3K9me3 TASOR KO/WT = -0.5; FDR significance = 0.05; determined after a comparative assessment of counts between conditions (n = 2) using negative binomial generalized linear models as implemented in edgeR and corrected for multiple comparisons using FDR method). 104 of them were ZNF genes (including 8 ZNF pseudogenes) and the rest were inspected in IGV to determine the most probable reason for HUSH-repression e.g. overlap with L1 elements or other HUSH targets, pseudogene, retrogene, genes with signal over long exons, novel transcripts or antisense lncRNAs or loci with bidirectional promoter. The 11 resulting genes remained unannotated, either excluded because of low, background H3K9me3 over region or mapping artifacts or the reason for repression was unclear. Fraction of pseudogenes and retrogenes transcribed (average RNA-seq signal of all samples above > 0.1 RPKM or within transcriptionally active gene) (right) f, Genome browser tracks showing HUSH-dependent H3K9me3, HUSH/MORC2-occupancy and RNA-seq in WT and HUSH KO K562 cells at representative long exons. Data from ref. 2. Source data

Fig 3: HUSH repression of the L1 transgene is independent of the integration mode and site and is due to the L1 ORF2 sequence.a, Schematic of L1-iRFP reporter. The single mRNA transcript generates two proteins due to peptide bond skipping at the P2A sequence: the ORF1 (ORF1p) and iRFP (iRFPp). Changes in reporter transcription (e.g. due to H3K9me3-mediated silencing) affect iRFP expression. b, Western blot validating TASOR KO in HeLa cells with decreased levels of HUSH subunits periphilin and MPP8 due to TASOR depletion. ß-actin is the loading control. c,d, Effect of reverse transcriptase inhibitor 3TC on the expression from L1 lentivirus (L1lenti) and L1 reporter integrated by transposase (L1pb). To validate that our reporters monitor expression only from the initial L1 integration, and no subsequent retrotransposition activity, we compared reporter expression in the presence and absence of reverse transcriptase inhibitor 3TC, which prevents retrotransposition and new L1 insertions. If expression from new insertions contributes to the iRFP signal, 3TC should decrease iRFP, in particular at time points =3 days. No such decrease in iRFP signal is observed with L1lenti and L1pb upon 3TC treatment, demonstrating that both reporters monitor expression from the initial L1 integration. As lentiviral integration requires reverse transcription, the 3TC was added 12h post transduction when reverse transcription will be complete. c, Flow cytometry histograms of expression of L1 lentivirus upon 50µM 3TC treatment in WT or TASOR KO cells (left). 3TC was added 12 h post transduction and expression measured at day 5 post transduction. As a positive control, 3TC was used at the time of transduction (c – right hand panel). The absence of iRFP signal confirms inhibition of RT activity. Quantification of expression using geometric mean fluorescence intensity (gMFI) (right). d, Flow cytometry histograms of expression of L1pb reporter in the absence or presence of 50µM 3TC after 5 days of dox induction (left). Quantification of expression using gMFI (right). e, Establishment of repression of L1 reporter lentivirus in WT and HUSH KOs: TASOR, MPP8 or periphilin KO HeLa cells, and KOs of HUSH-effectors MORC2 and SETDB1. HUSH/HUSH effector KO (all GFP+) were mixed with WT cells and transduced with the L1-iRFP reporter at high multiplicity of infection (MOI). Expression of the reporter was measured by flow cytometry 72h post transduction. Western blot validating KOs with p97 as a loading control (bottom panel). * marks non-specific band. f, Schematic of lentiviral vector driven by dox-responsive promoter for expression of Human Immunodeficiency virus 2 (HIV-2) viral protein X (Vpx) to induce TASOR depletion (left panel). Western blot validating TASOR depletion 6 days after Vpx induction by dox with tubulin as a loading control (right panel). Flow cytometry histogram showing expression of integrated L1 lentivirus reporter before and after TASOR depletion by Vpx (bottom panel). g, Expression of L1 reporter lentivirus in: WT HeLa (WT+reporter) (grey histogram), HeLa cells in which TASOR was depleted after the integration of L1 lentivirus (+TASOR KD) (purple histogram) and re-expression of mCherry-TASOR (+TASOR KD +mCherry-TASOR) (grey, dotted histogram). This experiment was repeated in Jurkat cells with similar results. h, Northern blot showing increased mRNA from L1pb reporter in TASOR KO cells 24 h post dox induction using iRFP probe. Full-length L1pb mRNA is the predominant RNA produced from the L1pb reporter. i, The expression of reporters with ORF2 or ORF1 sequences placed downstream of GFP in WT or TASOR-depleted (TASOR KD; see Extended Data Fig. 2a) HeLa cells measured by flow cytometry (upper panel). HUSH-mediated repression of reporters with ORF2 or ORF1 sequences placed upstream of GFP. CRISPR–Cas9 of TASOR (TASOR KO) after reporter integration (bottom panel). j, Northern blot analysis of mRNAs produced from L1pb reporter in which ORF2 (4kb) sequence was replaced by 4 tandem repeats of ORF1 (1kb). RNA was isolated from the mix of WT and TASOR KO cells and iRFP probe was used to detect reporter mRNA (Fig. 1d).