Extended Data Fig. 4: Characterization of L1 effectors and the IFN-I response.

a, Expression of TREX1 was determined by RT–qPCR and immunoblotting. For gel source data, see Supplementary Fig. 1. b, Expression of RB family genes was compared by RT–qPCR. Primer pairs for all genes were verified to be of equivalent efficiency. c, Enrichment of H3K9me3 and H3K27me3 on L1 elements was examined by ChIP–qPCR (PCR primers illustrated in Fig. 1b were used: 5′ UTR, amplicon A; ORF1, amplicon E; ORF2, amplicon F). d, ChIP–seq data from ENCODE were investigated for transcription factors that bind to the L1 consensus sequence. The fold change, log₂(enrichment), relative to input controls is shown for the indicated cell lines. The binding of YY1 to the L1 promoter has been documented⁶⁹ and was used as a positive control. CEBPB was used as a negative control. A schematic illustrating L1 coordinates and relevant features is shown above. Amplicons A–E are the same as shown in Fig. 1b. e, Transcriptional activity of the intact L1 5′ UTR or a UTR lacking the FOXA1-binding site (UTR−Δ) was determined using sense and antisense reporters cotransfected into early passage LF1 cells either with a FOXA1 expression plasmid or empty vector. f, FOXA1 was knocked down in senescent cells with shFOXA1 (a) (see also Fig. 2e and Extended Data Fig. 5a) and binding to the L1 5′ UTR (amplicon B) was determined by ChIP–qPCR. g, Knockdown of RB1, TREX1 and ectopic expression of FOXA1 were performed in early passage cells in all single (1×), double (2×) and triple (3×) combinations and assessed by RT–qPCR using poly(A)-purified RNA for activation of L1, IFNA and IFNB1 expression (primers for amplicon F). Three controls are shown: cells infected with irrelevant shRNA (shGFP), expression construct (LacZ), or uninfected early passage cells. h, L1 5′ UTR occupancy of RB1 and FOXA1 in 3× cells was determined by ChIP–qPCR performed as in Fig. 2a, b. Primers for amplicons A and B were used for RB1 and FOXA1, respectively. For comparison, single interventions in early passage cells with shRB1 (a) or FOXA1 cDNA expression (EP FOXA1-OE) are also shown. i, Confirmation of full-length L1 mRNA expression in 3× cells using RT–qPCR with primers for amplicons A and F on poly(A)-purified RNA. CTR, cells infected with irrelevant shRNA (shGFP). j, Heat map representation showing all biological replicates for the 67 genes significantly changing expression in SEN and/or 3× cells (Fig. 2h, Supplementary Table 6). Column clustering was calculated as 1 − Pearson correlation. Rows have been grouped into functional subsets of the IFN-I response. k, Venn diagram showing the overlap between the 67 significantly changing genes. n = 3 independent biological samples, repeated in two independent experiments (a–f, h); n = 3 independent experiments (g, i).) Data are mean ± s.d. *P ≤ 0.05, **P ≤ 0.01, unpaired two-sided t-test. Exact P values can be found in the accompanying Source Data.

Source Data