Fig 2: SPOC1 protein interacts with KAP-1 and enzymatically active H3K9 KMTs. (A) In GST-pulldown experiments with GST-SPOC1 wt, GST-SPOC1 (amino acids 1–100) or GST alone and chromatin extracts from U2OS cells, only the GST-SPOC1-full-length protein precipitated the KAP-1 protein, as detected on the immunoblot (lower panel). Amounts of GST proteins used were visualized by Amido black staining of the blots (upper panel). Asterisks mark the positions of full-length GST-proteins. (B) LUMIER-assay for mapping the KAP-1/SPOC1 interaction. The indicated combinations of SPOC1 and KAP-1, ProteinA (ProtA) and luciferase (Luc) fusion proteins were coexpressed. Luc activity coprecipitated with IgG-beads was measured (Z-scores). Significant KAP-1-interaction is only evident between both full-length proteins, or the C-terminal half of SPOC1 and the N-terminal 400 amino acids of KAP1, as detected by significant Luc activity in the corresponding pellets. Z-score = 0.5 indicates an interaction. Mean values ± SD of three independent experiments each performed in triplicate are given. (C) Immunoprecipitation of SPOC1 from HeLa nuclear extracts with anti-SPOC1 (lane 2) or with control antibodies (lane 1) and analysis of pelleted material by immunoblotting shows specific coprecipitation of G9a and SETDB1 with SPOC1. (D) Immunoprecipitation (IP) of endogenous GLP or SUV39H1 from C2C12 cell extracts and immunoblotting with the indicated Abs (IB Abs) against GLP (PP-B0422-00), SUV39H1 (07-550), SETDB1 (ab12317) and SPOC1 (6F6). Unlike the control Ab, GLP and SUV39H1 Abs precipitated all associated KMTs as well as SPOC1. (E) Nuclear extracts of control C2C12 cells (lane 1) or cells expressing HA-FLAG-tagged SETDB1 wt (lane 2) or SETDB1 H1224K mutant (lane 3) were analyzed on immunoblots (IB Ab.) with antibodies against FLAG (F1804), G9A (D141-3), GLP (PP-B0422-00), SUV39H1 (07-550) and SPOC1 (6F6). After anti-FLAG-SETDB1 immunoprecipitation (IP) the immunoblot detected specific coprecipitation of SPOC1 by wt SETDB1 (lane 5) but much less by enzymatically inactive SETDB1 H1224K (lane 6). (F) In vitro analysis of histone H3 methlylation by proteins immunoprecipitated by control, SPOC1, G9A, or SETDB1 antibodies. Left panel: autoradiography and SimplyBlue staining of the assay products separated by SDS–PAGE. Right panel: immunoblot which demonstrates that SPOC1, G9A and SETDB1 were immunoprecipitated specifically from the HeLa nuclear extracts. (G) In total lysates from HeLa-S3 cell lines constitutively expressing HA-FLAG-SETDB1 (HA-SETDB1), lentiviral-mediated SPOC1wt expression elevates H3K9me3 levels compared to control cells (REV), as detected by immunblotting with Abs against the indicated proteins (left). The SPOC1 PHD mutant (SPOC1mut) unable to bind to H3K4me3 only marginally increased H3K9me3 levels compared to SPOC1 wt.

Fig 3: HIF1a-AS1 interacts directly with the HUSH complex member MPP8.a Volcano plot of HIF1a-AS1 protein interaction partners after RNA pulldown assay and ESI-MS/MS measurements with fold enrichment and p value. n = 5. Significant proteins are shown above the line (p < 0.05). b List of proteins enriched after RNA pulldown assay, their p value (p, unpaired t test, two-tailed) and absolute fold change (FC). c RIP with MPP8 antibodies and qPCR for HIF1a-AS1 TFR2. IgG served as negative control. n = 4 independent experiments, Mann–Whitney test. *(p = 0.0286). d RIP with histone3-lysine9-trimethylation antibodies and qPCR for HIF1a-AS1 TFR2. IgG served as negative control. n = 3 independent experiments, Paired t test. *(p = 0.0162). e Scheme of the different HIF1a-AS1 RNAs used for in vitro RNA immunoprecipitation. E1, Exon1; E2, Exon2. f Scheme of the different MPP8 mutants used for in vitro RNA-immunoprecipitation. IDR intrinsically disordered region, ANK Ankyrin repeat; Chromo, Chromodomain. g RT-qPCR after in vitro binding assay of purified MPP8 with in vitro transcribed HIF1a-AS1 RNAs. MPP8 antibodies were used for RNA immunoprecipitation (RIP). An T7-MCS in vitro transcribed RNA served as negative control (CTL). FL, full length; E1, Exon1; E2, Exon2. ? indicates the deleted nt from HIF1a-AS1 full length. n = 4 independent experiments, One-Way ANOVA with Dunnett’s test. *FL (p = 0.018), *?E2 117-652 (p = 0.0453). h RT-qPCR after in vitro binding assay of different in vitro translated His-tagged MPP8 mutants with in vitro transcribed HIF1a-AS1 full length. Anti-His was used for RNA immunoprecipitation. n = 4 independent experiments, One-Way ANOVA with Dunnett’s test. *(p = 0.0029). i, j Proximity ligation assay of HUVECs with antibodies against MPP8 and H3K9me3 (i) or MPP8 and SETDB1 (j). The individual antibody alone served as negative control. Red dots indicate polymerase amplified interaction signals. Scale bar indicates 20 µm (i) or 10 µm (j). Images were representative of three independent experiments. Data are presented as mean values ± SEM.