Fig 2: RREB1 recruits a SIN3A-KDM1A silencing complex to target promoters.a Schematic of the protocol for isolation of the RREB1-SIN3A binary complex. RREB1-VC and SIN3-VN interaction reconstitute Venus (VC + VN) as the bait for IP-MS. b BioPlex2.0 analysis of the RREB1-SIN3A complex components identified by MS. Bait proteins are represented by a circle and prey proteins with a square. Proteins also identified in the RREB1-BioID are color coded based on SpecSum counts described previously. c Western blot analysis of protein fractions isolated by glycerol gradient (60–10%) ultracentrifugation. Blots were probed with the indicated antibodies for endogenously expressed RREB1 complex components. Dashed lines indicate different membranes (n = 3). d Expression of the indicated mRNAs in HEK293 cells expressing control siRNA (siCon) or siRNAs targeting SIN3A or KDM1A (n = 6). Box plots indicate the IQR of the data and the central line shows the median. e ChIP analysis of RREB1-SIN3A complex binding at the indicated promoter regions. Upper panels show the gene track view of SIN3A ChIP-seq (ENCODE, CistromeDB 63655, MCF-7 cells) and KDM1A ChIP-seq (ENCODE, CistromeDB 64823, K562 cells) at the indicated loci. Gene bodies are shown above the track sets for orientation. Red arrows point to the RRE. Lower panels show SIN3A, KDM1A and H3K4me2 ChIP-qPCR in HEK293 cells with a tet-inducible RREB1 transgene treated with DMSO (low RREB1) or tetracycline (high RREB1) (n = 3, error bars presented as mean values ± SD). f Gene track view of Sin3a ChIP-seq (ENCODE, Stanford/Yale TFBS, MEL cells) at the murine Fgfr4, Hras, and Map2k2 proximal promoters. Red arrows point to the putative murine Rreb1 binding elements. g ChIP analysis of Sin3a and Kdm1a at the indicated promoters in WT and Rreb1 + /− MEFs and h WT and Rreb1 + /− 6 month hearts (n = 3; *p < 0.01, **p < 0.001, ***p < 0.0001 two-tailed student’s t-test, error bars presented as mean values ± SD). Source data are provided as a Source Data file.

Fig 3: KDM3A mediates H3K9me2 modification on SP1 promoter to regulate its expression. A genes positively correlated with KDM3A in sarcoma tissues in UALCAN system; B, C KEGG pathway enrichment analysis on the candidate target genes of KDM3A; D expression profiles of SIN3A and SP1 in sarcoma in the GEPIA database; E expression patterns of SIN3A and SP1 in Ewing Sarcoma and OS in the GEO GSE73166 dataset; F, G mRNA (F) and protein (G) expression of SIN3A and SP1 in OS cells transfected with sh-KDM3A examined by RT-qPCR and western blot analysis, respectively; H IHC score of SP1 in the collected tumor and peritumor bone tissue samples; I correlation between KDM3A and SP1 IHC scores in OS tissues; J enrichment of KDM3A on SP1 promoter; K KDM3A and H3K9me2 modification levels on SP1 promoter in OS cells following KDM3A inhibition examined by the ChIP-qPCR assay. Three independent experiments were performed. Differences were analyzed by the unpaired t test (E), paired t test (H), and two-way ANOVA (F, G and K) followed by Tukey’s test. Correlation between variables was analyzed by the Pearson’s correlation analysis (I). *p < 0.05

Fig 4: TET1s increases CX40 expression by inhibiting histone deacetylation on the promoter of CX40. (A-B, D-E) p-HUVECs were transfected with TET1s-overexpressing adenovirus and negative control adenovirus and further tested after 48 h. (A) The global protein levels of ac-H3K27 and H3K27 in p-HUVECs were tested by Western blot (n=6 per group). (B) Sin3a interaction with TET1s and TET1-FL was analyzed by Co-IP (n=3 per group). (C) Schematic of human CX40 promoter and CHIP-qPCR products. TS indicates transcriptional start; P1-P5 indicates primer 1-primer 5; F indicates forward primer, R indicates reversed primer. (D-E) ChIP-qPCR was used to test Sin3a and ac-H3K27 enrichment in the CX40 promoter (-550 bp to +43 bp) (n=4 per group). (F-G) p-HUVECs were transfected with TET1s-overexpressing adenovirus and negative control adenovirus for 48 h and added HATI2 to media. (F) ChIP-qPCR was used to test ac-H3K27 enrichment in the CX40 promoter. (G) The CX40 mRNA levels were tested by RT-qPCR (n=4 per group). All data were shown as the mean ± SD.

Fig 5: Improved CRISPRi for MYC-regulated noncoding genes. (A) Pooled MYCncLibrary screen using the ZNF10 KRAB domain fused to dCas9 for CRISPRi. CPM, counts per million of CRISPRi sample; CPM0, counts per million of control sample (RAMOS cells, no dCas9). (B) Schematic depiction of KRAB-mediated recruitment of the corepressor TRIM28 and histone-modifying enzymes (HMEs) to induce transcriptional repression (Upper), and co-IP experiments to confirm TRIM28 binding to dCas9-KRAB and dCas9-kPOGO (Lower). Two lanes of the blot were removed (position indicated by dotted line); the original blot is shown in the SI Appendix, Fig. S2D. IB, immunoblot; IP, immunoprecipitation; kPOGO, KRAB domain of POGK. (C) Pooled MYCncLibrary screen using kPOGO fused to dCas9 for CRISPRi. (D) Schematic depiction of SID-mediated recruitment of the corepressor SIN3A and HMEs to induce transcriptional repression (Upper), and co-IP experiments to confirm SIN3A binding to dCas9-SID (Lower). SID, SIN3A-interacting domain. (E) Pooled MYCncLibrary screen using dCas9-SID for CRISPRi in RAMOS and P493-6 cells.