Fig 2: DBC1 is required for SE-associated gene expression in CRC cells. (A) Identification of SEs based on H3K27ac ChIP-seq signals in SW480 WT and D1KO cells using the ROSE algorithm. (B) Venn diagram showing the intersection of SEs identified in SW480 WT and D1KO cells. (C) MEME-ChIP motif analysis reveals the top enriched motifs within lost SEs in SW480 D1KO cells. (D) Bar plot showing enriched Hallmark pathways for 321 lost SE-associated genes in SW480 D1KO cells. (E) GSEA of gene expression in human CRC versus normal colon tissues (GSE50760) using the lost SE-associated gene set. (F) GSEA showing negative and positive enrichment of lost and gained SE-associated gene sets, respectively, in SW480 D1KO compared with WT cells. (G, H) qRT–PCR analyses of mRNA (G) and seRNA (H) expression of lost and gained SE-associated genes in SW480 WT and D1KO cells. Data are expressed as fold change relative to the WT and are means ± SD (n = 3). (I) Snapshots of ChIP-seq tracks for H3K27ac, H3K4me3, H3K4ac, H3K9/K14ac, H3K4me1 and TCF7L2 at SEs of PROX1, ASB4 and NTSR1 genes in SW480 WT and D1KO cells.

Fig 3: DBC1 facilitates SE-associated histone modifications by coordinating the interplay of p300 and KMT2D. (A) Protein levels in SW480 cells depleted of KMT2D or treated with SGC-CBP30 were monitored by immunoblot using the indicated antibodies. (B) ChIP-qPCR assays. Sheared chromatin from SW480 cells infected with lentiviruses expressing either a control (NS) or KMT2D shRNA or SW480 WT cells treated with 5 μM SGC-CBP30 for 1 h was immunoprecipitated with the indicated histone modification antibodies. qPCR analyses were performed using primers specific for SEs of the PROX1, ASB4, FSCN1 and NTSR1 genes. ChIP signals were normalized with inputs and shown as fold change relative to control IgG signal, and are means ± SD (n = 3). *P < 0.05, **P < 0.01. (C, D) In vitro HMT assays using recombinant nucleosomes, immunopurified HA-KMT2D, GST–p300-Br-HAT and GST–DBC1 as indicated (C) and in vitro HAT assays using recombinant nucleosomes, immunopurified FLAG-KMT2D SET, GST–p300-Br-HAT and GST–DBC1 as indicated (D). The reactions were analyzed by immunoblot using the indicated antibodies. (E) 293T cells were transfected with the indicated constructs, and HA-p300 immunoprecipitates were analyzed by immunoblots with the indicated antibodies. (F) In vitro translated HA-KMT2D SET, HA-DBC1 or both were incubated with GST–p300-Br-HAT bound to beads as indicated. Bound proteins were analyzed by immunoblot with anti-HA antibody. (G) Proximity biotinylation assays. SW480 WT and D1KO cells transfected with 3×FLAG-TurboID-KMT2D SET or 3×FLAG-TurboID were treated with biotin as indicated. Biotinylated endogenous proteins were pulled down using streptavidin beads and analyzed by immunoblot with the indicated antibodies. (H) The PROX1 locus is schematically depicted with locations of H3K27ac peaks in SW480 WT and D1KO, sgRNAs (arrow) and ChIP-PCR primers (arrowhead). (I) SW480 D1KO cells were infected with lentiviruses encoding the dCas9–DBC1 fusion protein and sgRNAs targeting the PROX1 SE. ChIP-qPCR assays using SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were performed with the indicated antibodies. **P < 0.01. (J) Relative levels of DBC1 mRNA and PROX1 seRNA in SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were determined by qRT–PCR. **P < 0.001. (K) Protein levels of PROX1 and DBC1 in SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were monitored by immunoblot using the indicated antibodies.

Fig 4: A proposed model in which DBC1 plays a critical role in regulating the AE and SE landscapes. DBC1 is targeted to SEs by the Wnt-regulated transcription factor TCF4/LEF1 and β-catenin. DBC1 facilitates the recruitment of and interaction between KMT2D and p300 by directly binding to their catalytic domains (SET and HAT) and promotes independent and cooperative effects of KMT2D and p300 on H3K4 methylation and H3 acetylation by facilitating KMT2D complex formation and enhancing p300 autoacetylation.

Fig 5: REGγ interacts with DBC1 and is phosphorylated by Chk2. (A) DBC1 is present in REGγ immunocomplexes from U2OS cells. (B) DBC1 was immunoprecipitated from U2OS cells before and after etoposide exposure. The presence of REGγ was determined by immunoblot. WCE, total cell extracts; IP, immunoprecipitates; PC, pre-cleared negative control. (C) In vitro kinase assays with recombinant active GST-Chk2 and full length or deletion mutants GST-REGγ as substrates. GST-Cdc25C was used as positive control. (D) Phos-Tag gel analyses of REGγ molecular shifts in response to etoposide and in the absence or presence of the Chk2 inhibitor VRX. (E) U2OS cells were transfected with vectors encoding FLAG-REGγWT and FLAG-REGγS247A and treated or untreated with etoposide. Molecular shifts of the ectopic proteins were analyzed by phos-tag gel.