Fig 2: Identification of the TINTIN complex in human cells, composed of BRD8–MRGBP–MRG15/X and new subunit EP400NL.A, purification of native NuA4/TIP60 complex from K562 cells. Strep elutions of indicated purifications were analyzed on 4 to 12% SDS-PAGE followed by silver staining. MRGBP and spliced variants of BRD8 (1BRD, one bromodomain, 2BRD, two bromodomains, and CRISPR for N-terminal-tagged BRD8) are linked to NuA4/TIP60 complex. B, Western blots of purified complexes in (A) confirmed the association with the NuA4/TIP60 complex and the new subunit EP400NL. C, Western blots of gel filtration fractions 17 to 33 (calibrated Superose 6, FPLC) with FLAG elutions from EPC1, BRD8, and MRGBP AAVS1 K562 cells (EPC1 purified fraction is used as a control reflecting the native NuA4–TIP60 complex (64)). Arrows point to specific annotated complexes with different elution profiles based on size. D, 4 to 12% SDS-PAGE followed by silver staining of annotated fractions from (C) showing the NuA4/TIP60 complex (17), the TINTIN complex (23), and a dimer MRGBP–MRG15 or MRGX (33). See also supplemental Table S5 for mass spectrometry results. EP400NL, EP400 N-terminal like; MRGBP, MRG-binding protein; NuA4, nucleosome acetyltransferase of H4; TINTIN, Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes; TIP60, Tat interactive protein 60 kDa.

Fig 3: Human TINTIN is implicated in the expression of specific genes.A, Venn diagrams showing that 161 genes are commonly downregulated (left) and 124 are upregulated in U2OS cells depleted (siRNA-mediated KDs) of BRD8, MRGBP, and MRG15 but not in cells depleted of Tip60/KAT5. Genes showing high changes in expression, using a cutoff of twofold difference (Log2 [fold change values]), were tabulated between the selected KDs and control (siLuc). B and C, Gene Ontology analysis for downregulated (B) and upregulated genes (C) showing a significant enrichment (p < 0.01) using DAVID 6.8. D, RT–qPCR to validate target genes of TINTIN. RFX8, HHIP, SYTL5, and CD180 are found downregulated, and AKT2 is upregulated when TINTIN subunits are depleted. PRRG4 and PCDHB9 are used as control. Values represent means relative to control (siLuc). Error bars represent the range from two biological replicates. E, Venn diagrams of downregulated (left) and upregulated (right) transcripts (versus genes) specifically regulated by BRD8, MRGBP, MRG15, or MRGX after exclusion of transcripts regulated by KAT5/Tip60, using a cutoff of twofold difference. siLuc was used as control. Two replicates of each KD were performed in U2OS cells. F, model of BRD8–MRGBP–MRG15/X function during transcription. BRD8 brings MRG proteins into the acetyltransferase NuA4/TIP60 complex because of its direct interaction with EP400 N-terminal region. This association is important for expression of specific gene targets. In addition, these subunits form a new independent functional module, called TINTIN, when BRD8 interacts instead with a new factor, EP400NL. TINTIN is also important for regulation of specific genes, maybe during the transcription elongation stage. BRD8, bromodomain 8; EP400NL, EP400 N-terminal like; KAT5, lysine acetyltransferase 5; KD, knockdown; MRG15, MORF-related gene on chromosome 15; MRGBP, MRG-binding protein; NuA4, nucleosome acetyltransferase of H4; qPCR, quantitative PCR; siLuc, siLuciferase; TINTIN, Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes; TIP60, Tat interactive protein 60 kDa; U2OS, human osteosarcoma epithelial cell.