Fig 2: Model for cell cycle-dependent transcriptional regulation of early and late cell cycle genesIn G0 or upon activation of p53, early and late cell cycle genes are repressed by DREAM. This complex can be recruited to early expressed genes via interactions of E2F4/DP with E2F sites. Interaction of LIN54 with CLE sites can support binding of DREAM. Early cell cycle genes can also be repressed by complexes of E2F1-3, DP, and pRB. CLE sites cannot support binding of pRB complexes. The interaction of LIN54 with CHRs is sufficient to recruit DREAM to late cell cycle gene promoters. CDE sites can support binding through interaction with E2F4/DP. pRB does not bind to CHR or CDE/CHR sites. In proliferating cells, early expressed genes are activated in S phase by E2F1-3/DP complexes interacting with E2F sites. These complexes cannot bind to CHR or CDE/CHR sites of late cell cycle genes. Late cell cycle genes are bound by the MYB-MuvB complex (MMB) in S phase and FOXM1-MuvB which results in maximum expression during G2 phase and mitosis.

Fig 3: Conserved sequences in LIN37 are essential for nuclear localization and interaction with MuvB complex components.(A) Structure and evolutionary conservation of Lin37. Conserved domains are highlighted in yellow and introduced mutations in red (NLS: K75A/R77A, CD1: D101A/R102G/S103A, CD2: C119A/R120G). Red arrows mark target sites for CRISPR/Cas9 mutagenesis. Wild-type and mutant Lin37 fused with N-terminal FLAG tags or C-terminal EGFP tags were expressed in (B) NIH3T3 cells or (C) HCT116 cells (input, 10 µg whole cell extract) and precipitated with anti-Flag antibodies or anti-EGFP antibodies (IP). The MuvB complex components Lin9, Lin54 and B-Myb were tested for interaction with Lin37 wild-type (WT) and mutants (NLS, CD1, CD2, CD1 +2). (D) Fusions of Lin37 variants with EGFP were expressed in NIH3T3 cells and tested for their subcellular localization (green). Nuclei were stained with Hoechst33342 (blue).

Fig 4: Different promoter elements in early and late cell cycle genes recruit MuvB components, pRB, or activating E2Fs in vitro(A) DREAM components (p130, E2F4, LIN54, and LIN37) as well as pRB and activating E2Fs (E2F1, E2F3) were purified from nuclear extracts of serum-starved T98G cells by DNA affinity purification and detected by western blot. Binding to wild-type promoter probes (WT) was compared with binding to mutant probes (E2F, CLE, CDE, and CHR). “CDE” and “CLE” represent non-functional elements. Background protein binding was determined with a probe of the Gapdhs promoter (CTRL). (B) Binding of the MMB components B-MYB and the activating E2F3 was analyzed by DNA affinity purification with nuclear extracts from proliferating T98G cells.

Fig 5: LIN9 and LIN37 scaffold the MuvB complex.a Domain architecture of human LIN9 and LIN37, with regions of predicted and validated structure shown as blocks. The conserved LIN37 CRAW domain and LIN9 DIRP domain structures are determined here. MBD is the MYB-binding domain. b Schematic model for subunit interactions within MuvB. c The indicated tagged subunits or MuvB complex (GST-LIN52, Strep-RBAP48, His-LIN54504–749, GST-LIN37, and GST-LIN994–542) were expressed in Sf9 cells and extracts were precipitated with resin capturing the indicated tag. Proteins were visualized with coomassie staining. *Indicates impurities or degradation observed in some RBAP48 expressions. These bands are not pulled out from the tandem purification. The experiment was repeated three times with similar results. d HCT116 cells were transfected with plasmids encoding the indicated FLAG-tagged mouse protein. FLAG-tagged proteins were precipitated from extracts using anti-FLAG antibody and visualized with anti-FLAG immunoblotting and immunoblotting with antibodies that recognize RBAP48, LIN37, LIN54, and p130. A biological replicate of the experiment was performed, and results were similar.