The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes

The cell cycle is tightly controlled by factors that regulate entry into and exit from each phase of the cycle. Most of these cell cycle proteins are transcriptionally regulated by MuvB (multi-vulva class B) complexes, RB (retinoblastoma) family members, or E2F (adenovirus early gene 2 binding factor) transcription factor family members.¹

MuvB complexes rotate between acting as transcriptional repressors during G0 and G1 and as transcriptional activators during S, G2, and M phases. Irrespective of function, all MuvB complexes share a conserved protein core composed of LIN9, LIN37, LIN52, LIN54, and RBBP4. Transcriptional repression is dependent on the formation of the DREAM (dimerization partner (DP), RB-like, E2F, and MuvB) complex where MuvB interacts with p130/p107, DP1/2, and E2F4/5. Entry into G1 triggers phosphorylation of p130/p107, initiating the disassembly of the DREAM complex. As G1 reaches completion, MuvB has dissociated from the DREAM complex and binds with B-MYB to create the MMB complex during S phase. This interaction is required for binding to FOXM1 during G2 resulting in transcriptional activation of cell cycle genes during the G2 and M phases.^1,2

The functions of the MuvB core complex proteins LIN52, LIN54, and RBBP4 have been elucidated, but little is known about the structure and function of LIN9 or LIN37. Neither protein has much if any homology to other known structural domains.

LIN9 and LIN37 Structural Modeling

LIN9 has three structured regions: amino acids 94-278 contain a Tudor domain, amino acids 333-421 form the MuvB binding region, and the C-terminal region is a predicted helix. Amino acids 95-126 of LIN37 contain a sequence indicative of MuvB binding and gene repression (CRAW domain). These predicted MuvB binding regions and previous studies showing binding to MuvB and MuvB interacting proteins influenced the hypothesis that LIN9 is a scaffolding protein for the other components of MuvB. Co-expression and precipitation experiments with all five of the MuvB complex core proteins resulted in co-precipitation and formation of the MuvB complex. However, co-expression of only LIN52, LIN54 and RBBP4 were not sufficient for co-precipitation of these proteins. These data along with previous work showing that LIN37 was not necessary for MuvB complex formation, supported the hypothesis that LIN9 is a scaffolding protein for the MuvB complex. Expression of different regions of LIN9 showed that the N-terminal region (1-300 amino acids) interacts with RBBP4 and LIN37, whereas the C-terminal region (300-542 amino acids) interacts with LIN52 and LIN54.

Co-expression of RBBP4 with LIN9^94-278 and LIN37^92-130 yielded a MuvB subcomplex - designated MuvBN - that was stable through affinity purification and size exclusion chromatography. This complex was crystalized to determine its structure and give insight into the structure of the classical MuvB complex. Using the known structure of RBBP4 as a starting point, a model of the MuvBN complex was created. LIN9 wraps around RBBP4 and creates a binding site for LIN37, which interacts with both LIN9 and RBBP4.

Previous studies of the structures of RBBP4 and RBBP7 show two common protein binding sites. One site interacts with PHF6, Fog1, and histone H3. The other site interacts with Suz12, Mta1, and histone H4. Based on the MuvBN model, interactions between RBBP4, LIN9, and LIN38 likely inhibit binding of histone H4 to RBBP4, but histone H3 likely still has access to the surface of the beta-propeller.

Tudor domains have been found to serve as histone readers, but while LIN9 has an exposed/available Tudor domain, alignments with other Tudor domains show that the LIN9 Tudor domain is likely unable to bind histones.

MuvB interacts with histone H3

To directly assess histone binding by these domains in the MuvB and MuvBN complexes fluorescence polarization experiments were performed. Consistent with the structural analysis, MuvB and MuvBN were able to bind histone H3 but not histone H4. While MuvBN has slightly lower affinity for histone H3 that MuvB, the authors concluded that the majority of the significant interactions between MuvB and histone H3 were still present in the MuvBN subcomplex. The MuvB complex was able to bind the H3 mark of transcriptionally silent heterochromatin (H3K9me3). MuvB was also found to bind to nucleosomes independent of histone H3 tail binding, creating a model by which MuvB interacts with histone H3 tails and/or the folded octamer to bind nucleosomes through RBBP4.

MuvB nucleosome binding directs cell cycle gene transcription

Examination of late cell-cycle genes showed higher nucleosome density downstream from transcriptional start sites of DREAM target genes. Since MuvB binds nucleosomes, the authors questioned whether MuvB directly increased nucleosome density on late cell-cycle gene promoters. Metal-shadowing electron microscopy illustrated more nucleosomes associated with a DNA sequence representing a minimal promoter region of a late cell-cycle gene when MuvB is present. Even though this interaction can occur in the absence of the CHR site, simultaneous engagement of the CHR site and nucleosome by MuvB increases the stability of the promoter nucleosome complex.

MNase-CHIP experiments were then used to detect MuvB association with nucleosomes in cells. MuvB binds and stabilizes nucleosomes in the 1+ position in DREAM promoters, which is indicative of repression of cell cycle genes. When Lin37 is absent, cell cycle genes are no longer repressed, and nucleosome patterning is consistent with genes undergoing transcription. While MuvB can still bind to nucleosomes under this knockout condition, the complex is widely distributed across the DNA rather than concentrated at the 1+ position.  When both Lin37 and RBBP4 are missing from cells, the MuvB complex can associate with DNA, but not nucleosomes. Together these data outline the critical roles of Lin37 and RBBP4 in nucleosome binding and transcription repression.   

The MuvB complex is a critical component for control of the cell cycle. The MuvB interacting partners change throughout the cell cycle leading to induction and inhibition of cell cycle gene transcription to promote or inhibit cell proliferation. This balancing act makes MuvB a potential cancer therapeutic target of interest³, and this current study increases our understanding of how MuvB inhibits transcription of cell cycle genes. The authors found that co-expression of RBBP4 with LIN9^94-278 and LIN37^92-130 was sufficient to induce this repressive phenotype. Lin9 is a critical scaffolding protein required to create the MuvB complex and Lin37 is critical for RBBP4 binding to nucleosomes in the repressive location.

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References

1. Fischer M, Müller GA. Cell cycle transcription control: DREAM/MuvB and RB-E2F complexes. Crit Rev Biochem Mol Biol. 2017;52(6):638-662. doi:10.1080/10409238.2017.1360836
2. Asthana A, Ramanan P, Hirschi A, et al. The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes. Nat Commun. 2022;13(1):526. doi:10.1038/s41467-022-28094-1
3. Iness AN, Litovchick L. MuvB: A Key to Cell Cycle Control in Ovarian Cancer. Front Oncol. 2018;8. doi:10.3389/fonc.2018.00223