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DREAM interrupted: severing LIN-35-MuvB association in Caenorhabditis elegans impairs DREAM function but not its
Paul D Goetsch1,2, Susan Strome2
1Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA.
Abstract:
The mammalian pocket protein family, which includes the Retinoblastoma protein (pRb) and Rb-like pocket proteins p107 and p130, regulates entry into and exit from the cell cycle by repressing cell cycle gene expression. Although pRb plays a dominant role in mammalian systems, p107 and p130 are the ancestral pocket proteins. The Rb-like pocket proteins interact with the highly conserved 5-subunit MuvB complex and an E2F-DP transcription factor heterodimer, forming the DREAM (for Dp, Rb-like, E2F, and MuvB) complex. DREAM complex assembly on chromatin culminates in repression of target genes mediated by the MuvB subcomplex. Here, we examined how the Rb-like pocket protein contributes to DREAM formation and function by disrupting the interaction between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit LIN-52 using CRISPR/Cas9 targeted mutagenesis. A triple alanine substitution of LIN-52's LxCxE motif severed LIN-35-MuvB association and caused classical DREAM mutant phenotypes, including synthetic multiple vulvae, high-temperature arrest, and ectopic expression of germline genes in the soma. However, RNA-sequencing revealed limited upregulation of DREAM target genes when LIN-35-MuvB association was severed, as compared with gene upregulation following LIN-35 loss. Based on chromatin immunoprecipitation, disrupting LIN-35-MuvB association did not affect the chromatin localization of E2F-DP, LIN-35, or MuvB components. In a previous study, we showed that in worms lacking LIN-35, E2F-DP, and MuvB chromatin occupancy was reduced genome-wide. With LIN-35 present but unable to associate with MuvB, our study suggests that the E2F-DP-LIN-35 interaction promotes E2F-DP's chromatin localization, which we hypothesize supports MuvB chromatin occupancy indirectly through DNA. Altogether, this study highlights how the pocket protein's association with MuvB supports DREAM function but is not required for DREAM's chromatin occupancy.
Insights
The pocket protein LIN-35
Area of Science:
- Cell cycle regulation
- Molecular biology
- Genetics
Background:
- The pocket protein family, including pRb, p107, and p130, regulates cell cycle progression.
- These proteins form the DREAM complex with MuvB and E2F-DP to repress gene expression.
- LIN-35 is the sole pocket protein in Caenorhabditis elegans.
Purpose of the Study:
- To investigate the role of pocket protein interaction with MuvB in DREAM complex formation and function.
- To understand how LIN-35's association with MuvB influences DREAM complex assembly and target gene repression in C. elegans.
Main Methods:
- CRISPR/Cas9 targeted mutagenesis to disrupt LIN-35 and LIN-52 interaction.
- Phenotypic analysis of mutant worms.
- RNA-sequencing to assess gene expression changes.
- Chromatin immunoprecipitation to determine protein localization on chromatin.
Main Results:
- Disrupting LIN-35-MuvB association caused DREAM mutant phenotypes but limited DREAM target gene upregulation.
- LIN-35-MuvB disruption did not affect the chromatin localization of DREAM components.
- LIN-35's interaction with E2F-DP promotes E2F-DP chromatin localization, indirectly supporting MuvB occupancy.
Conclusions:
- Pocket protein association with MuvB is crucial for DREAM complex function, not its chromatin occupancy.
- The LIN-35-E2F-DP interaction is key for recruiting E2F-DP to chromatin.
- This study clarifies the distinct roles of pocket protein-MuvB interaction versus overall DREAM complex chromatin localization.
