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The CRL4B E3 ubiquitin ligase complex regulates mitosis by recruiting phospho-specific DCAFs

ABSTRACT: The cullin scaffolds CUL4A and CUL4B assemble E3-ligase complexes regulating multiple mostly chromatin-associated cellular functions. Although they are structurally similar, we found that the unique N-terminal extension of CUL4B is heavily phosphorylated during mitosis, and this pattern is perturbed in CUL4B-P50L XLID patients. Indeed, phenotypic characterization and mutational analysis revealed that CUL4B phosphorylation is required for efficient progression through mitosis, controlling spindle positioning and cortical tension. Interestingly, while CUL4B phosphorylation triggers chromatin exclusion, it also promotes binding to actin regulators and two unconventional DCAFs, LIS1 and WDR1. Indeed, LIS1 and WDR1 directly bind DDB1, but their binding is enhanced by phosphorylation-dependent interactions with the unique amino terminal domain of CUL4B. Together, our studies uncover specific functions of CRL4B in mitosis, and identify previously unrecognized DCAFs that bind CUL4B by a phosphorylation-dependent mechanism.

INSTRUMENT(S): Q Exactive Plus, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Hela Cell

SUBMITTER: Petra Beli

LAB HEAD: Petra Beli

PROVIDER: PXD035329 | Pride | 2023-06-15

REPOSITORIES: Pride

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Project description:Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that they could provide transcriptional memory through mitosis. To obtain the first genome-wide view of the dynamics of chromatin structure during mitosis, we compared the DNase sensitivity of interphase and mitotic chromatin at two stages of cellular maturation in a rapidly dividingmurine erythroblastmodel. Despite global chromosome condensation visible during mitosis at the microscopic level, the chromatin accessibility landscape is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility, whereas distal regulatory elements preferentially lose accessibility. Promoters with the highest degree of accessibility preservation in mitosis tend to also be accessible across many murine tissues in interphase. Transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but does not visibly influence mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis and are modulated at the level of individual genes and regulatory elements. Dnase-Seq data is integrated with Chip-seq [GSE36589, GSE30142] and RNA-seq to examine epigentic changes in mitosis. We performed DNase-seq on two cell lines, G1E and G1E-ER4, both on an asynchronus population, and on a sample of cells in mitosis; each of the 4 experiments in triplicate.

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The CRL4B E3 ubiquitin ligase complex regulates mitosis by recruiting phospho-specific DCAFs

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