VPS72/YL1 function in nuclear re-assembly

• Applicant: Professor Dr. Wolfram Antonin
• Subject Area: Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 515940343

The nucleus reorganizes structurally and functionally considerably during mitosis. In animal cells, the nuclear envelope breaks down at the beginning of mitosis, the chromatin massively condenses to individualized chromosomes, which are captured and segregated by the spindle apparatus - processes that have been and still are intensively studied in many laboratories. We know much less how at the end of mitosis the interphase state of the nucleus is reestablished, competent for its manifold functions. For this, the highly condensed mitotic chromosomes are decompacted. Around the decondensing chromatin the nuclear envelope re-assembles, nuclear substructures like nucleoli reform and nuclear transport re-establishes compartmentalization. These processes are indispensable for reinitiating transcription and the perpetuation of genomic information and thus of central importance in the cellular life cycle. Despite its significance to basic research as well as its potential medical implications, nuclear reformation during mitotic exit is in molecular terms still ill defined. We have identified VPS72/YL1, a chaperone of the histone variant H2A.Z, and its conserved YL-1 domain as a crucial factor for nuclear re-assembly: In Xenopus egg extracts, where nuclear reformation can be conveniently reconstituted, depletion of VPS72 blocks H2A.Z integration into chromatin and leads to nuclear structure defects. The chromatin appears disorganized and the nuclear envelope is irregular and reaches into areas usually occupied by chromatin. The YL-1 domain, in turn, is not required for bulk H2A.Z chromatin loading but has a specific, yet unknown, function in nuclear re-assembly. Interestingly, this domain interacts with the spindle assembly checkpoint components MAD2 and p31 and a less defined factor, WDR83. Depletion of p31 and MAD2 compromises the nuclear structure as VPS72 depletion indicating a non-canonical function of the checkpoint proteins p31 and MAD2. Here, we will define the function of the YL-1 domain and its binding partners MAD2, p31 and WDR83 in nuclear re-assembly using Xenopus egg extracts and cellular assays. The second includes CRISPR/CAS based knockouts or inducible degradation of target proteins in tissue culture cells combined with life cell imaging. We will characterize the function of the VPS72/MAD2/p31/WDR83 interaction network for nuclear assembly and define which aspects of nuclear reformation are compromised if these components are missing. We will test the hypothesis that the YL-1 domain is required to load H2A.Z to or recognize specific chromatin loci, which in turn are crucial for proper nuclear re-assembly. For this, we use CHIP experiments in Xenopus egg extracts, to be established in this system, and in tissue culture cells. By a combination of cell-free and cellular assays, we will shed light on an ill-defined but important cell biological process at the end of mitosis vital to reestablish nuclear structure and function.

• DFG Programme: Research Grants