Characterization of the PRDM9 zinc-finger multimer formation
Sprache des Vortragstitels:
Englisch
Original Tagungtitel:
EMBO Conference on Meiosis 2017
Sprache des Tagungstitel:
Englisch
Original Kurzfassung:
PRDM9 has been identified as a meiosis specific protein that plays a key role in determining the location of meiotic recombination hotspots by recognizing hotspot-specific DNA motifs. PRDM9 is an epigenetic modifier, which binds DNA via its long C-terminal zinc-finger (ZnF) array, consisting of several tandem zinc-fingers (e.g. 11 zinc-finger repeats for the murine CAST allele), and directs in its close vicinity double strand breaks (DSBs) necessary for the initiation of recombination. Recently, it was shown that PRDM9 acts as a multimer in-vivo; however, to date it is still unclear what factors drive the multimerization and the number of units in an active multimer. Using in-vitro binding studies, we characterized the number of PRDM9 units forming an active multimeric complex with a high-affinity DNA target. Specifically, we first established that the molecular weight (MW) of a protein-DNA complex can be inferred from a native polyacrylamide gel in an electrophoretic mobility shift assay (EMSA). For this purpose, we used DNA sequences of increasing lengths containing one or two specific target sites for PRDM9. The relative increase in MW with the change in migration distance of two protein complexes bound to DNA compared to one was used to infer the MW of the protein in the complex. Next, we assessed which domains of PRDM9 drive the observed multimerization using different PRDM9 constructs missing distinct domains and parts of the ZnF. We observe that all tested PRDM9 constructs (full-length PRDM9, ZnF domain consisting of 5-11 zinc-fingers) form functional multimeric complexes of at least two or more monomer units that are mediated solely within the ZnF domain. Our results suggest that the number of zinc-fingers within an array might play an important role in the overall affinity of PRDM9, and could explain the dominance of one Prdm9 allele over the other in a heterozygous PRDM9 complex, as has been observed for human and mice crosses.