Understanding the Dynamics of PRDM9- dependent and independent Meiotic Recombination Hotspots at the single cell level

Abstract

Meiotic recombination takes place in a specialised organisation of chromatin into loops and axes, which brings together the hotspots and the DSB forming machinery. In mammals such as mice, the location of the hotspots is tightly regulated by the meiosis-specific protein, PRDM9. However, even in the absence of this protein, breaks can be formed, which are redirected towards the default break sites (DDSBs) located at functional elements like promoters and enhancers, which also have H3K4 trimethylation epigenetic marks. Here, we try to provide a mechanistic understanding of how PRDM9 can efficiently recruit the DSB machinery proteins, such as MEI4 and the axis proteins, such as SYCP3, to the hotspots. To do so, we have set up a technique combining immunofluorescence and fluorescence in situ hybridisation (FISH) that allows us to track the hotspot and the DDSBs, along with the proteins MEI4 and SYCP3, in a single cell. Additionally, we also provide evidence for the genomic localisation of REC114, a component present in the same complex as MEI4.