Establishing a system to characterize the Zinc Finger Binding Interactions of PRDM9 with the DNA of Recombination Hotspots

Description

In the past few years, PRDM9 has been recognized as a major player in meiosis of mammals. Explicitly, PRDM9 specifies the genomic regions that will undergo meiotic recombination. It is believed that PRDM9 recognizes specific DNA sequences via its tandem array of zinc fingers and then epigenetically marks the local chromatin by its histone methyltransferase activity. Yet, simple DNA sequences recognized by the Zn finger array do not capture all the aspects of the binding site information and we still do not fully understand the binding determinants of PRDM9 to DNA. For this reason, we are developing an in-vitro system to analyze PRDM9-DNA interactions using recombinantly expressed PRDM9 and 30-100bp double-stranded DNA oligos known to bind PRDM9. The large size of the protein and the repetitive sequences of the Zn finger array pose a serious problem for multiple expression hosts. Thus, we tested different expression systems including cell-free in-vitro expression (IVE), mammalian cell and bacterial expression, as well as insect cell expression that could potentially minimize PRDM9's high cellular toxicity, high protein aggregation and low solubility. None of these expression systems yielded a purifiable form of PRDM9, but IVE and bacterial expression provided enough protein yields to perform binding assays with crude extracts using Microscale Thermophoresis (MST), as well as, electrophoretic mobility shift assays (EMSAs). MST monitors the thermophoretic motion in a microscopic temperature gradient in absence or presence of a ligand, which we used to measure affinity constants of fluorescently labeled PRDM9 directly in crude cell extracts. We corroborated the binding dynamics measured in MST using EMSAs, in which the binding was visually examined by the presence of shifted bands formed by the PRDM9-DNA complex. With the current system we can now further characterize the nature of PRDM9 binding.

Period	02 Feb 2014
Event title	XVI. Annual Linz Winter Workshop
Event type	Conference
Location	AustriaShow on map