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Epigenetics
30.04.2018

Novel technique allows comprehensive analysis of protein/DNA binding

Together with Dutch colleagues, scientists of Helmholtz Zentrum München present a new molecular method in 'Nature Communications': It allows the proteome-wide analysis of binding affinities between proteins and DNA.

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DNA is wrapped around proteins called histones, which serve to package the DNA inside the cell nucleus. The basic unit of DNA packaging is the nucleosome, which is made up of eight histone proteins with DNA wrapped around them. The assembly of DNA and histone proteins is termed chromatin. Interactions of these molecules are essential for important processes like transcription, replication and DNA repair to take place. Understanding the location and nature of these interactions is therefore of significant interest for understanding the regulatory networks of these important cellular processes.

In this study, Till Bartke’s team and colleagues from Radboud University in The Netherlands, present a technique through which the binding affinities of proteins that bind to chromatin can be determined on a proteome-wide scale. This is a significant advance on previous techniques which only give an ‘on-off’ indication of binding and little information alluding to the strength of binding. “We have been discussing for a long time whether it would be possible to use proteomic approaches to quantify affinities of chromatin-binding proteins at a large scale, and we have finally managed to do so” explains Till Bartke, Deputy Director of the Institute of Functional Epigenetics at Helmholtz Zentrum München.

The quantitative technique works by exposing short sequences of DNA (oligonucleotides) to nuclear extracts. Following these ‘pulldown’ experiments, proteins that interact with the DNA sequence are then identified and their abundances determined by mass spectroscopy. By using different amounts of the oligonucleotide ‘bait’ and thus testing a range of different concentrations, the researchers could calculate the binding affinities of many DNA interacting proteins.  

In addition, they performed similar experiments using nucleosome complexes containing modified and unmodified histones as bait. As histone modifications can change chromatin structure and lead to changes in gene expression, this use of the technique allows the identification of biologically relevant interactions under different conditions. For example, the Histone 3 protein can be modified by acetyl groups on lysine residues 9 and 14 (H3K9AcK14Ac), a form of histone modification linked to active gene transcription. Using two connected nucleosomes containing H3K9AcK14Ac as bait, the researchers identified preferential binding of an important nucleosome remodelling complex (called SWI/SNF) to the modified version compared to unmodified.

“Understanding how interactions between biomolecules regulate a cell requires not only knowledge about what molecules interact with each other but also how strong these interactions are. The technique that we have developed now allows us to quantify binding events such as the interaction of nucleosome remodelling complexes with nucleosomes depending on the modifications that these are decorated with. This is important if we want to describe dynamic molecular processes that regulate biological events” says Till Bartke.

Not only is the technique capable of revealing such novel observations, but it can do so on a large-scale; determining affinity measures for tens to hundreds of nuclear proteins simultaneously. This technique is thus novel in its revelation of the proteome-wide binding landscape of proteins that recognise particular DNA sequences or nucleosome states.

Further Information

Original Publication:
Makowski, M. et al. (2018): Global profiling of protein-DNA and protein-nucleosome binding affinities using quantitative mass spectroscopy. Nature Communications, DOI: 10.1038/s41467-018-04084-0

As German Research Center for Environmental Health, Helmholtz Zentrum München pursues the goal of developing personalized medical approaches for the prevention and therapy of major common diseases such as diabetes mellitus, allergies and lung diseases. To achieve this, it investigates the interaction of genetics, environmental factors and lifestyle. The Helmholtz Zentrum München has about 2,500 staff members and is headquartered in Neuherberg in the north of Munich. Helmholtz Zentrum München is a member of the Helmholtz Association, a community of 19 scientific-technical and medical-biological research centers with a total of about 37,000 staff members. 

The Institute for Functional Epigenetics (IFE) is concerned with the packaging of genes. The focus is on the so-called "histone proteins" on which the DNA strands are wound and which can determine whether a gene can be read or not. In addition, the scientists are examining the connections between common diseases and distortions in DNA packing. Cutting edge methods have enabled them to follow changes of these processes even in individual cells.