Group leader: Antal Kiss

Email: kiss.antal[at]brc.hu

Group website:

http://group.szbk.u-szeged.hu/sysbiol/kiss-antal-lab-index.html

Group members

Name

Title

 

 

Antal KISS

Pál Albert

Nikolett Zsibrita

Ildikó Karcagi

Bence Varga

professor emeritus

junior research associate

junior research associate

research associate

Ph.D. student

publications

publications

publications

publications

publications

 

CV

CV

CV

CV

CV

Research                                  

Engineering research tools for targeted DNA methylation

Cytosine-5 methylation of genomic CG-sites is an important component of epigenetic regulation in mammals, which has key roles in a number of biological phenomena such as X-chromosome inactivation and genomic imprinting. The pattern of genomic DNA methylation correlates with the function of the cell and with tissue-specific gene expression. There is an increasing body of evidence supporting the role of aberrant DNA methylation in the pathogenesis of several diseases including cancer. Although it is generally accepted that methylation of promoters of mammalian genes leads to gene silencing, we are far from understanding of how expression of individual genes is affected by DNA methylation, and how DNA methylation is integrated into the complex network of epigenetic regulation. Targeted DNA methylation, i.e. methylation of predetermined genomic sites could be a useful experimental tool for addressing these questions. The approaches to targeted DNA methylation share the principle of the pioneering work (1): A CG-specific C5-DNA methyltransferase is covalently linked to a sequence-specific DNA binding protein, which binds to the intended genomic site, and the fused DNA methyltransferase preferentially methylates the sterically close CG sites. Earlier works mainly used zinc finger proteins as targeting modules, whereas in more recent approaches the zinc fingers were replaced with the catalytically inactive dCas9 protein (2). Despite of improvements, the technique is generally considered to suffer from unacceptable level of off-target methylation (2,3).

We study how different factors, such as catalytic activity and DNA binding affinity of the methyltransferase influence the specificity of targeted DNA methylation. We address these questions in an Escherichia coli model system using the CG-specific bacterial DNA methyltransferase M.SssI linked to zinc finger protein or CRISPR-dCas9 targeting domains.

In some mammalian cell types C5-cytosine methylation occurs, besides the predominant CG, also in non-CG context (CH), mainly in CA. The biological role of non-CG methylation is currently unknown (4). We aim to create variants of the CG-specific bacterial methyltransferase M.MpeI, which can methylate cytosines in CH (CA/CC/CT), but not in CG context. We chose M.MpeI for this work because of its high activity and the availability of an X-ray structure (5). The desired CH-specific M.MpeI mutants could be useful research tools in the study of the biological role of non-CG-specific DNA methylation. Moreover, analysis of the mutant enzymes will complement the information derived from the X-ray structure of the M.MpeI-DNA complex (5), and will likely provide insights into the mechanism of sequence specific DNA-protein interactions.

           

I-Block: A simple assay for studying sequence-specific DNA-protein interactions in E. coli

Sequence-specific DNA-protein interactions are central to the control of gene expression. For  characterization of DNA binding proteins it is important to test whether the protein can bind to a particular DNA sequence, and to determine how alterations in the protein and/or in the DNA affect the binding strength and/or specificity. The published methods have drawbacks: the in vitro methods need purified proteins, whereas the E. coli-based in vivo techniques require construction of protein fusions.

We have developed a simple method, called I-Block assay, which can detect sequence-specific binding of proteins to DNA in E. coli. The method works by measuring the indirect result of competition between the tested protein and RNA polymerase for binding to overlapping target sites in a modified lacI promoter. Binding of the tested protein to its target site interferes with transcription of the lacI gene leading to an increase of β-galactosidase activity (6).

The I-Block assay, in its current state, can test wether a protein can bind to a particular DNA sequence in E. coli. We are trying to develop the method into a high throughput technique, which can be used to find the best binding protein variant for a particular DNA sequence, or to identify the best binding DNA sequence for a protein.

 

References

  1. Xu, G.L. and Bestor, T.H. (1997) Cytosine methylation targetted to pre-determined sequences. Nat Genet, 17, 376-378.
  2. Lei, Y., Huang, Y.H. and Goodell, M.A. (2018) DNA methylation and de-methylation using hybrid site-targeting proteins. Genome Biol, 19, 187.
  3. Galonska, C., Charlton, J., Mattei, A.L., Donaghey, J., Clement, K., Gu, H., Mohammad, A.W., Stamenova, E.K., Cacchiarelli, D., Klages, S. et al. (2018) Genome-wide tracking of dCas9-methyltransferase footprints. Nat Commun, 9, 597.
  4. He, Y. and Ecker, J.R. (2015) Non-CG Methylation in the Human Genome. Annu Rev Genomics Hum Genet, 16, 55-77.
  5. Wojciechowski, M., Czapinska, H. and Bochtler, M. (2013) CpG underrepresentation and the bacterial CpG-specific DNA methyltransferase M.MpeI. Proc Natl Acad Sci U S A, 110, 105-110.
  6. Szentes, S., Zsibrita, N., Koncz, M., Zsigmond, E., Salamon, P., Pletl, Z. and Kiss, A. (2020) I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins. Nucleic Acids Res, 48, e28.