I explored experiments to pull-down QAPs using tagged small molecule G4 ligands but this proved to have limited efficiency, probably due to competition with endogenous QAPs for the same binding sites. In an alternative antibody-based approach, I adapted and optimized a recently developed method to locate G4 in the cellular genome called G4 ChIP-seq. The advanced protocol was used to map G4 landscapes in four different cancer cell lines and revealed conserved sites as well as substantial cell-type specificity. Furthermore, combining this with proteomics technologies revealed the enrichment of various proteins involved in gene expression and the organisation of DNA in the nucleus (so called transcription factors and chromatin-binding proteins) at G4 sites.
In addition I investigated a potential link between G4 formation and DNA methylation. In mammals, addition of a methyl group to cytosine is a fundamental ‘epigenetic’ mechanism used to control gene expression and inheritance. This methylation occurs predominantly at CpG dinucleotides and is installed and maintained by enzymes called DNA methyltransferases (DNMTs), which plays a critical role for gene regulation. Most CpGs tend to be highly methylated, but regions rich in CG denisty, so-called CpG islands (CGIs), are mostly depleted of DNA methylation. I showed that DNMT1 selectively binds to DNA G4s with considerably higher affinity than double strand-DNA substrates and that this interaction inactivates the enzymatic activity in vitro. In human chromatin, DNMT1 is preferentially located at CGIs low in methylation and suggests a model by which G4 structures recruit and sequester DNMT1 to shape the epigenetic landscape such that G4s protect CGIs from DNA methylation to promote gene expression
A study on the direct interaction of transcription factors and G4 structures is currently still on-going and will provide new mechanistic details on how G4s directly contribute to the regulation of gene expression and highlight potential for chemical intervention in cancer therapy.
So far, the main findings have be disseminated in two peer-review publications in Nature Protocols (doi: 10.1038/nprot.2017.150) and in Nature Structural and Molecular Biology (doi: 10.1038/s41594-018-0131-8) as well as in multiple seminar talks. Genomic data acquired from next-generation-sequencing was deposited in the Gene Expression Omnibus (GEO) repository under GSE99205 and GSE107690.
