Artificial Transcription Factors – A new class of epigenetic drug
Aberrant epigenetic status resulting in dysregulation of gene expression is a hallmark of many cancers and other diseases. In the last decade, countless time and energy has been spent in efforts to develop drugs targeting DNA methylation and histone modifications. To date however, none of these epigenetic drugs are sequence specific: they target epigenome-regulating proteins rather than the DNA itself. Thus, although drug treatment may alleviate symptoms of epigenetic dysregulation at a certain region of DNA, the drug’s genome-wide influence may actually cause dysregulation at other regions.
In contrast, transcription factors are proteins that are able to bind to specific DNA sequences and recruit additional machinery. In efforts to increase the specificity of interactions between epigenetic drugs and their target regions, Artificial Transcription Factors (ATFs) are currently being developed for clinical applications. These ATF proteins can be engineered, in theory, to modulate expression of specific target genes by binding to a specific DNA region and recruiting epigenetic machinery such as TETs, DNMTs, and histone modifying complexes in order to modify the local epigenetic landscape.
A recent study by Grimmer et al. examines the genome-wide binding patterns and influences of two ATFs that target the SOX2 promoter, which plays a large role in maintaining cell self-renewal and has been implicated in several cancers.
Transgenic MCF7 breast cancer cell lines were created to contain ATF-expressing plasmids, simulating drug treatment. Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) was used to isolate regions of DNA which the ATF was bound. Parallel whole genome bisulfate sequencing, ChIP-seq targeting histone modifications, and RNA-seq analysis were performed to show how the DNA bound-ATF may influence epigenetic changes and regulate transcription at each binding site. The group looked at four different six-finger ATFs that specifically recognized one of two unique 18 nt region of the SOX2 promoter and either contained or lacked an SKD domain – a transcriptionally repressive domain which has been shown to recruit a histone methyltransferase which trimethylates histone H3 on lysine 9. ChIP-seq results indicated that the ATFs did in fact bind to their target region with the SOX promoter, but also that binding was not restricted to this region! ATF-DNA binding occurred at several regions throughout the genome, mostly within promoters. ATFs containing an SKD domain had, roughly, a 5 fold increase in the number of binding sites, possibly due to protein-protein interactions between the SKD domain and other transcription factors. Comparison of the binding regions revealed a lack of binding specificity, implying that rather than all six zinc fingers binding to the full 18 nt, subsets of zinc fingers may be used for multiple genomic interactions.
Despite the widespread ATF binding, RNA-seq analysis showed that very few genes had changes in their expression profile. Additionally, down regulation of genes caused by ATF-SKD binding did not seem to be due to the loss of H3Kme3 on the repressed promoters. Gimmer et al. suggests that non-specific binding may be less of an issue than is the lack of function upon binding, and that an ATF-SKD may only be able to act as a transcriptional repressor under certain conditions, for example when several ATFs complexes bind close together or if binding occurs at the transcription start site (TSS).
Although ATFs represent an exciting advancement in epigenetic drug development, it seems that a better understanding is needed of how zinc fingers work together to bind DNA and how transcription factors may use different subsets to bind different sequences. This study offers a critical evaluation of current specificity and effectiveness of ATFs – the newest class of epigenetic drugs.
Grimmer MR, Stolzenburg S, Ford E, Lister R, Blancafort P, & Farnham PJ (2014). Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation. Nucleic acids research, 42 (16), 10856-68 PMID: 25122745