Epigenetic analyses demonstrate that gene promoters with high levels of DNA methylation frequently exhibit a low level of activity. Histone modifications have also been associated with altered transcription levels, with acetylated histones commonly linked to high levels of promoter activity. One model proposes that DNA methylation and histone modifications co-operate in order to repress transcription. A possible intermediary factor in this model is the site-specific DNA binding protein Kaiso, which has been shown in vitro to bind to methylated DNA sequences. Peggy Farnham, professor of biochemistry at the University of Southern California, and colleagues set about testing this model, revealing surprising results published in a recent study in Epigenetics & Chromatin. Farnham explained more about the findings their in vivo analyses yielded.
What does your article set out to investigate?
We are interested in the relationship between different forms of transcriptional repression. In particular, we wanted to test the model that site-specific transcription factors can bind to methylated promoters and then recruit histone deacetylases, resulting in cooperation between DNA methylation and repressive histone modifications to silence gene expression. Using in vitro assays, the transcription factor Kaiso (ZBTB33) has been shown to have high affinity for a DNA binding motif in which the central two CpGs are methylated. We therefore thought that this factor would be a good choice to begin our studies.
What most surprised you when you started looking at the data in your study?
We were very surprised to find that Kaiso preferred to bind to unmethylated promoters in vivo. Using chromatin from different cell types, we were able to observe that DNA methylation could block binding of Kaiso, even though this is the opposite of what is seen using in vitro assays.
Why do you think your in vivo studies have produced such differing results to previous in vitro studies?
We feel that the disconnect between the in vitro and the in vivo studies is due to the influence of chromatin landscape. We believe that although Kaiso may ‘prefer’ to bind to methylated DNA, this is not an option in the context of chromatin. Under conditions in which the CpGs in a promoter are highly methylated, the entire promoter region is often densely wrapped around nucleosomes and in a repressive heterochromatic state. The absence of a nucleosome-free region prevents access of transcription factors to the promoter region and hence prevents the access of Kaiso to its methylated motif. Being denied access to the preferred methylated motif, Kaiso is left with no option but to bind to the same motif in its unmethylated state.
How does your project fit in with the wider context of ENCODE?
The goals of ENCODE are to map all the functional elements in the human genome and to provide the community with some understanding of how these elements may contribute to cell growth, differentiation, and risk of disease. By integrating the maps of transcription factor binding sites, modified histones, and DNA methylation in a variety of different cell types, we hope to provide insights into how these factors contribute to the biology of a particular cell type and/or organ system.
You utilize ChIP-seq technology in your study. What do you think the next stage in development for this technology will be and how will this further your research?
Expanding the ChIP assay to a genome-wide scale has revealed many new insights about transcription factor binding that simply could not be determined using in vitro assays. Examples include the large numbers of binding sites that most factors display and that some factors prefer to bind to proximal promoter regions, whereas others bind in clusters with other factors at sites far from coding regions. I think that we are pretty good at tabulating these binding events; the next big step will be to develop high throughput methods to understand the functional significance of the binding. For this, we are developing genomic engineering strategies that will allow us to investigate the functional significance of distal regulatory regions in their natural chromatin environment.
What hurdles or caveats do ChIP-seq and related technologies present?
There are ~1400 site-specific DNA binding transcription factors and a vast number of cell types (especially when considering developmental stages and environmental influences). One complaint that I often hear from colleagues is that no one has studied their particular factor or mapped the binding of a certain factor in the cell type most important for their research. In most cases, this lack of data is due to the difficulty in obtaining the requisite numbers of cells for ChIP-seq assays and/or the low expression of that specific transcription factor. Studying specialized factors and cell systems will be difficult and will require teams of cell biologists and genomicists (and funding mechanisms that recognize the need for these studies).
What got you interested in research into transcriptional regulation and genomics?
I’ve always been interested in gene regulation, beginning with my thesis work on the regulation of attenuation in the trp operon of E. coli. However, the need for an assay that allows a genome-wide, unbiased investigation of transcription factor binding became very clear as cloning efforts revealed that transcription factors were grouped into families of highly related members, many of which bound to the same motifs in vitro. I realized that we could not understand the function of a particular transcription factor using in vitro assays, but instead we needed to examine binding in the context of chromatin.
What’s next for your research?
We are currently expanding our analyses to include transcription factors that are highly related to Kaiso and other factors which have been shown to bind methylated DNA in vitro.
Epigenetics & Chromatin 2013, 6:13
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