How does DamID work?
DamID is based on the creation of a fusion protein consisting of Escherichia coli DNA adenine methyltransferase (Dam) and a chromatin protein or transcription factor of interest. Dam methylates adenines in the sequence GATC. Endogenous methylation of adenines is absent in most eukaryotes. Upon expression of the fusion protein in cultured cells or in an intact organism, Dam will be targeted to the native binding sites of the chromatin protein. This will then result in local methylation of adenine residues. Hence, the sequences near a binding site of the protein will be marked with a unique methylation tag, which can be detected using restriction enzymes (DpnI and DpnII) that are methylation sensitive. Protocols have been developed to combine this with genomic tiling microarrays or next generation sequencing methods.
Is it really that simple?
Almost, but there’s one complication. The Dam-fusion protein is never perfectly targeted to the native binding sites — a fraction of the fusion protein molecules is inevitably diffusing around in the nucleus. This will cause considerable background methylation of non-target sites. To make things worse, some GATCs in the genome are more accessible to Dam than others, and as a result the non-specific background methylation is not homogeneous throughout the genome. Thus, one might easily mistake a non-target GATC in a very “open” chromatin region for a target of the chromatin protein.
Luckily, it turns out that one can correct for this non-targeted background methylation by measuring in a parallel control experiment the methylation levels by unfused Dam. For each GATC the methylation levels obtained with the fusion protein are then compared to the methylation levels obtained with Dam only. In practice, this comparison can be done by calculating the ratio methylation by Dam-fusion protein: methylation by Dam. We found that in this way the variation in chromatin accessibility is normalized for.
In which organisms can DamID be used? Does it work in mammalian cells?
Genome-wide DamID has been extensively used and validated in various organisms, including S. pombe (Stechlig, 2012), C. elegans (Schuster, 2010), D. melanogaster (Vogel, 2009), A. thaliana (Zhang, 2007), and mouse and human cell lines (Vogel, 2006; Peric-Hupkes, 2010). In Drosophila Kc cells we have generated genome-wide binding maps for more than 100 different chromatin proteins, including many proteins that were not previously known to be part of chromatin (Filion, 2010; van Bemmel, 2013).
There are many more citations but we do not keep a list of DamID publications.
What’s the trickiest part in the procedure?
Our work in Drosophila (flies and cell lines) suggests that it is very important to keep the expression level of the Dam-fusion protein very low, to avoid saturating methylation levels. We use the Drosophila heat-shock promoter for expression of our Dam fusions, but do the actual experiments in the absence of heat-shock. In human cells we also use an inducible promoter, but again for DamID we only use the leaky expression in the absence of induction. Under these conditions we cannot detect the fusion proteins themselves (by western blotting or immunofluorescence microscopy), but there is specific methylation of target sequences. Our interpretation is that only trace amounts of the Dam proteins are present, but that — thanks to the high enzymatic activity of Dam — this level is just right to obtain detectable but non-saturating levels of methylation. After induction the Dam-fusion proteins themselves could be detected easily, but at the same time the targeted methylation levels had reached saturation, and background methylation had become so high that no target sequences could be identified.
Thus, the trick is to keep the expression levels of the Dam (fusion) proteins low. In flies we therefore suggest to work with the (uninduced) hsp70 promoter or some derivative. In human cells we suggest that you use our vectors, which contain the inducible promoter from the pIND system. This low expression level has the additional advantage that the Dam fusion protein is unlikely to perturb the function(s) of the endogenous chromatin protein or its targets, since it is only present in trace amounts.
Does DamID work with next generation sequencing methods?
In the past we used NimbleGen and Agilent genomic tiling arrays. However, they are no longer available. DamIDseq combined with Illumina HiSeq technology works fine. Some protocols have been published (Luo, 2011; Wu, 2013) and we have developed some variants that appear to work well (manuscript in preparation).
How does DamID compare to chromatin immunoprecipitation?
Compared to ChIP, DamID has both advantages and disadvantages. It really depends on the specific application and on the protein of interest.
DamID may be good choice if you do not have a ChIP-grade antibody. In Drosophila cells, we found that DamID works for > 70-80% of all tested proteins (transcription factors, chromatin proteins, etc.), without ever having to adjust the protocol. Dam-fusion proteins may also be expressed specifically in one cell type of a mixed tissue (Southall, 2013), which makes it possible to generate a binding profile for that particular cell type without having to somehow purify or sort this cell type. DamID is extremely sensitive, so it can be done with very small numbers of cells: recently we have even generated genome-wide DamID maps from single cells (manuscript in preparation)!
Several side-by-side comparisons of ChIP-on-chip and DamID have been done. They showed that data are generally in good agreement (Moorman, 2006; Negre, 2006; Tolhuis, 2006; van Bemmel, 2010; Yin, 2011;).
That said, the mapping resolution of ChIP is somewhat better than that of DamID. Targeted methylation ‘spreads’ in cis from the protein binding site. We don’t know exacly how this happens, but it does limit the resolution of DamID. We estimate that this resolution is roughly 1kb.
Wolffe and Leblanc (2000) have suggested that DamID may work well for proteins that interact only transiently with their DNA targets, because targeted Dam could leave a permanent mark even after a brief interaction. Indeed, by visualizing m6A-tags in live cells, we found strong evidence that this is the case for interactions of the genome with lamins ( Kind, 2013).
Where can I find more information? Where can I get DamID (-seq) protocols?
Step-by-step protocols can be found on this website. More detailed discussion of DamID are published in Methods in Enzymology (2006) and Nat Protocols (2007). We hope to publish DamID-seq protocols soon.