It is no secret that DNA organisation in the nucleus is not random. Chromatin architecture is a highly dynamic structure and as it’s been long known in biology, changes in structure relate to changes in function. Indeed, various DNA binding proteins can affect chromatin folding and alter gene expression^[1]. Hence, identifying protein-DNA interactions is important for understanding chromatin rearrangement which occurs during normal development and disease progression, and how it affects gene expression. Amongst other tools, a technique called DNA adenine methyltransferase identification, or DamID, has been developed to study such interactions in real-time on a single-cell level.

In November 2019, Jop Kind visited IGMM to give a talk about DamID and its applications in studying genome organisation. Jop Kind leads a Developmental Epigenomics group at Hubrecht Institute, Netherlands. His group is focused on understanding how spatial and temporal changes in genome architecture affect gene expression. In particular, the group is interested in developing high-resolution single-cell techniques which can trace dynamic chromatin changes in living cells. Jop Kind began his talk by introducing the DamID technique and its applications, followed by the demonstration of how his group is using DamID to study changes in genome architecture during the development of early mouse embryos and presented some of their recent results.

DamID is a molecular technique developed to identify interactions between DNA and its binding proteins. DamID utilises bacterial DNA methyltransferase which, unlike its eukaryotic counterpart, does not methylate cytosine but instead establishes a methyl mark at the adenine residues (m6A) found in the GATC motif. Therefore, a fusion of the bacterial DNA methyltransferase (Dam) to the DNA-binding protein will result in the methylation of GATC motifs nearby the protein’s target. Next, DNA methylated by Dam can be enriched by using m6A-sensitive endonuclease, amplified by PCR and prepared for sequencing^[2]. Jop Kind has also mentioned the development of other tools based on DamID such as scDam&T. scDam&T is a fusion method capturing protein-DNA interactions and transcriptomics on a single-cell level. This tool could be used to establish a link between the binding of the epigenetic regulators and transcription. In addition, Jop Kind group has further expanded the DamID toolkit by fusing it to GFP to allow live visualisation of chromatin dynamics while fusion to the degradation tags allowed conditional Dam expression at different developmental stages.

A particularly interesting DamID application mentioned by Jop Kind was an observation of spatial chromatin reorganisation of the pre-implantation mouse embryos. Upon fertilisation, zygotic genome undergoes major changes involving genome rearrangement and erasure and re-establishment of epigenetic marks. However, the exact mechanism behind the chromatin rearrangement and its role in the developmental programme remain unclear. Jop Kind group utilised DamID to map genomic interactions with nuclear lamina in the fertilised zygote, 2-cell and 8-cell stage embryos. The results demonstrate that lamina-associated domains (LADs) are established de novo in the zygote before the transcriptional activation and therefore are not inherited from the maternal genome. Once established, LADs undergo further repositioning with distinct patterns observed at different developmental stages which correlate with transcriptional changes. For example, at the 2-cell stage newly established inter-LADs coincide with upregulation of gene expression, while genes found in previously established LADs are lowly transcribed ^[3]. Hence, the obtained results suggest that nuclear reorganisation observed during development is tightly linked to the transcriptional changes. It would be interesting to see what future studies will tell us about the mechanisms behind the establishment of LADs and their relationship with epigenetic marks such as histone modifications and DNA methylation.

Protein-DNA interactions have been of interest to the researchers for many decades and many tools for studying chromatin dynamics have been developed. However, DamID offers several advantages compared to other methods such as ChIP. First of all, it does not rely on the use of antibodies and therefore avoids the problem of poor antibody specificity or lack thereof. Additionally, it can identify previous protein’s binding locations instead of capturing binding position at a given time. Moreover, DamID allows observation of genome rearrangement in vivo on a single-cell level. Last, but not least, its versatility provides an opportunity for the development of the new adaptations by fusing Dam to other molecular tools. So far, DamID has proved its application is studying chromatin dynamics on its own and in combination with other tools and it would be interesting to see further expansion of the DamID-based toolkit and all the exciting discoveries it will bring.

References

1. Sivakumar, A., de las Heras, J. and Schirmer, E. (2019). Spatial Genome Organization: From Development to Disease. Frontiers in Cell and Developmental Biology, 7.
2. Aughey, G., Cheetham, S. and Southall, T. (2019). DamID as a versatile tool for understanding gene regulation. Development, 146(6), p.dev173666.
3. Borsos, M., Perricone, S., Schauer, T., Pontabry, J., de Luca, K., de Vries, S., Ruiz-Morales, E., Torres-Padilla, M. and Kind, J. (2019). Genome–lamina interactions are established de novo in the early mouse embryo. Nature, 569(7758), pp.729-733.

DamID: Where does it bind? / Institute of Genetics and Cancer by blogadmin is licensed under a Creative Commons Attribution CC BY 3.0

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