Researchers have developed a method that combines proximity labeling with mass spectrometry-based proteomics and DNA sequencing to capture both protein environments and genomic localization in a single experiment.
The approach, from the Andalusian Centre for Molecular Biology and Regenerative Medicine (CABIMER), Spain, and dubbed PLAMseq, offers a unified framework for studying chromatin-associated proteins without the need for target-specific antibodies.
Here, Román González Prieto, lead author of the study, discusses the analytical and computational considerations behind PLAMseq, the challenges of working with complex genomic regions, and the potential of integrated workflows to advance the study of epigenetic regulation.
What initially motivated your team to develop the new method?
When I started my own laboratory three years ago, I initially set out to study the role of SUMO and Ubiquitin in genome biology. I’d been intrigued by the functioning of histone SUMOylation for quite some time, as histones are inarguably the most important proteins associated with DNA in eukaryotes. However, the lack of specific antibodies for SUMOylated substrates – and the fact that my former postdoctoral supervisor had already tried to raise some with very limited success (and more than ten times my budget) – meant I went into it without much confidence.
Nevertheless, a few years ago, Rosa Barrio´s laboratory published the SUMO-ID method for identifying SUMOylation-dependent interactors for proteins. I thought the approach was brilliant, and due to my prior knowledge in proximity-labelling mass spectrometry experiments, it made identification of the specific readers of SUMOylated histones quite straightforward. Because of this, I was aware that the labelling protein itself was always the most highly enriched protein by several orders of magnitude.
I had just moved back to an institute where lots of labs do genomics (I come from more of a proteomics background myself). After reviewing their protocols and methods, I realized that they were fully compatible with the proximity labelling-MS methods, meaning I could combine them all and, crucially, obtain all of the information in one go. By avoiding the need for antibodies, this proved to be far more cost-effective than the ChIP-seq. It also bypassed the most critical bottleneck, as the approach would otherwise have required a specific ChIP-grade antibody, which was already virtually impossible to obtain.
In addition to its lower cost, the approach also eliminates the need for animals to raise the antibodies, meaning it’s also considered to be more ethical. With all this in mind, I decided to give it a chance.
Could you explain, in a nutshell, how PLAMseq works?
PLAMseq functions by labelling a protein of interest – as well as its environment – with a biotin spray, before it is then frozen at its genomic loci by formaldehyde crosslinking. This enables them to co-purify everything together, followed by identification using MS-based proteomics and DNA sequencing. This bypasses the need for specific antibodies, which traditionally represents a major constraint.
Were there any results that surprised you?
Well, to my knowledge, PLAMseq has enabled the first direct study of the relevance of a SUMOylation mark in genome biology. Truthfully, we were surprised at just how effective it proved to be! We obtained novel insight that actually made lots of sense, with no shift, change or paradigm of note to consider.
What major challenges did your team have to overcome?
The main technical challenge came when analyzing our genomic data. It so happens that SUMOylated histone H1 resides in repetitive regions and is not represented in the reference genomes we were using for mapping. We addressed this by bringing in collaborators with the necessary expertise.
The real “eureka” moment came once when we saw the data from the CTCF PLAMseq, which we had chosen to evaluate the power of the approach. We had not anticipated it would be so effective, and that’s when we became fully aware of the method’s capabilities.
How do you anticipate PLAMseq changing the way researchers investigate epigenetic regulation?
I think PLAMseq finally allows us to study chromatin-associated proteins in a direct and systematic way. Their study has remained elusive up to this point, due either to the absence of specific antibodies, or the transient nature of their association with chromatin. A clear example is transcription factors (TFs): ChIP experiments targeting TFs are notoriously challenging, even though TF misregulation is a major driver of many diseases and disorders.
Looking ahead, what are the next steps for your team?
We are now applying PLAMseq to characterize SUMOylation of other types of histones – and beyond that, to characterize protein interactions, co-localizations and interactions that occurred in the genome. To date, their study has been performed using indirect or ineffectual approaches.
This way, we can now disentangle molecular mechanisms, enabling us to tackle new challenges and design novel strategies to improve human health.
Román González Prieto is a Principal Investigator at the Andalusian Centre for Molecular Biology and Regenerative Medicine (CABIMER), Spain.
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