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UVA Team Uses Olink's PLA Method to Investigate Histone Modifications at Single-Cell Level

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Researchers at the University of Virginia School of Medicine have devised a method based on Olink Bioscience's proximity ligation assay to observe histone modifications at specific gene loci in single cells.

Detailed in a study published this week in Nature Methods, the technique combines in situ hybridization with PLA to detect histone modifications at single-cell resolution, and could prove broadly useful in epigenetic research, Delphine Gomez, a UVA researcher and first author on the paper, told ProteoMonitor this week.

Much epigenetics work is done via chromatin immunoprecipitation, or ChIP, wherein chromatin is pulled down by antibodies specific for a given histone modification and then analyzed via microarrays or sequencing to determine links between particular modifications and specific genomic regions.

Due to issues of tissue heterogeneity, however, it has been difficult to analyze epigenetic marks in individual cell types. As Gomez and her co-authors noted, populations of individual cells can be obtained through sorting techniques, but this results in the loss of tissue localization information and introduces the possibility that epigenetic marks might be altered during the sorting process.

Cell culture methods are also an option, but cells grown this way may differ from their in vivo counterparts.

By combining ISH and PLA, the UVA researchers were able to devise what Gomez said is essentially a single-cell ChIP assay capable of analyzing histone modifications in formalin-fixed, paraffin-embedded tissue sections.

PLA – which in addition to being sold by Olink is offered by Life Technologies as part of Applied Biosystems' TaqMan product line – uses pairs of antibodies attached to unique DNA sequences to detect a protein of interest. When the antibodies bind their target, the attached DNA strands are brought into proximity and ligate, forming a new DNA amplicon that can then be quantified.

In the UVA assay, the researchers first identified the promoter of interest using a biotin-labeled ISH probe. They followed this with a PLA in which one antibody targeted this ISH probe's biotin label, while the other targeted the histone modification of interest, allowing them to look at links between specific modifications and genomic loci.

In the Nature Methods paper, Gomez and her colleagues used the approach to investigate histone modifications that are markers of smooth muscle cell lineage, looking specifically at dimethylation of lysine 4 of histone H3 at the MYH11 locus.

However, the technique could also be applicable to a variety of other epigenetic questions, she said, noting cancer research in particular.

"We think this could be a really powerful technique in the cancer field [for studying] histology cross sections to get an idea of the epigenetic reprogramming of cancer cells," Gomez said. "The limitation of ChIP is that doing ChIP on a biopsy specimen you have a mixture of healthy and cancer cells. Doing the ISH-PLA [method] with a cancer tumor section, you could get a really precise idea of the epigenetic mechanisms involved in the development of cancer."

Gomez and her colleagues are not the first to find a use for PLA in epigenetics research. In August, a team led by researchers at Kimmel Cancer Center at Thomas Jefferson University in Philadelphia published a paper in Cell using the technique to explore precisely what epigenetic marks were responsible for passing on epigenetic information during cell replication (PM 8/24/2012).

To address this question, the researchers developed a series of assays to investigate in Drosophila what proteins were present in different stages of DNA replication, using the PLA technique to simultaneously detect, for instance, biotin-labeled nascent DNA and various proteins associated with the new strand.

In 2011, Uppsala University researcher Ulf Landegren, a founder of Olink and inventor of the PLA technique, published a paper in PNAS detailing an expanded PLA called 4PLA capable of confirming the simultaneous presence of up to four protein targets (PM 5/13/2011).

The researchers in that paper used the technique to isolate prostasomes – microvesicles secreted by prostate acinar cells that could be useful as prostate cancer biomarkers. However, other outside scientists suggested that the method had potential for epigenetic research, as well.

In particular, University of Pennsylvania researcher Ben Garcia, who was then assistant professor of molecular biology at Princeton University, noted that it could be particularly useful for chromatin biology, which focuses significantly on the post-translational modification patterns of histone proteins. 4PLA using antibodies targeting modification sites of interest could allow researchers to identify proteins with specific modification patterns.

"I think this could have immediate impact in the chromatin biology field," Garcia told ProteoMonitor at the time.

"Mass spec is [currently] really the only way to detect which combinations of modifications on histones — methylations, acetylations — are present," he said. "This could be a great potential affinity-capture type of system that could be used to look at combinatorial modification patterns, either just to detect them or to affinity-purify proteins with certain combinatorial patterns."

According to Gomez, the UVA researchers have no immediate plans to apply the 4PLA technique to their work, but, she said, they plan to continue their research with the ISH-PLA approach, and in fact have used it to look at a variety of additional histone modifications involved in smooth muscle cell lineage.

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