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Kimmel Cancer Center Researchers ID HMT Proteins as Possible 'Epigenetic Marks' Via PLA-Based Study


A team led by researchers at the Kimmel Cancer Center at Thomas Jefferson University this week published a study in Cell suggesting that histone-modifying proteins such as histone methyltransferases could be responsible for passing on epigenetic information during cell replication.

The findings, said Alexander Mazo, a Kimmel researcher and author on the paper, run counter to conventional wisdom in the field, which has typically held that the modified histones themselves – not the modifying proteins – are responsible for transfer of epigenetic information.

Maintenance of gene expression patterns over the course of the cell cycle relies on "epigenetic marks" – protein or nucleic acid modifications that work to reestablish the parent cells' chromatin structure in the nucleosomes of the resulting daughter cells.

One such mark is DNA methylation. A second kind of mark is thought likely to be encoded in chromatin-associated proteins — modified histones, in particular.

"It's been assumed that [in DNA replication] all the [chromatin-associated] proteins are getting kind of dispersed from the DNA, and that these modified histones are kind of jumping over the [DNA replication] fork," allowing them to be incorporated into the newly formed nucleosome, Mazo told ProteoMonitor.

This model, however, he said, has little actual experimental evidence backing it.

The idea that "[modified histone] proteins jump over the [DNA replication] fork... is not supported by experimental evidence," he said. "People just assume this."

The notion, meanwhile, that histones carry epigenetic information comes from experimental studies, Mazo said. However, he noted, this experimental evidence is indirect. Researchers have shown that histone-modifying proteins like HMTs can recognize histone modifications, which, Mazo said, has led to the theory that modified histones are retained throughout DNA replication and then recruit the necessary HMTs to the newly formed nucleosome, "restoring the full extent of the histone modification on both strands" of DNA.

Inherent in the theory, though, Mazo noted, is something of a chicken-and-egg question. Do the modified histones remain associated with the DNA throughout replication and then recruit the necessary modifying proteins to the new nucleosomes? Or do the modifying proteins remain associated with the DNA and then modify newly recruited histones to reconstitute the parent cell's epigenetic information?

To address this question, Mazo and his colleagues developed a series of assays based on Olink Biosciences' proximity ligation assay technology to investigate in Drosophila what proteins were present in various stages of DNA replication.

PLA – which is also 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.

Using the PLA technique, the researchers were able to detect the presence of target proteins during different DNA replication events by simultaneously targeting both analytes known to be linked to stages of replication and the proteins under investigation.

For instance, proliferating cell nuclear antigen is a key protein in DNA synthesis, and, the authors wrote, "any protein that is near the [replication fork] will be close to PCNA in vivo." Using this knowledge, they created PLAs targeting PCNA along with proteins, including methylated histone H3, that they thought might function as an epigenetic mark.

If a PLA for PCNA and a given protein generated signal, that indicated that the two were in close proximity and that that protein was retained during replication.

The Thomas Jefferson team performed similar experiments throughout the replication process, using, in addition to PLAs to PCNA, PLAs to biotin-labeled nascent DNA, which enabled them to track at what time points in DNA synthesis target proteins are associated with the new strand.

"In all these approaches we consistently see – no matter if [the nascent DNA] is 200 [base pairs] or 300 bp or 10 kb or through the whole S phase – we do not see methylated histones [associated with] DNA," Mazo said. "They do not show up until later when we are getting out of S phase into the next transcriptional phase."

PLAs showed that the HMTs thrithorax – Trx – and enhancer-of-zeste – E(z) – on the other hand, were consistently associated with DNA throughout replication – a fact, Mazo said, that suggests they, and not the modified histones – function as the epigenetic marks.

"These proteins were found to remain stably associated with DNA," he said, noting that the researchers had chosen them as potential candidates because "they are known to be specifically associated with very specific regions in the genome, and they are known to be important in transcription."

Mazo said the researchers aim to use the techniques detailed in the Cell paper to investigate a wide number of proteins and their associations with DNA during replication.

"We're now involved in testing a whole bunch of other proteins – either those with histone methylation or demethylation activities or nucleosome remodeling activities," he said. "So we're extending this in a rather big way in Drosophila, and we're planning to do it in human cells also."

Mazo also noted that the researchers are interested in expanding the number of proteins they can investigate simultaneously. Although not currently offered commercially, an expanded PLA capable of confirming the presence of up to four targets was published last year by Uppsala University researcher Ulf Landegren, a founder of Olink (PM 5/13/2011).