A new MIT study reveals that encoding memories in engram cells is controlled by large-scale remodeling of the proteins and DNA that make up cells’ chromatin. In this image of the brain, the hippocampus is the large yellow structure near the top. Green indicates neurons that were activated in memory formation; red shows the neurons that were activated in memory recall; blue shows the DNA of the cells; and yellow shows neurons that were activated in both memory formation and recall, and are thus considered to be the engram neurons. [Image courtesy of the researchers]

Studies in mice by a Massachusetts Institute of Technology (MIT)-led team have uncovered an epigenomic mechanism that appears to play a key role in how the brain forms and recalls memories. The studies suggest that neurons called engram cells encode the details of a new experience, and are then reactivated when the memory of that experience is recalled. The newly reported research indicates that this process is controlled by the large-scale remodeling of cells’ chromatin, the compressed structure that comprises DNA and histone proteins, which can control the activity of specific genes within a cell.

This remodeling, which allows specific genes involved in storing memories to become more active, takes place in multiple stages spread out over several days. “This paper is the first to really reveal this very mysterious process of how different waves of genes become activated, and what is the epigenetic mechanism underlying these different waves of gene expression,” said senior study author Li-Huei Tsai, PhD, director of MIT’s Picower Institute for Learning and Memory. Tsai, together with MIT postdoc and first author, Asaf Marco, PhD, and colleagues, reported their findings in nature neuroscience, in a paper titled “Mapping the epigenomic and transcriptomic interplay during memory formation and recall in the hippocampal engram ensemble.”

The formation and preservation of long-term memories depends on coordinated gene expression and synthesis of synaptic proteins, the authors explained. “These molecular processes act within a specific population of neurons, referred to as engram cells.” Engram cells are found in the hippocampus as well as other parts of the brain, and recent studies have shown that they form networks that are associated with particular memories, and that these networks are activated when that memory is recalled. However, the molecular mechanisms underlying the encoding and retrieval of these memories are not well understood.

“Recent approaches using activity-dependent expression of reporters provided a framework for exploring the engram ensemble, but the molecular mechanisms that govern memory storage and retrieval remain poorly understood,” the team continued. “Specifically, epigenetic modifications and the 3D genome architecture are emerging as key factors in the dynamic regulation of gene expression, and there is an increasing appreciation of their importance in neuronal function, development, and disease.”

Neuroscientists do know that in the very first stage of memory formation, genes known as immediate early genes are turned on in engram cells, but these genes soon return to normal activity levels. The MIT team wanted to explore what happens later in the process to coordinate the long-term storage of memories.

“The formation and preservation of memory is a very delicate and coordinated event that spreads over hours and days, and might be even months—we don’t know for sure,” Marco said. “During this process, there are a few waves of gene expression and protein synthesis that make the connections between the neurons stronger and faster.”

Tsai and Marco hypothesized that these waves could be controlled by epigenomic modifications, which are chemical alterations of chromatin that control whether a particular gene is accessible or not. Previous studies from Tsai’s lab had shown that when enzymes that make chromatin inaccessible are too active, they can interfere with the ability to form new memories.

To study epigenomic changes that occur in individual engram cells over time, the researchers used the targeted recombination in active populations (TRAP) mouse model—in which they could permanently tag engram cells in the hippocampus with a fluorescent protein when a memory is formed. “In this study, we used an activity-dependent tagging system in mice to determine the epigenetic state, 3D genome architecture, and transcriptional landscape of engram cells over the lifespan of memory formation and recall,” they explained.

These mice received a mild foot shock that they learned to associate with the cage in which they received the shock. When this memory forms, the hippocampal cells encoding the memory begin to produce a yellow fluorescent protein marker. “Then we can track those neurons forever, and we can sort them out and ask what happens to them one hour after the foot shock, what happens five days after, and what happens when those neurons get reactivated during memory recall,” Marco commented.

The results of the team’s experiments showed that right after a memory is formed, many regions of DNA underwent chromatin modifications. In these regions, the chromatin effectively becomes looser, allowing the DNA to become more accessible. To the researchers’ surprise, nearly all of these regions were in stretches of DNA where no genes are found, but which contain noncoding sequences called enhancers, which interact with genes to help turn them on. The researchers also found that in this early stage of memory formation, the chromatin modifications did not have any effect on gene expression.

When they next analyzed engram cells five days after memory formation, they found that as memories were consolidated over that period, the 3D structure of the chromatin surrounding the enhancers changed, bringing the enhancers closer to their target genes. This still didn’t turn on those genes, but it primed them to be expressed when the memory was recalled. Next, the researchers placed some of the mice back into the chamber where they received the foot shock, reactivating the fearful memory. In engram cells from those mice, the researchers found that the primed enhancers interacted frequently with their target genes, leading to a surge in the expression of those genes.

“Our findings reveal that memory encoding leads to an epigenetic priming event, marked by increased accessibility of enhancers without the corresponding transcriptional changes,” the team noted. Memory consolidation subsequently results in spatial reorganization of large chromatin segments and promoter–enhancer interactions. Finally, with reactivation, engram neurons use a subset of de novo long-range interactions, where primed enhancers are brought in contact with their respective promoters to upregulate genes involved in local protein translation in synaptic compartments.”

Many of the genes turned on during memory recall are involved in promoting protein synthesis at the synapses, helping neurons to strengthen their connections with other neurons. The researchers also found that the neurons’ dendrites—branched extensions that receive input from other neurons—developed more spines, offering further evidence that their connections were further strengthened.

The authors acknowledged that their studies did have some limitations. Nevertheless, they concluded, “… our study provides the first comprehensive landscape of 3D genome architecture during different phases of memory formation, as we showed re-localization of large chromatin segments and specific promoter–enhancer interactions dynamic within these compartments that enable fine-tuning of different transcriptional programs … Collectively, it appears that engram cells are marked at the epigenetic level, and the interplay among chromatin accessibility, 3D genome architecture, and promoter–enhancer interactions describes a well-coordinated system that leads to a delayed transcriptional surge during engram reactivation.”

Marco further noted that the study is the first to show that memory formation is driven by epigenomically priming enhancers to stimulate gene expression when a memory is recalled. “This is the first work that shows on the molecular level how the epigenome can be primed to gain accessibility. First, you make the enhancers more accessible, but the accessibility on its own is not sufficient. You need those regions to physically interact with the genes, which is the second phase. We are now realizing that the 3D genome architecture plays a very significant role in orchestrating gene expression.”

Although the researchers did not explore how long these epigenomic modifications last, Marco believes they may remain for weeks or even months. He now hopes to study how the chromatin of engram cells is affected by Alzheimer’s disease. Previous work from Tsai’s lab has shown that treating a mouse model of Alzheimer’s with an HDAC inhibitor, a drug that helps to reopen inaccessible chromatin, can help to restore lost memories.

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