Imaging method reveals DNA’s packaging in three dimensions

A new technique creates a detailed picture of chromatin — the coiled complex of DNA and proteins — in individual brain cells. Using the method in mouse brain tissue, researchers have discovered fresh details about how loss of the MeCP2 protein, which causes Rett syndrome, affects the structure of the genome.

The most striking finding, the researchers say, is that MeCP2’s absence affects chromatin structure in only certain types of neurons. Chromatin in the affected neurons is more tightly packed than usual, and chemical tags that are typically confined to small swaths of chromatin are redistributed to other areas. These changes make the DNA less accessible, and may turn off genes.

The findings, described 24 September in Cell, highlight a pitfall of traditional techniques, which estimate chromatin structure from a mishmash of neurons¹. Understanding cell-type-specific differences in chromatin structure may have important implications for treating Rett syndrome, the researchers say.

“If you want to fix this disease, you have to take into account that heterogeneity of responses due to loss of MeCP2,” says lead investigator Gail Mandel, senior scientist at the Vollum Institute at Oregon Health & Science University in Portland. “It’s going to be tough to fix the entire brain with one solution.”

The new method may also help researchers better understand the effects of autism-linked mutations in genes other than MeCP2.

“A lot of genes implicated in autism and neurodevelopmental disorders are chromatin-remodeling factors,” says Jeffrey Neul, chair of child neurology at the University of California, San Diego, who was not involved in the study. “This is a way to start looking at these and how they change chromatin.”

Capturing chromatin:

The new method is based on array tomography, a technique originally designed to visualize synapses, the points of contact between neurons. With this approach, researchers typically shave brain tissue into ultrathin slices — about 100 times thinner than the width of a human hair — and then label synaptic proteins with fluorescent molecules. A computer stitches the images together to reconstruct the three-dimensional structure of synapses.

Mandel and her colleagues adapted the technique to visualize chromatin by fluorescently labeling DNA. Tightly packed chromatin shows up as bright spots in the nucleus, whereas unraveled regions appear faint. This allowed the researchers to gauge the density of chromatin packing in individual cells.

To examine the effects of MeCP2 on chromatin structure, the researchers used brain slices from adult female mice lacking one copy of the gene. The remaining copy is randomly silenced in half of the mice’s cells through a natural process called X inactivation. This means that half of neurons are completely devoid of MeCP2 protein.

Mandel and her team first looked at pyramidal neurons in the hippocampus, a brain region involved in learning and memory. In these cells, loss of MeCP2 increases the amount of DNA in the tightly packed chromatin by 20 percent.

The researchers then looked at neurons in the cerebellum, which coordinates movement. Certain neurons in this region do not show changes in chromatin structure with the loss of MeCP2. The reason for this cell-type specificity is unclear.

Tag team:

The researchers also labeled the brain slices with fluorescent markers for five types of chemical tags that can cause chromatin to become tightly packed, turning off gene expression. They visualized each tag sequentially using a different marker for each tag — an improvement over having to probe the locations of each tag in a different sample.

“Before, we could ask, ‘Where is a particular mark?’ These guys can ask about one mark after another over and over again,” says Erez Aiden, assistant professor of genetics at Baylor College of Medicine in Houston, who was not involved in the study.

In the absence of MeCP2, one chemical tag is redistributed to regions of chromatin in hippocampal neurons that it doesn’t normally occupy, the researchers found. It also leads to a slight drop in gene expression.

“Loss of MeCP2 is causing the boundary of that repressive mark to disappear so that it moves into another territory,” Mandel says.

Missing the mark: A chromatin tag that represses gene expression (red) shifts to new areas of the nucleus of a neuron lacking MeCP2 (right) compared with one that includes the protein (left).

Mandel and her colleagues examined only a few of the many chemical tags that can modify chromatin, and only those that repress gene expression. The next step would be to look at other repressive tags as well as those that activate genes.

The researchers plan to use the method to assess chromatin density around specific genes. More generally, the technique could help to answer fundamental questions about how certain parts of the genome compartmentalize within the nucleus.

“This is a very valuable piece of work that pushes the ball forward on an extremely important set of questions,” Aiden says.