Howard Hughes Medical Institute (HHMI) scientists have profiled key features of the genetic material inside three types of brain cells and found vast differences in the patterns of chemical modifications that affect how the genes in each type of neuron are regulated. The analysis was made possible by a new method of collecting and purifying the nuclei of specific kinds of cells. Doing this type of study on cells in brain tissue has been challenging because the cells are densely packed and intimately intertwined.

A team of scientists led by Jeremy Nathans, an HHMI investigator at Johns Hopkins University, and Joseph Ecker, an HHMI investigator at the Salk Institute for Biological Studies, published the findings June 18, 2015, in the journal Neuron. Nathans and Ecker collaborated on the studies with researchers in the labs of HHMI investigator Terrence Sejnowski at Salk and Sean Eddy, a group leader at HHMI’s Janelia Research Campus. The researchers say the new method for obtaining cell-type specific nuclei, an adaptation of technology previously used in plants, will enable a wide range of studies in mammalian tissues.

Nathans is a neuroscientist who studies the how cells in the retina—the light-absorbing structure at the back of the eye, which is considered part of the brain—assume their correct identities, and how those cells respond to injury and disease. In 2010, he and Alisa Mo, an MD/PhD student in his lab, learned of a new methods scientists were using to separate genetic material according to cell type. Rather than disentangling entire cells, HHMI investigator Steven Henikoff and postdoctoral researcher Roger Deal at the Fred Hutchinson Cancer Research Center had simplified the problem by isolating only the nuclei of the cells they were interested in. The bulk of a cell's DNA is contained in its nucleus, as well as enough RNA for analyses of gene activity. Henikoff and Deal had developed the method, which they called INTACT, for plants, but Nathans and Mo thought it could be adapted for use in mammals.

Henikoff's team genetically manipulated the cells in their plants so that the cells they wanted to study --and only those cells -- produced a particular protein that protruded from the nucleus. They could then break up the plant cells and fish out the nuclei they were interested in with an antibody that stuck specifically to the nuclear protein they had introduced.

“It's a very clever strategy,” Nathans says. “We got to thinking about this in the context of the brain, where it's not so easy to isolate intact cells.”

Gilbert Henry, a research specialist in Eddy's lab, was already engineering INTACT to work in fruit flies, and offered insight that helped Mo develop a similar system in mice. Mo engineered mice that carried a gene for a tagged nuclear membrane protein Henry had designed, SUN-1, which would be turned on only in cells that made another protein, Cre. Taking advantage of mice that produce Cre in specific cell types, they could breed animals in which their nuclear tag was limited to the cells they wanted to study. Then they could use an antibody that clung to a tag on the SUN-1 protein to retrieve nuclei of those particular cells.

“We built this to be potentially expressable in any cell,” Nathans says. “It just needs the Cre reaction to turn it on, and there are many mouse lines available that express Cre in different cell types.”

For Nathans and Ecker, the first application of the technology was to map out epigenetic modifications in various cell types. Epigenetic modifications are chemical changes to DNA that affect how genes are regulated. They are widespread and important in shaping cells' identities and function.

Journal Link: Neuron, June 18, 2015