How does the nose remember that it is a nose? Or does the eye remember that it is an eye?
When scientists investigate the question of how cells remember what type of cells they are supposed to be, or their genetic lineage, it is important to understand how cells express different genes without changing the DNA sequence itself.
But studying this topic is tricky: Researchers can purify the proteins that drive gene expression, put them in a test tube and watch them correlate. But doing so inside the cells’ nucleus, their native environment, has hitherto been impossible.
Now, a team of researchers at three University of Michigan laboratories has been able to trace how the protein binds to a chromatin substrate within a living cell by creating a collaboration that combines the latest high-resolution imaging technology with a synthetic protein. Computational design and modeling. Their results were published in Science Advances.
“The biological question we ask is, ‘How do cells actually remember past experiences? And how these experiences also lead to cells creating distinct identities, like in the case of the human body where you have lineages of cells that make up neurons or blood cells or brain cells, all of which actually maintain their identities for generations,” said lead author Kaushik Ragunathan, professor of biological chemistry. At UM College of Medicine.
“One example I like to think of is that if you cut off your nose, you wouldn’t get a hand growing there, even though the genomes in your nose and the genomes in your hand are exactly the same.”
Cells control how genes are expressed and which copy of the DNA sequence contained within each cell, even though this sequence is the same across all cells in the body. One of the ways they control expression is by changing how tightly the DNA is assembled within the nucleus using proteins called ‘histones’. Histones can be modified by adding small chemical tags that regulate how tightly the DNA is wound around and thus whether genes can be expressed.
Proteins that have the ability to read, write and erase these histone tags explore DNA within the cell nucleus very quickly — on the order of milliseconds, according to Rajunathan. Ultimately, all this epigenetic information must be inherited across generations, but recognition of these marks is a complex process that involves chromatin binding and proteins that meet and interact with each other amid the chaos of all other potential competing interactions within the cell.
The ability to understand each step of the process — and thus enable control over how epigenetic information is inherited — intrigued co-author Julie Payten, professor of chemistry and biophysics.
Biteen uses single-molecule fluorescence imaging to track individual proteins within cells. Her lab can see where these proteins bind to chromatin, and Raghunathan’s expertise is in the molecular mechanisms that underpin how histone modifications and histone-associated proteins interact. These two worlds must come together so that the biochemistry of what’s going on in a test tube outside cells can be tested to see what’s going on inside.
“The timing of this process is very important to ensure that the right genes are silenced in the right place at the right time,” Pettin said. “What caught me about this project is that in the lab — in a test tube — you can purify two proteins, watch them bind, and see how well that binds, or what the affinity is for each other. That tells you what. Can It happens in cells, but not what Do It occurs in cells.
Biteen and Ragunathan worked with Peter Freddolino, assistant professor of biological chemistry, computational medicine and bioinformatics at UM School of Medicine, to combine computational modeling with their experimental results.
“This is where our collaboration gets really strong,” Petten said. “On the other hand, seeing the molecules is very useful and knowing how fast the molecules are moving helps a lot in terms of understanding what is possible inside the cell, but here we can take a leap forward by jamming the system even in unnatural ways in order to understand what these different motions of the molecules mean. actually in the cell.”
While epigenetic marks are extremely important for the maintenance of different tissues in complex organisms such as humans, they also play an important role in regulating the genes of single-celled organisms such as yeast. The team focused on a type of HP1 protein in yeast cells called Swi6. This family of proteins is associated with a specific type of histone modification in the cell to enforce gene silencing. By fusing fluorescent labels with Swi6, Bitee’s lab watched Swi6 move inside the cell nucleus.
As Swi6 searches for the correct binding site on the DNA, Petten said, it is moving quickly. When it finds its target, it slows down noticeably. The movement of the protein inside the cell is like gears in a car and things can move at different speeds depending on who the proteins are interacting with.
“By these spaghetti tracks that we enter inside the cell, we determine how much time they spend searching and how much time they spend laying down constraints,” Petten said. “The amount of time they spend in motion tells us how strongly they interact and their biochemical properties.”
And while Betaine’s lab can measure movements in a cell on a scale of tens of milliseconds, much of the biochemistry that happens in a cell happens faster, she said. Freddolino took this experimental information and developed models to estimate the ability of Swi6 proteins to jump between binding states identified in the experiments.
Freddolino’s modeling took into account experimental measurements and potential biochemical properties, which include how Swi6 molecules interact in the cell. These interactions include molecules floating freely in the cell solution, molecules bound to DNA, and molecules “holding hands” with each other, he said.
“My lab wanted to come up with a more accurate model that estimated what was likely a set of molecular states for proteins and their ability to jump between those states, which would then lead to the imaging data generated by the Vineyard Lab,” Fridolino said.
“Having this numerical model allows us to do computational experiments on what happens if protein binding is twice as fast as we think. What if it’s 10 times faster as we think? Or 10 times slower? Could that turn up the data? Fortunately, in these case, we were able to show that the relevant processes were indeed captured in fluorescence microscopy.”
After determining the binding properties of natural Swi6, the researchers tested their findings by redesigning Swi6 from its components to see if they could replicate some of its biochemical properties, Raghunathan said. This allowed the researchers to determine that the imaging and modeling conducted in the first part of the paper reflected how the protein bound in its native environment.
“Can we do what nature has done over millions of years and make a protein that in many ways has properties similar to those of Swi6 in cells?” Raghunathan said. “In in vivo biochemistry, which is what we decided to call this, was not something that was thought to be possible inside living cells, but we have shown that this is completely possible using imaging as a method. We are using this project as a basis for understanding how these epigenetic states can be created and maintained via generations”.