Kenneth Zaret is the Joseph Leidy Professor at the Smilow Center for Translational Research at the University of Pennsylvania. His lab works to understand how genes are activated and different cell types are specified in embryonic development. They investigate the molecular signaling pathways that commit an undifferentiated embryonic cell, the endoderm, to a particular cell type fate, using the specification of liver and pancreas cells as a model. In addition, the laboratory investigates ways that gene regulatory proteins control the packaging of DNA in the cell nucleus, to control gene activity. He discussed his recent research with SCINQ.
SCIENTIFIC INQUIRER: Let’s start this off with some background. What led researchers to assume that gene expression was turned off during cell division? What did they believe happened before, during, and after division?
KEN ZART: For decades, methods with relatively low sensitivity had suggested that genes become silent during the period when cells physically divide (mitosis). This made sense because during mitosis, the chromosomes condense markedly, they line up, and are divided equally among the daughter cells after cell division. It seemed reasonable that genes were silenced during the period when the chromosomes were so busy undergoing proper segregation into daughter cells. In addition, work from my lab and many others had shown that proteins that regulate gene expression were often, but not always, lost from the chromosomes during mitosis.
SI: What made you suspect things were not so quiet after all?
KZ: We initially assumed that genes were silent in mitosis and developed very sensitive technology to detect gene expression, in order to assess how the genes come back on when cells exit the mitotic period. We wondered if all the genes came on at the same time, which would suggest that existing gene regulatory proteins in the cell just re-bound the sites they bound prior to mitosis, or if there was a cascade of events where different genes were turned on at different times during mitotic exit, which would suggest a coordinated, regulatory process at work.
We found the latter; that is, that only about half the genes come on right away during mitotic exit, and there is an exquisite network by which some of those genes then get turned down and other genes come on later. All of this suggests interesting regulatory processes at work. But in addition, and more surprisingly, we discovered that many genes in fact do not become silent in mitosis; that they are expressed at a low level.
SI: Your approach to investigating gene expression during cell division involved reframing the question being asked. Rather than trying to find the best time to manipulate a cell’s fate, you explored how a cell shifts from non-expression to full expression. Why?
KZ: We are interested in gene regulatory mechanisms and my lab had previously published that a gene regulatory protein of interest to us remained bound to the chromosomes during mitosis, suggesting it has a “bookmarking” function to retain the memory of genes that were expressed in the previous cell cycle. By looking at the genes and their expression before, during, and after mitosis, we thought we could link the bookmarking phenomenon to changes in the expression of genes that are targeted by the bookmarking factors. But instead we saw something much more generally interesting and unexpected.
SI: How was gene expression monitored during division?
KZ: Many prior studies had fixed cells with chemicals, in order to monitor how genes are expressed. It has become clear that the cell fixing methods can be fraught with artifacts and in general the methodology lacks a good dynamic range. We adapted a method to feed cells precursors to RNA, as a pulse during mitosis, and ask how efficiently the precursors were incorporated into RNA. Thus the method worked on live cells.
SI: What did you discover was actually taking place?
KZ: We discovered that very many genes (over 8,000) in the cell are expressed at a low level in mitosis. They are also expressed with a low dynamic range, which differs greatly once cells exit mitosis and express different genes at very different levels.
Since it is known that gene expression is stimulated strongly by distant regulatory sequences called enhancers, and that long-range interactions across the chromosome are broken down in mitosis, it suggests that enhancers are not influencing gene activity during mitosis.
We therefore speculated that the low level expression seen in mitosis may largely reflect the activity of the local “promoter” elements right at the gene. This model shifts the attention to what is happening at promoters, rather than distant enhancers, to keep genes on at a low level during mitosis.
SI: What genes were still being expressed and to what level? Can you speculate as to why some genes are expressed and others suppressed?
KZ: So many genes are still expressed in mitosis that there is not a particular subset that stands out; the expressed genes represent most of the networks of the cell. More interestingly, during mitotic exit, when cells ramp gene activity back up to normal, we found that the first genes to be activated are largely involved in rebuilding the cell. This makes sense because the cells have to complete cell division and make new organelles, structures, etc. Later genes to turn on relate to cell specialization.
SI: What are the broader implications of the study’s findings? How can it potentially be applied practically?
KZ: The study suggests that to understand how cells retain their gene expression “memory” during the period of low activity in mitosis, it probably is good to focus on the local gene regulatory sequences at promoters. Also, during mitotic exit, when there are cascades of different gene expression programs turning on at different times, there may be more complex regulation that needs to be understood to learn how cells restore their gene regulatory network between periods of cell division.
This is an example of basic untargeted research; we really can’t predict the applications yet. By understanding how cells normally function, genetically, I anticipate that it will help us figure out how to control the process as needed, e.g. to reprogrammed cells from one type to another.
SI: How did you come to a life in science? Did you always want to be a scientist?
KZ: I definitely liked science in high school and give a lot of credit to two teachers I had in math and biology who reinforced my interests. My bio teacher recommended me for a summer NSF program during junior and senior year and that really opened my eyes about how enjoyable I found science. I heard that Reagan eventually cut that program to provide tax breaks for rich people and I hope that our current tax plan doesn’t make things worse for young kids like I was.
SI: What are the most important traits for a researcher to have?
KZ: Curiosity, persistence, and being able to look at data in a way that you didn’t expect.
SI: Who are your biggest professional influences?
I get a lot out of my colleagues at work and members of my lab. I try to cultivate an intense but open environment in the lab where we learn from each other; and input from my faculty colleagues at Penn is invaluable as well. I keep it local.
For more information about Ken Zaret’s research, visit his lab page.