Runways of reprogramming

What's the genomic traffic control for embryonic stem cells?

Waiting for clearance at the end of a runway, an airplane readies for its slot among the tens of thousands of flights each day in the United States. Every move the plane will make, from takeoff throughout its flight path toward a safe landing, will be controlled by an immensely intricate air traffic control network that is built on highly precise maps and navigation systems.

“If you didn’t have those maps and navigation tools, you wouldn’t be able to land in Boston on a foggy night,” notes Whitehead Member Richard Young, an accomplished pilot. “It just would not be possible.”

“In biomedicine, one key problem is that we don’t have anything like these maps for cell regulatory circuitry,” Young says. “But in the past several years we have developed technology and concepts that have enabled us to create richer maps and better identify how gene regulators are working together to control cell state.”

He and his colleagues are mapping the circuitry of embryonic stem cells (ESCs) across the entire genome, tracing the control pathways that such cells employ to maintain their embryonic state or, if instructed to do so, assume a new identity.

Grown in the lab from cells harvested from days-old embryos, ESCs are poised on the runway of development, waiting for clearance.

These cells hold unique powers. With proper care, they can renew themselves indefinitely, or they can develop into almost any kind of tissue type—an attribute called pluripotency.

Actually, these powers are not unique to ESCs—they are shared with the recently discovered “induced pluripotent stem” (iPS) cells, ESC-like cells created by genetically reprogramming adult cells.

Understanding the intricate circuitry of ESC and cells promises huge advances in basic biomedical research and eventual therapies. Such research will provide clues into development of both normal and diseased cells and tissues, allow new kinds of models for illnesses and potentially lead to new disease diagnostics and therapeutics.

But a whole lot of mapping must come first.

Masters of the genome universe
“The sequencing of the human genome gave us the static information about the building blocks involved in making cells throughout the human body, but it didn’t provide the intricate information about how genes are turned on or off,” says Alex Marson, formerly an MD/PhD student in the Young lab and now an MD candidate at Harvard Medical School. “But in the last few years new technologies have enabled us to trace the molecular switches upstream that are actually controlling gene expression.”

Working in conjunction with colleagues in the labs of Whitehead Member Rudolf Jaenisch and Affiliate Member David Gifford, Young lab researchers several years ago began probing ESCs to identify their “master transcription factors”—a handful of key regulators that direct and switch thousands of genes on or off in the cells.

Transcription factors are proteins that bind to specific areas of the genome to activate and deactivate specific genes. Master transcription factors are the linchpins for this process. Studying ESCs, the researchers located a surprisingly small set of these key players, including Oct4, Sox2, Nanog and Tcf3. (Other cell types have different sets.)

“Master transcription factors are so powerful they can reprogram a whole genome,” says Young. “But the problem is that master transcription factors are known for only a small number of cell types.”

To complicate matters, several other kinds of regulators work together with master transcription factors to drive gene expression within all cell types.

Signaling molecules, for example, can exert regulatory influences from outside the cell. Chromatin regulators, a set of proteins that help to package DNA for transcription, play an important role in development. A couple of years ago, work by the Young and Jaenisch labs revealed that a key set of chromatin regulators called Polycomb group proteins act in concert to silence transcription until it’s time for development to begin.

“During development, the cell detects neighboring cells on all sides, and this information helps determine the fate of the cells,” explains Young. “Master transcription factors respond to the signals from neighboring cells and cause the packaging material to change around active or silent genes.”

This summer, he and his colleagues shed light on how these signaling molecules, master transcription factors and chromatin regulators work together—and they illuminated the role of one more player in gene regulation, microRNAs.

Help 'Wnt'ed
In the August issue of Cell Stem Cell, scientists from the Young and Jaenisch labs connected the Wnt pathway, a major network of signaling molecules found in many organisms, directly to the heart of the control circuitry of ESCs where master transcription factors bind to DNA. This signaling pathway governs many cellular processes and has been implicated in a wide range of diseases, including cancer.

“We were surprised to find the Wnt pathway at the center of the gene regulation universe for ESCs,” says Young. “We knew that the Wnt pathway was involved in early development and somehow played a role in these cells, but until now we weren’t quite sure how it fit into the regulatory circuitry.”

The findings connect the Wnt pathway directly to the master transcription factor Tcf3 and reveal that Tcf3 in turn can control the levels of the pluripotency master regulators Oct4, Sox2 and Nanog. Moreover, a protein within this pathway known as Wnt3a promotes the conversion of adult cells into iPS cells.

“We show that the Wnt pathway helps to control pluripotency by regulating Oct4, Sox2 and Nanog,” says Marson. “By manipulating this pathway, we can help stem cells maintain their pluripotency and can modify and enhance the reprogramming process.”

Currently iPS cells can be created by reprogramming adult cells through the use of viruses to transfer four genes (Oct4, Sox2, c-Myc and Klf4) into the cells’ DNA. The activated genes then override the adult state and convert the cells to iPS cells.

However, this method wouldn’t work in the clinic, since the viruses typically employed in the process, called retroviruses, can insert DNA almost anywhere in the cell’s genome, potentially triggering the expression of cancer-causing genes.

To complicate matters, c-Myc itself is a known cancer-causing gene. And while earlier research has shown that c-Myc is not strictly required for the generation of iPS cells, its absence makes the
reprogramming process time-consuming and highly inefficient.

To bypass these obstacles, the Whitehead researchers replaced the c-Myc retrovirus with the protein produced by the Wnt3a gene. When added to the fluid surrounding the cells being reprogrammed, Wnt3a proteins enhanced the conversion of adult cells to iPS cells.

“iPS cells hold great potential for future medicine, but we must learn how to generate these cells in a manner that is safe for clinical therapies,” says Young. “This advance in reprogramming is one key step toward that goal.”

Mapping microRNAs
In a second paper reported in August, this one in Cell, Young and colleagues added yet another new dimension to the map—pinpointing where members of a fourth class of gene regulators, tiny snippets of RNA known as microRNAs, interact with the master transcription factors.

“This had been a missing piece of the map,” says Young. “Transcription factors and microRNAs help to keep stem cells in a pluripotent state and determine whether they differentiate into a specific cell type—whether it’s a liver or brain cell or another type of cell.”

It had been known that microRNA machinery is important in maintaining embryonic stem cells in their embryonic state. But previous studies only offered partial views of how microRNA genes fit in with the overall gene regulation circuitry.

Filling in these gaps required mapping the sites in the genome from which microRNA genes start, explains Stuart Levine, co-lead author on the paper and a postdoctoral scientist in Young’s lab.

The researchers then mapped where the four master transcription factors (Oct4, Sox2, Nanog and Tcf3) bind to DNA and launch gene expression, allowing them to identify the DNA sites where transcription factors overlap the microRNA genes.

“Knowing where genes start is essential to understanding their control,” says Levine. “Based on our knowledge of microRNAs’ gene start sites, we discovered how these genes are controlled by the master transcription factors.”

They found that the four core transcription factors are interacting with both active and repressed microRNA genes. The repressed genes are silenced by Polycomb proteins.

“We now have a list of which microRNAs are important in embryonic stem cells,” says Marson, also co-lead author on the paper. “This gives us clues about which microRNAs you might want to target to direct an embryonic stem cell into another type of cell. For example, you might be able to harness a microRNA to help drive an embryonic stem cell to become a neuron, aiding with neurodegenerative disease or spinal cord injury.”

Moreover, the results give scientists a better platform for analyzing microRNA gene expression in cancer and other diseases.

“Cancer stem cells, which are capable of promoting tumor growth, have gene expression programs surprisingly similar to embryonic stem cells,” notes Young. “By understanding ESCs we may understand how cancer stem cells lose control and become tumor-promoting cells.”

Flying with new maps
As they learn more about how master transcription factors, chromatin regulators, signaling pathways and microRNAs work together to control the gene expression program in ESCs, the scientists hope they can better identify the genomic traffic controls for other cell types.

This approach promises dramatic advances both in modeling diseases and in potential regenerative therapies.

“By understanding the circuitry within healthy cells, we can better understand what happens within this circuitry to alter cell state and cause disease,” says Young.

“For example, severe auto immune diseases result when subtle mutations occur within FoxP3, the master regulator for T cells,” he says. “We are just beginning to understand exactly how FoxP3 is involved and how changes in the genes it regulates can result in disease. Understanding these mutations may help us to design small molecule drugs to reprogram the cells and counteract the effects of mutations occurring with FoxP3.” This potentially could lead to powerful treatments for autoimmune diseases.

Down the road as regenerative medicine begins to arrive, “each patient will need to have his or her own personalized, custom-designed set of cells that the immune system will recognize as friend and not foe,” says Young. “We need to continue discovering the design principles of each cell in order to safely reprogram cells for therapeutic purposes.”

“With better knowledge of cell circuitry, we may ultimately be able to take a person’s skin cells and reprogram them directly into a specific cell type, without necessarily turning the cell back into its embryonic state,” he adds.

Eventually, scientists may be able to fashion the perfect genetic runway—taking patient cells and redesigning their genetic cargo to learn about their disease, or to arrive safely at a cure.

For more on this research, including a short movie on work covered here, see www.whitehead.mit.edu/stemcells.