Scientists from the Center for Genomic. Regulation in Barcelona, Spain have discovered a genetic regulatory network that revolves around a protein called Mel18. This regulatory network acts as a genetic switch during the differentiation of embryonic stem cells into heart muscle cells.
Mel18 acts in combination with a vitally important set of proteins called the “Polycomb Regulatory Complexes” or PRCs. PRCs are probably one of the major repressors of genes in adult and embryonic stem cells, and in this paper, Luciano De Croce and his colleagues showed that Mel18 acts with the PRCs to suppress gene expression.
Beyond that, however, once differentiation occurs, Mel18 combines with other proteins to continue to shut off the expression of unnecessary genes, but during early cardiac development, Mel18 completely shifts and becomes a driver of gene expression. It shifts its function by forming new complexes with other proteins that regulate gene expression in various ways.
Thus Mel18 acts as a genetic switch that guides stem cells into the cardiac fate and eventually into the heart muscle cell lineage.
This fascinating work, which was published in the journal Cell Stem Cell, can help stem cell scientists grow better heart muscle from induced pluripotent stem cells in the laboratory. It could also elucidate the underlying causes of heart defects in congenital heart disease. They may also lead to new ways of controlling stem cells in the laboratory to grow cellular repair kits and patches for patients with damaged or sick hearts.
Cancer cells form when healthy cells accumulate mutations that either inactivate tumor suppressor genes or activate proto-oncogenes. Tumor suppressor genes work inside cells to put the brakes on cell proliferation. Proto-oncogenes work to drive cell proliferation. Loss-of-function mutations in tumor suppressor genes remove controls on cell proliferation, which causes cells to divide uncontrollably. Conversely activating mutations in proto-oncogenes removes the controls on the activity of proto-oncogenes, converting them into oncogenes and driving the cell to divide uncontrollably. If a cell accumulates enough of these mutations, they can grow in such an uncontrollable fashion that they start to gain extra chromosomes or pieces of chromosomes, which contributes to the genetic abnormality of the cell. Accumulation of more mutations allows the cell to break free from the original tumorous mass and spread to other tissues.
There are over 35 identified tumor suppressor genes and one of these, CHD5, has another role besides controlling cell proliferation. Researchers at Karolinska Institutet in Stockholm, Swede, in collaboration with other laboratories at Trinity College in Dublin and BRIC in Copenhagen has established a vital role for CHD5 in normal nervous development.
Once stem cells approach the final phase of differentiation into neurons, the CHD5 protein is made at high levels. CHD5 reshapes the chromatin structure into which DNA is packaged in cells, and in doing so, it facilitates or obstructs the expression of other genes.
Ulrika Nyman, postdoc researcher in Johan Holmberg’s laboratory, said that when they switched of CHD5 expression in stem cells from mouse embryos at the time when the brain develops, the CHD5-less stem cells were unable to turn off those genes that are usually expressed in other tissues, and equally unable to turn on those genes necessary for making mature neurons. Thus these CHD5-less stem cells were trapped in a nether-state between stem cells and neurons.
The gene that encodes the CHD5 protein is found on chromosome 1 (1p36) and it is lost in several different cancers, in particular neuroblastomas, a disease found mainly in children and is thought to arise during the development of the peripheral nervous system.
Neuroblastomas that lack this part of chromosome 1 that contains the CHD5 gene are usually more aggressive and more rapidly fatal.
Treatment with retinoic acid forces immature nerve cells and some neuroblastomas to mature into specialized nerve cells. However, when workers from Holmberg’s laboratory prevented neuroblastomas from turning up their expression of CHD5, they no longer responded to retinoic acid treatment.
Holmberg explained, “In the absence of CHD5, neural tumor cells cannot mature into harmless neurons, but continue to divide, making the tumor more malignant and much harder to treat. We now hope to be able to restore the ability to upregulate CHD5 in aggressive tumor cells and make them mature into harmless nerve cells.”
Embryonic stem cells have the ability to differentiate into one of the more than 200 cell types. Differentiation requires a strictly regulated program of gene expression that turns certain genes on at specific times and shuts other genes off. Loss of this regulatory circuit prevents stem cells from properly differentiating into adult cell types, and an inability to differentiate has also been linked to the onset of cancer.
Researchers at the BRIC, University of Copenhagen have identified a crucial role of the molecule Fbx110 in embryonic stem cell differentiation. Kristian Helin from the BRIC said, “Our new results show that this molecule is required for he function of one of the most important molecular switches that constantly regulated the activity of our genes. If Fbx110 is not present in embryonic stem cells, the cells cannot differentiate properly and this can lead to developmental defects.”
What is the function of Fbx110? Fbx110 recruits members of the “Polycomb” gene family to DNA. Polycomb proteins, in particular PRC1 & 2, are known to modulate the structure of chromatin, even though they do not bind DNA. Fbx110,, however binds DNA, but it also binds PRC1 . Therefore, Fbx110 seems to serve as an adapter that recruits Polycomb proteins to DNA.
Postdoctoral fellow Xudong Wu, who led the experimental part of this investigation, said, “Our results show that Fbx110 is essential for recruiting PRC1 to genes that are to be silenced in embryonic stem cells. Fbx110 binds directly to DNA and to PRC1, and this way it serves to bring PRC1 to specific genes. When PRC1 is bound to DNA it can modify the DNA associated proteins, which lead to silencing of the gene to which it binds.”
Timing of gene activity is crucial during development and must be maintained throughout the lifespan of any cell. Particular genes are active at a certain times and inactive at other times, and PRC1 seems to be part of the reason for this coordination of gene activity. PRC1 is dynamically recruited to and dissociates from genes according to the needs of the organism.
When cancer arises, this tight regulation of gene activity is often lost and the cells are locked in an inchoate state. This loss of terminal differentiation causes increases cell proliferation and the accumulation of other mutations that allow the cancer cells to undergo continuous self-renewal through endless cell divisions. Such an ability is denied to mature cells because of their tightly controlled programs of gene expression.
Wu added, “Given the emerging relationship between cancer and stem cells, our findings may implicate that an aberrant activity of Fbx110 can disturb PRC function and promote a lack of differentiation in our cells. This makes it worth studying whether blocking the function of Fbx110 could be a strategy for tumor therapy.”
In collaboration with a biotech company called EpiTherapeutics, the BRIC researchers want to develop Fbx110 inhibitors as potential novel therapies for cancer.
Type 1 diabetes results from an inability to produce sufficient quantities of the hormone insulin. Without insulin, the body does not receive the signal to take up sugar from the blood, and the result is high blood sugar levels, which are damaging to tissues, and a general wasting of tissues because they cannot take up enough sugar to feed them.
The cells in the pancreas that produce insulin are the beta cells, and animal studies have shown that transplantation of new beta cells into diabetic animals can reverse and even in some cases cure the diabetic animals. Therefore researchers have tried to make beta cells from pluripotent stem cells in order to make a source of beta cells for transplantation.
Unfortunately, beta cell production in the laboratory has been fraught with problems. The cells produced by differentiation of embryonic stem cells do not have the characteristics of mature beta cells and they produce little insulin and are not glucose responsive (D’Amour, et al., (2006) Nat Biotechnol 24, 1392-1401).
A different strategy, however, works much better. Instead of differentiating stem cells into beta cells, differentiate them into those cells that will form beta cells and other types of pancreatic cells in the embryo – immature endocrine cell precursors – and then transplant those into the pancreas of diabetic mice. In this case, the endocrine cell precursors differentiate in the bodies of the mice into pancreatic beta cells that greatly resemble normal beta cells.
Why don’t embryonic stem cells for beta cells in culture? This question was pursued by a collaboration between research team led by Maike Sander at UC San Diego and a company called ViaCyte, Inc.
When it comes to endocrine precursors transplanted into mice, Dr. Sander noted that, “We found that the endocrine cells retrieved from transplanted mice are remarkably similar to primary human endocrine cells.” He continued, “This shows that hESCs (human embryonic stem cells) can differentiate into endocrine cells that are almost indistinguishable from their primary human counterparts.”
Well, ESCs can make perfectly fine beta cells in the mouse body, but not in culture. What’s up with that?
Sander and her colleagues examined the gene expression patterns of embryonic stem cells as they were differentiated and compared them with the gene expression patterns in those cells that were transplanted into mice and allowed to differentiate inside the body of the mouse.
What Sander and her team found was astounding. As cells progress through their developmental program, particular genes are brought on-line and expressed, and then turned off as the cells passed through each stage of endocrine cell differentiation. The cellular machinery that shuts off genes after they have been activated consists of a family of proteins that remodel chromatin known as the Polycomb group (PcG). PcG-mediated repression of genes silenced those genes that were only turned on temporarily once they were no longer required.
In the transplanted endocrine precursors, Sanders and his team noted an orderly progression of genes that were turned on and then turned off once as needed. However, in the embryonic stem cells that were differentiated into beta cells in culture, they discovered that these cells failed to express the majority of genes critical for endocrine cell function. The main reason for this appeared to be that the PcG-mediated repression of genes was not fully eliminated when particular genes had to be expressed at specific developmental stages. Thus these cultured cells failed to fully eliminate PcG-mediated repression on endocrine-specific genes, which contributes to the abnormality of the culture-derived beta cells.
Sander commented: “This information will help devise protocols to generate functional insulin-producing beta cells in vitro. This will be important not only for cell therapies, but also for identifying disease mechanisms that underlie the pathogenesis of diabetes.”
Embryonic stem cells (ESCs) have an uncanny ability to perpetually divide in culture and differentiate into any cell type found in the adult body. The internal switches inside ESCs that keep them pluripotent or drive them to differentiate are incompletely understood at this. However new work from the Carnegie Institution for Science has opened a new doorway into this event.
Yixian Zheng and his research team has focused on the process by which ESCs stay in their pluripotent state. There are three protein networks within the cell that direct the self-renewal and differentiation aspects of cell behavior. These networks consist of 1) the pluripotent core, which includes the protein called Oct4 and its many co-workers; 2) the Myc-Arf network, which directs cell proliferation, and 3) the PRC2 or polycomb proteins, which repress genes necessary for differentiation. How these networks are integrated remains quite unclear. Zhen and his group have found a protein that seems to link all three of these networks together.
A protein called Utf1 seems to act as the cord that ties all three of these networks together. First, Utf1 limits the loading of PRC2 on the DNA and it also prevents PRC2 from modifying chromatin so that the DNA assumes a very tight, compact structure that prevents gene expression. Thus, Utf1 keeps the DNA somewhat poised and ready for gene expression, should the proper conditions come about that favor differentiation. Secondly,. for those genes that are not completely shut off by PRC2, Utf1 works through a protein complex called the DCP1a complex to degrade these mRNAs made these incompletely repressed genes. Finally, Utf1 downregulates the My-Afr feed pathway. The Myc and Arf work together to curtail cell proliferation, but the inhibition of this pathway ensures that the cell continues to divide properly.
According to Zheng, “We are slowly but surely growing to understand the physiology of embryonic stem cells. It is crucial that we continue to carrying out [sic] basic research on how these cells function.”
Zheng is a Howard Hughes Medical Institute Researcher at the National Institutes of Health and in the Department of Embryology at the Carnegie Institute for Science in Baltimore, Maryland.
This work was published in the journal Cell under the title, “Regulation of pluripotency and self-renewal of ESCs through epigenetic-threshold modulation and mRNA pruning.” Cell 2012 3:576.