August 1, 2015 (Vol. 35, No. 14)

Richard A. A. Stein M.D., Ph.D.

Cells Needn’t Be Bound to the Fates Woven into Their Epigenetic Fabric

One of the most fascinating processes in biology involves the fusion between two haploid gametocytes to form a diploid zygote, from which all the cell types of an adult organism originate. Cellular reprogramming plays crucial functions at several stages during the mammalian life cycle, including gametogenesis and fertilization.

Reprogramming has attracted considerable attention, particularly in recent years, ever since the overexpression of only four genes was found to reprogram adult mouse cells toward a more embryonic cell fate and form induced pluripotent stem cells. That such an intervention could alter cell fate so dramatically encouraged scientists to study the molecular mechanisms behind cellular reprogramming.

The deliberate unraveling of cellular fate that characterizes induced pluripotency is presumably less graceful than the spontaneous unraveling that occurs during certain phases of mammalian development. To close this gap—and thereby gain the ability to reprogram cells both deliberately and gracefully—scientists are determined to learn all they can about the molecular events that determine cellular fates.

Oocyte Presents a Model of Efficiency

“The sperm, an extremely differentiated cell type, needs special mechanisms to switch it very fast into an intensely active nucleus with almost 100% reliability, says Sir John B. Gurdon, Ph.D., a professor emeritus at the University of Cambridge, a distinguished group leader at the Wellcome Trust/CRUK Gurdon Institute, and a co-recipient of the 2012 Nobel Prize in Physiology or Medicine. “The egg has a way of bringing this about.”

Dr. Gurdon and colleagues have evaluated the egg’s fate-switching capabilities by making use of the nuclear transfer experimental system. This system presents several technical advantages in the study of transcriptional events during reprogramming.

Using RNA-Seq and time-course analyses at the single-nucleus level to examine somatic cell reprogramming after nuclear transplantation into the Xenopus oocyte, Dr. Gurdon and colleagues found that genome-wide reprogramming events occur in a hierarchical sequence of molecular events. This analysis also unveiled a major selective switch in gene expression patterns in the transplanted somatic nucleus during cell differentiation.

“We still do not have a clear idea of what mechanisms ensure the stability of cell differentiation and therefore prevent switches in cell-type from occurring in normal life,” admits Dr. Gurdon. Due to the relatively limited knowledge about the mechanistic basis of cellular reprogramming, it is still unclear to what degree these events are informative about processes that occur during the reprogramming of induced pluripotent stem cells.

“The process of nuclear reprogramming by nuclear transfer, either to eggs or oocytes, is likely to be different from what happens during induced pluripotent stem cell reprogramming,” Dr. Gurdon continues. “The major challenge in understanding and bringing about reprogramming in somatic cells is the low efficiency with which it works.”

To appreciate the scale of this challenge, consider that less than 1% of experimental cells are able to form induced pluripotent stem cells. Also, reprogramming can take several weeks.

“[There] has been remarkably little progress in understanding induced pluripotent stem cell reprogramming,” notes Dr. Gurdon. He adds, however, that “[reversing] the differentiated state of specialized cells with high efficiency—and this last point is critical—could have very valuable consequences for cell replacement.”

Histone Chaperone Leaves Its Mark

“Although work on cellular reprogramming has been expanding tremendously, advances in the field have not occurred as big leaps of knowledge, but rather as incremental changes,” says Jose B. Cibelli, D.V.M., Ph.D., professor of animal science and physiology at Michigan State University. One of the efforts in Dr. Cibelli’s lab focuses on the ability of the oocyte to reprogram sperm cells. “We have shown that a chaperone, studied until recently mostly in yeast and the fruit fly, is essential for the ability of oocytes to reprogram cells,” notes Dr. Cibelli.

This histone-remodeling chaperone, ASF1A, is enriched in human oocytes during metaphase II, and it is required for reprogramming human adult dermal fibroblasts into induced pluripotent stem cells. Dr. Cibelli and colleagues revealed that in adult dermal fibroblasts, exposure to ASF1A and OCT4, in the presence of the oocyte-specific paracrine growth factor GDF9, was sufficient for reprogramming.

In the fruit fly and the budding yeast, ASF1A emerged as a factor that shapes chromatin remodeling in pluripotent embryonic cells. In human oocytes, it promotes acetylation of lysine 56 on histone H3, mainly during the S phase of the cell cycle. This post-translational modification was positively correlated in human embryonic stem cells with the binding of the Nanog, Sox2, and Oct4 transcription factors to their target promoters. ASF1A downregulation decreased H3K56 acetylation and the expression of pluripotency markers, and increased the expression of cellular differentiation markers.

“We found that ASF1A interacts with Oct4 and with enzymes that acetylate certain histones,” Dr. Cibelli points out. “We are just beginning to figure out what other partners these chaperones have.” Dr. Cibelli and colleagues showed that ASF1 overexpression in embryonic stem cells or induced pluripotent stem cells leads to a several-fold increase in the expression of pluripotency genes.

Even though several reports revealed that oocytes or transcription factors can reprogram differentiated cells to a more embryonic phenotype, an understanding of the sequence of events during this process has been elusive. “We lack the understanding of processes that occur during very small windows of time during cellular reprogramming,” concedes Dr. Cibelli. “[Slowing] down the process to characterize these intermediate steps in a model to replicate the process is the biggest challenge.”

Small Molecules Hit a Nerve

“The main area of our research is using small molecules for reprogramming,” says Arshak R. Alexanian, V.M.D., Ph.D., CSO at Cell Reprogramming & Therapeutics. Small molecules that penetrate the cells can regulate various cellular processes, and one of these is cellular plasticity. In previous work, Dr. Alexanian and colleagues tested several epigenetic modifiers, including compounds that inhibit DNA methylation or histone methylation and acetylation.

“With these small molecules, we were able to make certain cells become more or less embryonic-like,” asserts Dr. Alexanian. “Our goal is to make more cell types become more immature or pluripotent, and at the same time expose them to differentiation factors that would turn them to a narrow developmental fate.”

By exposing mesenchymal stem cells to epigenetic modifiers and neural inducing factors, Dr. Alexanian and colleagues were able to reprogram them into neuronal-like cells. When exposed to fat-differentiating medium, these cells subsequently reverted to the mesenchymal cells of origin and formed adipocytes. After these adipocytes were re-exposed to the neural induction medium, they re-differentiated into neuronal-like cells.

“We have shown that with this small-molecule approach, by inducing specific cell-signaling modulators, it is possible to turn cell fate from one phenotype to another,” continues Dr. Alexanian. These findings, he adds, illustrate the possibility of manipulating cell plasticity by using small-molecule modulators of the chromatin-modifying enzymes.

“Our main goal is to use the small-molecule approach to generate very specific neuronal subtypes,” informs Dr. Alexanian. This promises opportunities for conducting targeted in vitro and in vivo studies and therapeutic interventions, and for medical conditions in which these neurons are selectively affected. “Generating dopaminergic cells would help establish an animal model of Parkinson’s disease; GABAergic neurons would facilitate the study of Alzheimer’s disease, Huntington’s disease, and neuropathic pain; and motor neurons present promises in treating spinal cord injury or ALS,” explains Dr. Alexanian.

Spliced Forms Make the Rounds

“We found 80 to 90 unique splicing variants that occur during cellular reprogramming,” says In-Hyun Park, Ph.D., assistant professor of genetics at Yale University School of Medicine. In a study that used RNA-Seq to dissect the transcriptional landscape of somatic cell reprogramming, Dr. Park and colleagues found splice variants of multiple genes that are uniquely expressed at different stages of reprogramming. One of these, pCCNE1, a splicing variant of CCNE1, was observed in cells as they acquired pluripotency.

“This gene encodes cyclin E, a protein involved in the G1-to-S transition of the cell cycle,” notes Dr. Park. The pCCNE1 splicing isoform lacks exon 9, which encodes two alpha helices and a loop that are important for cyclin E binding to its cellular target, CDK2. Overexpression of the splice variant of this gene improved reprogramming without affecting cell cycle progression.

“This is a previously unexplored role of cyclin E,” continues Dr. Park. “We are trying to understand more about the molecular mechanism of this function.” This finding suggests that a gene may not only promote cell cycle progression, by means of a broadly known gene product, but also participate in pluripotency, by means of a splicing variant.

“This splicing variant is present only in primates, including humans or monkeys, but not in mice,” points out Dr. Park. “Therefore it might be involved in reprogramming specific to humans.”

In-Hyun Park, Ph.D., and colleagues at Yale University School of Medicine are investigating splice variants that have been found to be uniquely expressed at different states of cell reprogramming. They are particularly interested in learning how the overexpression of splice variants can improve cell reprogramming without affecting cell cycle progression. For example, the pCCNE1 splicing isoform (blue) lacks exon 9,which encodes two alpha helices and a loop (yellow) that are important for cyclin E binding to its cellular target, CDK2 (purple).

In Vivo Approach Sidesteps Rejection

“Even though the mammalian brain has the ability to regenerate a few neurons after injury, those neurons usually do not make a big difference in terms of recovery because their number is too low,” says Gong Chen, Ph.D., professor of biology and Verne M. Willaman Endowed Chair of Life Sciences at Pennsylvania State University. In recent studies, stem cell therapy has emerged as a promising strategy to generate neurons. “But those neurons are often rejected immunologically in mouse models, unless the mice are immune deficient, which is not immediately applicable to humans,” notes Dr. Chen. “This is one of the big problems for stem cell therapy.”

Dr. Chen and colleagues recently reported a new strategy to regenerate neurons affected by brain injury or degenerative conditions. “Our method regenerates neurons within the brain, using the brain’s internal glial cells,” asserts Dr. Chen, “and this alleviates concerns about immune rejection.”

To examine the reprogramming of glial cells into neurons, Dr. Chen and colleagues used a retrovirus to deliver the proneural transcription factor NeuroD1, which participates in embryonic brain development and adult neurogenesis. As a result, astrocytes were reprogrammed into glutaminergic neurons and oligodendrocyte precursor NG2 cells into glutamatergic and GABAergic neurons.

“The cells originate from within the injury site,” explains Dr. Chen. “This in situ approach is the unique feature of our technology.”

Electrophysiological recordings from cortical slices revealed that the neurons may present spontaneous and evoked synaptic responses, suggesting that they have integrated into the local neural circuits. These results open the possibility that glial cells could be reprogrammed into functional neurons to replace cells damaged by injury or disease.

One of the key considerations when regenerating injured or damaged neurons is whether the new cells are able to establish a global neuronal network in a manner that is as precise and functional as the previously existing neuronal circuits. In studies conducted by Dr. Chen and colleagues, reprogrammed neurons were able to form synaptic connections within their local environment.

“Additionally, we saw that some of these neurons send their axons to remote sites, such as the contralateral brain hemisphere, and establish long-range projections to form a large global network,” details Dr. Chen. Another consideration is whether the reprogrammed neurons can rescue the behavioral deficits. “Functional behavior improvement, which is a measure of neuron functionality, is another area that we are exploring,” continues Dr. Chen. “Preliminary animal studies have shown some improvement, but we need additional work.”

At Pennsylvania State University, Gong Chen, Ph.D., and colleagues are investigating a new in situ strategy to regenerate neurons affected by brain injury or degenerative conditions. These scientists are regenerating hippocampal neurons within the mouse brain by reprogramming the brain’s internal glial cells.

Activate T-Cell Transdifferentiation, Resolve Inflammation

Researchers have shown that it is possible to coax T cells to alter their behaviors. “Now that we have observed that we can convert harmful cells into cells that are protective, one challenge is to try to do that pharmaceutically,” says Richard A. Flavell, Ph.D., Sterling Professor of Immunobiology at Yale University School of Medicine.

Decades ago, researchers identified T helper 1 (Th1) and Th2 cells, two subsets of CD4+ T lymphocytes with antagonistic effects. More recently, in 2005, researchers discovered a new group of CD4+ T cells, the Th17 cells. For Th17 cells, a link was found between their aberrant regulation and several inflammatory and autoimmune conditions. This link not only suggested a new framework for understanding the pathogenesis of these conditions, it also pointed to new strategies for designing therapeutics.

Th17 cells display two characteristics: instability and plasticity. “Instability” indicates that Th17 cells can stop secreting their signature cytokine, interleukin-17A. “Plasticity” indicates that Th17 cells can assume the functional characteristics of T cells from other lineages. These characteristics are not well understood at the molecular level, but they both appear to be important for modulating inflammation.

In a study that examined Th17 cell plasticity during a self-limiting inflammatory response in mice, Dr. Flavell and colleagues revealed that some Th17 cells convert to other regulatory T cells, a process known as transdifferentiation, and that during this process, Th17 cells acquire an anti-inflammatory phenotype and change their transcriptional profile.

Dr. Flavell’s lab determined that this process occurs physiologically during the course of fungal and acute bacterial infections. “In our multiple sclerosis-like animal model, using our treatment, we were able to convert cells that drive the pathogenesis into regulatory T cells,” details Dr. Flavell.

Understanding the mechanistic basis of Th17 cell transdifferentiation, a process that contributes to the resolution of inflammation, has broad implications for developing therapeutic strategies that could impact several inflammatory and autoimmune conditions in which Th17 cells play key roles.

“One specific challenge in developing therapeutics is that, in this case, we want to enable an event, which is pharmacologically much more difficult than blocking an event,” explains Dr. Flavell. One of the teachings that emerged from the experience with many therapeutic agents that were designed to act on cellular targets is that it is virtually always easier to design an enzyme inhibitor than a compound that is intended to activate an enzyme. “To be therapeutically useful,” asserts Dr. Flavell, “such a reagent should be selectively directed at the disease itself.”  

Pioneer Factors on the Epigenetic Frontier

“The need to shift from phenomenology, where we know that we can obtain effects when we treat cells in a certain way, to [mechanistic understanding] is the biggest obstacle in cell reprogramming,” says Kenneth S. Zaret, Ph.D., professor of cell and developmental biology at the University of Pennsylvania School of Medicine. This obstacle is particularly daunting in eukaryotes, where the interaction between transcription factors and the DNA is shaped by the organization of the genetic material into nucleosomes.

One of the developments from the past decade was the discovery and characterization of a class of transcription factors that are known as pioneer transcription factors. The main attribute of pioneer transcription factors is that, as opposed to most other transcription factors that require open chromatin to be able to bind DNA, they are able to overcome steric constraints and bind closed chromatin.

To better understand the binding of the Oct4, Sox2, Klf4, and c-Myc pioneer transcription factors to closed chromatin, Dr. Zaret and colleagues used in vitro and in vivo biochemical, genomic, and structural experimental approaches to measure their interaction with DNA and nucleosomes. These four transcription factors, required for reprogramming somatic cells to pluripotency, have long been known to have different contributions to reprogramming in different cell types.

“This work provides understanding of the earliest events in how reprogramming factors scan the genome,” explains Dr. Zaret. Dr. Zaret and colleagues revealed that these four pioneer transcription factors have a range of affinities for nucleosomes, and their experimental approach offers an unprecedented insight into the hierarchy of transcription factor recruitment during reprogramming and into the biochemical and structural biological basis of their interaction with closed chromatin.

“Understanding these events places us in a better position to design better reprogramming factors and understand how that process can be manipulated experimentally,” asserts Dr. Zaret. Dissecting the molecular processes involved in cellular reprogramming promises to unveil new approaches that could increase the efficiency and the specificity of reprogramming, which are key challenges in the field. “This would allow reprogramming to occur faster, more precisely, and in a greater population of cells,” insists Dr. Zaret, who adds that “it will be important for cellular therapeutics.” 

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