Final Report Summary - EPIRNAS (Small RNA Mediated Epigenetics in Vertebrates)
This project has addressed how tiny RNA molecules can affect how the DNA, that is present in every cell, is used differently in different cell types. We have analyzed specifically small RNA molecules named piRNAs in the cells that give rise to offspring, the so-called germ cells. In these cells we have found that piRNAs serve as instructors to identify regions in the genome that germ cells do not want to be used. These regions mostly are segments of the genome that only serve to replicate themselves, so-called selfish DNA. If not silenced, such selfish DNA will replicate quickly leading to a dysfunctional genome. Interestingly, following identification by piRNAs, these regions can become ‘silenced’ in an extremely stable manner. So stable, that their silencing does not need the original piRNAs anymore. This means, that piRNAs function as instructors, while different mechanisms exist that serve to maintain the silencing. We also identified part of this maintenance machinery. Interestingly, this is composed of another small RNA-based system that can enter the nucleus, where the DNA is, and modify that DNA, or more precisely the proteins around which the DNA winds. Normally, during germ cell development these modifications are removed, and we believe that the small RNA molecules we identified act as an extra-chromosomal ‘cash’-memory to re-establish these modifications in the germ cells of the next generation.
In addition, we identified a process in which small RNA molecules are modified during their lives in the cell. This process, named non-templated uridylation, signals that the modified small RNA needs to be degraded, and absence of this mechanism leads to inappropriate amounts of small RNAs that in turn lead to defects of how the genome is used.
In parallel, we have been looking at how a chemical modification of the DNA, named DNA methylation, changes during differentiation of a stem cell. DNA methylation is believed to be a signal that stably silences DNA, and the differentiation of a stem cell can be expected to be accompanied by many changes in DNA methylation, because many genes are turned on and off. In fact, DNA methylation is often described as ‘locking’ the silencing of the expression of genes. Unexpectedly, when we analyzed the DNA methylation pattern of stem cells from the small intestine of mice and compared that to the DNA methylation pattern of their differentiated descendant, only minor changes were observed. The changes that we did see were mostly indicative of DNA methylation being influenced by proteins binding to the DNA and not of DNA methylation patterns driving the differentiation process. This picture differs drastically with the pictures obtained from stem cells differentiating in vitro in cell cultures, and might be related to the fact that the differentiated daughters of intestinal stem cells only live shortly. Nevertheless, this work showed that changes in DNA methylation most likely do not drive differentiation of intestinal stem cells in mice.
In addition, we identified a process in which small RNA molecules are modified during their lives in the cell. This process, named non-templated uridylation, signals that the modified small RNA needs to be degraded, and absence of this mechanism leads to inappropriate amounts of small RNAs that in turn lead to defects of how the genome is used.
In parallel, we have been looking at how a chemical modification of the DNA, named DNA methylation, changes during differentiation of a stem cell. DNA methylation is believed to be a signal that stably silences DNA, and the differentiation of a stem cell can be expected to be accompanied by many changes in DNA methylation, because many genes are turned on and off. In fact, DNA methylation is often described as ‘locking’ the silencing of the expression of genes. Unexpectedly, when we analyzed the DNA methylation pattern of stem cells from the small intestine of mice and compared that to the DNA methylation pattern of their differentiated descendant, only minor changes were observed. The changes that we did see were mostly indicative of DNA methylation being influenced by proteins binding to the DNA and not of DNA methylation patterns driving the differentiation process. This picture differs drastically with the pictures obtained from stem cells differentiating in vitro in cell cultures, and might be related to the fact that the differentiated daughters of intestinal stem cells only live shortly. Nevertheless, this work showed that changes in DNA methylation most likely do not drive differentiation of intestinal stem cells in mice.