Parts of this section have been published in (Stuwe et al., 2014)
The cytoplasmic role of the piRNA pathway includes deposition of H3K9me3 on target loci, which is a classical epigenetic signal. Epigenetics generally describes the long-term maintenance of distinct gene expression profiles in cells despite their same genetic compo- sition (DNA sequence). Two major modes of epigenetic regulation have been proposed: self-maintaining networks of transcription factors and chromatin modifications. Expres- sion of particular sets of transcription factors that form gene regulatory networks can generate distinct patterns of gene expression that can be stable over long time periods and impact the identity of the cell. This was demonstrated by the observation that induc- ing expression of four transcription factors (the so-called Yamanaka factors) is sufficient to trigger a stable switch in cellular identity and to generate iPS cells from differentiated cells (Wernig et al., 2007; Yu et al., 2007; Nakagawa et al., 2008; Okita et al., 2007; Takahashi and Yamanaka, 2006; Takahashi et al., 2007)
A second mechanism of cell identity determination relies on maintenance of distinct chromatin states. Chromatin is generally defined as DNA in tight complex with histone and non-histone proteins. DNA itself can be marked by covalent modifications, for exam- ple through cytosine methylation in CpG dinucleotides in mammals (Hackett and Surani, 2013). Multiple residues in histone proteins undergo posttranscriptional modifications, like for example methylation, acetylation and phosphorylation. Early work demonstrated that DNA methylation as well as certain histone modifications correlate with the tran- scriptional status of genes (Jones and Takai, 2001; Kouzarides, 2007). Specifically, loci enriched in methylation of Lysine 4 of the histone H3 tail (H3K4me) are associated ac-
CHAPTER 2. INTRODUCTION
tively transcribed genes, whilst other marks such as methylation of Lysine 9 (H3K9me2/3) accumulate over repressed genes. These observations lead to the hypothesis that mainte- nance of a particular pattern of chromatin mark distribution provides the physical basis for an epigenetic memory of gene expression.
However, it is important to note that profiling of chromatin modifications only reveal correlations between a specific chromatin mark and the transcriptional status of genes. It can neither prove that a particular chromatin mark directly influences transcription nor that the mark is stably maintained through cellular divisions, two properties necessary to provide true epigenetic signal. In fact, it is likely that certain chromatin modifications are the consequence rather than the cause of transcriptional state.
The observation of a phenomenon called Position-Effect Variegation (PEV) more than 80 years ago (Muller, 1930) was the first clue that chromatin structure can influence gene expression of neighboring loci and be transmitted through cellular divisions. Chromoso- mal rearrangements in Drosophila melanogaster that placed the gene responsible for red eye pigmentation in the vicinity of heterochromatin resulted in flies that have intermin- gled patches of red and white cells in their eyes. Patches of cells without pigmentation (white color) indicate that the vicinity to heterochromatin causes repression of the gene, presumably by spreading of the repressive chromatin structure from heterochromatin into the gene environment. Importantly, large patches of white and red color suggest that the on- or off-state of gene expression was randomly established in a few cells in the developing eye and then maintained in the progeny of these cells through mitotic cell divisions.
The maintenance of a distinct cell identity shows how epigenetic signals can be stable throughout mitotic cell divisions within the same organism. There are, however, exam- ples for transmission of epigenetic signals not only through mitosis from one cell to its progeny, but from one generation to the next. These cases of trans-generational epigenetic phenomena are characterized by inheritance of a distinct phenotype that is not encoded by a genetic source (DNA sequence). One such example is the fur coloration in the Agouti mouse strain (Argeson et al., 1996; Morgan et al., 1999). In this inbred strain the ani- mals are genetically identical and still the fur color of individual animals can vary. The study shows that the differences in fur color depend on the DNA methylation status of a retrotransposon inserted close to the promoter of the Agouti gene, which is involved in determining fur color. The DNA methylation status of the Agouti locus is inherited from the mother and supports the role of DNA methylation as a trans-generational epigenetic mark. In other cases of trans-generational epigenetic inheritance the underlying mecha- nisms are not understood. In most animals the parental chromatin marks are erased and newly established after the haploid genomes fuse in the zygote. One possible explanation
2.7. piRNAs are trans-generational epigenetic factors
for trans-generational inheritance of epigenetic marks would be the incomplete erasure of parental chromatin marks, which would allow re-establishment of expression patterns in the progeny that are similar to those in the parent.
Though trans-generational epigenetic inheritance has been described in just a few cases, as the Agouti example illustrates, they can provide invaluable insights into the mechanisms that likely operate to maintain cellular identities.
The next section will describe how piRNAs can be viewed as epigenetic factors that establish a cellular memory that is maintained through cell divisions as well as from one generation to the next.