PIWI proteins are involved in RNA mediated gene regulation, and have a strong role in mitosis during Drosophila embryogenesis. PIWI proteins interact with chromatin binding factors, specifically those dealing with heterochromatin formation. Determining the function of these proteins gains importance as their role in cancer, specifically tumorigenesis, is being uncovered. Increased detection of these proteins has been observed in breast cancer, cervical cancer, colon cancer, pancreatic cancer and more human cancers.
Heterochromatin: tightly wound chromatin, transcriptionally inactive
Euchromatin: loosely wound chromatin, transcriptionally active
Histone: An octameric protein compound that DNA is wrapped around, to store and order DNA into nucleosomes. Histones have tails that are exposed to chemical changes.
DNA Methylation/De-Methylation: Through the use of DNA Methyl Transferases (DNMT), a methyl group is transferred to a [cytosine] nucleotide
, adding a positive charge. Because DNA is negatively charged, this chemical change tightens the DNA, eliminating availability to transcription factors and polymerases. A de-methylase replaces the CH3 group with a hydrogen, returning the structure to it’s initial charge and reversing the change.
Histone Modification: Histone tails are available to chemical modifications that can be both markers for activation or repression. Lysine methylation may lead to both, while arginine acetylation leads to activation. (Acetylation adds an acetyl group
with a negative charge, repelling the DNA and loosening the structure). Serine phosphorylation is associated with signaling pathways, specifically involving kinases.
PIWI proteins are essential for early Drosophila embryogenesis (Sneha Ramesh Mani, Heather Megosh, Haifan Li)(5)
ARGONAUTE/PIWI protein family: Involved in RNA-mediated gene regulation through a variety of similar mechanisms, as seen in Table 1 (5). The argonaute (AGO) proteins are expressed in all tissues and function through interactions with miRNA and siRNA, typically leading to degradation. PIWI, however, begins in the cytoplasm and migrates to the nucleus where it stays(5). PIWI is highly conserved and has shown to have effects on gametogenesis in mice, zebrafish, humans and Drosophila (5).
In Drosophila melanogaster, PIWI, AGO3 and Aubergine make up this protein family. While it’s importance is clear, the mechanisms and somatic role are not and are explored in this paper. PIWI is known to interact with HP1A by recruitment to chromatin, dependent on RNA. Hp1A is a serine phosphorylase and is involved in chromatin structure and signaling pathways. This paper focuses on early embryogenesis for these three reasons:
1. It is the most important stage of somatic development
2. All three proteins are expressed, in contrast to the adults where Aub and AGO3 are restricted to the germline.
3. piwi, aub and ago3 mutants are viable but infertile, showing clear roles of these proteins in embryogenesis.
1. Piwi, Aub, Ago3 localization using immunofluorescent staining: Immunofluorescence targets and visualizes proteins of interest by using fluorescent, protein-specific antibodies. During cycles 1-8, all proteins are diffused throughout the embryo. At cycle 11, Piwi enters the nucleus. From 11-14, Piwi appears in the somatic nucleus during interphase, and moves into the cytoplasm during mitosis. Aub is within the pole plasm on the posterior embryonic end, and remains there. Ago3 does as well. Both are equally present in both somatic and germline cells. Hp1a (heterochromatin protein 1a) is cytoplasmic until cycle 7, when it enters the nucleus and remains there.
2. Maternal Piwi, Aub, Ago3 mutant effects: Depleted embryos were produced by crossing heterozygotic males and virgin females, creating a transheterozygotic combination. Mutations in all three proteins prove lethal. Piwi deficient embryos arrest before gastrulation, but after entering mitosis. 51% of Aub and Ago3 mutants’ stages were unidentifiable, due to the severity of the morphological effects. These mutants also have nuclear abnormalities; improper chromosome condensation, abnormal ploidy and fragmentation. This indicates the importance of PIWI protein function during mitosis, where deficiency can be lethal.
3. Spindle formation assay: DAPI stains DNA blue, PH3 stains phosphorylated serine histone tails red while a green dye stains alpha tubulin, protofilaments asociated with microtubules. These dyes become fluorescent and allow for protein visualization. In all three mutants, microtubule organization is affected. Alpha tubulin is visualized in Figure 2. A-D. Serine phosphorylation on Histone 3 (PH3) is visualized in Figure 2. I-L. Huge asynchronies seen in mutants, with cells in completely different stages. Some nuclei do enter mitosis (arrows), but have abnormal chromosomes. This suggests that the mutants don’t prevent mitotic entry due to abnormal chromosomal structure.
4. Mitotic defect imaging of Aub and Ago3 mutants: Confocal micrographs construct 3D images, and functions using a lazer scanning microscope. Confocal micrographs of DAPI stained mutants were used to identify when the defects begin to take place. This places PIWI protein function in the realm of mitosis, as deficient mutants show abnormal structures in every stage.
PIWI proteins show a clear role in Drosophila embryogenesis, specifically in spindle formation and chromatin condensation. In support, there is increased chromatin fragmentation and chromatin bridge formation in both Aub and Ago3 mutants, seen in Figure 3. HP1A, a chromatin modifying marker, shows increased nuclear localization, possibly linked to increased chromatin condensation. Histone 3 lysine 9 methylation (H3K9me3) is a histone modification required for HP1A localization, and showed reduction and diffusion in PIWI-deficient proteins, with no detection in Aub- and Ago3- mutants. The inability of HP1A to diffuse leads to increased methylation of chromatin, therefore silencing gene expression through heterochromatin formation. Piwi, Aub- and Ago3- show importance in localization and maintenance of these localization factors dealing with chromatin modification. This decrease in proper chromatin structure and stability allows for proper mitosis during embryogenesis.
Piwi expression and function in cancer. (Ryusuke Suzuki, Shozo Honda, Yohei Kirino)(3) LINK
“Overexpression of PIWI proteins may contribute to tumorigenesis by transcriptionally silencing tumor-suppressing genes through epigenetic mechanisms.”
Ectopic Expression of Germline Genes Drives Malignant Brain Tumor Growth in Drosophila. (Ana Janic, Leire Mendizabal, Salud Llamazares, David Rossell, Cayetano Gonzalez)(4) LINK
“…PIWI and AUB contribute to tumor growth”
Notes on paper’s strengths and weaknesses: Mani et al. provide a comprehensive look into the role of PIWI proteins, beginning with localization, then moving into roles in mitosis, and potential mechanisms for these results. This study is thorough, and provides suggestions for functions of proteins that are typically overlooked. However, the title of the paper mentions epigenetics, while the paper itself focuses little on epigenetics and heavily on sole protein function. While the function is related to epigenetics, it is only briefly discussed with little implication or suggestion for further study, or even application. PIWI proteins have great relevance, as discussed above, and Mani et al. misses this importance. While the knowledge of these proteins is very necessary, the implications of the findings are equally important. This paper provides strong results to support their conclusions and is very strong overall, but could use focus on implications and importance outside of the model organism.
1.Walter Jahn. ” GENETICS 3: GENE REGULATION: HETEROCHROMATIN VS. EUCHROMATIN.” Video. Youtube. Youtube, 5 November 2012. Web. 15 April 2010.
2. Zamudio, Natasha M., Suyinn Chong, and Moira K. O’Bryan. “Epigenetic regulation in male germ cells.” Reproduction 136.2 (2008): 131-146.
3. Suzuki, Ryusuke, Shozo Honda, and Yohei Kirino. “PIWI expression and function in cancer.” Frontiers in genetics 3 (2012).
4. Janic, Ana, et al. “Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila.” Science 330.6012 (2010): 1824-1827.
5. Mani, Sneha Ramesh, Heather Megosh, and Haifan Lin. “PIWI proteins are essential for early Drosophila embryogenesis.” Developmental biology 385.2 (2014): 340-349.