**2. The PIWI-piRNA pathway**

stem cells (CSCs) and progenies. These cells harbor chromosomal abnormalities, alterations of suppressor genes (TSG) and oncogenes, and aberrant transcriptomic profiles generated by genetic and epigenetic alterations [1, 2]. These cancer cells are in close relationship with a tumor microenvironment (TME), composed of immune and nonimmune stromal cells and modified extracellular matrix. Reciprocal interactions between tumor cells and TME are pivotal in cancer progression, allowing remodeling of TME and reprogramming of cancer cells that develop adaptive strategies to adjust their phenotype to unfavorable environmental conditions. Recently, CSCs were implicated in a new paradigm accounting for tumor heterogeneity [3]. CSCs have the property of self-renewal, lack senescence, maintain an undifferentiated state, and proliferate rapidly. These properties are controlled by epigenetic mechanisms that induce changes in gene expression profiling of tumor cells. Opposite to aging cells that increase genomic and chromosomal instability during adulthood, nonaging immortal cells, such as germline, somatic, and cancer stem cells, harbor a genomic instability triggered by unrepaired mutations with either no or only limited number of genomic alterations [4]. Epigenetic abnormalities are early events in cancer progression, resulting from various environmental injuries, and associate heterogeneity of DNA methylation, posttranscriptional modifications of histones, and deregulation of noncoding RNAs (ncRNAs). Global DNA hypomethylation results in chromosomal instability, overexpression of oncogenes, and reactivation of transposable elements (TEs) [5]. Localized (genes promoters) or wide (>1 Mb) DNA hypermethylation initiates repression of TSGs and modification of epigenetic marks through histone alterations, resulting in occurrence of an aberrantly stemlike state of CSCs. These alterations of the genomic methylation during carcinogenesis allow reprogramming of atypical proliferative cells into highly malignant cells characterized by unlimited proliferation, epithelial-mesenchymal transition (EMT), invasion, and prometastatic properties [6].

234 Chromatin and Epigenetics

Until recently, RNAs were considered as epigenetic regulators and mediators of gene expression, functioning as intermediates of translation in the flow of genetic information from DNA to proteins [7]. Large-scale genomic technologies have provided an astonishing insight into human genome and transcriptome. Next-generation sequencing techniques combined with bioinformatics have revealed that more than 50% of mammalian genomes were composed of TEs and that more than 98% of the human genome was actively transcribed [8]. However, only 1.1% of the genome encodes proteins, and a majority of genes are noncoding RNAs (ncRNAs) [9]. NcRNAs play pivotal roles in developmental and homeostatic processes, and their alterations are implicated in the pathogenesis of many diseases, by modulating expression of numerous genes at epigenetic, transcriptional, and posttranscriptional levels [10]. Most importantly, ncRNAs are frequently deregulated in cancer and have crucial roles in tumor initiation, progression, and metastatic spread. NcRNAs are classified into housekeeper ncRNAs (rRNAs, tRNAs, and snoRNAs) and regulatory ncRNAs. Regulatory ncRNAs are divided into several subfamilies, depending on their size, biogenesis, and biological functions. Small ncRNAs are composed of transcripts shorter than 200 nucleotides (nt), whereas long noncoding RNAs (lncRNAs) comprise transcripts longer than 200 nt [11]. Small ncRNAs also differ by their precursor structure and their mechanisms of biogenesis. They comprise microRNAs (miRNAs), small interfering RNAs (siRNAs), and PIWI-interacting RNA (piRNAs) [12–15].

MiRNAs and siRNAs are generated from double-stranded precursors, whereas piRNAs are processed from long single-stranded precursors. The endoribonuclease Dicer is pivotal in the maturation of miRNAs and siRNAs, but not in the piRNAs processing [16]. Regulatory RNA interference (RNAi) is a widely conserved small-RNA-mediated gene-silencing mechanism involved in crucial homeostatic events of most eukaryotes [22–24]. Small regulatory RNAs of 20–32 nt, such as endogenous siRNAs, miRNAs, and piRNAs, modulate transcriptional and posttranscriptional repression through complementary RNA or DNA recognition by interacting with well-conserved proteins of 95 kDa belonging to the Argonaute family that cleave their targets [25, 27]. AGO proteins include a PAZ domain [P-element-induced wimpy testis (PIWI)-AGO-Zwille] located in the N terminal region, which binds small ncRNAs and a PIWI domain in the C-terminal region that functions as double-strand-specific RNA endonuclease [28]. Based on sequence homology and functional domains in different species, the AGO family of proteins is divided into three subfamilies: AGO proteins (homologous to *Arabidopsis thaliana* AGO1), PIWI proteins (homologous to *Drosophila melanogaster* Piwi), and WAGO (worm-specific Argonaute clade). The number of AGO family proteins varies considerably between species. Eight different proteins were present in humans, whereas 27 proteins were identified in *C. elegans* and only one protein was observed in fission yeast [29]. AGO proteins interact functionally with siRNAs and miRNAs, which are small single-stranded RNAs of 20–22 nt in length processed in a Dicer-dependent manner from double-stranded precursors, to induce posttranscriptional gene silencing in the cytoplasm [30–32]. Conversely, PIWI proteins are implicated in biogenesis of piRNAs and in their main function through transcriptionally and posttranscriptionally repressing TEs in the nucleus and the cytoplasm [33]. PIWI proteins and piRNAs edify ribonucleoproteins named PiRNA-induced silencing complexes (pi-RISCs). Pi-RISCs specificity is determined by piRNA sequence, whereas Argonaute PIWI protein mediates its effector function. PiRNAs associate with PIWI proteins and guide piRISCs to recognize complementary targets and achieve RNA silencing at transcriptional and posttranscriptional levels (**Figure 1**). Cytoplasmic PIWI-piRNA complexes silence their targets posttranscriptionally *via* piRNA-directed cleavage and the "ping-pong" amplification cycle, whereas nuclear PIWI proteins and piRNAs silence gene transcriptionally through epigenetic changes, including DNA methylation, implementation of H3K9me3 repressive marks, interactions with Mael and HP1 proteins, and repression of Pol II.

three functional domains: the PIWI-Argonaute-Zwille (PAZ) domain recognizing the 3′ end of the RNA, the middle domain (MID) providing a binding pocket for the 5′ end of guide strand RNA, and the PIWI domain containing catalytic residues that cleave target transcripts [47]. Unlike proteins of the AGO subclass, PIWI proteins comprise posttranslationally dimethylated arginine-rich motifs that allow interactions with Tudor proteins. These last proteins have pivotal role in functional activities of PIWI proteins by providing a scaffold for edification of higher-order molecular complexes located in *Drosophila* germ cells and mouse testis perinuclear granules named "nuage," similar to P-bodies [51–53]. The PIWI protein family is conserved in numerous organisms, including jellyfish, sponge, *planaria*, zebrafish (Ziwi, Zili), *Caenorhabditis elegans* (Prg1, Prg 2), *Drosophila melanogaster* (Piwi, Aub, Ago3), mouse (MIWI, MILI, MIWI2), and human (PIWIL1, PIWIL2, PIWIL3, PIWIL4) [51] (**Table 1**). PIWIL3 is observed only in human, and its functions are actually largely unknown. PIWI proteins expression is identified in a majority of organs such as liver, lung, heart, brain, pancreas, and kidney [54, 55]. PIWI proteins and piRNAs were first implicated in development, differentiation, and maintenance of germline cells [18, 56, 57]. However, mounting evidence has revealed that the PIWI-piRNA pathway is also instrumental in controlling gene expression

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This pathway has pivotal roles at all steps of oogenesis and spermatogenesis, but also in somatic cells such as ovary and testis of *Drosophila* [15]. This axis also controls, although at lower levels of expression, numerous biological processes implicated in homeostasis, including brain maturation [59] pancreatic function [55], fat metabolism [60], and regeneration [61]. Indeed, this pathway was initially studied in gonads and implicated in gene silencing of germinal cells [43]. Loss of function studies performed in zebrafish, *Drosophila*, *Caenorhabditis*, and mice have confirmed that the PIWI-piRNA pathway is involved in germline development, spermatogenesis, and maintenance of germline stem cells. Mutations in this pathway resulted in expansive TEs mobility, genomic instability, and sterility [44]. PIWI proteins have nonredundant functions in cell compartments. *Drosophila* PIWI proteins Aub and Ago3 cleaved TEs in the cytoplasm, whereas Piwi inactivated TEs in the nucleus [62, 63]. All mouse PIWI proteins MIWI, MILI, and MIWI2 were expressed during spermatogenesis, whereas only MILI was weakly expressed in female germinal cells [64, 65]. These mouse PIWI proteins not only silenced TEs posttranscriptionally but also inactivated TEs genes transcriptionally through CpG DNA methylation on TEs loci. Homozygous MIWI, MILI, and MIWI2 knockout male mice models were associated with propagation of LINE1 sequences, depleted spermatogenesis, and apoptosis of germinal cells [66]. Particularly, *Drosophila* PIWI mutants were correlated with derepression of TEs, absence of germline stem cell renewal, and depletion of gametes [67–69]. Actually, the main function of this pathway is maintaining germline and somatic genome integrity by silencing TEs at transcriptional and posttranscriptional levels [70]. However, only 20% of piRNAs are localized in TEs and other repeat genomic regions, suggesting that this pathway may have additional biological functions. In germinal cells, the PIWI-piRNA pathway prevents genomic instability of the next generation and sterility. In somatic nonaging cells, this pathway is pivotal in self-renewal, differentiation and maturation of stem cell, embryonic development, and whole body regeneration. In somatic tissues, the PIWI-piRNA pathway is implicated in chromosomal conformation, memory-related synaptic plasticity, transcriptional regulation of mRNAs with deadenylation, and transgenerational

both in germinal and somatic cells [58].

inheritance to preserve the memory of self and nonself [71–73].

**Figure 1.** Biogenesis of piRNA and PIWI-piRNA pathways and their function in maintaining genome integrity through transposable element (TE) in germline cells at transcriptional and posttranscriptional level. In Drosophila ovaries, the primary pathway (in the nucleus) operates in both germline and surrounding somatic cells, whereas the "ping-pong" cycle (in the cytoplasm) operates only in germline cells. In the nucleus, PIWI-piRNA complex can regulate HP1, H3K9 methylation, and DNA methylation to influence transposons.

PiRNAs are small single-stranded ncRNAs of 25–33 nt identified in various organisms ranging from sponges to higher vertebrates [34]. Experimental and bioinformatics studies have shown that piRNAs are the most abundant small ncRNAs expressed in mammalian species [35]. They are derived from long single-strand RNA precursors in a Dicer-independent manner. The human genome comprises more than 30,000 piRNAs in which 80% originate from intergenic sequences and 20% from introns and exons of pre-mRNAs [36]. Unlike miRNAs and endosiRNAs, production of piRNAs is not carried out in a precise manner and single strands of long primary precursor transcripts generate numerous piRNAs without a conserved sequence [37]. They comprise Uracil at their 5′ end and methylated 2′-O group at their 3′ end [38, 39]. PiRNAs were first identified in *Drosophila melanogaster* and were named repeat-associated small-interfering RNAs (rasiRNAs) because of their repetitive elements and TEs suppressing activity. RasiRNAs were later found to interact with Argonaute PIWI proteins and ultimately renamed piRNAs in 2006 [40–42]. PiRNAs were further investigated in *Caenorhabditis elegans*, zebrafish, mice, and more recently in humans. Their vast number present in numerous locations of the genome suggests that piRNAs may have potential crucial implications in the control of major biological processes. Indeed, PIWI-piRNA complexes silence TEs and control expression and activity of genes and proteins. They are also instrumental in genome rearrangement, germ stem cell maintenance, reproduction and fertility regulation, embryogenesis, and homeostasis [26, 43–48]. At the opposite of miRNAs and endosiRNAs, piRNAs function only through binding with PIWI proteins and harbor tissue-specific expression in various organs such as prostate and thyroid [49].

PIWI proteins were also initially identified in *Drosophila melanogaster* in which they play crucial roles in germline stem cell maintenance and self-renewal [50]. These proteins contain three functional domains: the PIWI-Argonaute-Zwille (PAZ) domain recognizing the 3′ end of the RNA, the middle domain (MID) providing a binding pocket for the 5′ end of guide strand RNA, and the PIWI domain containing catalytic residues that cleave target transcripts [47]. Unlike proteins of the AGO subclass, PIWI proteins comprise posttranslationally dimethylated arginine-rich motifs that allow interactions with Tudor proteins. These last proteins have pivotal role in functional activities of PIWI proteins by providing a scaffold for edification of higher-order molecular complexes located in *Drosophila* germ cells and mouse testis perinuclear granules named "nuage," similar to P-bodies [51–53]. The PIWI protein family is conserved in numerous organisms, including jellyfish, sponge, *planaria*, zebrafish (Ziwi, Zili), *Caenorhabditis elegans* (Prg1, Prg 2), *Drosophila melanogaster* (Piwi, Aub, Ago3), mouse (MIWI, MILI, MIWI2), and human (PIWIL1, PIWIL2, PIWIL3, PIWIL4) [51] (**Table 1**). PIWIL3 is observed only in human, and its functions are actually largely unknown. PIWI proteins expression is identified in a majority of organs such as liver, lung, heart, brain, pancreas, and kidney [54, 55]. PIWI proteins and piRNAs were first implicated in development, differentiation, and maintenance of germline cells [18, 56, 57]. However, mounting evidence has revealed that the PIWI-piRNA pathway is also instrumental in controlling gene expression both in germinal and somatic cells [58].

This pathway has pivotal roles at all steps of oogenesis and spermatogenesis, but also in somatic cells such as ovary and testis of *Drosophila* [15]. This axis also controls, although at lower levels of expression, numerous biological processes implicated in homeostasis, including brain maturation [59] pancreatic function [55], fat metabolism [60], and regeneration [61]. Indeed, this pathway was initially studied in gonads and implicated in gene silencing of germinal cells [43]. Loss of function studies performed in zebrafish, *Drosophila*, *Caenorhabditis*, and mice have confirmed that the PIWI-piRNA pathway is involved in germline development, spermatogenesis, and maintenance of germline stem cells. Mutations in this pathway resulted in expansive TEs mobility, genomic instability, and sterility [44]. PIWI proteins have nonredundant functions in cell compartments. *Drosophila* PIWI proteins Aub and Ago3 cleaved TEs in the cytoplasm, whereas Piwi inactivated TEs in the nucleus [62, 63]. All mouse PIWI proteins MIWI, MILI, and MIWI2 were expressed during spermatogenesis, whereas only MILI was weakly expressed in female germinal cells [64, 65]. These mouse PIWI proteins not only silenced TEs posttranscriptionally but also inactivated TEs genes transcriptionally through CpG DNA methylation on TEs loci. Homozygous MIWI, MILI, and MIWI2 knockout male mice models were associated with propagation of LINE1 sequences, depleted spermatogenesis, and apoptosis of germinal cells [66]. Particularly, *Drosophila* PIWI mutants were correlated with derepression of TEs, absence of germline stem cell renewal, and depletion of gametes [67–69]. Actually, the main function of this pathway is maintaining germline and somatic genome integrity by silencing TEs at transcriptional and posttranscriptional levels [70]. However, only 20% of piRNAs are localized in TEs and other repeat genomic regions, suggesting that this pathway may have additional biological functions. In germinal cells, the PIWI-piRNA pathway prevents genomic instability of the next generation and sterility. In somatic nonaging cells, this pathway is pivotal in self-renewal, differentiation and maturation of stem cell, embryonic development, and whole body regeneration. In somatic tissues, the PIWI-piRNA pathway is implicated in chromosomal conformation, memory-related synaptic plasticity, transcriptional regulation of mRNAs with deadenylation, and transgenerational inheritance to preserve the memory of self and nonself [71–73].

PiRNAs are small single-stranded ncRNAs of 25–33 nt identified in various organisms ranging from sponges to higher vertebrates [34]. Experimental and bioinformatics studies have shown that piRNAs are the most abundant small ncRNAs expressed in mammalian species [35]. They are derived from long single-strand RNA precursors in a Dicer-independent manner. The human genome comprises more than 30,000 piRNAs in which 80% originate from intergenic sequences and 20% from introns and exons of pre-mRNAs [36]. Unlike miRNAs and endosiRNAs, production of piRNAs is not carried out in a precise manner and single strands of long primary precursor transcripts generate numerous piRNAs without a conserved sequence [37]. They comprise Uracil at their 5′ end and methylated 2′-O group at their 3′ end [38, 39]. PiRNAs were first identified in *Drosophila melanogaster* and were named repeat-associated small-interfering RNAs (rasiRNAs) because of their repetitive elements and TEs suppressing activity. RasiRNAs were later found to interact with Argonaute PIWI proteins and ultimately renamed piRNAs in 2006 [40–42]. PiRNAs were further investigated in *Caenorhabditis elegans*, zebrafish, mice, and more recently in humans. Their vast number present in numerous locations of the genome suggests that piRNAs may have potential crucial implications in the control of major biological processes. Indeed, PIWI-piRNA complexes silence TEs and control expression and activity of genes and proteins. They are also instrumental in genome rearrangement, germ stem cell maintenance, reproduction and fertility regulation, embryogenesis, and homeostasis [26, 43–48]. At the opposite of miRNAs and endosiRNAs, piRNAs function only through binding with PIWI proteins and harbor tissue-specific expression in various organs such as

methylation, and DNA methylation to influence transposons.

236 Chromatin and Epigenetics

**Figure 1.** Biogenesis of piRNA and PIWI-piRNA pathways and their function in maintaining genome integrity through transposable element (TE) in germline cells at transcriptional and posttranscriptional level. In Drosophila ovaries, the primary pathway (in the nucleus) operates in both germline and surrounding somatic cells, whereas the "ping-pong" cycle (in the cytoplasm) operates only in germline cells. In the nucleus, PIWI-piRNA complex can regulate HP1, H3K9

PIWI proteins were also initially identified in *Drosophila melanogaster* in which they play crucial roles in germline stem cell maintenance and self-renewal [50]. These proteins contain

prostate and thyroid [49].

**Table 1.**

This table shows the structure and the network of human PIWI proteins. The human PIWI protein family includes PIWIL1, PIWIL2, PIWIL3, and PIWIL4. The

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general structure of Argonaute proteins depicting the PAZ domain (red) with the MID domain (blue), and PIWI domain (green). NCBI (http://www.ncbi.nlm.nih.gov) [74].

238 Chromatin and Epigenetics
