[Show abstract][Hide abstract] ABSTRACT: The microRNA (miRNA) pathway, as a fundamental mechanism of gene regulation, plays a key role in controlling the establishment, self-renewal, and differentiation of stem cells. Such regulation is manifested as fine tuning the temporal- and tissue-specificity of gene expression. This fine-tuning function is achieved by (1) miRNAs form positive and negative feedback loops with transcription factors and epigenetic factors to exert concerted control of given biological processes and/or (2) different miRNAs converge to control one or more mRNA targets in a signaling pathway. These regulatory mechanisms are found in embryonic stem cells, iPS cells, and adult tissue stem cells. The distinct expression profiles of miRNAs and their regulatory roles in various types of stem cells render these RNAs potentially effective tools for clinical diagnosis and therapy. WIREs Dev Biol 2012, 1:83-95. doi: 10.1002/wdev.5 For further resources related to this article, please visit the WIREs website.
[Show abstract][Hide abstract] ABSTRACT: Epigenetic research has been focused on cell-type-specific regulation; less is known about common features of epigenetic programming shared by diverse cell types within an organism. Here, we report a modified method for chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and its use to construct a high-resolution map of the Drosophila melanogaster key histone marks, heterochromatin protein 1a (HP1a) and RNA polymerase II (polII). These factors are mapped at 50-bp resolution genome-wide and at 5-bp resolution for regulatory sequences of genes, which reveals fundamental features of chromatin modification landscape shared by major adult Drosophila cell types: the enrichment of both heterochromatic and euchromatic marks in transposons and repetitive sequences, the accumulation of HP1a at transcription start sites with stalled polII, the signatures of histone code and polII level/position around the transcriptional start sites that predict both the mRNA level and functionality of genes, and the enrichment of elongating polII within exons at splicing junctions. These features, likely conserved among diverse epigenomes, reveal general strategies for chromatin modifications.
[Show abstract][Hide abstract] ABSTRACT: PIWI proteins and piRNAs have been linked to transposon silencing in the primordial mouse testis, but their function in the adult testis remains elusive. Here we report the cytological characterization of piRNAs in the adult mouse testis and the phenotypic analysis of Miwi(-/-); Mili(-/-) mice. We show that piRNAs are specifically present in germ cells, especially abundant in spermatocytes and early round spermatids, regardless of the type of the genomic sequences to which they correspond. piRNAs and PIWI proteins are present in both the cytoplasm and nucleus. In the cytoplasm, they are enriched in the chromatoid body; whereas in the nucleus they are enriched in the dense body, a male-specific organelle associated with synapsis and the formation of the XY body during meiosis. Moreover, by generating Miwi(-/-); Mili(-/-) mice, which lack all PIWI proteins in the adult, we show that PIWI proteins and presumably piRNAs in the adult are required only for spermatogenesis. Spermatocytes without PIWI proteins are arrested at the pachytene stage, when the sex chromosomes undergo transcriptional silencing to form the XY body. These results pinpoint a function of the PIWI protein subfamily to meiosis during spermatogenesis.
[Show abstract][Hide abstract] ABSTRACT: Small noncoding RNAs have emerged as potent regulators of gene expression, especially in the germline. We review the biogenesis and regulatory function of three major small noncoding RNA pathways in the germline: The small interfering RNA (siRNA) pathway that leads to the degradation of target mRNAs, the microRNA (miRNA) pathway that mostly represses the translation of target mRNAs, and the newly discovered Piwi-interacting RNA (piRNA) pathway that appears to have diverse functions in epigenetic programming, transposon silencing, and the regulation of mRNA translation and stability. The siRNA and miRNA pathways are present in the germline as well as many somatic tissues, whereas the piRNA pathway is predominantly confined to the germline. Investigation of the three small RNA pathways has started to reveal a new dimension of gene regulation with defining roles in germline specification and development.
Cold Spring Harbor perspectives in biology 06/2011; 3(9):a002717. · 9.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genomic imprinting causes parental origin-specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.
[Show abstract][Hide abstract] ABSTRACT: The nuage is a germline-specific perinuclear structure that remains functionally elusive. Recently, the nuage in Drosophila was shown to contain two of the three PIWI proteins - Aubergine and Argonaute 3 (AGO3) - that are essential for germline development. The PIWI proteins bind to PIWI-interacting RNAs (piRNAs) and function in epigenetic regulation and transposon control. Here, we report a novel nuage component, PAPI (Partner of PIWIs), that contains a TUDOR domain and interacts with all three PIWI proteins via symmetrically dimethylated arginine residues in their N-terminal domain. In adult ovaries, PAPI is mainly cytoplasmic and enriched in the nuage, where it partially colocalizes with AGO3. The localization of PAPI to the nuage does not require the arginine methyltransferase dPRMT5 or AGO3. However, AGO3 is largely delocalized from the nuage and becomes destabilized in the absence of PAPI or dPRMT5, indicating that PAPI recruits PIWI proteins to the nuage to assemble piRNA pathway components. As expected, papi deficiency leads to transposon activation, phenocopying piRNA mutants. This further suggests that PAPI is involved in the piRNA pathway for transposon silencing. Moreover, AGO3 and PAPI associate with the P body component TRAL/ME31B complex in the nuage and transposon activation is observed in tral mutant ovaries. This suggests a physical and functional interaction in the nuage between the piRNA pathway components and the mRNA-degrading P-body components in transposon silencing. Overall, our study reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway.
Development 03/2011; 138(9):1863-73. · 6.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MITOPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini (Zuc), the Drosophila homolog of MITOPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MITOPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MITOPLD is involved in piRNA biogenesis, we generated Mitopld mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MITOPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MITOPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.
[Show abstract][Hide abstract] ABSTRACT: Lin28 inhibits the expression of let-7 microRNAs but also exhibits let-7-independent functions. Using immunoprecipitation and deep sequencing, we show here that Lin28 preferentially associates with a small subset of cellular mRNAs. Of particular interest are those for ribosomal proteins and metabolic enzymes, the expression levels of which are known to be coupled to cell growth and survival. Polysome profiling and reporter analyses suggest that Lin28 stimulates the translation of many or most of these targets. Moreover, Lin28-responsive elements were found within the coding regions of all target genes tested. Finally, a mutant Lin28 that still binds RNA but fails to interact with RNA helicase A (RHA), acts as a dominant-negative inhibitor of Lin28-dependent stimulation of translation. We suggest that Lin28, working in concert with RHA, enhances the translation of genes important for the growth and survival of human embryonic stem cells.
[Show abstract][Hide abstract] ABSTRACT: Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways.
Journal of Biological Chemistry 02/2011; 286(5):3789-97. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Canalization, also known as developmental robustness, describes an organism's ability to produce the same phenotype despite genotypic variations and environmental influences. In Drosophila, Hsp90, the trithorax-group proteins and transposon silencing have been previously implicated in canalization. Despite this, the molecular mechanism underlying canalization remains elusive. Here using a Drosophila eye-outgrowth assay sensitized by the dominant Kr(irregular facets-1)(Kr(If-1)) allele, we show that the Piwi-interacting RNA (piRNA) pathway, but not the short interfering RNA or micro RNA pathway, is involved in canalization. Furthermore, we isolated a protein complex composed of Hsp90, Piwi and Hop, the Hsp70/Hsp90 organizing protein homolog, and we demonstrated the function of this complex in canalization. Our data indicate that Hsp90 and Hop regulate the piRNA pathway through Piwi to mediate canalization. Moreover, they point to epigenetic silencing of the expression of existing genetic variants and the suppression of transposon-induced new genetic variation as two major mechanisms underlying piRNA pathway-mediated canalization.
[Show abstract][Hide abstract] ABSTRACT: The topipotency of the germline is the full manifestation of the pluri- and multipotency of embryonic and adult stem cells, thus the germline and stem cells must share common mechanisms that guarantee their multipotentials in development. One of the few such known shared mechanisms is represented by Piwi proteins, which constitute one of the two subfamilies of the Argonaute protein family. Piwi proteins bind to Piwi-interacting RNAs (piRNAs) that are generally 26 to 31 nucleotides in length. Both Piwi proteins and piRNAs are most abundantly expressed in the germline. Moreover, Piwi proteins are expressed broadly in certain types of somatic stem/progenitor cells and other somatic cells across animal phylogeny. Recent studies indicate that the Piwi-piRNA pathway mediates epigenetic programming and posttranscriptional regulation, which may be responsible for its function in germline specification, gametogenesis, stem cell maintenance, transposon silencing, and genome integrity in diverse organisms.
Annual Review of Genetics 11/2010; 45:447-69. · 17.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cell research has been focused on niche signaling and epigenetic programming of stem cells. However, epigenetic programming of niche cells remains unexplored. We showed previously that Piwi plays a crucial role in Piwi-interacting RNA-mediated epigenetic regulation and functions in the niche cells to maintain germline stem cells (GSCs) in the Drosophila ovary. To investigate the epigenetic programming of niche cells by Piwi, we screened mutations in the Polycomb and trithorax group genes, and an enhancer of Polycomb and trithorax called corto, for their potential genetic interaction with piwi. corto encodes a chromatin protein. corto mutations restored GSC division in mutants of piwi and fs(1)Yb (Yb), a gene that regulates piwi expression in niche cells to maintain GSCs. Consistent with this, corto appears to be expressed in the niche cells and is not required in the germline. Furthermore, in corto-suppressed Yb mutants, the expression of hedgehog (hh) is restored in niche cells, which is likely responsible for corto suppression of the GSC and somatic stem cell defects of Yb mutants. These results reveal a novel epigenetic mechanism involving Corto and Piwi that defines the fate and signaling function of niche cells in maintaining GSCs.
[Show abstract][Hide abstract] ABSTRACT: Loss-of-function studies in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) via nonviral approaches have been largely unsuccessful. Here we report a simple and cost-effective method for high-efficiency delivery of plasmids and siRNAs into hESCs and iPSCs. Using this method for siRNA delivery, we achieve >90% reduction in the expression of the stem cell factors Oct4 and Lin28, and observe cell morphological and staining pattern changes, characteristics of hESC differentiation, as a result of Oct4 knockdown.
[Show abstract][Hide abstract] ABSTRACT: To examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of hESCs into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation-N1 (early initiation), N2 (neural progenitor), and N3 (early glial-like)-were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call isoform specialization. During neural differentiation, we observed differential expression of many types of genes, including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, neural progenitor cell identity maintenance, and the transition from a predominantly neuronal state into one with increased gliogenic potential.
Proceedings of the National Academy of Sciences 03/2010; 107(11):5254-9. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.
PLoS ONE 01/2010; 5(10):e13406. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yb regulates the proliferation of both germline and somatic stem cells in the Drosophila melanogaster ovary by activating piwi and hh expression in niche cells. In this study, we show that Yb protein is localized as discrete cytoplasmic spots exclusively in the somatic cells of the ovary and testis. These spots, which are different from all known cytoplasmic structures in D. melanogaster, are evenly electron-dense spheres 1.5 microm in diameter (herein termed the Yb body). The Yb body is frequently associated with mitochondria and a less electron-dense sphere of similar size that appears to be RNA rich. There are one to two Yb bodies/cell, often located close to germline cells. The N-terminal region of Yb is required for hh expression in niche cells, whereas the C-terminal region is required for localization to Yb bodies. The entire Yb protein is necessary for piwi expression in niche cells. A double mutant of Yb and a novel locus show male germline loss, revealing a function for Yb in male germline stem cell maintenance.
The Journal of Cell Biology 06/2009; 185(4):613-27. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Piwi proteins are essential for germline development, stem cell self-renewal, epigenetic regulation, and transposon silencing [1-7]. They bind to a complex class of small noncoding RNAs called Piwi-interacting RNAs (piRNAs) . Mammalian Piwi proteins such as Mili are localized in the cytoplasm of spermatogenic cells, where they are associated with a germline-specific organelle called the nuage or its derivative, the chromatoid body, as well as with polysomes . To investigate the molecular mechanisms mediated by Mili, we searched for Mili-interacting proteins. Here, we report that Mili specifically interacts with Tudor domain-containing protein 1 (Tdrd1), a germline protein that contains multiple Tudor domains [10, 11]. This RNA-independent interaction is mediated through the N-terminal domain of Mili and the N-terminal region of Tdrd1 containing the myeloid Nervy DEAF-1 (MYND) domain and the first two Tudor domains. In addition, Mili positively regulates the expression of the Tdrd1 mRNA. Furthermore, Mili and Tdrd1 mutants share similar spermatogenic defects. However, Tdrd1, unlike Mili, is not required for piRNA biogenesis. Our results suggest that Mili interacts with Tdrd1 in the nuage and chromatoid body. This interaction does not contribute to piRNA biogenesis but represents a regulatory mechanism that is critical for spermatogenesis.
Current biology: CB 05/2009; 19(8):640-4. · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The hallmark of a stem cell is its ability to self-renew and to produce numerous differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new dimension of gene regulation in controlling stem cell fate and behaviour.
[Show abstract][Hide abstract] ABSTRACT: The Argonaute/PIWI protein family consists of Argonaute and PIWI subfamilies. Argonautes function in RNA interference and micro-RNA pathways; whereas PIWIs bind to PIWI-interacting RNAs and regulate germ line development, stem cell maintenance, epigenetic regulation, and transposition. However, the role of PIWIs in mammalian stem cells has not been demonstrated, and molecular mechanisms mediated by PIWIs remain elusive. Here we show that MILI, a murine PIWI protein, is expressed in the cytoplasm of testicular germ line stem cells, spermatogonia, and early spermatocytes, where it is enriched in chromatoid bodies. MILI is essential for the self-renewing division and differentiation of germ line stem cells but does not affect initial establishment of the germ line stem cell population at 7 days postpartum. Furthermore, MILI forms a stable RNA-independent complex with eIF3a and associates with the eIF4E- and eIF4G-containing m7G cap-binding complex. In isolated 7 days postpartum seminiferous tubules containing mostly germ line stem cells, the mili mutation has no effect on the cellular mRNA level yet significantly reduces the rate of protein synthesis. These observations indicate that MILI may positively regulate translation and that such regulation is required for germ line stem cell self-renewal.
Journal of Biological Chemistry 01/2009; 284(10):6507-19. · 4.65 Impact Factor