Plzf Regulates Germline Progenitor Self-Renewal by Opposing mTORC1

SummaryHyperactivity of mTORC1, a key mediator of cell growth, leads to stem cell depletion, although the underlying mechanisms are poorly defined. Using spermatogonial progenitor cells (SPCs) as a model system, we show that mTORC1 impairs stem cell maintenance by a negative feedback from mTORC1 to receptors required to transduce niche-derived signals. We find that SPCs lacking Plzf, a transcription factor essential for SPC maintenance, have enhanced mTORC1 activity. Aberrant mTORC1 activation in Plzf −/− SPCs inhibits their response to GDNF, a growth factor critical for SPC self-renewal, via negative feedback at the level of the GDNF receptor. Plzf opposes mTORC1 activity by inducing expression of the mTORC1 inhibitor Redd1. Thus, we identify the mTORC1-Plzf functional interaction as a critical rheostat for maintenance of the spermatogonial pool and propose a model whereby negative feedback from mTORC1 to the GDNF receptor balances SPC growth with self-renewal

Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway.

Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.We thank the Cold Spring Harbor Laboratory Microscopy Shared Resources for assistance, which are funded in part by Cancer Center Support Grant 5P30CA045508. This work was supported in part by a grant from the STARR Cancer Consortium, grants from the National Institutes of Health (NIH MERIT Award, R37GM062534 to G. J.H.), and a generous gift from Kathryn W. Davis to G.J.H. N.P. and G.J.H. are or were Investigators of the Howard Hughes Medical Institute. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Cell lines have been deposited by A.S. at the Drosophila Genomics Resource Center (NIH 2P40OD010949-10A1). G.J.H. is supported by Cancer Research UK and is a Wellcome Trust Investigator.This is the final version of the article. It first appeared from Cold Spring Harbor Press at http://dx.doi.org/10.1101/gad.284927.116

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming

piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.This work was supported by the National Institutes of Health R37 grant GM062534-14 to G.J.H. iTRAQ was performed with assistance from the Cold Spring Harbor Laboratory Proteomics Shared Resource, which is supported by Cancer Center support grant 5P30CA045508. W.S.S.G. is a McClintock Fellow of the Watson School of Biological Sciences and is supported by the NSS Scholarship from the Agency for Science, Technology and Research, Singapore. O.H.T. is supported by a fellowship of the Human Frontier Science Program. R.B. is supported by the Starr Centennial Scholarship from the Watson School of Biological Sciences. G.J.H. is a Howard Hughes Medical Institute Investigator.This is the final version of the article. It first appeared from Cold Spring Harbor Laboratory Press via http://dx.doi.org/10.1101/gad.260455.11

piRNA-guided co-transcriptional silencing coopts nuclear export factors.

The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. Here we show that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. We mapped the protein domains crucial for the Nxf2/Panx complex formation and show that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.This work is supported by National Institutes of Health Grants R37 GM062534 (to G.J.H.) and R01-HG006677 (to M.S.); National Science Foundation Grant DBI-1350041 (to M.S.); and a Swiss National Science Foundation Grant 31003A-143732 (to L.S.). This work was performed with assistance from Cold Spring Harbor Laboratory Shared Resources, which are funded, in part, by Cancer Center Support Grant 5P30CA045508.This is the final version of the article. It first appeared from PNAS via http://dx.doi.org/10.1073/pnas.151671811