Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory.

PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.

The roles of microRNAs in mouse development.

Hundreds of microRNAs (miRNAs) are expressed in distinct spatial and temporal patterns during embryonic and postnatal mouse development. The loss of all miRNAs through the deletion of critical miRNA biogenesis factors results in early lethality. The function of each miRNA stems from their cumulative negative regulation of multiple mRNA targets expressed in a particular cell type. During development, miRNAs often coordinate the timing and direction of cell fate transitions. In adults, miRNAs frequently contribute to organismal fitness through homeostatic roles in physiology. Here, we review how the recent dissection of miRNA-knockout phenotypes in mice as well as advances related to their targets, dosage, and interactions have collectively informed our understanding of the roles of miRNAs in mammalian development and adaptive responses.

microRNA control of mouse and human pluripotent stem cell behavior.

In the past decade, significant progress has been made in understanding both microRNA function and cellular pluripotency. Here we review the intersection of these two exciting fields. While microRNAs are not required for the establishment and maintenance of pluripotency in early development and cell culture, respectively, they are critically important in the regulation of the cell cycle structure of pluripotent stem cells as well as the silencing of the pluripotency program upon differentiation. Pluripotent cells, both in vivo and in vitro, dominantly express a single family of microRNAs, which can promote the reprogramming of a somatic cell back to a pluripotent state. Here, we review the known mechanisms by which these and other microRNAs regulate the different aspects of the pluripotent stem cell program in both mouse and human.

Regulation of microRNA function in somatic stem cell proliferation and differentiation.

microRNAs (miRNAs) are important modulators of development. Owing to their ability to simultaneously silence hundreds of target genes, they have key roles in large-scale transcriptomic changes that occur during cell fate transitions. In somatic stem and progenitor cells--such as those involved in myogenesis, haematopoiesis, skin and neural development--miRNA function is carefully regulated to promote and stabilize cell fate choice. miRNAs are integrated within networks that form both positive and negative feedback loops. Their function is regulated at multiple levels, including transcription, biogenesis, stability, availability and/or number of target sites, as well as their cooperation with other miRNAs and RNA-binding proteins. Together, these regulatory mechanisms result in a refined molecular response that enables proper cellular differentiation and function.

Posttranscriptional crossregulation between Drosha and DGCR8.

The Drosha-DGCR8 complex, also known as Microprocessor, is essential for microRNA (miRNA) maturation. Drosha functions as the catalytic subunit, while DGCR8 (also known as Pasha) recognizes the RNA substrate. Although the action mechanism of this complex has been intensively studied, it remains unclear how Drosha and DGCR8 are regulated and if these proteins have any additional role(s) apart from miRNA processing. Here, we report that Drosha and DGCR8 regulate each other posttranscriptionally. The Drosha-DGCR8 complex cleaves the hairpin structures embedded in the DGCR8 mRNA and thereby destabilizes the mRNA. We further find that DGCR8 stabilizes the Drosha protein via protein-protein interaction. This crossregulation between Drosha and DGCR8 may contribute to the homeostatic control of miRNA biogenesis. Furthermore, microarray analyses suggest that a number of mRNAs may be downregulated in a Microprocessor-dependent, miRNA-independent manner. Our study reveals a previously unsuspected function of Microprocessor in mRNA stability control.

MicroRNA-dependent inhibition of PFN2 orchestrates ERK activation and pluripotent state transitions by regulating endocytosis.

Profilin2 (PFN2) is a target of the embryonic stem cell (ESC)-enriched miR-290 family of microRNAs (miRNAs) and an actin/dynamin-binding protein implicated in endocytosis. Here we show that the miR-290-PFN2 pathway regulates many aspects of ESC biology. In the absence of miRNAs, PFN2 is up-regulated in ESCs, with a resulting decrease in endocytosis. Reintroduction of miR-290, knockout of Pfn2, or disruption of the PFN2-dynamin interaction domain in miRNA-deficient cells reverses the endocytosis defect. The reduced endocytosis is associated with impaired extracellular signal-regulated kinase (ERK) signaling, delayed ESC cell cycle progression, and repressed ESC differentiation. Mutagenesis of the single canonical conserved 3' UTR miR-290-binding site of Pfn2 or overexpression of the Pfn2 open reading frame alone in otherwise wild-type cells largely recapitulates these phenotypes. Taken together, these findings define an axis of posttranscriptional control, endocytosis, and signal transduction that is important for ESC proliferation and differentiation.

Epigenetic control of transcriptional regulation in pluripotency and early differentiation.

Pluripotent stem cells give rise to all cells of the adult organism, making them an invaluable tool in regenerative medicine. In response to differentiation cues, they can activate markedly distinct lineage-specific gene networks while turning off or rewiring pluripotency networks. Recent innovations in chromatin and nuclear structure analyses combined with classical genetics have led to novel insights into the transcriptional and epigenetic mechanisms underlying these networks. Here, we review these findings in relation to their impact on the maintenance of and exit from pluripotency and highlight the many factors that drive these processes, including histone modifying enzymes, DNA methylation and demethylation, nucleosome remodeling complexes and transcription factor-mediated enhancer switching.

The eutheria-specific miR-290 cluster modulates placental growth and maternal-fetal transport.

The vertebrate-specific ESCC microRNA family arises from two genetic loci in mammals: miR-290/miR-371 and miR-302. The miR-302 locus is found broadly among vertebrates, whereas the miR-290/miR-371 locus is unique to eutheria, suggesting a role in placental development. Here, we evaluate that role. A knock-in reporter for the mouse miR-290 cluster is expressed throughout the embryo until gastrulation, when it becomes specifically expressed in extraembryonic tissues and the germline. In the placenta, expression is limited to the trophoblast lineage, where it remains highly expressed until birth. Deletion of the miR-290 cluster gene (Mirc5) results in reduced trophoblast progenitor cell proliferation and a reduced DNA content in endoreduplicating trophoblast giant cells. The resulting placenta is reduced in size. In addition, the vascular labyrinth is disorganized, with thickening of the maternal-fetal blood barrier and an associated reduction in diffusion. Multiple mRNA targets of the miR-290 cluster microRNAs are upregulated. These data uncover a crucial function for the miR-290 cluster in the regulation of a network of genes required for placental development, suggesting a central role for these microRNAs in the evolution of placental mammals.

MicroRNA-29 regulates T-box transcription factors and interferon-γ production in helper T cells.

MicroRNA (miRNA)-deficient helper T cells exhibit abnormal IFN-γ production and decreased proliferation. However, the contributions of individual miRNAs to this phenotype remain poorly understood. We conducted a screen for miRNA function in primary T cells and identified individual miRNAs that rescue the defects associated with miRNA deficiency. Multiple members of the miR-17 and miR-92 families enhanced miRNA-deficient T cell proliferation whereas miR-29 largely corrected their aberrant interferon-γ (IFN-γ) expression. Repression of IFN-γ production by miR-29 involved direct targeting of both T-bet and Eomes, two transcription factors known to induce IFN-γ production. Although not usually expressed at functionally relevant amounts in helper T cells, Eomes was abundant in miRNA-deficient cells and was upregulated after miR-29 inhibition in wild-type cells. These results demonstrate that miR-29 regulates helper T cell differentiation by repressing multiple target genes, including at least two that are independently capable of inducing the T helper 1 (Th1) cell gene expression program.

Let-7 and miR-125 cooperate to prime progenitors for astrogliogenesis.

The molecular basis of astrocyte differentiation and maturation is poorly understood. As microRNAs have important roles in cell fate transitions, we set out to study their function during the glial progenitor cell (GPC) to astrocyte transition. Inducible deletion of all canonical microRNAs in GPCs in vitro led to a block in the differentiation to astrocytes. In an unbiased screen, the reintroduction of let-7 and miR-125 families of microRNAs rescued differentiation. Let-7 and miR-125 shared many targets and functioned in parallel to JAK-STAT signaling, a known regulator of astrogliogenesis. While individual knockdown of shared targets did not rescue the differentiation phenotype in microRNA-deficient GPCs, overexpression of these targets in wild-type GPCs blocked differentiation. This finding supports the idea that microRNAs simultaneously suppress multiple mRNAs that inhibit differentiation. MicroRNA-regulated transcripts exhibited concordant changes during in vivo differentiation and were enriched for a gene set upregulated in glioblastomas, consistent with validity of using the in vitro model to study in vivo events. These findings provide insight into the microRNAs and the genes they regulate in this important cell fate transition.

Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro.

Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1(+) and subsequent PDX1(+)/NKX6.1(+) pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1(+)/NKX6.1(+) progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells.

NF45 and NF90/NF110 coordinately regulate ESC pluripotency and differentiation.

While years of investigation have elucidated many aspects of embryonic stem cell (ESC) regulation, the contributions of post-transcriptional and translational mechanisms to the pluripotency network remain largely unexplored. In particular, little is known in ESCs about the function of RNA binding proteins (RBPs), the protein agents of post-transcriptional regulation. We performed an unbiased RNAi screen of RBPs in an ESC differentiation assay and identified two related genes, NF45 (Ilf2) and NF90/NF110 (Ilf3), whose knockdown promoted differentiation to an epiblast-like state. Characterization of NF45 KO, NF90 + NF110 KO, and NF110 KO ESCs showed that loss of NF45 or NF90 + NF110 impaired ESC proliferation and led to dysregulated differentiation down embryonic lineages. Additionally, we found that NF45 and NF90/NF110 physically interact and influence the expression of each other at different levels of regulation. Globally across the transcriptome, NF45 KO ESCs and NF90 + NF110 KO ESCs show similar expression changes. Moreover, NF90 + NF110 RNA immunoprecipitation (RIP)-seq in ESCs suggested that NF90/NF110 directly regulate proliferation, differentiation, and RNA-processing genes. Our data support a model in which NF45, NF90, and NF110 operate in feedback loops that enable them, through both overlapping and independent targets, to help balance the push and pull of pluripotency and differentiation cues.

Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition.

Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events that are driving these transitions. Here we used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA-binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation. Using this strategy, we identified highly specific patterns of mRNA recruitment to the polysomes that are synchronized with the cell cycle. A large number of the mRNAs recovered with translating ribosomes contain motifs for the RNA-binding proteins DAZL (deleted in azoospermia-like) and CPEB (cytoplasmic polyadenylation element-binding protein). Although a Dazl role in early germ cell development is well established, no function has been described during oocyte-to-embryo transition. We demonstrate that CPEB1 regulates Dazl post-transcriptionally, and that DAZL is essential for meiotic maturation and embryonic cleavage. In the absence of DAZL synthesis, the meiotic spindle fails to form due to disorganization of meiotic microtubules. Therefore, Cpeb1 and Dazl function in a progressive, self-reinforcing pathway to promote oocyte maturation and early embryonic development.

Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.

MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.

A miR-372/let-7 Axis Regulates Human Germ Versus Somatic Cell Fates.

The embryonic stem cell cycle (ESCC) and let-7 families of miRNAs function antagonistically in the switch between mouse embryonic stem cell self-renewal and somatic differentiation. Here, we report that the human ESCC miRNA miR-372 and let-7 act antagonistically in germline differentiation from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). hESC and iPSC-derived primordial germ cell-like cells (PGCLCs) expressed high levels of miR-372 and conversely, somatic cells expressed high levels of let-7. Manipulation of miRNA levels by introduction of miRNA mimics or knockdown with miRNA sponges demonstrated that miR-372 promotes whereas let-7 antagonizes PGCLC differentiation. Knockdown of the individual miR-372 targets SMARCC1, MECP2, CDKN1, RBL2, RHOC, and TGFBR2 increased PGCLC production, whereas knockdown of the let-7 targets CMYC and NMYC suppressed PGCLC differentiation. These findings uncover a miR-372/let-7 axis regulating human primordial germ cell (PGC) specification. Stem Cells 2016;34:1985-1991.

DGCR8 is essential for tumor progression following PTEN loss in the prostate.

In human prostate cancer, the microRNA biogenesis machinery increases with prostate cancer progression. Here, we show that deletion of the Dgcr8 gene, a critical component of this complex, inhibits tumor progression in a Pten-knockout mouse model of prostate cancer. Early stages of tumor development were unaffected, but progression to advanced prostatic intraepithelial neoplasia was severely inhibited. Dgcr8 loss blocked Pten null-induced expansion of the basal-like, but not luminal, cellular compartment. Furthermore, while late-stage Pten knockout tumors exhibit decreased senescence-associated beta-galactosidase activity and increased proliferation, the simultaneous deletion of Dgcr8 blocked these changes resulting in levels similar to wild type. Sequencing of small RNAs in isolated epithelial cells uncovered numerous miRNA changes associated with PTEN loss. Consistent with a Pten-Dgcr8 association, analysis of a large cohort of human prostate tumors shows a strong correlation between Akt activation and increased Dgcr8 mRNA levels. Together, these findings uncover a critical role for microRNAs in enhancing proliferation and enabling the expansion of the basal cell compartment associated with tumor progression following Pten loss.

Opposing microRNA families regulate self-renewal in mouse embryonic stem cells.

When embryonic stem cells (ESCs) differentiate, they must both silence the ESC self-renewal program and activate new tissue-specific programs. In the absence of DGCR8 (Dgcr8(-/-)), a protein required for microRNA (miRNA) biogenesis, mouse ESCs are unable to silence self-renewal. Here we show that the introduction of let-7 miRNAs-a family of miRNAs highly expressed in somatic cells-can suppress self-renewal in Dgcr8(-/-) but not wild-type ESCs. Introduction of ESC cell cycle regulating (ESCC) miRNAs into the Dgcr8(-/-) ESCs blocks the capacity of let-7 to suppress self-renewal. Profiling and bioinformatic analyses show that let-7 inhibits whereas ESCC miRNAs indirectly activate numerous self-renewal genes. Furthermore, inhibition of the let-7 family promotes de-differentiation of somatic cells to induced pluripotent stem cells. Together, these findings show how the ESCC and let-7 miRNAs act through common pathways to alternatively stabilize the self-renewing versus differentiated cell fates.

DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal.

The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation.

Sox1 marks an activated neural stem/progenitor cell in the hippocampus.

The dentate gyrus of the hippocampus continues generating new neurons throughout life. These neurons originate from radial astrocytes within the subgranular zone (SGZ). Here, we find that Sox1, a member of the SoxB1 family of transcription factors, is expressed in a subset of radial astrocytes. Lineage tracing using Sox1-tTA;tetO-Cre;Rosa26 reporter mice shows that the Sox1-expressing cells represent an activated neural stem/progenitor population that gives rise to most if not all newly born granular neurons, as well as a small number of mature hilar astrocytes. Furthermore, a subpopulation of Sox1-marked cells have long-term neurogenic potential, producing new neurons 3 months after inactivation of tetracycline transactivator. Remarkably, after 8 weeks of labeling and a 12-week chase, as much as 44% of all granular neurons in the dentate gyrus were derived from Sox1 lineage-traced adult neural stem/progenitor cells. The fraction of Sox1-positive cells within the radial astrocyte population decreases with age, correlating with a decrease in neurogenesis. However, expression profiling shows that these cells are transcriptionally stable throughout the lifespan of the mouse. These results demonstrate that Sox1 is expressed in an activated stem/progenitor population whose numbers decrease with age while maintaining a stable molecular program.

Small RNAs in early mammalian development: from gametes to gastrulation.

Small non-coding RNAs, including microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), play essential roles in mammalian development. The function and timing of expression of these three classes of small RNAs differ greatly. piRNAs are expressed and play a crucial role during male gametogenesis, whereas endo-siRNAs are essential for oocyte meiosis. By contrast, miRNAs are ubiquitously expressed in somatic tissues and function throughout post-implantation development. Surprisingly, however, miRNAs are non-essential during pre-implantation embryonic development and their function is suppressed during oocyte meiosis. Here, we review the roles of small non-coding RNAs during the early stages of mammalian development, from gamete maturation through to gastrulation.