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Abstract: In recent years, PVRL2 (also known as Nectin-2 or CD112), a member of the Nectin family, has emerged as a crucial regulator of anti-tumor immune responses. PVRL2 interacts with the co-inhibitory receptor PVRIG that is expressed on T cells and NK cells, inhibiting their function. This interaction has thus become the presumed mechanism for PVRL2’s immunoinhibitory function. Therapeutic development targeting the PVRL2 pathway has primarily focused on the receptor PVRIG, with two anti-PVRIG antibodies currently undergoing Phase 1/2 clinical trials (NCT03667716, NCT04570839, NCT05746897). However, to date, very few studies have directly investigated the role of PVRL2, and whether PVRL2 indeed acts through PVRIG hasn’t been formally tested. Our recent work reveals the crucial role of PVRL2 in suppressing anti-tumor immune responses across multiple syngeneic mouse models. CRISPR-directed deletion of PVRL2 robustly reduced tumor growth in an immune-dependent manner, with an even greater effect than seen with the deletion of the prominent checkpoint, PD-L1. Specifically, our findings suggest that PVRL2 functions by suppressing CD8 T and NK cells in the tumor microenvironment. Interestingly, the impact of PVRL2 on tumor growth in our study is certainly much more significant than observed in previous work on PVRIG. To follow up on this interesting finding and determine whether the PVRL2’s impact on immune cells acts through PVRIG, we generated Pvrig KO mice. Surprisingly, we found that PVRL2’s function is largely independent of PVRIG, as it still robustly promotes tumor growth even in the absence of PVRIG, indicating PVRIG-independent mechanisms. We further demonstrate that PVRL2 operates in parallel to the PVR-TIGIT pathway, another important immunoregulatory pathway in the Nectin family. Combined deletion of PVR or PVRIG with PVRL2 loss did not further suppress tumor growth, while combined TIGIT blockade with PVRL2 loss resulted in the most substantial reduction in tumor growth. These findings strongly indicate the existence of additional unknown receptors mediating PVRL2’s function that remain to be discovered. Importantly, our study uncovers PVRL2’s central role in suppressing anti-tumor immune responses through mechanism outside its presumed receptor PVRIG, providing a compelling rationale for directly targeting PVRL2 itself. Combining PVRL2 and TIGIT targeting results in a near-complete block in tumor growth, offering a novel and promising therapeutic approach for cancer immunotherapy through the combinatorial targeting of PVRL2 and TIGIT. Citation Format: Jiuling Yang, Li Wang, James Byrnes, Lisa Kirkemo, Hannah Driks, Cassandra Belair, Oscar Aguilar, Lewis Lanier, Jim Wells, Lawrence Fong, Robert Blelloch. PVRL2 suppresses antitumor immune responses through PVRIG- and TIGIT-independent pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB237.

Poliovirus receptor-related 2 (PVRL2, also known as nectin-2 or CD112) is believed to act as an immune checkpoint protein in cancer; however, most insight into its role is inferred from studies on its known receptor, poliovirus receptor (PVR)-related immunoglobulin domain protein (PVRIG, also known as CD112R). Here, we study PVRL2 itself. PVRL2 levels were found to be high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer showed a dramatic reduction in tumor growth that was immune dependent. This effect was even greater than that seen with deletion of PD-L1. PVRL2 was shown to function by suppressing CD8+ T and natural killer cells in the tumor microenvironment. The loss of PVRL2 suppressed tumor growth even in the absence of PVRIG. In contrast, PVRIG loss showed no additive effect in the absence of PVRL2. T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade combined with PVRL2 deletion resulted in a near complete block in tumor growth. This effect was not recapitulated by the combined deletion of PVRL2 with its paralog, PVR, which is the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the antitumor immune response with functions beyond that of its known receptor PVRIG. Moreover, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy.

Coincident transcription and DNA replication causes replication stress and genome instability. Rapidly dividing mouse pluripotent stem cells are highly transcriptionally active and experience elevated replication stress, yet paradoxically maintain genome integrity. Here, we study FOXD3, a transcriptional repressor enriched in pluripotent stem cells, and show that its repression of transcription upon S phase entry is critical to minimizing replication stress and preserving genome integrity. Acutely deleting Foxd3 leads to immediate replication stress, G2/M phase arrest, genome instability and p53-dependent apoptosis. FOXD3 binds near highly transcribed genes during S phase entry, and its loss increases the expression of these genes. Transient inhibition of RNA polymerase II in S phase reduces observed replication stress and cell cycle defects. Loss of FOXD3-interacting histone deacetylases induces replication stress, while transient inhibition of histone acetylation opposes it. These results show how a transcriptional repressor can play a central role in maintaining genome integrity through the transient inhibition of transcription during S phase, enabling faithful DNA replication.

BackgroundEnhancers are essential in defining cell fates through the control of cell-type-specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers including the monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27.ResultsHere we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early differentiation of mouse embryonic stem cells. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1 but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. However, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of these chromatin events from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers.ConclusionsCollectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription.

The human placenta contains two specialized regions: the villous chorion where gases and nutrients are exchanged between maternal and fetal blood, and the smooth chorion (SC) which surrounds more than 70% of the developing fetus but whose cellular composition and function is poorly understood. Here, we use single cell RNA-sequencing to compare the cell types and molecular programs between these two regions in the second trimester human placenta. Each region consists of progenitor cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs) with similar gene expression programs. While CTBs in the villous chorion differentiate into syncytiotrophoblasts, they take an alternative trajectory in the SC producing a previously unknown CTB population which we term SC-specific CTBs (SC-CTBs). Marked by expression of region-specific cytokeratins, the SC-CTBs form a stratified epithelium above a basal layer of progenitor CTBs. They express epidermal and metabolic transcriptional programs consistent with a primary role in defense against physical stress and pathogens. Additionally, we show that SC-CTBs closely associate with EVTs and secrete factors that inhibit the migration of the EVTs. This restriction of EVT migration is in striking contrast to the villous region where EVTs migrate away from the chorion and invade deeply into the decidua. Together, these findings greatly expand our understanding of CTB differentiation in these distinct regions of the human placenta. This knowledge has broad implications for studies of the development, functions, and diseases of the human placenta.

Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan-binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived small extracellular vesicles (EVs), leading to the robust quantification of 953 cell and EV surface annotated proteins. We identified a newly recognized subset of EV-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer EVs. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.

Pluripotent embryonic stem cells have a unique cell cycle structure with a suppressed G1/S restriction point and little differential expression across the cell cycle phases. Here, we evaluate the link between G1/S restriction point activation, phasic gene expression, and cellular differentiation. Expression analysis reveals a gain in phasic gene expression across lineages between embryonic days E7.5 and E9.5. Genetic manipulation of the G1/S restriction point regulators miR-302 and P27 respectively accelerates or delays the onset of phasic gene expression in mouse embryos. Loss of miR-302-mediated p21 or p27 suppression expedites embryonic stem cell differentiation, while a constitutive Cyclin E mutant blocks it. Together, these findings uncover a causal relationship between emergence of the G1/S restriction point with a gain in phasic gene expression and cellular differentiation.

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.

The placenta is the interface between mother and fetus in all eutherian species. However, our understanding of this essential organ remains incomplete. A substantial challenge has been the syncytial cells of the placenta, which have made dissociation and independent evaluation of the different cell types of this organ difficult. Here, we address questions concerning the ontogeny, specification, and function of the cell types of a representative hemochorial placenta by performing single nuclei RNA sequencing (snRNA-seq) at multiple stages of mouse embryonic development focusing on the exchange interface, the labyrinth. Timepoints extended from progenitor-driven expansion through terminal differentiation. Analysis by snRNA-seq identified transcript profiles and inferred functions, cell trajectories, signaling interactions, and transcriptional drivers of all but the most highly polyploid cell types of the placenta. These data profile placental development at an unprecedented resolution, provide insights into differentiation and function across time, and provide a resource for future study.

Differentiation of human embryonic stem cells into pancreatic β cells holds great promise for the treatment of diabetes. Recent advances have led to the production of glucose-responsive insulin-secreting cells in vitro, but resulting cells remain less mature than their adult primary β cell counterparts. The barrier(s) to in vitro β cell maturation are unclear. Here, we evaluated a potential role for microRNAs. MicroRNA profiling showed high expression of let-7 family microRNAs in vivo, but not in in vitro differentiated β cells. Reduced levels of let-7 in vitro were associated with increased levels of the RNA binding protein LIN28B, a negative regulator of let-7 biogenesis. Ablation of LIN28B during human embryonic stem cell (hESC) differentiation toward β cells led to a more mature glucose-stimulated insulin secretion profile and the suppression of juvenile-specific genes. However, let-7 overexpression had little effect. These results uncover LIN28B as a modulator of β cell maturation in vitro.

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 Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

MicroRNAs hold great promise as biomarkers of disease. However, there are few efficient and robust methods for measuring microRNAs from low input samples. Here, we develop a high-throughput sequencing protocol that efficiently captures small RNAs while minimizing inherent biases associated with library production. The protocol is based on early barcoding such that all downstream manipulations can be performed on a pool of many samples thereby reducing reagent usage and workload. We show that the optimization of adapter concentrations along with the addition of nucleotide modifications and random nucleotides increases the efficiency of small RNA capture. We further show, using unique molecular identifiers, that stochastic capture of low input RNA rather than PCR amplification influences the biased quantitation of intermediately and lowly expressed microRNAs. Our improved method allows the processing of tens to hundreds of samples simultaneously while retaining high efficiency quantitation of microRNAs in low input samples from tissues or bodily fluids.

The enhancer landscape of pluripotent stem cells undergoes extensive reorganization during early mammalian development. The functions and mechanisms behind such reorganization, however, are unclear. Here, we show that the transcription factor GRHL2 is necessary and sufficient to activate an epithelial subset of enhancers as naive embryonic stem cells (ESCs) transition into formative epiblast-like cells (EpiLCs). Surprisingly, many GRHL2 target genes do not change in expression during the ESC-EpiLC transition. Instead, enhancers regulating these genes in ESCs diminish in activity in EpiLCs while GRHL2-dependent alternative enhancers become activated to maintain transcription. GRHL2 therefore assumes control over a subset of the naive network via enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency. These data evoke a model where the naive pluripotency network becomes partitioned into smaller, independent networks regulated by EpiLC-specific transcription factors, thereby priming cells for lineage specification.

Translation and mRNA degradation are intimately connected, yet the mechanisms that link them are not fully understood. Here, we studied these mechanisms in embryonic stem cells (ESCs). Transcripts showed a wide range of stabilities, which correlated with their relative translation levels and that did not change during early ESC differentiation. The protein DHH1 links translation to mRNA stability in yeast; however, loss of the mammalian homolog, DDX6, in ESCs did not disrupt the correlation across transcripts. Instead, the loss of DDX6 led to upregulated translation of microRNA targets, without concurrent changes in mRNA stability. The Ddx6 knockout cells were phenotypically and molecularly similar to cells lacking all microRNAs (Dgcr8 knockout ESCs). These data show that the loss of DDX6 can separate the two canonical functions of microRNAs: translational repression and transcript destabilization. Furthermore, these data uncover a central role for translational repression independent of transcript destabilization in defining the downstream consequences of microRNA loss.

Mouse oocyte maturation, fertilization, and reprogramming occur in the absence of transcription, and thus, changes in mRNA levels and translation rate are regulated through post-transcriptional mechanisms [1]. Surprisingly, microRNA function, which is a major form of post-transcriptional regulation, is absent during this critical period of mammalian development [2, 3]. Here, we investigated the mechanisms underlying the global suppression of microRNA activity. In both mouse and frogs, microRNA function was active in growing oocytes but then absent during oocyte maturation. RNA sequencing (RNA-seq) of mouse oocytes uncovered that the microRNA effector protein AGO2 is predominantly expressed as an alternative isoform that encodes a truncated protein lacking all of the known essential domains. Full-length Ago2 as well as the related Argonautes (Ago1, Ago3, and Ago4) were lowly expressed in maturing mouse oocytes. Reintroduction of full-length AGO2 together with an exogenous microRNA in either mouse or frog oocytes restored translational repression of a target reporter. However, levels of endogenous transcripts remained unchanged. Consistent with a lack of microRNA activity, analysis of transcripts with alternative polyadenylation sites showed increased stability of transcripts with a longer 3' UTR during oocyte maturation. Redundant mechanisms protecting endogenous transcripts and the conserved loss of microRNA activity suggest a strong selection for suppressing microRNA function in vertebrate oocytes.

Micropatterned cocultures are a useful experimental tool for the study of cell-cell interactions. Patterning methods often rely on sequential seeding of different cell types or removal of a barrier separating two populations, but it is difficult to pattern sharp interfaces between pure populations with low cross-contamination when using these approaches. Patterning by the use of reconfigurable substrates can overcome these limitations, but such methods can be costly and challenging to employ in a typical biology laboratory. Here, we describe a low-cost and simple-to-use reconfigurable substrate comprised of a transparent elastic material that is partially cut to form a slit that opens when the device is stretched. The slit seals back up when released, allowing two initially separate, adherent cell populations to be brought together to form a contact interface. Fluorescent imaging of patterned cocultures demonstrates the early establishment of a sharp cellular interface. As a proof of principle, we demonstrate the use of this device to study competition at the interface of two stem cell populations.

B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.

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.

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.

The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15, in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development.

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.

© 2015 Macmillan Publishers Limited. All rights reserved.The embryonic stem cell (ESC)-enriched miR-294/302 family and the somatic cell-enriched let-7 family stabilizes the self-renewing and differentiated cell fates, respectively. The mechanisms underlyin

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.

Embryonic stem cells (ESCs) have adopted an accelerated cell-cycle program with shortened gap phases and precocious expression of cell-cycle regulatory proteins, including cyclins and cyclin-dependent kinases (CDKs). We examined the effect of CDK inhibition on the pathways regulating proliferation and survival of ESCs. We found that inhibiting cyclin-dependent kinase 1 (CDK1) leads to activation of the DNA damage response, nuclear p53 stabilization, activation of a subset of p53 target genes including NOXA, and negative regulation of the anti-apoptotic protein MCL1 in human and mouse ESCs, but not differentiated cells. We demonstrate that MCL1 is highly expressed in ESCs and loss of MCL1 leads to ESC death. Finally, we show that clinically relevant CDK1 inhibitors prevent formation of ESC-derived tumors and induce necrosis in established ESC-derived tumors. Our data demonstrate that ES cells are uniquely sensitive to CDK1 inhibition via a p53/NOXA/MCL1 pathway.

Embryonic stem cells (ESCs) have adopted an accelerated cell-cycle program with shortened gap phases and precocious expression of cell-cycle regulatory proteins, including cyclins and cyclin-dependent kinases (CDKs). We examined the effect of CDK inhibitio

Establishment, maintenance, and exit from pluripotency require precise coordination of a cell's molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells.

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.

Ectopic expression of specific factors such as Oct4, Sox2, and Klf4 (OSK) is sufficient to reprogram somatic cells into induced pluripotent stem cells (iPSCs). In this study, we examine the paths taken by cells during the reprogramming process by following the transcriptional activation of two pluripotent miRNA clusters (mir-290 and mir-302) in individual cells in vivo and in vitro with knockin reporters. During embryonic development and embryonic stem cell differentiation, all cells sequentially expressed mir-290 and mir-302. In contrast, during OSK-induced reprogramming, cells activated the miRNA loci in a stochastic, nonordered manner. However, the addition of Sall4 to the OSK cocktail led to a consistent reverse sequence of locus activation (mir-302 then mir-290) and increased reprogramming efficiency. These results demonstrate that cells can follow multiple paths during the late stages of reprogramming, and that the trajectory of any individual cell is strongly influenced by the combination of factors introduced.

Serum microRNAs hold great promise as easily accessible and measurable biomarkers of disease. In prostate cancer, serum miRNA signatures have been associated with the presence of disease as well as correlated with previously validated risk models. However, it is unclear whether miRNAs can provide independent prognostic information beyond current risk models. Here, we focus on a group of low-risk prostate cancer patients who were eligible for active surveillance, but chose surgery. A major criteria for the low risk category is a Gleason score of 6 or lower based on pre-surgical biopsy. However, a third of these patients are upgraded to Gleason 7 on post surgical pathological analysis. Both in a discovery and a validation cohort, we find that pre-surgical serum levels of miR-19, miR-345 and miR-519c-5p can help identify these patients independent of their pre-surgical age, PSA, stage, and percent biopsy involvement. A combination of the three miRNAs increased the area under a receiver operator characteristics curve from 0.77 to 0.94 (p

Cells are constantly changing their state of equilibrium in response to internal and external stimuli. These changes in cell identity are driven by highly coordinated modulation of gene expression. This coordinated regulation is achieved in large part due to changes in the structure and composition of the chromatin, driven by epigenetic modulators. Recent discoveries in cellular and genomic reprogramming have highlighted the importance of chromatin modifications to reach and uphold the fidelity of target cell states. In this review, we focus on the latest work addressing the mechanisms surrounding the epigenetic regulation of various types of reprogramming, including somatic cell nuclear transfer (SCNT), cell fusion and transcription factor-induced and microRNA-induced pluripotency. The studies covered herein showcase the interplay between these epigenetic pathways, and highlight the importance of furthering our understanding of these connections to form a clearer picture of the mechanisms underlying stable cell fate transitions.

Individual microRNAs (miRNAs) can target hundreds of mRNAs forming networks of presumably cooperating genes. To test this presumption, we functionally screened miRNAs and their targets in the context of dedifferentiation of mouse fibroblasts to induced plu

The miR-294 and miR-302 microRNAs promote the abbreviated G1 phase of the embryonic stem cell (ESC) cell cycle and suppress differentiation induced by let-7. Here, we evaluated the role of the retinoblastoma (Rb) family proteins in these settings. Under normal growth conditions, miR-294 promoted the rapid G1-S transition independent of the Rb family. In contrast, miR-294 suppressed the further accumulation of cells in G1 in response to nutrient deprivation and cell-cell contact in an Rb-dependent fashion. We uncovered five additional miRNAs (miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218) that silenced ESC self-renewal in the absence of other miRNAs, all of which were antagonized by miR-294 and miR-302. Four of the six differentiation-inducing miRNAs induced an Rb-dependent G1 accumulation. However, all six still silenced self-renewal in the absence of the Rb proteins. These results show that the miR-294/miR-302 family acts through Rb-dependent and -independent pathways to regulate the G1 restriction point and the silencing of self-renewal, respectively.

Epithelial-mesenchymal transition (EMT) and the reverse process, mesenchymal-epithelial transition (MET), are essential during development and in the regulation of stem cell pluripotency, yet these processes are also activated in pathological contexts, such as in fibrosis and cancer progression. In EMT and MET, diverse signaling pathways cooperate in the initiation and progression of the EMT and MET programs, through regulation at transcriptional, post-transcriptional, translational, and post-translational levels. MicroRNAs recently emerged as potent regulators of EMT and MET, with their abilities to target multiple components involved in epithelial integrity or mesenchymal traits. By affecting EMT and MET processes, microRNAs are involved in the regulation of stem cell pluripotency and the control of tumor progression.

Regulatory T cells (Treg) are integral for immune homeostasis. Here we demonstrate that canonical microRNAs (miRNAs) are required for Treg function because mice with DGCR8-deficient Treg cells spontaneously develop a scurfy-like disease. Using genetic lineage marking we show that absence of miRNAs leads to reduced FoxP3 expression in Treg cells in vivo. In vitro culture of purified DGCR8-deficient Treg leads to a loss of FoxP3 expression. We conclude that canonical miRNAs are essential to maintain stable FoxP3 expression and Treg function. Thus, signals interfering with miRNA homeostasis might contribute to autoimmune diseases.

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

MicroRNAs (miRNAs) are critical regulators of nervous system function, and in vivo knockout studies have demonstrated that miRNAs are necessary for multiple aspects of neuronal development and survival. However, the role of miRNA biogenesis in the formation and function of synapses in the cerebral cortex is only minimally understood. Here, we have generated and characterized a mouse line with a conditional neuronal deletion of Dgcr8, a miRNA biogenesis protein predicted to process miRNAs exclusively. Loss of Dgcr8 in pyramidal neurons of the cortex results in a non-cell-autonomous reduction in parvalbumin interneurons in the prefrontal cortex, accompanied by a severe deficit in inhibitory synaptic transmission and a corresponding reduction of inhibitory synapses. Together, these results suggest a vital role for miRNAs in governing essential aspects of inhibitory transmission and interneuron development in the mammalian nervous system. These results may be relevant to human diseases such as schizophrenia, where both altered Dgcr8 levels as well as aberrant inhibitory transmission in the prefrontal cortex have been postulated to contribute to the pathophysiology of the disease.

The vast majority of animals mate more or less promiscuously. A few mammals, including humans, utilize more restrained mating strategies that entail a longer term affiliation with a single mating partner. Such pair bonding mating strategies have been resistant to genetic analysis because of a lack of suitable model organisms. Prairie voles are small mouse-like rodents that form enduring pair bonds in the wild as well as in the laboratory, and consequently they have been used widely to study social bonding behavior. The lack of targeted genetic approaches in this species however has restricted the study of the molecular and neural circuit basis of pair bonds. As a first step in rendering the prairie vole amenable to reverse genetics, we have generated induced pluripotent stem cell (IPSC) lines from prairie vole fibroblasts using retroviral transduction of reprogramming factors. These IPSC lines display the cellular and molecular hallmarks of IPSC cells from other organisms, including mice and humans. Moreover, the prairie vole IPSC lines have pluripotent differentiation potential since they can give rise to all three germ layers in tissue culture and in vivo. These IPSC lines can now be used to develop conditions that facilitate homologous recombination and eventually the generation of prairie voles bearing targeted genetic modifications to study the molecular and neural basis of pair bond formation.

Abstract Background Neuronal phenotypes associated with hemizygosity of individual genes within the 22q11.2 deletion syndrome locus hold potential towards understanding the pathogenesis of schizophrenia and autism. Included among these genes is Dgcr8, which encodes an RNA-binding protein required for microRNA biogenesis. Dgcr8 haploinsufficient mice (Dgcr8+/-) have reduced expression of microRNAs in brain and display cognitive deficits, but how microRNA deficiency affects the development and function of neurons in the cerebral cortex is not fully understood. Results In this study, we show that Dgcr8+/- mice display reduced expression of a subset of microRNAs in the prefrontal cortex, a deficit that emerges over postnatal development. Layer V pyramidal neurons in the medial prefrontal cortex of Dgcr8+/- mice have altered electrical properties, decreased complexity of basal dendrites, and reduced excitatory synaptic transmission. Conclusions These findings demonstrate that precise microRNA expression is critical for the postnatal development of prefrontal cortical circuitry. Similar defects in neuronal maturation resulting from microRNA deficiency could represent endophenotypes of certain neuropsychiatric diseases of developmental onset.

Recent prostate-specific antigen-based screening trials indicate an urgent need for novel and noninvasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Noncoding microRNAs (miRNA) in the serum and plasma have been shown to have potential as noninvasive markers for physiologic and pathologic conditions. To identify serum miRNAs that diagnose and correlate with the prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR method involving the purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA-deficient) mouse ES cells as the benchmark, we confirmed the validity of our technique and uncovered a considerable lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA scores, we identified miRNA signatures that allow us to diagnose cancer patients and correlate with a prognosis. These serum signatures include oncogenic and tumor-suppressive miRNAs, suggesting functional roles in prostate cancer progression.

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.

BackgroundThe Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript.Methodology/principal findingsTo determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8.Conclusion/significanceWe conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.