Explore the vast range of titles available.

Key Points Insights gained from population-based and single-cell studies reveal two major phases during reprogramming. OSK (OCT4, SOX2 and KLF4) factors act as 'pioneer' factors that open chromatin regions and allow the activation of those genes that are essential for establishment and maintenance of the pluripotent state. This promiscuous binding of OSK is also essential for the initiation of crucial processes for the reprogramming process such as proliferation and mesenchymal-to-epithelial transition (MET). We present evidence supporting a model in which the reprogramming process contains an early stochastic phase that leads to the instigation of a second more deterministic phase that starts with the activation of Sox2 . How similar are induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs)? The available evidence has not settled whether the alterations seen in iPSCs are the result of the reprogramming process or whether they are due to pre-existing genetic and epigenetic differences among parental fibroblasts. Our understanding of the molecular steps that occur during reprogramming somatic cells to induced pluripotent stem cells has recently been improved through analyses of cell populations and single cells. Here the authors consider the phases of reprogramming, models for describing the process and the roles of reprogramming factors. Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic-stem-cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodelling the genome, including new insights into the function of MYC, and describe the different phases, markers and emerging models of reprogramming.

Detailed analyses of siRNA delivery by lipid nanoparticles reveal major pathways of siRNA internalization and endosomal escape. Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein–small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

Human trophoblast stem cells (hTSCs) can be derived from embryonic stem cells (hESCs) or be induced from somatic cells by OCT4, SOX2, KLF4 and MYC (OSKM). Here we explore whether the hTSC state can be induced independently of pluripotency, and what are the mechanisms underlying its acquisition. We identify GATA3, OCT4, KLF4 and MYC (GOKM) as a combination of factors that can generate functional hiTSCs from fibroblasts. Transcriptomic analysis of stable GOKM- and OSKM-hiTSCs reveals 94 hTSC-specific genes that are aberrant specifically in OSKM-derived hiTSCs. Through time-course-RNA-seq analysis, H3K4me2 deposition and chromatin accessibility, we demonstrate that GOKM exert greater chromatin opening activity than OSKM. While GOKM primarily target hTSC-specific loci, OSKM mainly induce the hTSC state via targeting hESC and hTSC shared loci. Finally, we show that GOKM efficiently generate hiTSCs from fibroblasts that harbor knockout for pluripotency genes, further emphasizing that pluripotency is dispensable for hTSC state acquisition. In this work Naama et al. describe and deeply characterize a direct approach to produce human induced trophoblast stem cells from fibroblasts and show that it produces superior hiTSCs when compared to OSKM-hiTSCs.

Following fertilization, it is only at the 32-64-cell stage when a clear segregation between cells of the inner cell mass and trophectoderm is observed, suggesting a ‘T’-shaped model of specification. Here, we examine whether the acquisition of these two states in vitro, by nuclear reprogramming, share similar dynamics/trajectories. Using a comparative parallel multi-omics analysis (i.e., bulk RNA-seq, scRNA-seq, ATAC-seq, ChIP-seq, RRBS and CNVs) on cells undergoing reprogramming to pluripotency and TSC state we show that each reprogramming system exhibits specific trajectories from the onset of the process, suggesting ‘V’-shaped model. We describe in detail the various trajectories toward the two states and illuminate reprogramming stage-specific markers, blockers, facilitators and TSC subpopulations. Finally, we show that while the acquisition of the TSC state involves the silencing of embryonic programs by DNA methylation, during the acquisition of pluripotency these regions are initially defined but retain inactive by the elimination of H3K27ac. Ectopic transcription factor expression can reprogram mouse fibroblasts to pluripotent or trophoblast stem cells. Here the authors apply multi-omics analyses to the induction of pluripotency and trophoblast stem cell state from fibroblasts, comparing these with the changes in transcriptome of early embryonic cells.

The cell-type-specific function of transcription factors (TFs) is crucial for determining cellular identity. However, it is unclear how a single TF can function specifically in different cell types. Here, we define the molecular features that enable OCT4 to reprogram somatic cells into pluripotent or trophoblast stem cells, maintain the self-renewal of embryonic stem cells (ESCs), and drive lineage commitment during early embryonic development. Embedded within the intrinsically disordered regions (IDRs) of OCT4, we uncover s hort li near p eptides that are e ssential for r eprogramming (SLiPERs) but dispensable for ESC self-renewal. SLiPERs adopt a quasi-ordered state and, during reprogramming, recruit a unique set of proteins to closed chromatin that are unnecessary for ESC self-renewal. Interestingly, SLiPERs are essential for embryos to develop beyond late gastrulation. Removing SLiPERs leads to aberrant OCT4 binding, derailing the regular transition of ESCs out of pluripotency. Our findings identify modules within IDRs that contribute to the functional versatility and specificity of TFs. Here they perform a systematic dissection of OCT4 and reveal how intrinsically disordered regions can be used to serve specific functions during reprogramming and embryonic development. This can be exploited to engineer more efficient and specific reprogramming factors.

Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so. This distinction between undifferentiated and differentiated cells arises during cell differentiation, and can be reversed by reprogramming of gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b in tandem with DNMT3a). Overall, the findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, thus underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus. Gene-editing at the DM1 mutant locus revealed a fundamental difference between undifferentiated and differentiated cell states: abnormal epigenetic modifications cannot be repaired after differentiation.

Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 \"cancer initiating genes\", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification.

Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild‐type p53. These miRNAs are repressed by p53 in an E2F1‐mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi‐gene transcriptional and post‐transcriptional feed‐forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network. Synopsis Precise regulation of gene expression is crucial for maintaining homeostasis in healthy tissues and for the execution of cellular programs such as proliferation, differentiation and cell death. In the last decade, microRNAs (miRNAs) have been uncovered as an expanding family of gene expression regulators. These short non‐coding RNAs regulate gene expression at the post‐transcriptional level by promoting translational inhibition or mRNA degradation (Bartel, 2004 ). Similar to protein‐coding genes, the expression of miRNAs is also regulated by transcription factors (TFs), and induction or repression of miRNAs has been demonstrated to play a role in physiological processes such as immune response (Thai et al , 2007 ) and apoptosis (Chang et al , 2007 ; Raver‐Shapira et al , 2007 ). Accordingly, deregulation of miRNAs is associated with diverse types of diseases, including a variety of cancers (Esquela‐Kerscher and Slack, 2006 ; Volinia et al , 2006 ). In an earlier computational study, we predicted the presence of several types of regulatory network motifs that involve TFs and miRNAs (Shalgi et al , 2007 ), and may provide a mechanism for fine‐tuned coordination between transcriptional and post‐transcriptional regulation of gene expression. Here, we describe and experimentally demonstrate one such regulatory motif, termed feed‐forward loop (FFL), which involves the TF E2F1, a set of miRNAs, and their common targets (Figure 8 ). In this FFL, E2F1, a key regulator of cell‐cycle progression, transcriptionally activates a family of 15 miRNAs that are organized in three paralogous polycistrons on three different chromosomes. These miRNAs silence a group of antiproliferative regulators including the pocket proteins pRb and p130 and the CDK inhibitors p21 and p57. Importantly, these genes are themselves transcriptional targets of E2F1. Thus, a TF activates a set of genes as well as a set of miRNAs, which in turn post‐transcriptionally regulate that set of genes. Increasing the complexity of this regulatory FFL, many of the shared targets of E2F1 and the miRNAs function as regulators of the cell cycle; some negatively regulate E2F itself. For example, the pocket proteins pRB and p130 are the major components that regulate the activity of E2F family members throughout the phases of the cell cycle through direct protein–protein interaction. The TF p53 is regarded as one of the key proteins that prevent malignant transformation (Ryan et al , 2001 ), and deactivating mutations of this tumor suppressor are highly common in a wide variety of tumors (Hussain and Harris, 1999 ). A hallmark activity of p53 is the inhibition of proliferation and the induction of cellular senescence on diverse types of stress signals with oncogenic potential, including DNA damage, telomere shortening and oncogene activation. There are several known mechanisms by which p53 negatively regulates proliferation, the key one being the transcriptional activation of the CDK inhibitor p21, which indirectly inhibits the activity of E2F family members. Another recently discovered mechanism for inhibiting proliferation by p53 is the induction of miRNAs from the miR‐34 family, which also modulate the E2F pathway (He et al , 2007 ; Tarasov et al , 2007 ; Tazawa et al , 2007 ; Kumamoto et al , 2008 ). Additionally, direct and indirect transcriptional repression by p53 is considered important for its ability to inhibit proliferation (Ho and Benchimol, 2003 ). Using miRNA microarrays, we discovered that p53 activation during cellular senescence in primary human fibroblasts leads to a decrease in the expression of the above‐mentioned family of miRNAs, including members of the miR‐17‐92, miR‐106b/93/25 and miR‐106a‐92 polycistronic miRNA clusters. A similar decrease in miRNA expression was observed in human breast cancer specimens that harbor wild‐type p53 as compared with those that harbor mutant forms of p53. We further investigated the mechanism by which p53 represses the expression of this group of miRNAs, and found that activation of p53 leads to a dramatic reduction of E2F1 mRNA, protein and activity levels, which in turn leads to a decrease in the E2F1‐dependent transcriptional activation of these miRNAs. To study the consequence of deregulation of this FFL and importance of its inhibition by p53, we ectopically expressed representative members from the set of p53‐repressed miRNAs, namely the miR‐106b/93/25 polycistron, in primary human fibroblasts. Consequently, these cells acquired an enhanced proliferative phenotype manifested by increased growth rate, increased colony formation efficiency and delayed entry into replicative senescence. These results position the repression of this set of miRNAs as a novel mechanism by which p53 inhibits proliferation and controls cell fate. Here we identified a group of 15 co‐regulated paralogous miRNAs which are transcriptionally activated by E2F1. This group includes the miR‐17‐92, miR‐106a‐92 and miR‐106b/93/25 polycistronic miRNAs. These miRNAs silence anti‐proliferative genes, which themselves are E2F1 targets and function as negative regulators of proliferation. Thus, E2F1 and this group of microRNAs cooperate in a feed‐forward loop that involves transcriptional and post‐transcriptional modes of regulation. The key tumor suppressor p53 disrupts this feed‐forward loop by inactivating E2F1 in senescent cells and in human cancers. This inhibition serves as another arm of p53's tight control of proliferation.

Combined hormone drugs are the basis for orally administered contraception. However, they are associated with severe side effects that are even more impactful for women in developing countries, where resources are limited. The risk of side effects may be reduced by non-hormonal small molecules which specifically target proteins involved in fertilization. In this study, we present a virtual docking experiment directed to discover molecules that target the crucial fertilization interactions of JUNO (oocyte) and IZUMO1 (sperm). We docked 913,000 molecules to two crystal structures of JUNO and ranked them on the basis of energy-related criteria. Of the 32 tested candidates, two molecules (i.e., Z786028994 and Z1290281203) demonstrated fertilization inhibitory effect in both an in vitro fertilization (IVF) assay in mice and an in vitro penetration of human sperm into hamster oocytes. Despite this clear effect on fertilization, these two molecules did not show JUNO–IZUMO1 interaction blocking activity as assessed by AVidity-based EXtracellular Interaction Screening (AVEXIS). Therefore, further research is required to determine the mechanism of action of these two fertilization inhibitors.