Delineating the signaling pathways that underlie ESC pluripotency is paramount for development of ESC applications in both the research and clinical settings. In culture pluripotency is maintained by leukemia inhibitory factor (LIF) stimulation of two separate signaling axes: Stat3/Klf4/Sox2 and PI3K/Tbx3/Nanog, which converge in the regulation of Oct4 expression. However, LIF signaling is not required in vivo for self-renewal, thus alternate signaling axes likely mediate these pathways. Additional factors that promote pluripotency gene expression have been identified, including the direct regulation of Oct4 by liver receptor homolog-1 (Lrh-1) and β-catenin regulation of Nanog. Here, we present genetic, molecular, and pharmacological studies identifying a signaling axis in which β-catenin promotes pluripotency gene expression in an Lrh-1-dependent manner. Furthermore, Lrh-1 was identified as a novel β-catenin target gene, and Lrh-1 regulation is required for maintaining proper levels of Oct4, Nanog, and Tbx3. Elucidation of this pathway provides an alternate mechanism by which the primary pluripotency axis may be regulated in vivo and may pave the way for small molecule applications to manipulate pluripotency or improve the efficiency of somatic cell reprogramming. Stem Cells 2010;28:1794–1804

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyse the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterised genes to well-studied pathways and identifies targets relevant for therapeutic intervention.

Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.

SummaryTHAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression.

Alpha‐1 antitrypsin deficiency (AATD) liver disease is characterized by marked heterogeneity in presentation and progression, despite a common underlying gene mutation, strongly suggesting the involvement of other genetic and/or epigenetic modifiers. Variation in clinical phenotype has added to the challenge of detection, diagnosis, and testing of new therapies in patients with AATD. We examined the contribution of DNA methylation (5‐methylcytosine [5mC]) to AATD liver disease heterogeneity because 5mC responds to environmental and genetic cues and its deregulation is a major driver of liver disease. Using liver biopsies from adults with early‐stage AATD and the ZZ genotype, genome‐wide 5mC patterns were interrogated. We compared DNA methylation among patients with early AATD, and among patients with normal liver, cirrhosis, and hepatocellular carcinoma derived from multiple etiologic exposures, and linked patient clinical/demographic features. Global analysis revealed significant genomic hypomethylation in AATD liver–impacting genes related to liver cancer, cell cycle, and fibrosis, as well as key regulatory molecules influencing growth, migration, and immune function. Further analysis indicated that 5mC changes are localized, with hypermethylation occurring within a background of genome‐wide 5mC loss and with patients with AATD manifesting distinct epigenetic landscapes despite their mutational homogeneity. By integrating clinical data with 5mC landscapes, we observed that CpGs differentially methylated among patients with AATD disease are linked to hallmark clinical features of AATD (e.g., hepatocyte degeneration and polymer accumulation) and further reveal links to well‐known sex‐specific effects of liver disease progression. Conclusion: Our data reveal molecular epigenetic signatures within this mutationally homogeneous group that point to ways to stratify patients for liver disease risk.

The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c-Cbl--known for its roles in regulating lymphocyte signalling--as a modulator of dendritic cell activation. In c-Cbl-deficient dendritic cells, Toll-like receptor-induced expression of proinflammatory factors, such as interleukin-12, is increased, correlating with a greater potency of dendritic-cell-based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)-jB activity. In addition, c-Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF-jB. Our data indicate that c-Cbl has a crucial, RING-domain-dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.

About 23.6 million people or 7.8% of the population in the USA are afflicted with diabetes. Another 57 million have prediabetes, people who are prone to develop frank diabetes in the coming years. The total costs of diagnosed diabetes were estimated to be $174 billion in 2007, which included $116 billion for direct medical costs and $58 billion for indirect costs (resulting from disability, work loss, and premature mortality). 1 Type 2 diabetes accounts for some 90% of patients whereas type 1a (autoimmune) diabetes accounts for most of the other diabetic patients. Individuals with type 1 diabetes have absolute insulin deficiency, whereas those with type 2 disease, relative insulin deficiency usually in the presence of insulin resistance. Insulin injections are the standard therapy for type 1 diabetes. Unfortunately, insulin secretion is exquisitely sensitive to the minute to minute changes in blood glucose, and glycemia stimulated insulin secretion (GSIS) cannot be mimicked by exogenous insulin injections. Pancreas transplantation is an effective mode of therapy, but is associated with considerable operative morbidity and mortality. Whilst the initial success with the Edmonton protocol2 popularized pancreatic islet transplantation as a form of treatment, this option has been found to have some severe limitations3. Donor availability limits the number of transplants that can be done; the need for lifetime immunosuppression is another problem. Moreover, though short-term experiments indicate that in good centers, up to ~80% of transplant recipients can become free of insulin injections shortly after the procedure, long-term follow up indicates that the vast majority of them are back on insulin again within five years.4 These difficulties have prompted research into the development of innovative methods to generate new β cells in the body.There are two experimental approaches to generate new β cells: [1] reprogramming of non-β cells to β cells using gene therapy in vivo, or [2] reprogramming of isolated non-β cells to β cells in vitro. To determine if true reprogramming has occurred, stringent criteria must be fulfilled to exclude hybdrid cells or cell mimicry. 5 Whilst insulin production is a necessary criterion, it is not sufficient. The newly reprogrammed cells should be fully characterized to determine how closely they resemble functional β cells by their morphology, expression profile, and whether they exhibit GSIS. Not all non-β cells are equally susceptible to reprogramming. Below we review the different strategies and types of cells that have been studied with respect to the generation of new β cell by reprogramming.

Extensive research has been devoted to the goal of understanding how a single cell of embryonic origin can give rise to every somatic cell type and the germ cell lineage, a hallmark defined as "pluripotency." The aggregate of this work supports fundamentally important roles for the gene transcription networks inherent to the pluripotent cell. Transcription networks have been identified that are both required for pluripotency, as well as sufficient to reprogram somatic cells to a naive pluripotent state. Several members of the nuclear receptor (NR) superfamily of transcription factors have been identified to play diverse roles in the regulation of pluripotency. The ligand-responsive nature of NRs coupled with the abundance of genetic models available has led to a significant advance in the understanding of NR roles in embryonic stem cell pluripotency. Furthermore, the presence of a ligand-binding domain may lead to development of small molecules for a wide range of therapeutic and research applications, even in cases of NRs that are not known to respond to physiologic ligands. Presented here is an overview of NR regulation of pluripotency with a focus on the transcriptional, proteomic, and epigenetic mechanisms by which they promote or suppress the pluripotent state.