RARγ is required for mesodermal gene expression prior to gastrulation in Xenopus

The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here, we show in Xenopus that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets upregulated by RA receptor signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the genes encoding the RA-synthesizing enzyme Aldh1a2 and the RA-degrading enzyme Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of Myod protein in the presomitic mesoderm, but ectopic, persistent expression of Myod protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation

RARγ is required for mesodermal gene expression prior to gastrulation in Xenopus

The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here, we show in Xenopus that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets upregulated by RA receptor signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the genes encoding the RA-synthesizing enzyme Aldh1a2 and the RA-degrading enzyme Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of Myod protein in the presomitic mesoderm, but ectopic, persistent expression of Myod protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation

Relevance of iPSC-derived human PGC-like cells at the surface of embryoid bodies to prechemotaxis migrating PGCs

Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38+ hPGCLCs [∼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38+ hPGCLCs and CD38− EB cells significantly expressed PRDM1 and TFAP2C, although PRDM1 mRNA in CD38− cells lacked the 3′-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of T, NANOS3, and SOX17) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs on Matrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis

Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers.

In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs

Ascorbic Acid Prevents Loss of Dlk1-Dio3 Imprinting and Facilitates Generation of All-iPS Cell Mice from Terminally Differentiated B Cells

The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells, which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor–mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs

HD CAGnome: A Search Tool for Huntingtin CAG Repeat Length-Correlated Genes

Background: The length of the huntingtin (HTT) CAG repeat is strongly correlated with both age at onset of Huntington’s disease (HD) symptoms and age at death of HD patients. Dichotomous analysis comparing HD to controls is widely used to study the effects of HTT CAG repeat expansion. However, a potentially more powerful approach is a continuous analysis strategy that takes advantage of all of the different CAG lengths, to capture effects that are expected to be critical to HD pathogenesis. Methodology/Principal Findings We used continuous and dichotomous approaches to analyze microarray gene expression data from 107 human control and HD lymphoblastoid cell lines. Of all probes found to be significant in a continuous analysis by CAG length, only 21.4% were so identified by a dichotomous comparison of HD versus controls. Moreover, of probes significant by dichotomous analysis, only 33.2% were also significant in the continuous analysis. Simulations revealed that the dichotomous approach would require substantially more than 107 samples to either detect 80% of the CAG-length correlated changes revealed by continuous analysis or to reduce the rate of significant differences that are not CAG length-correlated to 20% (n = 133 or n = 206, respectively). Given the superior power of the continuous approach, we calculated the correlation structure between HTT CAG repeat lengths and gene expression levels and created a freely available searchable website, “HD CAGnome,” that allows users to examine continuous relationships between HTT CAG and expression levels of ∼20,000 human genes. Conclusions/Significance: Our results reveal limitations of dichotomous approaches compared to the power of continuous analysis to study a disease where human genotype-phenotype relationships strongly support a role for a continuum of CAG length-dependent changes. The compendium of HTT CAG length-gene expression level relationships found at the HD CAGnome now provides convenient routes for discovery of candidates influenced by the HD mutation

Tailoring Adjuvant Endocrine Therapy for Postmenopausal Breast Cancer: A CYP2D6 Multiple-Genotype-Based Modeling Analysis and Validation

Purpose: Previous studies have suggested that postmenopausal women with breast cancer who present with wild-type CYP2D6 may actually have similar or superior recurrence-free survival outcomes when given tamoxifen in place of aromatase inhibitors (AIs). The present study established a CYP2D6 multiple-genotype-based model to determine the optimal endocrine therapy for patients harboring wild-type CYP2D6. Methods: We created a Markov model to determine whether tamoxifen or AIs maximized 5-year disease-free survival (DFS) for extensive metabolizer (EM) patients using annual hazard ratio (HR) data from the BIG 1-98 trial. We then replicated the model by evaluating 9-year event-free survival (EFS) using HR data from the ATAC trial. In addition, we employed two-way sensitivity analyses to explore the impact of HR of decreased-metabolizer (DM) and its frequency on survival by studying a range of estimates. Results: The 5-year DFS of tamoxifen-treated EM patients was 83.3%, which is similar to that of genotypically unselected patients who received an AI (83.7%). In the validation study, we further demonstrated that the 9-year EFS of tamoxifentreated EM patients was 81.4%, which is higher than that of genotypically unselected patients receiving tamoxifen (78.4%) and similar to that of patients receiving an AI (83.2%). Two-way sensitivity analyses demonstrated the robustness of the results

CATCHprofiles: Clustering and Alignment Tool for ChIP Profiles

Chromatin Immuno Precipitation (ChIP) profiling detects in vivo protein-DNA binding, and has revealed a large combinatorial complexity in the binding of chromatin associated proteins and their post-translational modifications. To fully explore the spatial and combinatorial patterns in ChIP-profiling data and detect potentially meaningful patterns, the areas of enrichment must be aligned and clustered, which is an algorithmically and computationally challenging task. We have developed CATCHprofiles, a novel tool for exhaustive pattern detection in ChIP profiling data. CATCHprofiles is built upon a computationally efficient implementation for the exhaustive alignment and hierarchical clustering of ChIP profiling data. The tool features a graphical interface for examination and browsing of the clustering results. CATCHprofiles requires no prior knowledge about functional sites, detects known binding patterns “ab initio”, and enables the detection of new patterns from ChIP data at a high resolution, exemplified by the detection of asymmetric histone and histone modification patterns around H2A.Z-enriched sites. CATCHprofiles' capability for exhaustive analysis combined with its ease-of-use makes it an invaluable tool for explorative research based on ChIP profiling data

Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics

Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics

In Vivo Imaging of HIF-Active Tumors by an Oxygen-Dependent Degradation Protein Probe with an Interchangeable Labeling System

Hypoxia-inducible factor (HIF) functions as a master transcriptional regulator for adaptation to hypoxia by inducing adaptive changes in gene expression for regulation of proliferation, angiogenesis, apoptosis and energy metabolism. Cancers with high expression of the alpha subunit of HIF (HIFα) are often malignant and treatment-resistant. Therefore, the development of a molecular probe that can detect HIF activity has great potential value for monitoring tumor hypoxia. HIF prolyl hydroxylases (HPHDs) act as oxygen sensors that regulate the fate of HIFα protein through its oxygen-dependent degradation (ODD) domain. We constructed a recombinant protein PTD-ODD-HaloTag (POH) that is under the same ODD regulation as HIFα and contains protein transduction domain (PTD) and an interchangeable labeling system. Administration of near-infrared fluorescently labeled POH (POH-N) to mouse models of cancers allowed successful monitoring of HIF-active regions. Immunohistochemical analysis for intratumoral localization of POH probe revealed its specificity to HIF-active cells. Furthermore, lack of the PTD domain or a point mutation in the ODD domain abrogated the specificity of POH-N to HIF-active cells. Overall results indicate that POH is a practical probe specific to HIF-active cell in cancers and suggest its large potential for imaging and targeting of HIF-related diseases