Julian Gingold

Zfp281 mediates Nanog autorepression through recruitment of the NuRD complex and inhibits somatic cell reprogramming

The homeodomain transcription factor Nanog plays an important role in embryonic stem cell (ESC) self-renewal and is essential for acquiring ground-state pluripotency during reprogramming. Understanding how Nanog is transcriptionally regulated is important for further dissecting mechanisms of ESC pluripotency and somatic cell reprogramming. Here, we report that Nanog is subjected to a negative autoregulatory mechanism, i.e., autorepression, in ESCs, and that such autorepression requires the coordinated action of the Nanog partner and transcriptional repressor Zfp281. Mechanistically, Zfp281 recruits the NuRD repressor complex onto the Nanog locus and maintains its integrity to mediate Nanog autorepression and, functionally, Zfp281-mediated Nanog autorepression presents a roadblock to efficient somatic cell reprogramming. Our results identify a unique transcriptional regulatory mode of Nanog gene expression and shed light into the mechanistic understanding of Nanog function in pluripotency and reprogramming.

Modeling Familial Cancer with Induced Pluripotent Stem Cells

In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs.

A Genome-wide RNAi Screen Identifies Opposing Functions of Snai1 and Snai2 on the Nanog Dependency in Reprogramming

Nanog facilitates embryonic stem cell self-renewal and induced pluripotent stem cell generation during the final stage of reprogramming. From a genome-wide small interfering RNA screen using a Nanog-GFP reporter line, we discovered opposing effects of Snai1 and Snai2 depletion on Nanog promoter activity. We further discovered mutually repressive expression profiles and opposing functions of Snai1 and Snai2 during Nanog-driven reprogramming. We found that Snai1, but not Snai2, is both a transcriptional target and protein partner of Nanog in reprogramming. Ectopic expression of Snai1 or depletion of Snai2 greatly facilitates Nanog-driven reprogramming. Snai1 (but not Snai2) and Nanog cobind to and transcriptionally activate pluripotency-associated genes including Lin28 and miR-290-295. Ectopic expression of miR-290-295 cluster genes partially rescues reprogramming inefficiency caused by Snai1 depletion. Our study thus uncovers the interplay between Nanog and mesenchymal factors Snai1 and Snai2 in the transcriptional regulation of pluripotency-associated genes and miRNAs during the Nanog-driven reprogramming process.

Combining competition assays with genetic complementation strategies to dissect mouse embryonic stem cell self-renewal and pluripotency

Substantial scientific interest has been dedicated recently to the crucial factors that control the pluripotent state of stem cells. To gain a comprehensive understanding of the molecular mechanisms regulating mouse embryonic stem cell (mESC) self-renewal and lineage differentiation, we have developed a robust method for studying the role of a particular gene in these processes. This protocol describes detailed procedures for the design and generation of the complementation rescue system and its application in dissecting the network of pluripotency-associated factors, using mESCs as a model. Specifically, three main procedures are described: (i) screening pluripotency-associated factors by competition assay; (ii) setting up an inducible complementation rescue system; and (iii) dynamically studying the pluripotency network response to target depletion. Completion of the competition assay and complementation rescue system takes 35 and 30 d, respectively, and an additional 16 d to study the dynamic molecular effects of a gene of interest in the pluripotency network.

Tbx3 Controls Dppa3 Levels and Exit from Pluripotency toward Mesoderm

Summary Tbx3, a member of the T-box family, plays important roles in development, stem cells, nuclear reprogramming, and cancer. Loss of Tbx3 induces differentiation in mouse embryonic stem cells (mESCs). However, we show that mESCs exist in an alternate stable pluripotent state in the absence of Tbx3. In-depth transcriptome analysis of this mESC state reveals Dppa3 as a direct downstream target of Tbx3. Also, Tbx3 facilitates the cell fate transition from pluripotent cells to mesoderm progenitors by directly repressing Wnt pathway members required for differentiation. Wnt signaling regulates differentiation of mESCs into mesoderm progenitors and helps to maintain a naive pluripotent state. We show that Tbx3, a downstream target of Wnt signaling, fine tunes these divergent roles of Wnt signaling in mESCs. In conclusion, we identify a signaling-TF axis that controls the exit of mESCs from a self-renewing pluripotent state toward mesoderm differentiation.

Rational design and characterization of d-phe-pro-d-arg-derived direct thrombin inhibitors.

The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH2. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH2, competitively inhibits α-thrombins cleavage of the S2238 chromogenic substrate with a Ki of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′.

Osteosarcoma: Molecular Pathogenesis and iPSC Modeling

Rare hereditary disorders provide unequivocal evidence of the importance of genes in human disease pathogenesis. Familial syndromes that predispose to osteosarcomagenesis are invaluable in understanding the underlying genetics of this malignancy. Recently, patient-derived induced pluripotent stem cells (iPSCs) have been successfully utilized to model Li-Fraumeni syndrome (LFS)-associated bone malignancy, demonstrating that iPSCs can serve as an in vitro disease model to elucidate osteosarcoma etiology. We provide here an overview of osteosarcoma predisposition syndromes and review recently established iPSC disease models for these familial syndromes. Merging molecular information gathered from these models with the current knowledge of osteosarcoma biology will help us to gain a deeper understanding of the pathological mechanisms underlying osteosarcomagenesis and will potentially aid in the development of future patient therapies.

Genomic Profiling and Metabolic Homeostasis in Primary Liver Cancers

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), the two most common primary liver cancers, represent the second most common cancer-related cause of death worldwide, with most cases being diagnosed at an advanced stage. Recent genome-wide studies have helped to elucidate the molecular pathogenesis and genetic heterogeneity of liver cancers. This review of the genetic landscape of HCC and iCCA discusses the most recent findings from genomic profiling and the current understanding of the pathways involved in the initiation and progression of liver cancer. We highlight recent insights gained from metabolic profiling of HCC and iCCA. This knowledge will be key to developing clinically useful diagnostic/prognostic profiles, building targeted molecular and immunologic therapies, and ultimately curing these complex and heterogeneous diseases.

Li–Fraumeni Syndrome Disease Model: A Platform to Develop Precision Cancer Therapy Targeting Oncogenic p53

Li-Fraumeni syndrome (LFS) is a rare hereditary autosomal dominant cancer disorder. Germline mutations in TP53, the gene encoding p53, are responsible for most cases of LFS. TP53 is also the most commonly mutated gene in human cancers. Because inhibition of mutant p53 is considered to be a promising therapeutic strategy to treat these diseases, LFS provides a perfect genetic model to study p53 mutation-associated malignancies as well as to screen potential compounds targeting oncogenic p53. In this review we briefly summarize the biology of LFS and current understanding of the oncogenic functions of mutant p53 in cancer development. We discuss the strengths and limitations of current LFS disease models, and touch on existing compounds targeting oncogenic p53 and in vitro clinical trials to develop new ones. Finally, we discuss how recently developed methodologies can be integrated into the LFS induced pluripotent stem cell (iPSC) platform to develop precision cancer therapy.

Minimally Invasive Approaches to Myoma Management

Patients affected by the presence of leiomyomas may incur a substantial physical, emotional, social, and financial toll as well as losses in their quality of life. Although many myomas are not amenable to medical therapy or hysteroscopic resection, many others are amenable to minimally invasive surgical approaches. In patients who prefer to retain their fertility, laparoscopic myomectomy should be considered the intervention of choice. In this review, we expand on the surgical techniques of both conventional laparoscopic and robotic-assisted myomectomies. We discuss port placement, enucleation of myomas, tissue extraction, minimization of blood loss, adhesion prevention, and the technique for closure of uterine incisions. Finally, we discuss the available data supporting the use of these 2 approaches as the preferred, safe, and effective fertility-sparing surgical option. We also briefly discuss the emerging technologies of uterine artery embolization, ultrasound surgery, and radiofrequency ablation.