Study of the human SOX17 locus and its genetic determinants in definitive endoderm

Embryonic development and organogenesis depend on the precise spatiotemporal expression of specific sets of genes. Precisely controlled gene expression ensures cell state transitions, especially in the early stages of development, as gastrulation. These complex multi-layered cellular processes are orchestrated by the interfacing of the epigenome, 3-dimensional (3D) nuclear organization, cis-regulatory elements (CREs) with transcription factors (TF), and long non-coding RNAs (lncRNAs). In the gastrulating embryo, definitive endoderm is specified from the pluripotent epiblast following a series of regulatory events, including the activation of SOX17, a key TF of that particular germ layer. Although SOX17 has been extensively studied in early embryonic development, the precise control of its activation, the locus, and the epigenetic rules governing its genetic regulatory network (GRN) remains poorly investigated. In my thesis, I in-depth characterized the human SOX17 locus, exploring the relevance and regulatory impact of 3D nuclear organization, its distal CREs, and their activity. I applied a series of loss of function (LOF) and transgenic experiments to dissect the locus at a satisfactory resolution. In particular, I showed SOX17 among a subset of developmental regulators topologically isolated within CTCF-CTCF loop domains and highlighted the importance of gene control in 3D within this type of domain. I pinpointed the relevance of SOX17’s distal CREs and their definitive endoderm-specific interaction and showed this interaction to be highly dependent on CTCF-CTCF loop-formation to guarantee proper gene control. I found CRE-dependent SOX17 gene deregulation associated with poor definitive endoderm differentiation outcome and a stalled “mesendodermal-like” phenotype. Assessing the genetic identity of different CREs, I divulged the presence of a novel lncRNA within the locus, namely LNCSOX17. I fully characterized LNCSOX17 and established its identity as a bona fide lncRNA through a series of genetic perturbations. I demonstrated the importance of LNCSOX17 for forming definitive endoderm and the lack of participation in SOX17 cis-acting gene control. I associated the loss of LNCSOX17 RNA but not its active transcription at the locus with an aberrant endodermal transcriptome, a lack of epithelial-to-mesenchymal transition (EMT), and the hyperactivity of the detrimental definitive endoderm JNK/JUN/AP1 signaling pathway. I found definitive endoderm lacking LNCSOX17 to be functionally impeded in the generation of pancreatic progenitor populations. The studies within this thesis serve as valuable examples to support the functional relevance of 3D nuclear organization and its importance for developmental gene control in cis via CTCF-CTCF loop domain-mediated CRE-promoter contact facilitation. They associate developmental gene expression levels with various phenotypes, identify a so far unknown developmental lncRNA molecule, and imply its relevance for the formation of definitive endoderm. The outlined results advance our knowledge of developmental TF gene-control and its importance for the development of human definitive endoderm

T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation

Topological isolation of developmental regulators in mammalian genomes

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression

Generation of two human isogenic iPSC lines from fetal dermal fibroblasts

Two isogenic hiPSC lines, ZIPi013-B and ZIPi013-E, were generated by reprogramming fetal dermal fibroblasts with episomal vectors. Previously, the same fetal fibroblasts were reprogrammed multiple times in a study comparing other reprogramming methods. As a consequence, the genomes have been sequenced multiple times. Both new cell lines offer the opportunity to study basic stem cell biology and model human disease. They can be applied as reference cell lines for creating isogenic clones bearing disease mutations on a well-characterized genomic background, as both cell lines have demonstrated excellent differentiation capacity in multiple labs.Resource tableUnlabelled TableUnique stem cell lines identifierZIPi013-BZIPi013-EAlternative names of stem cell linesZIP13K2 (ZIPi013-B)ZIP13K5 (ZIPi013-E)InstitutionZentrum für Integrative Psychiatrie gGmbH, Kiel, GermanyContact information of distributorPD Dr. Franz-Josef Müller, [email protected] of cell linesiPSCOriginhumanCell SourceFibroblastsClonalityClonalMethod of reprogrammingTransgene free, episomalMultiline rationaleIsogenic clonesGene modificationNOType of modificationN/AAssociated diseaseN/AGene/locusN/AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateStock date ZIPi013-B 8th December 2017, stock date ZIPi013-E 12th December 2017Cell line repository/bankN/AEthical approvalhttps://www.sciencellonline.com/technical-support/ethical-statement.htmlEthikkommission der medizinischen Fakultät der Christian-Albrechts-Universität zu Kiel, approval number A145/1

Radiotherapy promotes tumor-specific effector CD8+ T cells via dendritic cell activation

Radiotherapy is an important treatment for cancer. The main mode of action is thought to be the irreversible damage to tumor cell DNA, but there is evidence that irradiation mobilizes tumor-specific immunity, and recent studies showed that the efficacy of high-dose radiotherapy depends on the presence of CD8(+) T cells. We show in this study that the efficacy of radiotherapy given as a single, high dose (10 Gy) crucially depends on dendritic cells and CD8(+) T cells, whereas CD4(+) T cells or macrophages are dispensable. We show that local high-dose irradiation results in activation of tumor-associated dendritic cells that in turn support tumor-specific effector CD8(+) T cells, thus identifying the mechanism that underlies radiotherapy-induced mobilization of tumor-specific immunity. We propose that in the absence of irradiation, the activation status of dendritic cells rather than the amount of tumor-derived Ag is the bottleneck, which precludes efficient anti-tumor immunity

NY-ESO-1-specific immunological pressure and escape in a patient with metastatic melanoma

During cancer progression, malignant cells may evade immunosurveillance. However, evidence for immunological escape in humans is scarce. We report here the clinical course of a melanoma patient whose initial tumor was positive for the antigens NY-ESO-1, MAGE-C1, and Melan-A. Upon immunization with a recombinant vaccinia/fowlpox NY-ESO-1 construct, the patient experienced a mixed clinical response and spreading of the NY-ESO-1 epitopes in the CD4+ T cell compartment. After NY-ESO-1 protein + CpG immunization, the patient's anti-NY-ESO-1 IgG response increased. Over the following years, progressing lesions were resected and found to be NY-ESO-1-negative while being positive for MAGE-C1, Melan-A, and MHC-I. The fatal, inoperable brain metastasis was analyzed after his death and also proved to be NY-ESO-1-negative, while being positive for MAGE-C1 and Melan-A, as well as MHC-I. We propose that cancer control and cancer escape in this patient were governed by NY-ESO-1-specific immunological pressure. Our findings provide evidence for the existence of immunoediting and immunoescape in this cancer patient

A novel human-derived antibody against NY-ESO-1 improves the efficacy of chemotherapy

We investigated whether antibodies against intracellular tumor-associated antigens support tumor-specific immunity when administered together with a treatment that destroys the tumor. We propose that released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic cells. We cloned the first human monoclonal antibodies against the Cancer/Testis (CT) antigen, NY-ESO-1. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates cross-presentation of a NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells, and whether this results in the maturation of dendritic cells in vitro. We investigated the efficacy of 12D7 in combination with chemotherapy using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1. Human dendritic cells that were incubated with NY-ESO-1:12D7 immune complexes efficiently stimulated NY-ESO-1(157-165)/HLA-A2-specific human CD8+ T cells to produce interferon-γ, whereas NY-ESO-1 alone did not. Furthermore, the incubation of dendritic cells with NY-ESO-1:12D7 immune complexes resulted in the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil (5-FU) plus 12D7 was significantly more effective than chemotherapy alone. We propose systemic injection of monoclonal antibodies (mAbs) against tumor-associated antigens plus a treatment that promotes the local release of those antigens resulting in immune complex formation as a novel therapeutic modality for cancer

T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation