Haploid Human Cells Research Articles

A Reporter Screen in a Human Haploid Cell Line Identifies CYLD as a Constitutive Inhibitor of NF-κB

The development of forward genetic screens in human haploid cells has the potential to transform our understanding of the genetic basis of cellular processes unique to man. So far, this approach has been limited mostly to the identification of genes that mediate cell death in response to a lethal agent, likely due to the ease with which this phenotype can be observed. Here, we perform the first reporter screen in the near-haploid KBM7 cell line to identify constitutive inhibitors of NF-κB. CYLD was the only currently known negative regulator of NF-κB to be identified, thus uniquely distinguishing this gene. Also identified were three genes with no previous known connection to NF-κB. Our results demonstrate that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors.

Abstract PR03: Genome-wide screens for Wnt signaling in human haploid cells

Abstract Wnt signaling is at the core of human development and stem cell biology. Defective Wnt signaling leads to several forms of cancer, most notably colorectal cancer. Using HAP1 cells, a human haploid cell line, we conducted a set of genome-wide forward genetic screens to identify new Wnt pathway components. We constructed a HAP1 line harboring a GFP reporter driven by the Wnt-responsive TCF promoter, we mutagenized the cells by insertion of a gene-trap retrovirus throughout the haploid genome, and we sorted the mutagenized population for cells exhibiting phenotypes of interest using FACS. In order to find 1. negative regulators, 2. positive regulators and 3. feedback modulators of the pathway, we sorted for 1. cells expressing GFP in the absence of Wnt, 2. cells with reduced GFP expression after strong Wnt stimulation and 3. cells with enhanced GFP expression after mild Wnt stimulation, respectively. Deep sequencing of amplified genomic DNA flanking the retroviral insertion sites in the sorted cell populations revealed genes whose disruption may have led to the phenotype of interest. Among the most frequently disrupted genes (some with over 150 unique retroviral insertions) we found many of the known components of the canonical Wnt pathway, including LRP6, casein kinase I, APC, β-catenin, DOT1-L, TCF4 and the recently discovered negative feedback regulator ZNRF3. Unexpectedly, the screen for positive regulators revealed retroviral insertions in AXIN2 (a scaffold protein found in the β-catenin destruction complex) predicted to yield a C-terminally truncated form that appears to act as a dominant negative. We are currently assessing hits not previously known to be involved in Wnt signaling. This abstract is also presented as Poster A40. Citation Format: Andres Lebensohn, Casey Hughes, Caleb Marceau, Rajat Rohatgi, Jan Carette. Genome-wide screens for Wnt signaling in human haploid cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR03.

Abstract IA18: Gene-drug interaction screens in cancer using isogenic cell models

Abstract With the complete molecular characterization of the major cancer types in sight, it is clear that only a minority of the cancer genes represent readily accessible drug targets. Furthermore, mechanistic biomarkers that predict response to targeted therapies remain scant. To address these challenges, we employ large-scale chemical genetic screens in isogenic cells to systematically uncover gene-drug interactions. For instance, using a panel of engineered cell lines we have recently identified several known and novel breast cancer vulnerabilities and a mechanism of resistance to drugs inhibiting the PI3K/mTOR pathway. We have performed similar screens in an isogenic lung cancer model that attempts to capture some of the higher order gene-drug interactions. In addition, I will present our efforts concerning the generation of a collection of haploid human knockout cells covering the majority of expressed genes that represents a powerful platform empowering haploid genetics in human cells. Citation Format: Sebastian Nijman. Gene-drug interaction screens in cancer using isogenic cell models. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr IA18.

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.

P051 Mapping the TNF-induced necroptosis pathway by experimental test-tube genetics in human haploid cells

Introduction TNF signaling can result in NF-kB and MAPK activation, apoptosis or necrosis depending on cell type and cell state. Until recently necrosis has been seen as an uncontrolled mode of cell death when compared to the programmed apoptosis. The identification of RIP3 as a crucial mediator of TNF-induced necrosis clearly demonstrates that in many cases necrosis is controlled by a dedicated signaling cascade, leading to the definition of necroptosis [3] , [4] , [6] . Increasing evidences demonstrate that RIP3-mediated necroptosis participates in inflammation and in both innate and adaptive immunity. Despite this, the signaling events downstream of RIP3 kinase activation remain largely obscure. Methods Here we describe the design of a haploid genetic screen in human KBM7 cells that aims to identify new components of TNF-induced RIP3-dependent necroptosis signaling pathways. The KBM7 chronic myeloid leukemia cell line is haploid for all chromosomes except chromosome 8. This allows gene inactivation through random insertional mutagenesis using a retroviral gene-trap vector and subsequent insertion site mapping [1] , [2] . Results Our data demonstrate that the TNF signaling pathway is functional in KBM7 cells resulting in NF-kB and MAPK activation, apoptosis or necroptosis depending on defined experimental settings. Under necroptosis inducing conditions, cell death occurs without detectable caspase activation and is suppressed by the RIP1 kinase inhibitor necrostatin-1. Based on these results, mutagenized KBM7 cells will be submitted to TNF-induced necroptosis and resistant clones mapped to identify genes required for necroptotic cell death. Mixed lineage kinase domain-like protein (MLKL) has been recently shown to participate in TNF-induced necroptosis [5] . An MLKL-deficient KBM7 cell clone is completely resistant to TNF-induced necroptosis while retaining sensitivity to TNF-induced apoptosis. This finding confirms by gene inactivation in human cells that MLKL is crucial for TNF-necroptosis and, importantly, validates our screening approach. Conclusion We expect the screen to genetically define the essential molecular elements required for necroptosis. Identified components will then be characterized using affinity-purification coupled to mass spectrometry. Our study should thus contribute significantly to the molecular understanding of necroptosis, a process whose relevance in inflammation, innate and adaptive immunity has just began to be fully appreciated.

Fluorescence-Based Phenotypic Selection Allows Forward Genetic Screens in Haploid Human Cells

The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

Chlamydia trachomatis is a pathogen responsible for a prevalent sexually transmitted disease. It is also the most common cause of infectious blindness in the developing world. We performed a loss-of-function genetic screen in human haploid cells to identify host factors important in C. trachomatis L2 infection. We identified and confirmed B3GAT3, B4GALT7, and SLC35B2, which encode glucuronosyltransferase I, galactosyltransferase I, and the 3'-phosphoadenosine 5'-phosphosulfate transporter 1, respectively, as important in facilitating Chlamydia infection. Knockout of any of these three genes inhibits Chlamydia attachment. In complementation studies, we found that the introduction of functional copies of these three genes into the null clones restored full susceptibility to Chlamydia infection. The degree of attachment of Chlamydia strongly correlates with the level of sulfation of the host cell, not simply with the amount of heparan sulfate. Thus, other, as-yet unidentified sulfated macromolecules must contribute to infection. These results demonstrate the utility of screens in haploid cells to study interactions of human cells with bacteria. Furthermore, the human null clones generated can be used to investigate the role of heparan sulfate and sulfation in other settings not limited to infectious disease.

The nature and origin of binucleate cells in human preimplantation embryos: relevance to placental mesenchymal dysplasia

Cleavage-stage embryos often have nuclear abnormalities, one of the most common being binucleate blastomeres, which may contain two diploid or two haploid nuclei. Biopsied cells from preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) cycles were studied to determine the relative frequency of binucleate cells with two haploid versus two diploid nuclei. The frequency of mononucleate haploid biopsied blastomeres was also recorded. In the chromosomal PGD cycles 45.2% of the biopsied binucleate cells were overall diploid and 38.7% were overall tetraploid, compared with 50.0% and 29.2% for the PGS group, respectively. Placental mesenchymal dysplasia is a rare condition associated with intrauterine growth restriction, prematurity and intrauterine death. Recent work suggests that androgenetic diploid/haploid mosaicism may be a causal mechanism. There are two possible origins of haploid nuclei, either the cell contained only one parental genome initially or they may be derived from the cytokinesis of binucleate cells with two haploid nuclei. Binucleate formation therefore may be a way of doubling up the haploid genome, to produce diploid cells of androgenetic origin as seen in placental mesenchymal dysplasia. Since the early studies in human embryos haploid cells were seen whose origin was unknown. Preimplantation embryos often have nuclear abnormalities, one of the most common being binucleate blastomeres, i.e. cells that contain two nuclei. These nuclei could be both diploid but many binucleate cells contain two haploid nuclei. Biopsied cells from preimplantation genetic diagnosis and preimplantation genetic screening cycles were studied to determine the relative frequency of binucleate cells with two haploid versus two diploid nuclei. The frequency of mononucleate haploid biopsied blastomeres was also recorded. There are two possible origins of haploid nuclei, either the cell contained only one parental genome initially or they may be derived from the cytokinesis of binucleate cells with two haploid nuclei. Binucleate formation therefore may be a way of doubling up the haploid genome, to produce diploid cells of androgenetic origin as seen in placental mesenchymal dysplasia, a rare condition associated with intrauterine growth restriction, prematurity and intrauterine death.

Human Haploid Cells Differentiated from Meiotic Competent Clonal Germ Cell Lines That Originated from Embryonic Stem Cells

Early germ-like cells (GLCs) derived from human embryonic stem cells (hESCs) have presented new opportunities to study germ cell differentiation in vitro. However, differentiation conditions that facilitate the formation of haploid cells from the derived GLCs have eluded the field. The inability to propagate GLCs in culture is a further limitation, resulting in inconsistent rederivations of GLCs from hESCs with relatively few GLCs in these heterogeneous populations. Here we found in vitro conditions that enrich for DDX4/POU5F1+ GLCs (∼60%) and that has enabled continual propagation for >50 passages without loss of phenotype. Clonal isolation of single GLCs from these mixed cultures generated 3 GLC (>90% DDX4/POU5F1+) and 2 hESC (<0.1% DDX4+) lines that could be continually expanded without loss of phenotype. Differentiation of clonal GLC lines in serum resulted in expression of postmeiotic markers and >11% were haploid, ∼5-fold higher than previous studies. The robust clonal meiotic competent and incompetent GLC lines will be used to understand the factors controlling human germ cell meiosis and postmeiotic maturation.

DNA methylation in the Alu sequences of diploid and haploid primary human cells.

We have investigated DNA methylation in human Alu sequences, both in general and in specific Alu sequences associated with the genes for alpha 1 globin, tissue plasminogen activator (tPA), adrenocorticotropic hormone (ACTH) and angiogenin. We studied DNAs from lymphocytes, granulocytes, brain, heart muscle and sperm, and from the human HeLa and KB cell lines by using cleavage with methylation-sensitive restriction enzymes combined with Southern blot hybridization and by using genomic sequencing. The results can be summarized as follows. (i) In differentiated primary human cells, Alu elements are often highly methylated even when they are in very 5'-CG-3'-rich regions. This finding is not consistent with the notion that hypermethylation would be a sufficient condition in itself for 5'-CG-3' sequences to undergo loss of 5-methyl-deoxycytidine (5-mC) due to deamination and subsequent mutation. (ii) There are distinct differences in the levels of methylation in the specific Alu sequences. (iii) Alu elements in the DNA of haploid spermatozoa are much less methylated than in diploid cells. Preliminary data indicate that spermatozoa contain Alu-specific RNAs. (iv) The results of cell-free transcription experiments with Alu elements suggest that the in vitro transcription of Alu elements can be inhibited by 5'-CG-3' methylation. High levels of 5'-CG-3' methylation in Alu elements could contribute to their general transcriptional inactivity. (v) The patterns of methylation observed in the Alu elements and in the surrounding sequences are characterized by cell type specific interindividual concordance.

Fibrillar center distribution in nucleoli of PHA-stimulated human lymphocytes

We have utilized acidic toluidine blue staining for RNA and immunofluorescence staining for RNA polymerase 1 to visualize the distribution of fibrillar centers (FCs) in nucleoli of PHA-stimulated human lymphocytes. At 0 h, there is a single large fibrillar center in each nucleolus which splits into smaller and more numerous FCs until the number of FCs reaches five, the number of nucleolus organizers in normal haploid human cells. With time, each FC then "unwinds" to form linear arrays of smaller FCs until the maximum number of FCs approaches the ribosomal gene copy number of 200 at 48 h in culture. It is hypothesized that in the most active state, each nucleolar FC visualized by RNA polymerase 1 staining actually represents a single transcription unit and the distance between adjacent FCs is occupied by the nontranscribed spacer region. We conclude that the number of fibrillar centers per nucleolus can be used as a direct quantitative measure of nucleolar transcriptional activity.