Identification of Novel Regulators of TNF-alpha Signaling using Genome-wide RNAi Screens

Signaling pathways are crucial for multicellular organisms: they are necessary for cell communication and development, they enable cells to specialize and act together. TNF-alpha signaling is one such pathway crucial for orchestrating the body's response to cellular stress or invasion by pathogens. TNF- signaling mediates inflammation, a key event for an efficient innate immune response. As inflammation has to be tightly controlled in order to avoid damage to the host, deregulation of TNF-alpha signaling has been implicated in the pathogenesis of many inflammatory diseases and cancer. TNF-alpha exerts inflammatory effects by binding to its receptor, TNFR1. Upon binding, several proteins including DD (death domain) proteins are recruited to TNFR1 to form the TNFR complex, resulting in the activation of the IkappaB kinase complex (IKK). Subsequently, inhibitor of kappaB (IkappaB) proteins are phosphorylated and degraded, releasing transcription factors of the NF-kappaB family. NF-kappaB then controls the expression of hundreds of different genes required for inflammation and innate immunity. The aim of my PhD project was to identify new factors required for TNF-alpha signaling. In order to monitor NF-kappaB transcriptional activity upon stimulation with TNF-alpha, I established a cell-based dual luciferase assay. This assay was suited to measure TNF-alpha signaling activity in miniaturized format necessary for large-scale experiments. For finding novel regulators of TNF-alpha signaling, I used the cell-based dual luciferase assay in two genome-wide RNAi screens. These screens identified several candidates potentially implicated in NF-kappaB activation by TNF-alpha. I next established secondary assays for confirming the requirement of these candidates in TNF-signaling. On the basis of the results of these secondary assays, I selected three candidates, SPP1, GAB3 and CASP4, for further characterization. Epistasis experiments revealed that SPP1, GAB3 and CASP4 are required for the activation of the IkappaB kinase complex. Further experiments demonstrated that the candidates are not essential for proper TNFR1 cell surface expression. SPP1, a multifunctional protein, has been described to interact with the DD protein MyD88 during Toll-like receptor signaling. It could thus interact with DD proteins present in the TNFR complex. GAB3 belongs to a family of scaffold proteins of which one member, GAB2, has been shown to interact with RANK, a TNFR family member. Analogously, GAB3 could act as a scaffold at TNFR1 supporting the recruitment of signaling molecules. CASP4 is an inflammatory caspase. My results indicate that CASP4 catalytical activity is dispensable for its role in TNF-alpha signaling. CASP4 could thus serve as another scaffold protein in the TNFR complex as described for other caspases. Future experiments will identify the interaction partners of SPP1, GAB3 and CASP4, clarifying the molecular details of their mode of action in TNF-alpha-induced activation of NF-kappaB

Sequencing of the IL6 gene in a case–control study of cerebral palsy in children

BACKGROUND: Cerebral palsy (CP) is a group of nonprogressive disorders of movement and posture caused by abnormal development of, or damage to, motor control centers of the brain. A single nucleotide polymorphism (SNP), rs1800795, in the promoter region of the interleukin-6 (IL6) gene has been implicated in the pathogenesis of CP by mediating IL-6 protein levels in amniotic fluid and cord plasma and within brain lesions. This SNP has been associated with other neurological, vascular, and malignant processes as well, often as part of a haplotype block. METHODS: To refine the regional genetic association with CP, we sequenced (Sanger) the IL6 gene and part of the promoter region in 250 infants with CP and 305 controls. RESULTS: We identified a haplotype of 7 SNPs that includes rs1800795. In a recessive model of inheritance, the variant haplotype conferred greater risk (OR = 4.3, CI = [2.0-10.1], p = 0.00007) than did the lone variant at rs1800795 (OR = 2.5, CI = [1.4-4.6], p = 0.002). The risk haplotype contains one SNP (rs2069845, CI = [1.2-4.3], OR = 2.3, p = 0.009) that disrupts a methylation site. CONCLUSIONS: The risk haplotype identified in this study overlaps with previously identified haplotypes that include additional promoter SNPs. A risk haplotype at the IL6 gene likely confers risk to CP, and perhaps other diseases, via a multi-factorial mechanism

In depth comparison of an individuals DNA and its lymphoblastoid cell line using whole genome sequencing.

BACKGROUND: A detailed analysis of whole genomes can be now achieved with next generation sequencing. Epstein Barr Virus (EBV) transformation is a widely used strategy in clinical research to obtain an unlimited source of a subjects DNA. Although the mechanism of transformation and immortalization by EBV is relatively well known at the transcriptional and proteomic level, the genetic consequences of EBV transformation are less well understood. A detailed analysis of the genetic alterations introduced by EBV transformation is highly relevant, as it will inform on the usefulness and limitations of this approach. RESULTS: We used whole genome sequencing to assess the genomic signature of a low-passage lymphoblastoid cell line (LCL). Specifically, we sequenced the full genome (40X) of an individual using DNA purified from fresh whole blood as well as DNA from his LCL. A total of 217.33 Gb of sequence were generated from the cell line and 238.95 Gb from the normal genomic DNA. We determined with high confidence that 99.2% of the genomes were identical, with no reproducible changes in structural variation (chromosomal rearrangements and copy number variations) or insertion/deletion polymorphisms (indels). CONCLUSIONS: Our results suggest that, at this level of resolution, the LCL is genetically indistinguishable from its genomic counterpart and therefore their use in clinical research is not likely to introduce a significant bias

Sequencing of the IL6 gene in a case¿control study of cerebral palsy in children

Abstract Background Cerebral palsy (CP) is a group of nonprogressive disorders of movement and posture caused by abnormal development of, or damage to, motor control centers of the brain. A single nucleotide polymorphism (SNP), rs1800795, in the promoter region of the interleukin-6 (IL6) gene has been implicated in the pathogenesis of CP by mediating IL-6 protein levels in amniotic fluid and cord plasma and within brain lesions. This SNP has been associated with other neurological, vascular, and malignant processes as well, often as part of a haplotype block. Methods To refine the regional genetic association with CP, we sequenced (Sanger) the IL6 gene and part of the promoter region in 250 infants with CP and 305 controls. Results We identified a haplotype of 7 SNPs that includes rs1800795. In a recessive model of inheritance, the variant haplotype conferred greater risk (OR = 4.3, CI = [2.0-10.1], p = 0.00007) than did the lone variant at rs1800795 (OR = 2.5, CI = [1.4-4.6], p = 0.002). The risk haplotype contains one SNP (rs2069845, CI = [1.2-4.3], OR = 2.3, p = 0.009) that disrupts a methylation site. Conclusions The risk haplotype identified in this study overlaps with previously identified haplotypes that include additional promoter SNPs. A risk haplotype at the IL6 gene likely confers risk to CP, and perhaps other diseases, via a multi-factorial mechanism

Sequencing of the IL6 gene in a case¿control study of cerebral palsy in children

Abstract Background Cerebral palsy (CP) is a group of nonprogressive disorders of movement and posture caused by abnormal development of, or damage to, motor control centers of the brain. A single nucleotide polymorphism (SNP), rs1800795, in the promoter region of the interleukin-6 (IL6) gene has been implicated in the pathogenesis of CP by mediating IL-6 protein levels in amniotic fluid and cord plasma and within brain lesions. This SNP has been associated with other neurological, vascular, and malignant processes as well, often as part of a haplotype block. Methods To refine the regional genetic association with CP, we sequenced (Sanger) the IL6 gene and part of the promoter region in 250 infants with CP and 305 controls. Results We identified a haplotype of 7 SNPs that includes rs1800795. In a recessive model of inheritance, the variant haplotype conferred greater risk (OR = 4.3, CI = [2.0-10.1], p = 0.00007) than did the lone variant at rs1800795 (OR = 2.5, CI = [1.4-4.6], p = 0.002). The risk haplotype contains one SNP (rs2069845, CI = [1.2-4.3], OR = 2.3, p = 0.009) that disrupts a methylation site. Conclusions The risk haplotype identified in this study overlaps with previously identified haplotypes that include additional promoter SNPs. A risk haplotype at the IL6 gene likely confers risk to CP, and perhaps other diseases, via a multi-factorial mechanism

In depth comparison of an individual's DNA and its lymphoblastoid cell line using whole genome sequencing

Abstract Background A detailed analysis of whole genomes can be now achieved with next generation sequencing. Epstein Barr Virus (EBV) transformation is a widely used strategy in clinical research to obtain an unlimited source of a subject’s DNA. Although the mechanism of transformation and immortalization by EBV is relatively well known at the transcriptional and proteomic level, the genetic consequences of EBV transformation are less well understood. A detailed analysis of the genetic alterations introduced by EBV transformation is highly relevant, as it will inform on the usefulness and limitations of this approach. Results We used whole genome sequencing to assess the genomic signature of a low-passage lymphoblastoid cell line (LCL). Specifically, we sequenced the full genome (40X) of an individual using DNA purified from fresh whole blood as well as DNA from his LCL. A total of 217.33 Gb of sequence were generated from the cell line and 238.95 Gb from the normal genomic DNA. We determined with high confidence that 99.2% of the genomes were identical, with no reproducible changes in structural variation (chromosomal rearrangements and copy number variations) or insertion/deletion polymorphisms (indels). Conclusions Our results suggest that, at this level of resolution, the LCL is genetically indistinguishable from its genomic counterpart and therefore their use in clinical research is not likely to introduce a significant bias