Project description:In this study we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo. By a dual fusion of heterochromatin protein 1 (HP1a) and Krüppel associated box (KRAB) to MS2 coat protein and recruitment to a nuclease competent CRISPR complex, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 (Myd88) gene in vivo. We report that this strategy can effectively modulate host immune response against adeno-associated virus-mediated gene therapy and influence the course of septicemia.
Project description:Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma and immunosuppression-related lymphomas. These B-cell malignancies arise by distinct transformation pathways and utilize divergent viral and host expression programs. To identify host dependency factors elicited by EBV latent-infection states, we performed parallel genome-wide CRISPR/Cas9 screens in Burkitt lymphoma (BL) and lymphoblasotid cell lines (LCL). Our results highlighted 57 BL and 87 LCL genes selectively critical for their growth and survival. LCL hits were enriched for EBV-induced genes, including viral super-enhancer targets and multiple kinases. We uncovered key CD19/CD81 roles in EBV membrane protein-driven PI3K/AKT activation and mechanisms by which EBV evades tumor suppressor responses to its growth program. LMP1-induced cFLIP was critical for LCL defense against TNFa-mediated programmed cell death, while EBV-induced BATF/IRF4 were critical for LCL BIM suppression and MYC induction. EBV super-enhancer targeted IRF2 protected LCLs against BLIMP1 responses. Collectively, our results identify host/pathogen interaction-driven synthetic lethal targets for therapeutic intervention.
Project description:Many genes determining cell identity are regulated by a set of enhancer elements collectively referred to as super-enhancers. It has been suggested that super-enhancers represent a new class of cis-element, in which the assembly of enhancers confers an emergent property from the extended regulatory domain. To investigate this, we used published criteria to define one of the strongest super-enhancers in mouse erythroid cells – a 24kb region regulating α-globin expression, which comprises five enhancer-like components. Using homologous recombination, we deleted each component of this super-enhancer, singly and in informative combinations, and examined hematologic phenotype, gene expression, chromatin structure and chromosome conformation. Each component behaves independently, in an additive rather than synergistic manner. We conclude that the sub-classification of enhancers is unjustified beyond a description of their strength, which is defined by the number of lineage-specific transcription factors they bind. These findings ask afresh why enhancer-like elements cluster at key genes.
Project description:Many genes determining cell identity are regulated by a set of enhancer elements collectively referred to as super-enhancers. It has been suggested that super-enhancers represent a new class of cis-element, in which the assembly of enhancers confers an emergent property from the extended regulatory domain. To investigate this, we used published criteria to define one of the strongest super-enhancers in mouse erythroid cells – a 24kb region regulating α-globin expression, which comprises five enhancer-like components. Using homologous recombination, we deleted each component of this super-enhancer, singly and in informative combinations, and examined hematologic phenotype, gene expression, chromatin structure and chromosome conformation. Each component behaves independently, in an additive rather than synergistic manner. We conclude that the sub-classification of enhancers is unjustified beyond a description of their strength, which is defined by the number of lineage-specific transcription factors they bind. These findings ask afresh why enhancer-like elements cluster at key genes.
Project description:Many genes determining cell identity are regulated by a set of enhancer elements collectively referred to as super-enhancers. It has been suggested that super-enhancers represent a new class of cis-element, in which the assembly of enhancers confers an emergent property from the extended regulatory domain. To investigate this, we used published criteria to define one of the strongest super-enhancers in mouse erythroid cells – a 24kb region regulating α-globin expression, which comprises five enhancer-like components. Using homologous recombination, we deleted each component of this super-enhancer, singly and in informative combinations, and examined hematologic phenotype, gene expression, chromatin structure and chromosome conformation. Each component behaves independently, in an additive rather than synergistic manner. We conclude that the sub-classification of enhancers is unjustified beyond a description of their strength, which is defined by the number of lineage-specific transcription factors they bind. These findings ask afresh why enhancer-like elements cluster at key genes.
Project description:Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.
Project description:This model was used to describe the behaviour of the synthetic open loop circuit in yeast, published in [1]. The accompanying script will simulate the model as used in main Figure 2c. Additional scaling and preprocessing was done to compare the model with data used in the experiment and can be obtained from the authors upon request.
[1] Gnügge, Robert, et al. "An Orthogonal Permease–Inducer–Repressor Feedback Loop Shows Bistability." ACS synthetic biology 5.10 (2016): 1098-1107.