Project description:To understand the function of MSI1 in pluripotent stem cells, RNA-seq assays were performed on mouse embryonic stem cells R1, MSI1 knockout cell line R1-C5, human embryonic stem cells H9, RRM knockout cell line H9-C8, MSI1 full-length overexpression cell line H9-MSI1OE, MSI1C variant overexpression cell line H9-MSI1 (138-362) OE , H9-MSI1(272-362)OE. RNA bound by MSI1 in R1 and H9, and MSI1C variants MSI1 (138-362), MSI1(272-362) were detected using RIP-seq.
Project description:Transcription regulation requires many protein interactions on chromatin, and only a subset of transcription factors have well-defined activation or repression domains. The Arabidopsis transcription factor HY5 controls critical growth-related gene expression programs during plant development, but it’s primary activity in regulating transcription remains unclear. To address this question, we generated constitutive repressor and activator HY5 fusion proteins to direct the expression of HY5 target genes. We used RNA-seq, ChIP-seq, and multiple phenotypes to demonstrate that HY5 depends on accessory factors to promote transcription and identify high confidence direct targets of HY5. We suggest that this strategy can be used broadly to define the transcription regulation activity and direct targets of transcription factors. Interestingly, this approach also revealed a mechanism by which HY5 promotes the accumulation of its own negative regulators. We show that HY5 directly regulates components of the COP1 E3-ubiquitin ligase complex, and by uncoupling this feedback loop we can induce partial de-etiolation in the dark. This provides a system by which plants can quickly repress growth upon light exposure. Lastly, we show that modulating this system can generate significant phenotypic diversity and provide proof of concept that these fusion proteins can modulate growth in tomato, opening a novel path toward selecting desirable traits in crop species.
Project description:mRNA-seq and ribosome profiling of neural stem cells overexpressing or knocked out for Musashi RNA-binding proteins Study of the global effects of Musashi (Msi) proteins on the transcriptome of embryonic neural stem cells. Neural stem cells were derived from brains of E12.5 or E13.5 embryos engineered to have inducible Msi1 or Msi2 genes, or from embryos with double floxed alleles of Msi1 and Msi2 carrying a Tamoxifen-induclble Cre (CreER). The overexpression mice were made using the Flp-in system (OpenBioSystems), where a cDNA of interest (in this case Msi1 or Msi2) is knocked into the Collagen (Col1A1) locus. The expression of the cDNA of interest is driven by m2rTTA that is knocked into the Rosa26 locus (R26). KH2 describes a strain containing the R26-m2rTTA but lacking Msi1 or Msi2 cDNA. MSI1 describes a strain containing R26-m2rTTA and Msi1 cDNA in Col1A1. MSI2 describes a strain containing R26-m2rTTA and Msi2 cDNA in Col1A1. C1 describes a strain lacking the CreER allele but containing double floxed alleles of Msi1/Msi2 (used as Tamoxifen control). C4 describes a strain carrying the CreER allele and double floxed alleles of Msi1/Msi2.
Project description:Musashi1 (Msi1) is a highly conserved RNA binding protein that is required during the development of the nervous system. Msi1 has a role in neural stem cells, controlling the balance between self-renewal and differentiation. Msi1 has also been implicated in cancer, being highly expressed in multiple tumor types. In this study, we analyzed Msi1 expression in a large cohort of medulloblastoma samples and showed that Msi1 is highly expressed in tumor tissue compared to normal cerebellum and that high Msi1 expression is associated with a poor prognosis. Using a nude mouse xenograft model, we demonstrate that Msi1 is important for tumor growth. We then used RIP-chip (ribonucleoprotein immunoprecipitation followed by microarray analysis) to identify mRNA targets of Msi1 in medulloblastoma. In conclusion, our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target.
Project description:LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology and defective hormonal and stimulus responses. HY5 thus acts as an integrater that links various gene networks to coordinate plant development. Here we report an effort to experimentally map the HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate HY5 binds to over 9,000 genes, which detectably impact the expression of over 1,100 genes, either positively or negatively. Further, HY5 indirectly regulates many other genes through sub-networks mediated by other regulators. In particular, we show that HY5 regulates eight microRNA (miRNA) genes, which in turn control transcript abundance of specific target genes. Over-expressing the HY5-targeted miR408 resulted in phenotypes that are opposite to hy5 mutants. Together our results revealed both the transcriptional and post-transcriptional components of the HY5-mediated gene networks responsible for the phenotypic complexity in plants.
Project description:Musashi1 (Msi1) is a highly conserved RNA binding protein that is required during the development of the nervous system. Msi1 has a role in neural stem cells, controlling the balance between self-renewal and differentiation. Msi1 has also been implicated in cancer, being highly expressed in multiple tumor types. In this study, we analyzed Msi1 expression in a large cohort of medulloblastoma samples and showed that Msi1 is highly expressed in tumor tissue compared to normal cerebellum and that high Msi1 expression is associated with a poor prognosis. Using a nude mouse xenograft model, we demonstrate that Msi1 is important for tumor growth. We then used RIP-chip (ribonucleoprotein immunoprecipitation followed by microarray analysis) to identify mRNA targets of Msi1 in medulloblastoma. In conclusion, our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target. RIP-Chip analysis to identify mRNA preferentially associated with Msi1 protein. RIP-Chip experiments were performed on two biologically replicated samples. A total of 8 microarrays were carried on using technical replicates of Msi1 antibody vs. prebleed serum for each dye orientation. We prepared two biological replicates for two different arrays. Each array consisted of 4 microarrays with 2 replicates for each dye orientation.
Project description:RNA binding proteins (RBPs) function as master regulators of gene expression. Alterations in their expression levels and function are often observed in tumors with numerous oncogenic RBPs identified in recent years. Musashi1 (Msi1) is an RBP and stem cell marker gene that controls the balance between self-renewal and proliferation. High Msi1 levels have been observed in multiple tumor types including glioblastoma and are often associated with poor patient outcomes. Msi1 knockdown is known to affect numerous cancer phenotypes and to prevent growth of different tumor types. To dissect Msi1’s main contributions to glioblastoma development, we conducted a comprehensive genomic analysis. We identified a network of cell cycle/division and DNA replication genes and established these processes as its core functions. Msi1 controls this gene network via two mechanisms: direct interaction and indirect regulation mediated by the transcription factors E2F2 and E2F8. We corroborated Msi1’s impact on these processes and determined that glioblastoma lines with Msi1 KO display increased sensitivity to cell cycle and DNA replication inhibitors. As Msi1 inhibitors have been identified, a drug combination strategy (Msi1 + cell cycle/DNA replication inhibitors) could be a viable route to treat glioblastoma.
Project description:The stem cell marker Musashi1 (MSI1) is highly expressed during neurogenesis and in Glioblastoma (GBM). In cancer and non-malignant progenitor cells, MSI1 promotes self-renewal and impairs differentiation, involving the regulation of mRNA translation. However, a comprehensive understanding of MSI1’s role in promoting GBM-driving networks remains to be deciphered.
Project description:As a source of both energy and environmental information, the importance for plants to monitor the incoming light is evidenced by the highly integrated nature of the light signal transduction pathway with numerous other pathways throughout plant development. One of these signal integrators is the bZIP transcription factor HY5 which holds a key role as a positive regulator of light signalling in plants. Although HY5 is described to act as a DNA-binding transcriptional regulator, the lack of any apparent transactivation domain makes it unclear how HY5 is able to accomplish its many functions. We describe the identification of three B-box containing proteins (BBX20, 21 and 22) as essential partners for HY5 dependent modulation of hypocotyl elongation, anthocyanin accumulation and transcriptional regulation. The bbx202122 triple mutant mimics the phenotypes of hy5 in the light and its strong suppression of the cop1 phenotype in darkness. Furthermore, RNA-seq experiments show that 84% of genes differentially regulated in bbx202122 are also HY5 regulated, consistent with the B-box proteins and HY5 acting interdependently.