Project description:The aim of the experiment was to identify accessible (open) chromatin regions in the genome prior to and post induction of EBV's lytic phase by the BZLF1 protein. Two versions of BZLF1 were employed: full-length and activation-domain (AD)-truncated BZLF1 non-induced and induced for 15 h. The experiments were performed as triplicates.
Project description:The aim of the experiment was to identify accessible (open) chromatin regions in the genome prior to and post induction of EBV's BZLF1 protein. Two versions of BZLF1 were employed: full-length and activation-domain (AD)-truncated BZLF1 non-induced and induced for 15 h. The experiments were performed as triplicates.
Project description:(i) Transcripts of DG75 iBZLF1 cells were analyzed comparing non-induced cells and cells induced with doxycycline (100 ng/ ml) for 6 h. Doxycycline induces the expression of the full length viral transcription factor BZLF1. (ii) For control purposes, the transcripts of DG75 cells equipped with a doxycycline-regulated truncated BZLF1 allele lacking its transactivation domain (DG75 iBZLF1 AD-truncated) were analyzed prior to and after induction with doxycycline for 6 h. (iii) For an additional control, the transcripts of parental, unmodified DG75 cells were analyzed prior to and after adding doxycycline for 6 h. All experiments were performed as triplicates. The analysis is based on Homo sapiens (human) genome assembly GRCh37 (hg19) from Genome Reference Consortium. Artificial ERCC Spike-in RNAs (Thermo Scientific) were added as external controls.
Project description:Identification of specific chromatin interactions of 49 selected genes with the Capture-C technique in DG75 iBZLF1 cells prior to and 15 h after expression of EBV's protein BZLF1. The experiments were performed as triplicates.
Project description:To study the effect of GLI3 knockout on early brain organoid development, we collected single-cell multiome data from 18 day old brain organoids
Project description:In this project, we aimed to examine the transcriptional changes that occur after irradiation of intestinal organoid-derived subcutaneous heterotopic tumors over a 7 day period post-radiation treatment. AKPT (villinCreER;Apcfl/fl;KrasG12D/+;Trp53fl/fl;TgfbrIfl/fl) intestinal organoids were cultured, then suspended in a 50:50 phosphate buffered-saline and Matrigel mixture and subcutaneously implanted into male C57BL/6 mice. Two weeks after implantation, mice were given either a single dose of 15 Gy radiation or 3 doses of 7 Gy radiation. Tumours were harvested 4 hours, 24 hours, 3 days, and 7 days after receiving the single dose of 15 Gy or the final dose of 7 Gy radiation, as well as from corresponding non-irradiated controls. RNA was extracted from the collected tumours and processed for RNA sequencing.
Project description:Histone deacetylases (HDACs) have been widely pursued as targets for anti-cancer therapeutics. However, many of these targets are universally essential for cell survival, which may limit the therapeutic window that can be achieved by drug candidates. By examining large collections of CRISPR/Cas9-based essentiality screens, we discovered a genetic interaction between HDAC1 and HDAC2 wherein each paralog is synthetically lethal with hemizygous deletion of the other. This collateral synthetic lethality is caused by recurrent chromosomal translocations that occur in diverse solid and hematological malignancies, including neuroblastoma and multiple myeloma. Using genetic deletion or dTAG-mediated degradation, we show that HDAC2 disruption suppresses the growth of HDAC1-deficient neuroblastoma in vitro and in vivo. Mechanistically, we find that targeted degradation of HDAC2 in these cells prompts the degradation of several members of the nucleosome remodeling and deacetylase (NuRD) complex, leading to diminished chromatin accessibility at HDAC2/NuRD-bound sites of the genome and impaired control of enhancer-associated transcription. Furthermore, we reveal that several of the degraded NuRD complex subunits are dependencies in neuroblastoma and multiple myeloma, providing motivation to develop paralog-selective HDAC1 or HDAC2 degraders. Altogether, we identify HDAC1/2 collateral synthetic lethality as a new therapeutic target and reveal a novel mechanism for exploiting NuRD-associated cancer dependencies.
Project description:The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of N-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK and AKT. We find that each oncogene has somewhat different surfaceomes but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting their strong dependence on the MAPK pathway to propagate signaling. Using a recently developed glyco-proteomics method of activated ion electron transfer dissociation (AI-ETD) we found massive oncogene-induced changes in 142 N-linked glycans and differential increases in complex hybrid glycans especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems level view of how specific driver oncogenes remodel the surface glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.
Project description:To review an improved proximity-labeling strategy that uses the improved E. coli biotin ligase TurboID to characterize C. elegans protein complexes, the interactome of a presynaptic active zone protein, ELKS-1, was analyzed as proof of principle. A significant constraint on the sensitivity of TurboID-based proximity labeling is the presence of abundant, endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a co-factor. We developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry, by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography (IMAC).