7 research outputs found
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Genome-wide CRISPR screening identifies new regulators of glycoprotein secretion.
Background: The fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. Methods: With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen, followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. Results: We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5. Conclusions: This study has identified new factors for protein secretion involved in Golgi homeostasis
Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.
Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies
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Research data supporting 'Genome-wide CRISPR screening identifies new regulators of glycoprotein secretion'
Files S1 - S6 contain underlying data for figures published in the paper.
S1: sgRNA counts of unsorted and sorted population from the CRISPR screen detailed in the paper, which aimed to identify genes that regulate glycoprotein secretion. Sheet 1 is a table of sgRNA counts, including the gene name that each sgRNA targets. Sheet 2 includes full descriptions of the populations.
S2: MaGECK (version 0.5.7) analysis of sgRNA counts from the CRISPR screen. Both MaGECK-MLE and MaGECK-RRA results are shown. False discovery rates (FDR) from this table were used to create figure 1C-E
S3: Raw FCS files from flow cytometry staining of sorted samples, used to create figure 1B.
S4: Raw chemiluminescence data, used to create figure 2.
S5: Unedited image files used in Figures 3D, 4 and 5.
S6: Data output from Cell Profiler, showing total number of cells identified per image and number of cells classified as having fragmented or intact Golgi.
E1-E4 is extended data for figures and tables published in the paper.
E1: Figure of all genes analysed for having fragmented Golgi; top hits from this talbe are shown in Figure 3C. Percentage of cells with fragmented Golgi for all of the hits screened in the tertiary screen. As in Figure 3C, hits are arranged alphabetically and coloured by cell count, with darker blue spots representing more confluent wells.
E2: Sequence of P5 primers used for PCR amplification of sgRNA.
E3: Sequence of P7 primers used for PCR amplification of sgRNA.
E4: Details of the siGenome pooled siRNA library used in secondary screening
Recommended from our members
Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.
Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies