Project description:We performed a genome-scale screen for suppressors of interferon stimulated gene (ISG) expression in human haploid cells (HAP1). Ubiquitin specific peptidase 14 (USP14) was a significant hit. In order to validate USP14 as a regulator of ISG expression, we created knockouts of USP14 in HAP1 cells using CRISPR-Cas9 and performed RNA-seq on coding RNA from USP14 KO and WT cells. This data was used to determine if ISGs were upregulated in USP14 KO HAP1 cells.
Project description:We performed a genome-scale screen for suppressors of interferon stimulated gene (ISG) expression in human haploid cells (HAP1). DEAD-box helicase 6 (DDX6) was a significant hit. In order to validate DDX6 as a regulator of ISG expression, we created knockouts of DDX6 in HAP1 cells using CRISPR-Cas9 and performed RNA-seq on coding RNA from DDX6 KO and WT cells. This data was used to determine if ISGs were upregulated in DDX6 KO HAP1 cells.
Project description:We analysed the composition of histone acetylase 1 (HDAC1)-associated proteins in different cellular backgrounds using quantitative mass spectrometry (MS). First, we affinity purified (AP) C-terminally FLAG-tagged HDAC1 from the nearly haploid human cell line HAP1 (Andersson et al, 1987). To examine cross-species conservation in the composition of HDAC1 complexes we additionally analysed HDAC1-FLAG purifications from mouse embryonic fibroblasts (MEFs). Genetic suppressor element 1 (GSE1) was identified as a prominent interaction partner of HDAC1. Hence, we purified V5-tagged GSE1 from HAP1 cells and analysed co-precipitated proteins.
Project description:The human N-terminal acetyltransferase E (NatE) including its associated NatA co-translationally acetylates the N-terminus of about 40-60% of the proteome to mediate diverse biological processes including protein half-life, localization and protein interaction. In eukaryotes, the NatE complex contains the NAA50 catalytic subunit with substrate specificity for N-terminal methionine acetylation and NatA, which facilitates ribosomal targeting of the complex for co-translational activity. NatA, contains NAA10 catalytic and NAA15 auxiliary subunits, and forms a complex with a protein with intrinsic NAA10 inhibitory activity, HYPK. The molecular basis for how the human NAA10 and NAA50 catalytic subunits within NatE complex coordinate function and how HYPK regulates NatE activity is unknown. Here, we characterize the biochemical interplay between the human NAA10 and NAA50 catalytic subunits of NatE and its regulation by HYPK and correlate this to the cryo-EM structures of the human NatE and NatE/HYPK complexes. We show that NAA50 and HYPK exhibit negative cooperative binding to NatA in vitro and in human cells, by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contributes to NAA10 activity inhibition through structural alteration of the NAA10 substrate binding site. NatE is about 8-fold more active than NAA50, likely due to a reduced entropic cost for substrate binding through NatA tethering, but is inhibited by HYPK through structural alteration of the NatE substrate binding site. Taken together, these studies reveal the molecular basis for coordinated N-terminal acetylation by the NAA10 and NAA50 catalytic subunits of NatE and its modulation by HYPK.
Project description:The aim of the experiment is to assess the robustness of CRISPR-Cas9 based genetic engineering. Using RNA sequencing, we are able to quantify the abundance of transcripts corresponding to CRISPR-targets and thus the degree of nonsense mediated decay.
Project description:Chromosome-centric Human Proteomic Project (C-HPP) aims at identifying and characterizing protein products encoded from all protein-coding genes of human chromosomes. As evident in the recent nature papers, it is possible that those missing proteins may be low abundant in many cell types or expressed only in a few cell types. In order to identify missing proteins, we focused on extensively unexplored cell lines(HAP1, KBM-7).
Project description:Centrosomes are cytoplasmic membraneless organelles that comprise a pair of centrioles, and a pericentriolar material. We developed a new centrosome affinity capture method termed CAPture that achieves high-coverage proteomes from 20-30 million cells. Here we present CAPture-MS data from HAP1 cells (WT and two CEP83-KO clones) demonstrating the power and sensitivity of CAPture-MS to detect centrosomal distal appendages hierarchical interactions.