Biology subjects

Krogan, N.

Publications and source records attributed to Krogan, N..

6 recordsLinked to original sources

bioRxiv2018

Targeting MYC Overexpressing Leukemia with Cardiac Glycoside Proscillaridin Through Downregulation of Histone Acetyltransferases

Targeting MYC oncogene remains a major therapeutic goal in cancer chemotherapy. Here, we demonstrate that proscillaridin, a cardiac glycoside approved for heart failure treatment exhibit anticancer selectivity towards high MYC expressing leukemic cell lines and leukemia stem cells. At a clinically relevant concentration, proscillaridin induced a rapid downregulation of MYC protein level, due to a significant decrease in MYC protein half-life. Proscillaridin treatment induced a downregulation of gene sets involved in MYC pathway, and a concomitant upregulation of genes involved in hematopoietic differentiation. Proscillaridin induced a significant loss of lysine acetylation in histone H3 (K9, K14, K18 and K27) and in non-histone proteins such as MYC, MYC target proteins, and a series of histone acetylation regulators. Loss of lysine acetylation correlated with a rapid downregulation of histone acetyltransferase protein levels, involved in histone and MYC acetylation (CBP, P300, GCN5, TIP60, and MOZ), preferentially in MYC overexpressing leukemia as compared to other cancer cells. These results support the repurposing of proscillaridin in MYC overexpressing leukemia and propose a novel strategy to target MYC in cancer.

pharmacology and toxicology↗

bioRxiv2018

Unc93b1 recruits Syntenin-1 to dampen TLR7 signaling and prevent autoimmunity

Recognition of nucleic acids enables detection of diverse pathogens by a limited number of innate immune receptors but also exposes the host to potential autoimmunity. At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self RNA or DNA, respectively. Despite the structural and functional similarities between these receptors, their contribution to autoimmune diseases such as SLE can be quite different. However, mechanisms of negative regulation that differentiate between TLR7 and TLR9 have not been described. Here we report a new function for the TLR trafficking chaperone Unc93b1 that specifically limits TLR7 signaling and prevents TLR7-dependent autoimmunity. Unc93b1 is known to traffic TLRs from the endoplasmic reticulum to endosomes, but this new regulatory function does not affect TLR7 localization. Instead, Unc93b1 recruits Syntenin-1, which inhibits TLR7, but not TLR9, signaling. Syntenin-1 binding requires phosphorylation of two serine residues on Unc93b1, providing a mechanism for dynamic regulation of the activation threshold of TLR7. Disruption of the Unc93b1/Syntenin-1 interaction in mice results in TLR7-dependent autoimmunity. Thus, Unc93b1 not only enables proper trafficking of nucleic acid sensing TLRs but also sets the activation threshold of these potentially self-reactive receptors.

immunology↗

bioRxiv2018

Systematic characterization of genome editing in primary T cells reveals proximal genomic insertions and enables machine learning prediction of CRISPR-Cas9 DNA repair outcomes

The Streptococcus pyogenes Cas9 (SpCas9) nuclease has become a ubiquitous genome editing tool due to its ability to target almost any location in DNA and create a double-stranded break1,2. After DNA cleavage, the break is fixed with endogenous DNA repair machinery, either by non-templated mechanisms (e.g. non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ)), or homology directed repair (HDR) using a complementary template sequence3,4. Previous work has shown that the distribution of repair outcomes within a cell population is non-random and dependent on the targeted sequence, and only recent efforts have begun to investigate this further5-11. However, no systematic work to date has been validated in primary human cells5,7. Here, we report DNA repair outcomes from 1,521 unique genomic locations edited with SpCas9 ribonucleoprotein complexes (RNPs) in primary human CD4+ T cells isolated from multiple healthy blood donors. We used targeted deep sequencing to measure the frequency distribution of repair outcomes for each guide RNA and discovered distinct features that drive individual repair outcomes after SpCas9 cleavage. Predictive features were combined into a new machine learning model, CRISPR Repair OUTcome (SPROUT), that predicts the length and probability of nucleotide insertions and deletions with R2 greater than 0.5. Surprisingly, we also observed large insertions at more than 90% of targeted loci, albeit at a low frequency. The inserted sequences aligned to diverse regions in the genome, and are enriched for sequences that are physically proximal to the break site due to chromatin interactions. This suggests a new mechanism where sequences from three-dimensionally neighboring regions of the genome can be inserted during DNA repair after Cas9-induced DNA breaks. Together, these findings provide powerful new predictive tools for Cas9-dependent genome editing and reveal new outcomes that can result from genome editing in primary T cells.

cell biology↗

bioRxiv2018

The cellular NMD pathway restricts Zika virus infection and is targeted by the viral capsid protein

Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly1, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice2-4. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1)5. Endogenous UPF1 interacted with the viral capsid protein in co-immunoprecipitation experiments and capsid expression post-transcriptionally downregulated UPF1, a process that we confirmed occurs during de novo ZIKV infection. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures. We therefore propose that ZIKV, via the capsid protein, has evolved a strategy to dampen antiviral activities of NMD6,7, which subsequently contributes to neuropathology in vivo.

microbiology↗

bioRxiv2017

BRG1/BRM-associated factor complex subunit diversity promotes temporally distinct gene expression programs in cardiogenesis

Chromatin remodeling complexes instruct cellular differentiation and lineage specific transcription. The BRG1/BRM associated factor (BAF) complexes are important for several aspects of differentiation. We show that the catalytic subunit Brg1 has a specific role in cardiac precursors (CPs) to initiate cardiac gene expression programs and repress non-cardiac expression. Using immunoprecipitation with mass spectrometry (IP-MS), we determined the dynamic composition of BAF complexes during mammalian cardiac differentiation, and identified BAF60c (SMARCD3) and BAF170 (SMARCC2) as subunits enriched in CPs and cardiomyocytes (CM). Baf60c and Baf170 co-regulate gene expression with Brg1 in CPs, but in CMs control different gene expression programs, although still promoting a cardiac-specific gene set. BRG1, BAF60, and BAF170 all modulate chromatin accessibility, to either promote accessibility at activated genes, while closing up chromatin at repressed genes. BAF60c and BAF170 are required for proper BAF complex composition and stoichiometry, and promote BRG1 occupancy in CM. Additionally, BAF170 facilitates expulsion of BRG1-containing complexes in the transition from CP to CM. Thus, dynamic interdependent BAF complex subunit assembly modulates chromatin states and thereby directs temporal gene expression programs in cardiogenesis.\n\nSignificance statementBRG1/BRM associated factors (BAF) form multi-subunit protein complexes that reorganize chromatin and regulate transcription. Specific BAF complex subunits have important roles during cell differentiation and development. We systematically identify BAF subunit composition and find temporal enrichment of subunits during cardiomyocyte differentiation. We find the catalytic subunit BRG1 has important contributions in initiating gene expression programs in cardiac progenitors along with cardiac-enriched subunits BAF60c and BAF170. Both these proteins regulated BAF subunit composition and chromatin accessibility and prevent expression of non-cardiac developmental genes during precursor to cardiomyocyte differentiation. Mechanistically, we find BAF170 destabilizes the BRG1 complex and expels BRG1 from cardiomyocyte-specific genes. Thus, our data shows synergies between diverse BAF subunits in facilitating temporal gene expression programs during cardiogenesis.

developmental biology↗

bioRxiv2017

Evolutionary proteomics uncovers ciliary signaling components

Cilia are organelles specialized for movement and signaling. To infer when during animal evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from three organisms: sea urchins, sea anemones and choanoflagellates. From these ciliomes, we identified 437 high confidence ciliary candidate proteins conserved in mammals, including known regulators of Hh, GPCR and TRP channel signaling. The phylogenetic profiles of their ciliary association indicate that the Hh and GPCR pathways were linked to cilia before the origin of bilateria and TRP channels before the origin of animals. We demonstrated that some of the candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein that we identified in the cilia of all three organisms. In animals, ENKUR is expressed by cells with motile cilia, ENKUR localizes to cilia in diverse organisms and, in both Xenopus laevis and mice, ENKUR is required for patterning the left/right axis. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to Hh, GPCR and TRP channel signaling, and uncovers a novel ciliary protein that controls vertebrate development and human disease.

evolutionary biology↗