Project description:Two molecular subgroups of MF-iCC were identified based on differentially expressed genes between MF-iCC with a cholangiolocellular carcinoma (CLC) component and those without a CLC component.
Project description:Genomic DNA from 189 wild type Col x CLC, 191 msh2 Col x CLC or 187 msh2 Col x Ler F2 individuals was extracted using CTAB and used to generate sequencing libraries as described (Serra et al 2018 PNAS). Sequencing data was analysed to identify crossovers as previously reported, using the TIGER pipeline (Rowan et al., 2015 G3).
Project description:The ubiquitous CLC membrane transporters are the only transporter family known to exchange anions for cations. Despite extensive study, there is no model to completely explain the 2:1 Cl‒/H+ stoichiometric exchange mechanism. Here, we provide such a model. Using CLC-ec1, a bacterial homolog that has served as a paradigm for the family, we determined cryo-EM structures at pH 7, pH 4.5, and pH 3. Molecular dynamics simulations of the pH-3 structure reveal critical steps in the transport mechanism, including release of Cl- ions to the extracellular side, opening of the inner gate, and water wires that facilitate H+ transport. Water wires are observed frequently in both the canonical H+-transport pathway and in the Cl- pathway, where they had not been previously reported. We propose that tight coupling of Cl‒/H+ transport is maintained (uncoupled H+ transport is avoided) because H+ transfer from the water wires to the catalytic glutamate is favored only when Cl‒ is also present in the pathway . Mutations that weaken Cl‒ binding without changing the pathway structure exhibit functional properties consistent with this model.
Project description:ClC-2 is a broadly expressed Cl- channel of the CLC family of Cl- channels and transporters which is abundantly expressed in brain. Here it was proposed to participate in lowering the cytoplasmic Cl- concentration of neurons, a process that establishes an inhibitory response to the neurotransmitters GABA and glycine (Staley et al., 1996). Heterozygous mutations in CLCN2 (the gene encoding ClC-2) were recently reported in a few patients with three clinically distinct forms of epilepsy (Haug et al, 2003). However, the disruption of ClC-2 in mice (ClC-2 KO mouse) did not entail epilepsy (Bösl et al., 2001; Nehrke et al., 2002) but myelin vacuolation in fiber tracts of the central nervous system. We used a gene expression profiling of the ClC-2 KO mouse in brain to identify possible disease mechanism which cause the observed myelin phenotype. As these myelin vacuolation became apparent in the fiber tracts of ClC-2 KO cerebellum at P28 and increased with age, we analysed the cerebellum of ClC-2 KO mice at different postnatal ages, before (P14) and after (P35) the KO cerebellum has been affected by myelin vacuolation.
Project description:Lysosomal dysfunction is considered pathogenic in Alzheimer Disease (AD). Loss of Presenilin-1(PSEN1) function causing early onset AD impedes acidification via defective vATPase V0a1 subunit delivery to lysosomes. We report that isoproterenol and related β2-adrenergic agonists re-acidify lysosomes in PSEN1 KO cells and fibroblasts from PSEN1 familial AD(FAD) patients, restores lysosomal calcium homeostasis and proteolysis, and reverses impaired autophagy flux. We identify a novel rescue mechanism involving PKA-mediated facilitated delivery of ClC-7 to lysosomes, which stimulates chloride influx and reverses markedly lowered Cl- content of PSEN1 KO lysosomes. Notably, PSEN1 loss-of-function impedes ER-to-lysosome delivery of ClC-7, thus accounting for lysosomal Cl- deficits that compound pH deficits due to deficient vATPase function. Transcriptomics of PSEN1-deficient cells reveal strongly down-regulated ER-to-lysosome transport pathways and reversibility by isoproterenol. Our findings uncover a broadened PSEN1 role in lysosomal ion homeostasis and novel pH modulation of lysosomes through β-adrenergic regulation of ClC-7, which can be therapeutically modulated.
Project description:Identifying crossover locations in Arabidopsis thaliana wild type Col x CLC, msh2 Col x CLC and msh2 Col x Ler F2 populations using genotyping by sequencing.
Project description:To investigate drug-induced epigenetic resistance in AML, the K562 cell line was selected due to its responsiveness to anthracyclines and its ease of genetic manipulation. Daunorubicin (DNR) was chosen as a model drug because of its pivotal role in AML treatment. DNR treatment of K562 cells induced expression of all three ALDH1 isoforms. To explore the activation of cis-regulatory elements, ChIP-Seq with H3K27Ac was performed on K562 cells treated or not with DNR for 18 hours.
Project description:Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR) but not the nucleoside analog Cytarabine, the two main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 hours) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, accelerating deSUMOylation dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that proteins that are deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. Although DNR does not modify CTCF binding on chromatin in general and at NFKB2 promoter in particular, it leads to a SUMO-dependent reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with distal cis-regulatory regions.
Project description:Acute Myeloid Leukemia cell line HL-60 were treated with 1 M daunorubicin (DNR) or 2M cytarabin (Ara-C) or 1 M DNR + 10 M VAS2870 (NADPH oxidase inhibitor) for 2 hours. ChIP-Seq experiments were carried out with SUMO-2/3 antibodies. Immunoprecipitated DNA and corresponding inputs from 3 independent experiments were pooled and processed for deep-sequencing (Hi-SEq 2000, Illumina).