Trans-Binding Mechanism of Ubiquitin-like Protein Activation Revealed by a UBA5-UFM1 Complex

Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s). Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation

Pluripotent stem cell-derived models of neurological diseases reveal early transcriptional heterogeneity

Abstract Background Many neurodegenerative diseases develop only later in life, when cells in the nervous system lose their structure or function. In many forms of neurodegenerative diseases, this late-onset phenomenon remains largely unexplained. Results Analyzing single-cell RNA sequencing from Alzheimer’s disease (AD) and Huntington’s disease (HD) patients, we find increased transcriptional heterogeneity in disease-state neurons. We hypothesize that transcriptional heterogeneity precedes neurodegenerative disease pathologies. To test this idea experimentally, we use juvenile forms (72Q; 180Q) of HD iPSCs, differentiate them into committed neuronal progenitors, and obtain single-cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking down several differentially variable genes results in increased aggregate formation, a pathology associated with HD. We further validate the increased transcriptional heterogeneity in CHD8+/− cells, a model for autism spectrum disorder. Conclusions Overall, our results suggest that although neurodegenerative diseases develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value

Mesoscale modeling and single-nucleosome tracking reveal remodeling of clutch folding and dynamics in stem cell differentiation

Nucleosomes form heterogeneous groups in vivo, named clutches. Clutches are smaller and less dense in mouse embryonic stem cells (ESCs) compared to neural progenitor cells (NPCs). Using coarse-grained modeling of the pluripotency Pou5f1 gene, we show that the genome-wide clutch differences between ESCs and NPCs can be reproduced at a single gene locus. Larger clutch formation in NPCs is associated with changes in the compaction and internucleosome contact probability of the Pou5f1 fiber. Using single-molecule tracking (SMT), we further show that the core histone protein H2B is dynamic, and its local mobility relates to the structural features of the chromatin fiber. H2B is less stable and explores larger areas in ESCs compared to NPCs. The amount of linker histone H1 critically affects local H2B dynamics. Our results have important implications for how nucleosome organization and H2B dynamics contribute to regulate gene activity and cell identity.This work was supported by a University of Pennsylvania Epigenetics Pilot Award (M.L.); an NSF Center for Engineering and Mechanobiology (CEMB) Pilot Award (M.L.); a Linda Pechenik Montague Investigator Award (M.L.); the European Union’s Horizon 2020 Research and Innovation Programme (CellViewer grant 686637 to M.L., M.P.C., and E.M.); Ministerio de Ciencia, Innovación y Universidades ( BFU2017-86760-P [AEI/FEDER, UE]) and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya (AGAUR grant 2017 SGR 689 to M.P.C.); National NSFC grant 319711771003712 (M.P.C.); National Institutes of Health , National Institute of General Medical Sciences awards R01-GM055264 and R35-GM122562 ; People Program (Marie Curie Actions) FP7/2007–2013 under REA grant 608959 (M.V.N.); and Juan de la Cierva-Incorporación 2017 (M.V.N.

Pluripotent stem cell-derived models of neurological diseases reveal early transcriptional heterogeneity

Background: Many neurodegenerative diseases develop only later in life, when cells in the nervous system lose their structure or function. In many forms of neurodegenerative diseases, this late-onset phenomenon remains largely unexplained. Results: Analyzing single-cell RNA sequencing from Alzheimer’s disease (AD) and Huntington’s disease (HD) patients, we find increased transcriptional heterogeneity in disease-state neurons. We hypothesize that transcriptional heterogeneity precedes neurodegenerative disease pathologies. To test this idea experimentally, we use juvenile forms (72Q; 180Q) of HD iPSCs, differentiate them into committed neuronal progenitors, and obtain single-cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking down several differentially variable genes results in increased aggregate formation, a pathology associated with HD. We further validate the increased transcriptional heterogeneity in CHD8+/− cells, a model for autism spectrum disorder. Conclusions: Overall, our results suggest that although neurodegenerative diseases develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofReviewedFacult