ISWI catalyzes nucleosome sliding in condensed nucleosome arrays.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. We investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner qualitatively agree with our data. We speculate that 'monkey-bar' mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

ChromatoShiny: an interactive R/Shiny App for plotting chromatography profiles.

Background: Unicorn™ software on Äkta liquid chromatography instruments outputs chromatography profiles of purified biological macromolecules. While the plots generated by the instrument software are very helpful to inspect basic chromatogram properties, they lack a range of useful annotation, customization and export options. Methods: We use the R Shiny framework to build an interactive app that facilitates the interpretation of chromatograms and the generation of figures for publications. Results: The app allows users to fit a baseline, to highlight selected fractions and elution volumes inside or under the plot (e.g. those used for downstream biochemical/biophysical/structural analysis) and to zoom into the plot. The app is freely available at https://ChromatoShiny.bio.ed.ac.uk. Conclusions: It requires no programming experience, so we anticipate that it will enable chromatography users to create informative, annotated chromatogram plots quickly and simply.

A CDK-regulated chromatin segregase promoting chromosome replication.

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme-Yta7-entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

A critical role for linker DNA in higher-order folding of chromatin fibers.

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.

Longitudinal analyses of leukemia-associated antigen-specific CD8+ T cells in patients after allogeneic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment approach for patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). Graft versus leukemia (GVL) effects, which are exerted by donor T cells directed against leukemic-associated antigens (LAAs), are considered to play a crucial role in disease eradication. Although the expansion of cytotoxic T lymphocytes (CTLs) specific for cytomegalovirus (CMV) in response to an infection has been shown in multiple studies, data on CTLs mediating GVL effects are limited. To evaluate a potential increase or decrease of T lymphocytes specific for LAAs in the setting of allogeneic HSCT, we monitored leukemia-specific CD8+ T cells throughout the first year after HSCT in 18 patients using streptamer technology. A broad panel of promising LAAs was selected: Wilms tumor protein, proteinase 3, receptor for hyaluronan acid-mediated motility, apoptosis regulator Bcl-2, survivin, nucleophosmin, and fibromodulin. T cells specifically directed against AML- or CLL-associated antigens were found at very low frequencies in peripheral blood. Substantial frequencies of LAA-specific T cells could not be measured at any time point by flow cytometry. In contrast, abundant CMV-pp65-specific T cells were detected in CMV-seropositive patient-recipient pairs and an increase prompted by CMV infection could be demonstrated. In conclusion, T lymphocytes with specificities for the aforementioned LAAs can only be detected in minimal quantities in the early phase after allogeneic HSCT.