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.

A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini.

Despite the numerous sequencing methods available, the vast diversity in size and chemical modifications of RNA molecules makes the capture of the full spectrum of cellular RNAs a difficult task. By combining quasi-random hexamer priming with a custom template switching strategy, we developed a method to construct sequencing libraries from RNA molecules of any length and with any type of 3' terminal modification, allowing the sequencing and analysis of virtually all RNA species. Ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to comprehensively characterize changes in small non-coding RNAs and mRNAs simultaneously, with performance comparable to separate dedicated methods. With LIDAR, we comprehensively characterized the coding and non-coding transcriptome of mouse embryonic stem cells, neural progenitor cells, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared to traditional ligation-dependent sequencing methods, and uncovered the presence of tDRs with blocked 3' ends that had previously escaped detection. Our findings highlight the potential of LIDAR to systematically detect all RNAs in a sample and uncover new RNA species with potential regulatory functions.

Expanding the Epitranscriptomic RNA Sequencing and Modification Mapping Mass Spectrometry Toolbox with Field Asymmetric Waveform Ion Mobility and Electrochemical Elution Liquid Chromatography.

Post-transcriptional modifications of RNA strongly influence the RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in the fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before MS identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA MS increases both coverage and throughput, while EMLC LC-MS orthogonally separates RNA molecules of different lengths and charges. The combination of the two methods offers a broadly applicable platform to improve the length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.

Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications.

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.

Variation on a theme: Evolutionary strategies for H2A.Z exchange by SWR1-type remodelers.

Histone variants are a universal means to alter the biochemical properties of nucleosomes, implementing local changes in chromatin structure. H2A.Z, one of the most conserved histone variants, is incorporated into chromatin by SWR1-type nucleosome remodelers. Here, we summarize recent advances toward understanding the transcription-regulatory roles of H2A.Z and of the remodeling enzymes that govern its dynamic chromatin incorporation. Tight transcriptional control guaranteed by H2A.Z nucleosomes depends on the context provided by other histone variants or chromatin modifications, such as histone acetylation. The functional cooperation of SWR1-type remodelers with NuA4 histone acetyltransferase complexes, a recurring theme during evolution, is structurally implemented by species-specific strategies.

Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms.

Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In Drosophila, the single DOMINO chromatin regulator complex is thought to combine both activities via an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. Drosophila generates the same diversity by alternative splicing of a single gene.

Cells contain a large number of proteins that control the activity of genes in response to various signals and changes in their environment. Often these proteins work together in groups called complexes. In the fruit fly Drosophila melanogaster, one of these complexes is called DOMINO. The DOMINO complex alters gene activity by interacting with other proteins called histones which influence how the genes are packaged and accessed within the cell. DOMINO works in two separate ways. First, it can replace certain histones with other variants that regulate genes differently. Second, it can modify histones by adding a chemical marker to them, which alters how they interact with genes. It was not clear how DOMINO can do both of these things and how that is controlled; but it was known that cells can make two different forms of the central component of the complex, called DOM-A and DOM-B, which are both encoded by the same gene. Scacchetti et al. have now studied fruit flies to understand the activities of these forms. This revealed that they do have different roles and that gene activity in cells changes if either one is lost. The two forms operate as part complexes with different compositions and only DOM-A includes the TIP60 enzyme that is needed to modify histones. As such, it seems that DOM-B primarily replaces histones with variant forms, while DOM-A modifies existing histones. This means that each form has a unique role associated with each of the two known behaviors of this complex. The presence of two different DOMINO complexes is common to flies and, probably, other insects. Yet, in other living things, such as mammals and yeast, their two roles are carried out by protein complexes originating from two distinct genes. This illustrates a concept called convergent evolution, where different organisms find different solutions for the same problem. As such, these findings provide an insight into the challenges encountered through evolution and the diverse solutions that have developed. They will also help us to understand the ways in which protein activities can adapt to different needs over evolutionary time.

Loss of glucocorticoid receptor expression mediates in vivo dexamethasone resistance in T-cell acute lymphoblastic leukemia.

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.

CHRAC/ACF contribute to the repressive ground state of chromatin.

The chromatin remodeling complexes chromatin accessibility complex and ATP-utilizing chromatin assembly and remodeling factor (ACF) combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression remains unclear. Here, we explored the influence of Drosophila melanogaster chromatin accessibility complex/ACF on transcription by using complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of an Acf knock-out allele showed that only lowly expressed genes are derepressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together, our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.