RESUMO
Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication, RNA transcription, chromatin extrusion and nucleosome remodeling) occur on individual chromatin arrays remains unresolved. Here, to address this deficit, we present SAMOSA-ChAAT: a massively multiplex single-molecule footprinting approach to map the primary structure of individual, reconstituted chromatin templates subject to virtually any chromatin-associated reaction. We apply this method to distinguish between competing models for chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h: nucleosome-density-dependent spacing versus fixed-linker-length nucleosome clamping. First, we perform in vivo single-molecule nucleosome footprinting in murine embryonic stem cells, to discover that ISWI-catalyzed nucleosome spacing correlates with the underlying nucleosome density of specific epigenomic domains. To establish causality, we apply SAMOSA-ChAAT to quantify the activities of ISWI ATPase SNF2h and its parent complex ACF on reconstituted nucleosomal arrays of varying nucleosome density, at single-molecule resolution. We demonstrate that ISWI remodelers operate as density-dependent, length-sensing nucleosome sliders, whose ability to program DNA accessibility is dictated by single-molecule nucleosome density. We propose that the long-observed, context-specific regulatory effects of ISWI complexes can be explained in part by the sensing of nucleosome density within epigenomic domains. More generally, our approach promises molecule-precise views of the essential processes that shape nuclear physiology.
Assuntos
Cromatina , Nucleossomos , Animais , Camundongos , Histonas/metabolismo , DNA , Montagem e Desmontagem da Cromatina , Adenosina Trifosfatases/metabolismo , Mamíferos/genéticaRESUMO
Unlike other chromatin remodelers, INO80 preferentially mobilizes hexasomes, which can form during transcription. Why INO80 prefers hexasomes over nucleosomes remains unclear. Here, we report structures of Saccharomyces cerevisiae INO80 bound to a hexasome or a nucleosome. INO80 binds the two substrates in substantially different orientations. On a hexasome, INO80 places its ATPase subunit, Ino80, at superhelical location -2 (SHL -2), in contrast to SHL -6 and SHL -7, as previously seen on nucleosomes. Our results suggest that INO80 action on hexasomes resembles action by other remodelers on nucleosomes such that Ino80 is maximally active near SHL -2. The SHL -2 position also plays a critical role for nucleosome remodeling by INO80. Overall, the mechanistic adaptations used by INO80 for preferential hexasome sliding imply that subnucleosomal particles play considerable regulatory roles.
Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cromatina/metabolismo , Histonas/metabolismo , Nucleossomos/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The critical role of the INO80 chromatin remodeling complex in transcription is commonly attributed to its nucleosome sliding activity. Here, we have found that INO80 prefers to mobilize hexasomes over nucleosomes. INO80's preference for hexasomes reaches up to â¼60 fold when flanking DNA overhangs approach â¼18-bp linkers in yeast gene bodies. Correspondingly, deletion of INO80 significantly affects the positions of hexasome-sized particles within yeast genes in vivo. Our results raise the possibility that INO80 promotes nucleosome sliding by dislodging an H2A-H2B dimer, thereby making a nucleosome transiently resemble a hexasome. We propose that this mechanism allows INO80 to rapidly mobilize nucleosomes at promoters and hexasomes within gene bodies. Rapid repositioning of hexasomes that are generated in the wake of transcription may mitigate spurious transcription. More generally, such versatility may explain how INO80 regulates chromatin architecture during the diverse processes of transcription, replication, and repair.
Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Cromatina/genética , Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.
Assuntos
Cromatina/genética , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Nucleossomos/genética , Imagem Individual de Molécula , Acetilação , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , DNA/química , DNA/metabolismo , Epigênese Genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/metabolismo , Estudo de Prova de Conceito , Conformação Proteica , Processamento de Proteína Pós-Traducional , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Zinc finger protein Zscan4 is selectively expressed in mouse two-cell (2C) embryos undergoing zygotic genome activation (ZGA) and in a rare subpopulation of embryonic stem cells with 2C-like features. Here, we show that Zscan4 specifically recognizes a subset of (CA)n microsatellites, repeat sequences prone to genomic instability. Zscan4-associated microsatellite regions are characterized by low nuclease sensitivity and high histone occupancy. In vitro, Zscan4 binds nucleosomes and protects them from disassembly upon torsional strain. Furthermore, Zscan4 depletion leads to elevated DNA damage in 2C mouse embryos in a transcription-dependent manner. Together, our results identify Zscan4 as a DNA sequence-dependent microsatellite binding factor and suggest a developmentally regulated mechanism, which protects fragile genomic regions from DNA damage at a time of embryogenesis associated with high transcriptional burden and genomic stress.