Remodeling and Repositioning of Nucleosomes in Nucleosomal Arrays.

ATP-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit or evict nucleosomes or reposition them along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate. In vivo, however, nucleosomes form extended arrays with regular spacing. Here, we describe how regularly spaced nucleosome arrays can be reconstituted in vitro and how these arrays can be used to dissect remodeling in the test tube. We outline two assays. The first assay senses various structural changes to a specific nucleosome within the nucleosomal array whereas the second assay is specific toward detecting repositioning of nucleosomes within the array. Both assays exploit changes to the accessibility of DNA to restriction enzymes during the remodeling reaction.

PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3.

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

Computational study of remodeling in a nucleosomal array.

Chromatin remodeling complexes utilize the energy of ATP hydrolysis to change the packing state of chromatin, e.g. by catalysing the sliding of nucleosomes along DNA. Here we present simple models to describe experimental data of changes in DNA accessibility along a synthetic, repetitive array of nucleosomes during remodeling by the ACF enzyme or its isolated ATPase subunit, ISWI. We find substantial qualitative differences between the remodeling activities of ISWI and ACF. To understand better the observed behavior for the ACF remodeler, we study more microscopic models of nucleosomal arrays.

Rapid purification of recombinant histones.

The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

ISWI remodelling of physiological chromatin fibres acetylated at lysine 16 of histone H4.

ISWI is the catalytic subunit of several ATP-dependent chromatin remodelling factors that catalyse the sliding of nucleosomes along DNA and thereby endow chromatin with structural flexibility. Full activity of ISWI requires residues of a basic patch of amino acids in the N-terminal 'tail' of histone H4. Previous studies employing oligopeptides and mononucleosomes suggested that acetylation of the H4 tail at lysine 16 (H4K16) within the basic patch may inhibit the activity of ISWI. On the other hand, the acetylation of H4K16 is known to decompact chromatin fibres. Conceivably, decompaction may enhance the accessibility of nucleosomal DNA and the H4 tail for ISWI interactions. Such an effect can only be evaluated at the level of nucleosome arrays. We probed the influence of H4K16 acetylation on the ATPase and nucleosome sliding activity of Drosophila ISWI in the context of defined, in vitro reconstituted chromatin fibres with physiological nucleosome spacing and linker histone content. Contrary to widespread expectations, the acetylation did not inhibit ISWI activity, but rather stimulated ISWI remodelling under certain conditions. Therefore, the effect of H4K16 acetylation on ISWI remodelling depends on the precise nature of the substrate.

No need for a power stroke in ISWI-mediated nucleosome sliding.

Nucleosome remodelling enzymes of the ISWI family reposition nucleosomes in eukaryotes. ISWI contains an ATPase and a HAND-SANT-SLIDE (HSS) domain. Conformational changes between these domains have been proposed to be critical for nucleosome repositioning by pulling flanking DNA into the nucleosome. We inserted flexible linkers at strategic sites in ISWI to disrupt this putative power stroke and assess its functional importance by quantitative biochemical assays. Notably, the flexible linkers did not disrupt catalysis. Instead of engaging in a power stroke, the HSS module might therefore assist DNA to ratchet into the nucleosome. Our results clarify the roles had by the domains and suggest that the HSS domain evolved to optimize a rudimentary remodelling engine.

Nucleosome sliding mechanisms: new twists in a looped history.

Nucleosomes, the basic organizational units of chromatin, package and regulate eukaryotic genomes. ATP-dependent nucleosome-remodeling factors endow chromatin with structural flexibility by promoting assembly or disruption of nucleosomes and the exchange of histone variants. Furthermore, most remodeling factors induce nucleosome movements through sliding of histone octamers on DNA. We summarize recent progress toward unraveling the basic nucleosome sliding mechanism and the interplay of the remodelers' DNA translocases with accessory domains. Such domains optimize and regulate the basic sliding reaction and exploit sliding to achieve diverse structural effects, such as nucleosome positioning or eviction, or the regular spacing of nucleosomes in chromatin.

The ATPase domain of ISWI is an autonomous nucleosome remodeling machine.

ISWI slides nucleosomes along DNA, enabling the structural changes of chromatin required for the regulated use of eukaryotic genomes. Prominent mechanistic models imply cooperation of the ISWI ATPase domain with a C-terminal DNA-binding function residing in the HAND-SANT-SLIDE (HSS) domain. Contrary to these models, we show by quantitative biochemical means that all fundamental aspects of nucleosome remodeling are contained within the compact ATPase module of Drosophila ISWI. This domain can independently associate with DNA and nucleosomes, which in turn activate ATP turnover by inducing a conformational change in the enzyme, and it can autonomously reposition nucleosomes. The role of the HSS domain is to increase the affinity and specificity for nucleosomes. Nucleosome-remodeling enzymes may thus have evolved directly from ancestral helicase-type motors, and peripheral domains have furnished regulatory capabilities that bias the remodeling reaction toward different structural outcomes.

dMec: a novel Mi-2 chromatin remodelling complex involved in transcriptional repression.

The ATP-dependent chromatin remodeller Mi-2 functions as a transcriptional repressor and contributes to the suppression of cell fates during development in several model organisms. Mi-2 is the ATPase subunit of the conserved Nucleosome Remodeling and Deacetylation (NuRD) complex, and transcriptional repression by Mi-2 is thought to be dependent on its associated histone deacetylase. Here, we have purified a novel dMi-2 complex from Drosophila that is distinct from dNuRD. dMec (dMEP-1 complex) is composed of dMi-2 and dMEP-1. dMec is a nucleosome-stimulated ATPase that is expressed in embryos, larval tissues and adult flies. Surprisingly, dMec is far more abundant than dNuRD and constitutes the major dMi-2-containing complex. Both dNuRD and dMec associate with proneural genes of the achaete-scute complex. However, despite lacking a histone deacetylase subunit, only dMec contributes to the repression of proneural genes. These results reveal an unexpected complexity in the composition and function of Mi-2 complexes.