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

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

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

Side-by-side comparison of histone purities.

<p>Histones were purified according to the RHP protocol (RHP) and according to a published protocol that started with the preparation of inclusion bodies (IB; ref. 5). Both purification procedures started from the same amount of bacteria that were grown on the same day. (A) SDS-PAGE analysis. H2A showed the weakest overexpression (Fig. S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029.s001" target="_blank">File S1</a>) and is consequently the least pure. M: protein marker. (B) Purities.</p

Histone extraction.

<p>Whole cell extracts were prepared under denaturing conditions from bacteria expressing <i>Drosophila</i> H2B by French Press and sonication. Cell debris and residual insoluble material were pelleted by centrifugation. Efficiency of the histone extraction was analyzed on Coomassie-stained SDS gels by loading equivalent amounts of the supernatant containing the solubilized histones (SN) and the corresponding pellet fraction (P). Most H2B was present in the supernatant.</p

Histone purification by cation exchange chromatography.

<p>The whole cell extract from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone-0104029-g002" target="_blank">Figure 2</a> containing solubilized <i>Drosophila</i> H2B (SN) was filtered and applied to cation exchange chromatography under denaturing conditions. (A) Equivalent amounts of the filtered whole cell extract (Input) and the flow-through fraction of the cation exchange column were analyzed by SDS-PAGE. Most H2B bound to the chromatography resin. (B) H2B was eluted by a NaCl gradient as indicated. Fractions 4–8 were pooled and processed further as described in the main text.</p