Journal of Molecular Biology
Volume 381, Issue 4, 12 September 2008, Pages 816-825
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30 nm Chromatin Fibre Decompaction Requires both H4-K16 Acetylation and Linker Histone Eviction

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Abstract

The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concentration of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.

Introduction

The hierarchical packaging of eukaryotic DNA by histone proteins into chromatin represents a significant steric barrier to all cellular processes that involve DNA. Therefore, chromatin compaction (and decompaction) is necessarily part of the regulation of cellular processes such as transcription, replication, recombination and repair. Yet, the mechanism by which chromatin decompaction and dynamics are regulated is largely unknown.

The first level of DNA compaction is achieved by wrapping 147 bp DNA into 1.7 superhelical turns around a histone octamer, consisting of an H3–H4 tetramer and two H2A-H2B dimers.1, 2, 3 Whereas about 75% of the histone octamer mass forms the globular structure onto which the DNA is wrapped, the remaining 25% form long N-terminal tails rich in lysine and arginine residues.3 In all, eight N-terminal tails protrude from the surface of the nucleosome core where they are free to make numerous intermolecular contacts. The binding of the linker histone (H1 or H5), which is present at close to one molecule per nucleosome in the majority of eukaryotic organisms,4, 5, 6 organizes an additional 20 bp of DNA to complete the nucleosome.7, 8 In vitro, nucleosome arrays condense in a salt-dependent manner into compact filaments with a diameter of about 30 nm.9, 10, 11, 12, 13 X-ray diffraction and electron microscope studies have provided evidence for the presence of the 30 nm chromatin fiber in diverse eukaryotic nuclei.14, 15, 16

The critical role of the long and highly basic histone octamer N-terminal tails in the regulation of chromosomal processes such as transcription has become increasingly apparent with the identification of a plethora of different post-translational modifications, subsets of which correlate with regions of silenced or transcriptionally active chromatin.17, 18 Whilst it has become evident that many specific histone tail post-translational modifications function in the recruitment of regulatory proteins that are likely to affect chromatin structure indirectly,19, 20 numerous in vitro studies have provided evidence that the histone octamer N-terminal tails also play a direct role in the modulation of nucleosome array compaction.21, 22, 23 A central remaining question is whether certain histone tail modifications directly regulate chromatin compaction.

Acetylation of the histone octamer N-terminal histone tails24 is a prevalent and reversible histone modification whose levels correlate with transcriptionally active chromatin.25, 26 The effect of acetylating lysine residues by acetyltransferases27 is to reduce the net positive charge of the very basic histone tails and is therefore expected to modulate electrostatic histone tail interactions.28, 29 It is believed that acetylation causes the unfolding of the chromatin fiber that permits transcription, consistent with the results of in vitro experiments using randomly hyperacetylated histones.30 Of the many lysine residues subjected to acetylation, the specific acetylation of lysine 16 in the N-terminal tail of histone H4 (H4-K16Ac) is particularly prevalent and functionally important in a variety of organisms. In budding yeast, over 80% of H4 is acetylated at K16 and in vivo evidence shows that this modification has a role in maintaining or promoting gene transcription.31, 32, 33, 34 Similarly, in flies the enhancement of transcription from the male X-chromosome correlates with H4 K16 acetylation.35, 36 These biological observations suggest that this specific acetylation mark might have a unique role in regulating chromatin compaction. In two recent in vitro studies utilizing short tandem arrays of reconstituted nucleosome cores it was concluded that deletion of the H4 N-terminal tail and acetylation of H4 on K16 are sufficient to inhibit the formation of the 30 nm chromatin fiber.37, 38 In the first, Richmond and colleagues37 identified the region of the H4 N-terminal tail encompassing K16 (residues 14–19) as essential for salt-dependent compaction. Subsequently, Peterson and colleagues showed that compaction of the same nucleosome core array could be inhibited by H4-K16Ac.38 However, both of these studies suffer from the same three limitations. Firstly, the short nucleosome repeat length chosen in the construction of these nucleosome core arrays is relatively rare in nature,39 and constrains the folding into a structure40 that is distinct from the 30 nm chromatin fiber.13, 41 Secondly, the absence of bound linker histone in these two studies significantly limits the compaction of the reconstituted arrays and prevents the formation of the more relevant 30 nm chromatin fiber. Thirdly, the use of very short nucleosome arrays questions the reliability of such fibers in reflecting the compaction behavior of native chromatin.

Here, we present the results of an investigation aimed at defining the relative contribution of the linker histone and H4-K16Ac on the compaction states of chromatin. This study goes beyond previous studies as it is based on a model reconstituted nucleosome array that is very long and contains stoichiometric concentrations of bound linker histone and hence mimics the compaction and decompaction behavior of the 30 nm chromatin fiber. Nucleosome arrays were assembled from recombinant histone octamers containing different combinations of N-terminal tail truncations and modifications, onto a DNA array containing 61 tandem copies of 202 bp 601 DNA (202 bp × 61). The compaction properties of these arrays were analyzed using three complementary techniques: electrophoretic mobility in native agarose gels, analytical ultracentrifugation and visualization by electron microscopy. We find that the decompaction of the 30 nm chromatin fiber is not a simple single-step mechanism, but a two-step mechanism. Significantly, incorporation of histone H4 partially acetylated on K16 results in a cooperative decompaction. Visualization by electron microscopy shows that acetylation leads to the loss of fully compact chromatin fibers, consistent with abrogation of nucleosome–nucleosome contacts, but not full fiber unfolding. Further decompaction is achieved by the removal of the linker histone.

Section snippets

Reconstitution of long nucleosome arrays containing histone octamer N-terminal tail deletions

To minimize end-effects and hence better mimic the compaction behavior of native chromatin, we produced a very long DNA array consisting of 61 tandem copies of the 601 nucleosome positioning DNA sequence onto which the histone octamer positions uniquely.42 Chromatin fibers were assembled using our reconstitution system that produces nucleosome arrays containing stoichiometric concentrations of both histone octamer and linker histone H5.12, 43 At physiological salt conditions these nucleosome

Discussion

The biologically relevant starting point for understanding the regulatory mechanisms that determine the compaction state of the 30 nm chromatin fiber is an analysis of the folding behavior of chromatin arrays containing the linker histone. As such, our results go beyond previous studies using model nucleosome core arrays that did not include the linker histone in the reconstitutions.37, 38 Our conclusions are particularly reliable because they are derived from the use of three independent but

601 DNA array

The DNA array is based on the Widom 601 DNA nucleosome-positioning sequence,42 and contains 61 tandem copies of 202 bp of the 601 DNA fragment. Monomeric DNA fragments were ligated together in a tandem arrangement to form multimers and then cloned into pUC18 as described previously.12, 13, 43 Plasmids were grown in DH5α E. coli cells. The 601 DNA array was excised by digestion with EcoRV and purified. Mixed sequence competitor DNA (crDNA), about 147 bp in length, was obtained from nucleosome

Acknowledgements

We thank Tony Crowther for helpful discussions. We thank Peter Becker for providing recombinant baculoviruses for the expression of the MOF-MSL1-MSL3 complex. We thank Louise Fairall for help and John Widom for providing the 601 DNA sequence. We thank Sara Sandin for advice with electron microscopy and for discussions, and Jo Butler for advice on analytical ultracentrifugation. Work in the laboratory of R.G.R. was supported by grant from the NIH.

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