Linking Genome Structure and Function through ... - ACS Publications

Mar 17, 2006 - Department of Biochemistry and Molecular Biology, 1870 Campus Delivery, Colorado State University, Fort Collins, Colorado 80523. Effort...
0 downloads 0 Views 701KB Size
Linking Genome Structure and Function through Specific Histone Acetylation Jeffrey C. Hansen* Department of Biochemistry and Molecular Biology, 1870 Campus Delivery, Colorado State University, Fort Collins, Colorado 80523

E

fforts to understand histone acetyla­ tion lie at the crossroads of chemical and biological research. It is wellestablished that addition of a two-carbon acetyl group to a specific lysine side chain on one of the core histone N-terminal “tail” domains (NTDs) can stimulate an entire biological process such as gene transcrip­ tion (1, 2 ). However, we know almost nothing about the molecular mechanism(s) that transduce the change in the local chemistry of the histone tail domain into potent biological regulation. A very recent publication by Shagren-Knack et al. (3 ) addresses the molecular basis of acetyla­ tion function by deter­mining how specific acetylation of K16 of the H4 NTD influences the salt-dependent condensation of model nucleosomal arrays and native chromatin fibers in vitro. The exciting results obtained by Shagren-Knaak et al. (3 ) show that K16 acetylation completely inhibits the ability of the H4 NTD to mediate chroma­ tin fiber condensation and that H4 K16 acetylation is a potent modulator of the nucleosome–nucleosome interactions that drive chromatin fiber condensation. These findings strongly implicate changes in genome architecture as a primary mecha­ nism through which K16acet regulates processes such as transcription. At the molecular level, these results indicate that conversion of a single, strategically placed lysine side chain from a positively charged amine to a neutral hydrophobic moiety is sufficient to completely abol­ ish the ability of the H4 NTD to engage in nucleosome–nucleosome interactions. www.acschemicalbiolog y.o rg

Core Histone NTDs, Chromatin Fiber Condensation, and Genome Architecture. A schematic depiction of the hierarchical organization of interphase chromosomes is shown in Figure 1. Chromosomal DNA is assembled with repetitively spaced core histone octamers into nucleosomal arrays. A short stretch of nucleosomal arrays in many ways can be thought of as the “subunit” of an interphase chromosome. Nucleosomal arrays that are complexed with non-histone proteins are called chromatin fibers (hence, there are many different specific types of chromatin fibers embedded within chromosomes). Local nucleosome–nucleosome interactions result in formation of a highly condensed irregular helical structure traditionally termed “the 30 nm fiber” (4 ). Longer range organizational levels beyond 30 nm fibers have been well-documented in chromo­ somes (5 ), although little is known at the biochemical level about how they are assembled and maintained. In vitro studies of chromatin fiber dynamics have been performed for over 30 years. Early studies were limited by the use of heterogeneous endogenous chroma­ tin fragments and the at times staggering complexity of the system; even a short 12‑mer array of nucleosomes is composed of nearly 100 histone proteins and 2500 bp of DNA, has a mass of 3 million Da, and exists in many different conformational states in solution. Progress during the last 2 decades has been driven by the availability of model systems that can be assembled in vitro from defined sequence

A b s t r a c t A recent publication shows that a simple chemical event, acetylation of lysine 16 on the histone H4 N-terminal tail domain (NTD), completely abolishes the ability of the H4 NTD to mediate the nucleosome–nucleosome inter­actions involved in chromatin condensation. This result provides novel insight into the molecular mechanism of histone acetylation and also implicates H4 K16acet-dependent changes in chromatin fiber architecture as a central mechanism for generating transcriptionally active genomic domains.

*To whom correspondence should be addressed. E-mail: jeffrey.c.hansen@ colostate.edu.

Published online March 17,2006 10.1021/cb6000894 CCC: $33.50 © 2006 by American Chemical Society

VOL.1 NO. 2 • ACS CHEMICAL BIOLO GY

69

Maximally Folded Nucleosomal Array

Interphase Chromatid

Moderately Folded Nucleosomal Array

CH 3 | O=C |

NH |

K16

Core Histone Tails

Extended Nucleosomal Array

Nucleosome K16

DNA

|

NH |

O=C |

CH3

Figure 1. Schematic illustration of the interphase chromosome organization. Extended, moderately folded, and maximally folded nucleosomal arrays are discussed in detail in the text. The first nucleosome (lower right-hand corner) is shown as having lysine 16 acetylation on each H4 NTD. The acetyl groups are shown in blue.

DNA and pure histones, in effect yielding a homogenous preparation of length and compositionally defined chromatin fibers. Moreover, the recent use of native and mutant recombinant core histones (6–9 ) has opened the doors to a much better understanding of the histone contributions to chromatin fiber architecture. Model system studies have provided the following essential background for the Shagren-Knaak et al. article (3 ): (a) under physiological ionic conditions, nucleo­ somal arrays are in equilibrium between extended (“beads-on-a-string”), moderately folded, extensively folded, and oligo­ meric conformational states (4 ); (b) the extensively folded “30 nm” chromatin fiber appears to be a two-start helix (6), although the detailed structure remains a mystery; (c) assembly of both folded and oligomeric nucleosomal arrays requires cation-dependent DNA charge neutraliza­ tion and additional functions mediated by the core histone NTDs (4 ); (d) the H4 NTD mediates local nucleosome–nucleosome interactions by binding to a specific acidic domain formed by H2A/H2B on the surface of adjacent nucleosomes (7–9 ); and (e) a threshold level of nonspecific core histone acetylation inhibits the nucleosome– 70

ACS C H E M I C A L B I OLOGY • VOL.1 NO. 2

nucleosome interactions involved in array condensation (4 ). Other equally important studies have shown that H4 K16acet is associated with transcriptionally active euchromatic domains in vivo (10–12 ). With this as background, we can now better understand the importance of the central questions addressed by Shagren-Knaak et al. (3 ). Does H4 K16acet inhibit folding and/or oligomerization of model nucleo­ somal arrays and endogenous, transcrip­ tionally active chromatin fragments? Is specific acetylation a biochemical switch that destabilizes the repressive nucleo­ some–nucleosome interactions involved in chromatin condensation? Is modulation of genome architecture one of the mecha­ nisms through which specific acetylation accomplishes its biological functions? The Key to Success: Overcoming Technical Challenges. As is so often the case, surmounting technical barriers paved the way for success. Specifically, it was necessary to assemble prepara­ tions of defined nucleosomal arrays that uniformly contained H4 NTDs acetylated only on lysine 16. Shagren-Knaak et al (3 ). conquered this problem by using recom­ binant core histones together with a “native chemical ligation” strategy. In

this protocol, a peptide was synthesized that consisted of residues 1–22 of H4 and was acetylated on K16. This peptide was chemically ligated to a recombinantly expressed H4 fragment (residues 23–104) to yield full-length H4 that was acetylated exclusively on K16. This acetylated H4 was mixed with recombinant full-length H2A, H2B, and H3 using standard protocols to yield H4 K16acet core histone octamers. Defined 12‑mer nucleosomal arrays were assembled from core histone octamers and nucleosome positioning DNA using classical salt dialysis reconstitution. Three different types of histone octamers were used for the reconstitutions: wild-type, H4 K16acet, and H4 NTD– (octamers lack­ ing H4 residues 1–22). The latter provides a critical control that defined the condensa­ tion behavior of arrays that lack a functional H4 NTD. Whereas assembly of wild-type and H4 NTD– arrays has been accom­ plished previously and is straightforward (6–9 ), assembly of the model K16acet arrays represented an elegant solution to a very difficult problem, one that allowed direct determination of the structural effects of a histone modification that increased the mass of a 3 MDa nucleosomal array by roughly 1/100 of 1%! This technical rigor laid the foundation for the success of the subsequent biochemical and bio­ physical experiments discussed below. H4 K16acet Is a Potent Chemical Switch That Regulates Genome Architecture. To assay for salt-dependent folding, native and H4 K16acet and H4 NTD– nucleosomal arrays in the absence and presence of 1 mM Mg2+ were analyzed by sedimenta­ tion velocity in the analytical ultracentri­ fuge. Nucleosomal arrays (12-mer) undergo a change in sedimentation coefficient from ~30 to ~55 S as they progress from the fully extended to maximally folded conforma­ tions, making sedimentation velocity an ideal assay for defining the extent of compaction under any given set of solution conditions (4 ). The exciting result obtained w w w. a c s c h e m i ca l biology.org

by Shagren-Knaak et al. (3 ) was that H4 K16acet arrays sedimented identically as the H4 NTD– arrays and were incapable of forming the maximally folded 30 nm struc­ tures formed by the native nucleo­somal arrays under the same ionic conditions. Subsaturated arrays (i.e.,