New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.

Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. (3) Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity. Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, we will discuss 16 more proteins with native disorder. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles. Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence. The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.

Intrinsically Disordered Proteins

Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9 Å Resolution

The nucleosome, which is the primary building block of chromatin, is not a static structure: It can adopt alternative conformations. Changes in solution conditions or changes in histone acetylation state cause nucleosomes and nucleosomal arrays to behave with altered biophysical properties. Distinct subpopulations of nucleosomes isolated from cells have chromatographic properties and nuclease sensitivity different from those of bulk nucleosomes. Recently, proteins that were initially identified as necessary for transcriptional regulation have been shown to alter nucleosomal structure. These proteins are found in three types of multiprotein complexes that can acetylate nucleosomes, deacetylate nucleosomes, or alter nucleosome structure in an ATP-dependent manner. The direct modification of nucleosome structure by these complexes is likely to play a central role in appropriate regulation of eukaryotic genes.

Alteration of Nucleosome Structure as a Mechanism of Transcriptional Regulation

A positive role for histone acetylation in transcription factor access to nucleosomal DNA

Use of selectively trypsinized nucleosome core particles to analyze the role of the histone “tails” in the stabilization of the nucleosome

Role of the histone "tails" in the folding of oligonucleosomes depleted of histone H1.

DNA and protein determinants of nucleosome positioning have been examined after in vitro reconstitutions of native or modified histone octamers onto tandem repeats of 207- and 172-base-pair DNA sequences containing the Lytechinus variegatus 5S rRNA gene and onto monomeric sequences derived from these by digestion with various restriction endonucleases. In all cases, a major nucleosome position as well as a number of minor positions have been observed, which indicates that the generation of multiple positions is an inherent property of the 5S rRNA gene sequence. Interestingly, all positions observed differ by multiples of 10 base pairs. Data obtained under different reconstitution conditions demonstrate that the observed distributions of nucleosomes on these DNA templates are equilibrium distributions. This study has also examined the positioning of histone octamers from which histone "tails" had been removed by tryptic digestion. Results indicate that the histone tails are not determinants of nucleosome positioning. Although our results suggest that the mechanical properties of the 5S rDNA are the fundamental factors determining nucleosome positioning, they are insufficient to direct all nucleosomes into a single location.

https://www.pnas.org/content/pnas/87/15/5724.full.pdf

DNA and protein determinants of nucleosome positioning on sea urchin 5S rRNA gene sequences in vitro.

It is demonstrated that the histone (H3-H4)2 tetramer can find specific positions on DNA, even in the absence of other histones. Purified histone (H3-H4)2 tetramers were reconstituted onto 208-base-pair (bp) DNA molecules containing a nucleosome-positioning sequence by using salt-gradient dialysis. The stoichiometry of histone tetramer to DNA was shown to be 1:1. Digestion with micrococcal nuclease led to formation of protected DNA fragments of approximately 73 bp. Cleavage of the 73-bp DNA with restriction enzymes produced a small set of defined bands, demonstrating positioning of the (H3-H4)2 tetramer on DNA. Analysis of the restriction digests shows that the 73-bp DNA corresponds mainly to two fragments, one lying on either side of the pseudo-dyad axis of the major position adopted by complete histone octamers on this DNA. This result means that a single (H3-H4)2 histone tetramer can fold approximately 146 bp of DNA with the same positioning as the complete octamer but that a region near the pseudo-dyad is only weakly protected against micrococcal nuclease attack in the absence of histones H2A and H2B.

https://www.pnas.org/content/pnas/88/23/10596.full.pdf

Nucleosome positioning is determined by the (H3-H4)2 tetramer.

In order to better understand the conformational changes induced in the nucleosome core particle by changes in the ionic strength of the media in the range from 0.1 to 0.6 M NaCl, we have conducted a very detailed structural analysis, combining circular dichroism, DNase I digestion, and sedimentation equilibrium. The results of such analysis indicate that the secondary structure of both DNA and histones exhibits small (~5%) but noticeable changes as the salt increases within this range. In the case of DNA, the data are consistent with a trend toward a more relaxed secondary structure. The DNase I pattern of digestion is also altered by the salt and suggests a DNA relaxation around the flanking ends. From the hydrodynamic measurements, we also observe a significant change in the virial coefficients of the particle as the salt increases, which in turn are in very good agreement with the theoretically expected values. Furthermore, the preferential hydration parameter is also found to increase with the salt. We believe that the self-dependent conformational change of the nucleosome core particle is the result of the conjunction of all these subtle changes. Yet, from the present data, their exact relationship to the tertiary structure of the whole particle at the different ionic strengths cannot be exactly defined.

Feng Dong

Papers

Use of selectively trypsinized nucleosome core particles to analyze the role of the histone “tails” in the stabilization of the nucleosome

Role of the histone "tails" in the folding of oligonucleosomes depleted of histone H1.

DNA and protein determinants of nucleosome positioning on sea urchin 5S rRNA gene sequences in vitro.

Nucleosome positioning is determined by the (H3-H4)2 tetramer.

Analysis of the changes in the structure and hydration of the nucleosome core particle at moderate ionic strengths