Linker histone H1.0 interacts with an extensive network of proteins found in the nucleolus.

The H1 linker histones are abundant chromatin-associated DNA-binding proteins. Recent evidence suggests that linker histones also may function through protein-protein interactions. To gain a better understanding of the scope of linker histone involvement in protein-protein interactions, we used a proteomics approach to identify H1-binding proteins in human nuclear extracts. Full-length H1.0 and H1.0 lacking its C-terminal domain (CTD) were used for protein pull-downs. A total of 107 candidate H1.0 binding proteins were identified by LC-MS/MS. About one-third of the H1.0-dependent interactions were mediated by the CTD, and two-thirds by the N-terminal domain-globular domain fragment. Many of the proteins pulled down by H1.0 were core splicing factors. Another group of H1-binding proteins functions in rRNA biogenesis. H1.0 also pulled down numerous ribosomal proteins and proteins involved in cellular transport. Strikingly, nearly all of the H1.0-binding proteins are found in the nucleolus. Quantitative biophysical studies with recombinant proteins confirmed that H1.0 directly binds to FACT and the splicing factors SF2/ASF and U2AF65. Our results demonstrate that H1.0 interacts with an extensive network of proteins that function in RNA metabolism in the nucleolus, and suggest that a new paradigm for linker histone action is in order.

Dynamic fuzziness during linker histone action.

Linker histones are multi-domain nucleosome binding proteins that stabilize higher order chromatin structures and engage in specific protein-protein interactions. Here we emphasize the structural and functional properties of the linker histone C-terminal domain (CTD), focusing on its intrinsic disorder, interaction-induced secondary structure formation and dynamic fuzziness. We argue that the fuzziness inherent in the CTD is a primary molecular mechanism underlying linker histone function in the nucleus.

The linker region of macroH2A promotes self-association of nucleosomal arrays.

MacroH2A is a histone variant found in higher eukaryotes localized at the inactive X chromosome and is known to maintain heterochromatic regions in the genome. MacroH2A consists of a conserved histone domain and a macro domain connected by a linker region. To understand the contributions of the three domains to chromatin condensation, we incorporated various constructs of macroH2A into defined nucleosomal arrays and analyzed their impact on in vitro chromatin compaction. The folding and oligomerization properties of arrays containing full-length macroH2A (macroH2A(FL)), macroH2A(1-161) (encompassing the histone domain and linker region), and macroH2A(1-122) (histone domain only) were compared with major-type H2A arrays. Analytical ultracentrifugation and atomic force microscope imaging indicate that macroH2A(1-161)-containing arrays favor condensation under conditions where major-type arrays are nearly fully extended. In contrast, arrays with macroH2A(FL) exhibit behavior similar to that of major-type arrays. This suggests that the linker region of macroH2A facilitates array condensation and that this behavior is inhibited by the macro domain. Furthermore, chimeric major-type H2A arrays containing the macroH2A linker domain (H2A(ML)) exhibited the same condensation properties as macroH2A(1-161) arrays, thus emphasizing the intriguing behavior of the macroH2A linker region.

Multifunctionality of the linker histones: an emerging role for protein-protein interactions.

Linker histones, e.g., H1, are best known for their ability to bind to nucleosomes and stabilize both nucleosome structure and condensed higher-order chromatin structures. However, over the years many investigators have reported specific interactions between linker histones and proteins involved in important cellular processes. The purpose of this review is to highlight evidence indicating an important alternative mode of action for H1, namely protein-protein interactions. We first review key aspects of the traditional view of linker histone action, including the importance of the H1 C-terminal domain. We then discuss the current state of knowledge of linker histone interactions with other proteins, and, where possible, highlight the mechanism of linker histone-mediated protein-protein interactions. Taken together, the data suggest a combinatorial role for the linker histones, functioning both as primary chromatin architectural proteins and simultaneously as recruitment hubs for proteins involved in accessing and modifying the chromatin fiber.

Two regions of Bacillus subtilis transcription factor SpoIIID allow a monomer to bind DNA.

Nutrient limitation causes Bacillus subtilis to develop into two different cell types, a mother cell and a spore. SpoIIID is a key regulator of transcription in the mother cell and positively or negatively regulates more than 100 genes, in many cases by binding to the promoter region. SpoIIID was predicted to have a helix-turn-helix motif for sequence-specific DNA binding, and a 10-bp consensus sequence was recognized in binding sites, but some strong binding sites were observed to contain more than one match to the consensus sequence, suggesting that SpoIIID might bind as a dimer or cooperatively as monomers. Here we show that SpoIIID binds with high affinity as a monomer to a single copy of its recognition sequence. Using charge reversal substitutions of residues likely to be exposed on the surface of SpoIIID and assays for transcriptional activation in vivo and for DNA binding in vitro, we identify two regions essential for DNA binding, the putative recognition helix of the predicted helix-turn-helix motif and a basic region near the C terminus. SpoIIID is unusual among prokaryotic DNA-binding proteins with a single helix-turn-helix motif in its ability to bind DNA monomerically with high affinity. We propose that the C-terminal basic region of SpoIIID makes additional contacts with DNA, analogous to the N-terminal arm of eukaryotic homeodomain proteins and the "wings" of winged-helix proteins, but structurally distinct. SpoIIID is highly conserved only among bacteria that form endospores, including several important human pathogens. The need to conserve biosynthetic capacity during endospore formation might have favored the evolution of a small transcription factor capable of high-affinity binding to DNA as a monomer, and this unusual mode of DNA binding could provide a target for drug design.

Determinants of histone H4 N-terminal domain function during nucleosomal array oligomerization: roles of amino acid sequence, domain length, and charge density.

Mg(2+)-dependent oligomerization of nucleosomal arrays is correlated with higher order folding transitions that stabilize chromosome structure beyond the 30-nm diameter fiber. In the present studies, we have employed a novel mutagenesis-based approach to identify the macromolecular determinants that control H4 N-terminal domain (NTD) function during oligomerization. Core histones were engineered in which 1) the H2A, H2B, and H3 NTDs were swapped onto the H4 histone fold; 2) the length of the H4 NTD and the H2A NTD on the H4 histone fold, were increased; 3) the charge density of the NTDs on the H4 histone fold was increased or decreased; and 4) the H4 NTD was placed on the H2B histone fold. Model nucleosomal arrays were assembled from wild type and mutant core histone octamers, and Mg(2+)-dependent oligomerization was characterized. The results demonstrated that the H2B and H3 NTDs could replace the H4 NTD, as could the H2A NTD if it was duplicated to the length of the native H4 NTD. Arrays oligomerized at lower salt concentrations as the length of the NTD on the H4 histone fold was increased. Mutations that decreased the NTD charge density required more Mg(2+) to oligomerize, whereas mutants that increased the charge density required less salt. Finally, the H4 NTD functioned differently when attached to the H2B histone fold than the H4 histone fold. These studies have revealed new insights into the biochemical basis for H4 NTD effects on genome architecture as well as the protein chemistry that underlies the function of the intrinsically disordered H4 NTD.

The silent information regulator 3 protein, SIR3p, binds to chromatin fibers and assembles a hypercondensed chromatin architecture in the presence of salt.

The telomeres and mating-type loci of budding yeast adopt a condensed, heterochromatin-like state through recruitment of the silent information regulator (SIR) proteins SIR2p, SIR3p, and SIR4p. In this study we characterize the chromatin binding determinants of recombinant SIR3p and identify how SIR3p mediates chromatin fiber condensation in vitro. Purified full-length SIR3p was incubated with naked DNA, nucleosome core particles, or defined nucleosomal arrays, and the resulting complexes were analyzed by electrophoretic shift assays, sedimentation velocity, and electron microscopy. SIR3p bound avidly to all three types of templates. SIR3p loading onto its nucleosomal sites in chromatin produced thickened condensed fibers that retained a beaded morphology. At higher SIR3p concentrations, individual nucleosomal arrays formed oligomeric suprastructures bridged by SIR3p oligomers. When condensed SIR3p-bound chromatin fibers were incubated in Mg(2+), they folded and oligomerized even further to produce hypercondensed higher-order chromatin structures. Collectively, these results define how SIR3p may function as a chromatin architectural protein and provide new insight into the interplay between endogenous and protein-mediated chromatin fiber condensation pathways.

A beta-hairpin comprising the nuclear localization sequence sustains the self-associated states of nucleosome assembly protein 1.

The histone chaperone nucleosome assembly protein 1 (NAP1) is implicated in histone shuttling as well as nucleosome assembly and disassembly. Under physiological conditions, NAP1 dimers exist in a mixture of various high-molecular-weight oligomers whose size may be regulated by the cell cycle-dependent concentration of NAP1. Both the functional and structural significance of the observed oligomers are unknown. We have resolved the molecular mechanism by which yeast NAP1 (yNAP1) dimers oligomerize by applying x-ray crystallographic, hydrodynamic, and functional approaches. We found that an extended beta-hairpin that protrudes from the compact core of the yNAP1 dimer forms a stable beta-sheet with beta-hairpins of neighboring yNAP1 dimers. Disruption of the beta-hairpin (whose sequence is conserved among NAP1 proteins in various species) by the replacement of one or more amino acids with proline results in complete loss of yNAP1 dimer oligomerization. The in vitro functions of yNAP1 remain unaffected by the mutations. We have thus identified a conserved structural feature of NAP1 whose function, in addition to presenting the nuclear localization sequence, appears to be the formation of higher-order oligomers.

Novel mechanism of inhibition of rat kidney-type glutaminase by bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES).

The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during chronic neurodegenerative diseases may contribute to the propagation of glutamate excitotoxicity. Thus an inhibitor that selectively inactivates the released GA may limit the accumulation of excess glutamate and minimize the loss of neurological function that accompanies brain injury. The present study examines the mechanism of inactivation of rat KGA (kidney GA isoform) by the small-molecule inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide]. BPTES is a potent inhibitor of KGA, but not of the liver GA isoform, glutamate dehydrogenase or gamma-glutamyl transpeptidase. Kinetic studies indicate that, with respect to glutamine, BPTES has a K(i) of approx. 3 microM. Moreover, these studies suggest that BPTES inhibits the allosteric activation caused by phosphate binding and promotes the formation of an inactive complex. Gel-filtration chromatography and sedimentation-velocity analysis were used to examine the effect of BPTES on the phosphate-dependent oligomerization of KGA. This established that BPTES prevents the formation of large phosphate-induced oligomers and instead promotes the formation of a single oligomeric species with distinct physical properties. Sedimentation-equilibrium studies determined that the oligomer produced by BPTES is a stable tetramer. Taken together, the present work indicates that BPTES is a unique and potent inhibitor of rat KGA and elucidates a novel mechanism of inactivation.

Intrinsic disorder and autonomous domain function in the multifunctional nuclear protein, MeCP2.

To probe the tertiary structure and domain organization of native methyl CpG-binding protein 2 (MeCP2), the recombinant human e2 isoform was purified to homogeneity and characterized by analytical ultracentrifugation, CD, and protease digestion. The location of intrinsic disorder in the MeCP2 sequence was predicted using the FoldIndex algorithm. MeCP2 was found to be monomeric in low and high salt and over a nearly 1000-fold concentration range. CD indicated that the MeCP2 monomer was nearly 60% unstructured under conditions where it could preferentially recognize CpG dinucleotides and condense chromatin. Protease digestion experiments demonstrate that MeCP2 is composed of at least six structurally distinct domains, two of which correspond to the well characterized methyl DNA binding domain and transcriptional repression domain. These domains collectively are organized into a tertiary structure with coil-like hydrodynamic properties, reflecting the extensive disorder in the MeCP2 sequence. When expressed as individual fragments, the methyl DNA binding domain and transcriptional repression domain both could function as nonspecific DNA binding domains. The unusual structural features of MeCP2 provide a basis for understanding MeCP2 multifunctionality in vitro and in vivo. These studies also establish an experimental paradigm for characterizing the tertiary structures of other highly disordered proteins.

Nucleosome recognition by the Piccolo NuA4 histone acetyltransferase complex.

The mechanisms by which multisubunit histone acetyltransferase (HAT) complexes recognize and perform efficient acetylation on nucleosome substrates are largely unknown. Here, we use a variety of biochemical approaches and compare histone-based substrates of increasing complexity to determine the critical components of nucleosome recognition by the MOZ, Ybf2/Sas3, Sas2, Tip60 family HAT complex, Piccolo NuA4 (picNuA4). We find the histone tails to be dispensable for binding to both nucleosomes and free histones and that the H2A, H3, and H2B tails do not influence the ability of picNuA4 to tetra-acetylate the H4 tail within the nucleosome. Most notably, we discovered that the histone-fold domain (HFD) regions of histones, particularly residues 21-52 of H4, are critical for tight binding and efficient tail acetylation. Presented evidence suggests that picNuA4 recognizes the open surface of the nucleosome on which the HFD of H4 is located. This binding mechanism serves to direct substrate access to the tails of H4 and H2A and allows the enzyme to be "tethered", thereby increasing the effective concentration of the histone tail and permitting successive cycles of H4 tail acetylation.

Domain organization and quaternary structure of the Saccharomyces cerevisiae silent information regulator 3 protein, Sir3p.

The silent information regulator protein 3 (Sir3p) functions in the initiation, propagation, and maintenance of transcriptionally silenced chromatin in Saccharomyces cerevisiae. To better understand the physicochemical basis for its effects on chromatin architecture, recombinant full-length S. cerevisiae Sir3p has been purified to near homogeneity on the large-scale and characterized by circular dichroism, limited protease digestion, and analytical ultracentrifugation. Results indicate that the Sir3p monomer has a unique tripartite domain organization, including a nearly 300-amino-acid residue stretch of intrinsically disordered residues that lies internal to its structured N- and C-terminal regions. Sir3p self-associates extensively in moderate salt and at micromolar protein concentrations producing a broad range of oligomers that sediment from 8 to in excess of 85 S. These results provide new insight into Sir3p domain organization and quaternary structure and support a nucleosome bridging model for Sir3p-dependent regulation of chromatin architecture.

Chromatin architectural proteins.

The accessibility of eukaryotic DNA is dependent upon the hierarchical level of chromatin organization. These include (1) intra-nucleosome interactions, (2) inter-nucleosome interactions and (3) the influence of non-histone chromatin architectural proteins. There appears to be interplay between all these levels, in that one level can override another or that two or more can act in concert. In the first level, the stability of the nucleosome itself is dependent on the number and type of contacts between the core histones and the surrounding DNA, as well as protein-protein interactions within the core histone octamer. Core histone variants, post-translational modifications of the histones, and linker histones binding to the DNA all influence the organization and stability of the nucleosome. When nucleosomes are placed end-to-end in linear chromatin arrays, the second level of organization is revealed. The amino terminal tails of the histone proteins make contacts with adjacent and distant nucleosomes, both within the fiber and between different fibers. The third level of organization is imposed upon these 'intrinsic' constraints, and is due to the influence of chromatin binding proteins that alter the architecture of the underlying fiber. These chromatin architectural proteins can, in some cases, bypass intrinsic constraints and impart their own topological affects, resulting in truly unique, supra-molecular assemblages that undoubtedly influence the accessibility of the underlying DNA. In this review we will provide a brief summary of what has been learned about the intrinsic dynamics of chromatin fibers, and survey the biology and architectural affects of the handful of chromatin architectural proteins that have been identified and characterized. These proteins are likely only a small subset of the architectural proteins encoded within the eukaryotic genome. We hope that an increased understanding and appreciation of the contribution of these proteins to genome accessibility will hasten the identification and characterization of more of these important regulatory factors.

Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists nucleosome sliding.

Eukaryotic chromatin is highly dynamic and turns over rapidly even in the absence of DNA replication. Here we show that the acidic histone chaperone nucleosome assembly protein 1 (NAP-1) from yeast reversibly removes and replaces histone protein dimer H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange. Transient removal of H2A-H2B dimers facilitates nucleosome sliding along the DNA to a thermodynamically favorable position. Histone exchange as well as nucleosome sliding is independent of ATP and relies on the presence of the C-terminal acidic domain of yeast NAP-1, even though this region is not required for histone binding and chromatin assembly. Our results suggest a novel role for NAP-1 (and perhaps other acidic histone chaperones) in mediating chromatin fluidity by incorporating histone variants and assisting nucleosome sliding. NAP-1 may function either untargeted (if acting alone) or may be targeted to specific regions within the genome through interactions with additional factors.

Self-association of the yeast nucleosome assembly protein 1.

The self-association properties of the yeast nucleosome assembly protein 1 (yNAP1) have been investigated using biochemical and biophysical methods. Protein cross-linking and calibrated gel filtration chromatography of yNAP1 indicate the protein exists as a complex mixture of species at physiologic ionic strength (75-150 mM). Sedimentation velocity reveals a distribution of species of 4.5-12 Svedbergs (S) over a 50-fold range of concentrations. The solution-state complexity is reduced at higher ionic strength, allowing for examination of the fundamental oligomer. Sedimentation equilibrium of a homogeneous 4.5 S population at 500 mM sodium chloride reveals these species to be yNAP1 dimers. These dimers self-associate to form higher order oligomers at more moderate ionic strength. Titration of guanidine hydrochloride converts the higher order oligomers to the homogeneous 4.5 S dimer and then converts the 4.5 S dimers to 2.5 S monomers. Circular dichroism shows that guanidine-mediated dissociation of higher order oligomers into yNAP1 dimers is accompanied by only slight changes in secondary structure. Dissociation of the dimer requires a nearly complete denaturation event.

KIX-mediated assembly of the CBP-CREB-HTLV-1 tax coactivator-activator complex.

The HTLV-1 transcriptional activator Tax is required for viral replication and pathogenesis. In concert with human CREB, Tax recruits the human transcriptional coactivator and histone acetyltransferase p300/CBP to the HTLV-1 promoter. Here we investigate the structural features of the interaction between Tax and the KIX domain of p300/CBP. Circular dichroism spectroscopy, nuclear magnetic resonance chemical shift perturbation mapping, and sedimentation equilibrium analysis show that KIX binds a Tax subdomain corresponding to residues 59-98 of Tax (called Tax(59-98)). Circular dichroism spectroscopy suggests that Tax(59-98) is intrinsically disordered (natively unfolded) in isolation and adopts an ordered conformation upon binding KIX. The interaction is disrupted by a single amino acid variation of Tax(59-98) in which leucine 68 is substituted with proline. Chemical shift perturbation mapping reveals that the Tax-binding surface of KIX is distinct from that utilized by CREB, and corresponds to the site of KIX that interacts with the human transcription factors c-Jun and mixed lineage leukemia protein (MLL). Sedimentation equilibrium analysis shows that Tax and the phosphorylated KID domain of CREB can simultaneously bind KIX to form a ternary 1:1:1 complex. The results provide a molecular description of the concerted recruitment of p300/CBP via the KIX domain by Tax and phosphorylated CREB during Tax-mediated gene expression.

Preferential binding of the histone (H3-H4)2 tetramer by NAP1 is mediated by the amino-terminal histone tails.

The yeast nucleosome assembly protein 1 (yNAP1) participates in many diverse activities, such as the assembly of newly synthesized DNA into chromatin and the rearrangement of nucleosomes during transcriptional activation. yNAP1 does not require ATP hydrolysis to perform these functions and is a valuable tool for in vitro chromatin assembly. Using recombinant histone complexes, we show that yNAP1 has a preference for binding the (H3-H4)2 tetramer over the (H2A-H2B) dimer. We find that the loss of the histone tails abrogates this preference for H3 and H4, and we demonstrate a direct interaction between yNAP1 and the amino-terminal tails of H3 and H4. yNAP1 binds to one histone fold domain, thus specifying the stoichiometry of the complexes formed with the histone dimer and tetramer. Finally, we provide evidence that the acidic carboxyl-terminal region of yNAP1, although dispensable for nucleosome assembly in vitro, contributes to binding via structure-independent electrostatic interactions. Our results are consistent with recent mechanistic investigations of NAP1 and expand our understanding of the histone chaperone family of assembly factors.

p53 Transcriptional activity is mediated through the SRC1-interacting domain of CBP/p300.

The tumor suppressor p53 recruits the cellular coactivator CBP/p300 to mediate the transcriptional activation of target genes. In this study, we identify a novel p53-interacting region in CBP/p300, which we call CR2, located near the carboxyl terminus. The 95-amino acid CR2 region (amino acids 2055--2150) is located adjacent to the C/H3 domain and corresponds precisely with the minimal steroid receptor coactivator 1 (SRC1)-interacting domain of CBP (also called IBiD). We show that the region of p53 that participates in the CR2 interaction resides within the first 107 amino acids of the protein. p53 binds strongly to the CR2 domain of both CBP and the highly homologous coactivator p300. Importantly, an in-frame deletion of CR2 within the full-length p300 protein strongly compromises p300-mediated p53 transcriptional activation from a chromatin template in vitro. The identification of the p53-interacting CR2 domain in CBP/p300 prompted us to ask if the human T-cell leukemia virus (HTLV-I) Tax protein, which also interacts with CR2, competes with p53 for binding to this domain. We show that p53 and Tax exhibit mutually exclusive binding to the CR2 region, possibly contributing to the previously reported Tax repression of p53 function. Together, these studies identify and molecularly characterize a new p53 binding site on CBP/p300 that participates in coactivator-mediated p53 transcription function. The identity of the p53.CR2 interaction indicates that at least three distinct sites on CBP/p300 may participate in mediating p53 transactivation.