A Distinct Switch in Interactions of the Histone H4 Tail Domain upon Salt-dependent Folding of Nucleosome Arrays

The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes (1, 2). Likewise, H4 tail-H2A contacts stabilize array folding (3). However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.     

Epigenetic regulation of transcription by RNA polymerase III

Chromatin participates actively in all DNA transactions and all phenomena directly under the influence of chromatin are explained by epigenetic mechanisms. The genes transcribed by RNA polymerase (pol) III are generally found in regions free of nucleosomes, the structural units of chromatin. Yet, histone modifications and positions of nucleosomes in the gene flanking regions have been reported to show direct correlation with activity status of these genes. Gene-specific as well as genome-wide studies have also revealed association of several epigenetic components with pol III-transcribed genes. This review presents a summary of the research in past many years, which have gathered enough evidence to conclude that pol III-transcribed genes are important components of an epigenome.     

Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms

Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.     

The JmjN Domain of Jhd2 Is Important for Its Protein Stability,and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.     

Elevated levels of polyamines alter chromatin in murine skin and tumors without global changes in nucleosome acetylation

Polyamines affect nucleosome oligomerization and DNA conformation in vitro, yet little information exists regarding the influence of naturally synthesized polyamines on mammalian chromatin. Capitalizing on the relative inefficiency of a moderate ionic strength extraction buffer to dissociate histones, we obtained evidence of altered chromatin in transgenic mice that overexpress ornithine decarboxylase (ODC), which catalyzes polyamine synthesis. Dissociation of histones from chromatin in ODC transgenic mouse skin, as well as in tumors that develop spontaneously in ODC/Ras bigenic mice, is dramatically reduced relative to normal littermate skin. This could reflect tighter tethering of nucleosomes to DNA or a more compacted chromatin structure due to elevated intracellular concentrations of polyamines since this effect is reversible upon treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC enzymatic activity. Impeded release of nonhistone chromatin proteins HP-1beta and nucleophosmin, but not Lamin B, HDAC-1, HMGB, HMGN2, or HMGA1, suggests that polyamines exert selective effects on specific chromatin protein complexes. Moreover, overall acetylation, as well as specific methylation, of nucleosomes in ODC mice is unaffected, implying that access by histone modifying enzymes is not generally restricted. The abnormal chromatin environment fostered by elevated levels of polyamines may be a necessary prerequisite for epithelial tumor growth and maintenance.     

Temperature and osmotic stress dependence of the thermodynamics for binding linker histone H10, Its carboxyl domain (H10-C) or globular domain (H10-G) to B-DNA

Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. In contrast to the nucleosome core histone proteins, the role of H1 in compacting DNA is not clearly understood. In this study ITC was used to measure the binding constant, enthalpy change, and binding site size for the interactions of H1⁰, or its C-terminal (H1⁰-C) and globular (H1⁰-G) domains to highly polymerized calf-thymus DNA at temperatures from 288 K to 308 K. Heat capacity changes, ΔC_p, for these same H1⁰ binding interactions were estimated from the temperature dependence of the enthalpy changes. The enthalpy changes for binding H1⁰, H1⁰-C, or H1⁰-G to CT-DNA are all endothermic at 298 K, becoming more exothermic as the temperature is increased. The ΔH for binding H1⁰-G to CT-DNA is exothermic at temperatures above approximately 300 K. Osmotic stress experiments indicate that the binding of H1⁰ is accompanied by the release of approximately 35 water molecules.We estimate from our naked DNA titration results that the binding of the H1⁰ to the nucleosome places the H1⁰ protein in close contact with approximately 41 DNA bp. The breakdown is that the H1⁰ carboxyl terminus interacts with 28 bp of linker DNA on one side of the nucleosome, the H1⁰ globular domain binds directly to 7 bp of core DNA, and shields another 6 linker DNA bases, 3 bp on either side of the nucleosome where the linker DNA exits the nucleosome core.     

Identification of residues in chromodomain helicase DNA-binding protein 1 (Chd1) required for coupling ATP hydrolysis to nucleosome sliding

Chromatin remodelers are ATP-dependent machines responsible for directionally shifting nucleosomes along DNA. We are interested in defining which elements of the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler are necessary and sufficient for sliding nucleosomes. This work focuses on the polypeptide segment that joins the ATPase motor to the C-terminal DNA-binding domain. We identify amino acid positions outside the ATPase motor that, when altered, dramatically reduce nucleosome sliding ability and yet have only ～3-fold reduction in ATPase stimulation by nucleosomes. These residues therefore appear to play a role in functionally coupling ATP hydrolysis to nucleosome sliding, and suggest that the ATPase motor requires cooperation with external elements to slide DNA past the histone core.     

Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120 , a cold-specific gene in wheat

The Wcs120 gene encodes a highly abundant protein which appears to play an important role during cold acclimation of wheat. To understand the regulatory mechanism controlling its expression at low temperature, the promoter region has been characterized. Electrophoretic mobility shift assays using short promoter fragments revealed the presence in nuclear extracts from non-acclimated (NA) plants of multiple DNA-binding proteins which interact with several elements. In contrast, no DNA-binding activity was observed in the nuclear extracts from cold-acclimated (CA) plants. In vitro dephosphorylation of these CA nuclear extracts with alkaline phosphatase restored the binding activity. Moreover, okadaic acid (a potent phosphatase inhibitor) markedly stimulated the in vivo accumulation of the WCS120 family of proteins. This suggests that protein phosphatases PP1 and/or PP2A negatively regulate the expression of the Wcs120 gene. In addition, both Ca²⁺-dependent and Ca²⁺-independent kinase activities were found to be significantly higher in the CA nuclear extracts. Western analysis using antibodies directed against protein kinase C (PKC) isoforms showed that a PKCγ homolog (84 kDa) is selectively translocated into the nucleus in response to low temperature. Taken together, our results suggest that, in vivo, the expression of the Wcs120 gene may be regulated by nuclear factors whose binding activity is modulated by a phosphorylation/dephosphorylation mechanism. Received: 9 June 1997 / Accepted: 18 August 1997     

Development of a fluorescent probe for the study of nucleosome assembly and dynamics

To develop a probe for use in real-time dynamic studies of nucleosomes, core histones (from Drosophila) were conjugated to a DNA-intercalating dye, thiazole orange, by a reaction targeting Cys 110 of histone H3. In the absence of DNA, the conjugated histones are only very weakly fluorescent. However, upon reconstitution into nucleosomes by standard salt dialysis procedures, the probe fluoresces strongly, reflecting its ability to intercalate into the nucleosomal DNA. The probe is also sensitive to the nature of the DNA-histone interaction. Nucleosomes reconstituted by stepwise salt dialysis give a fluorescence signal quite different from that of the species formed when DNA and histones are simply mixed in low salt. In addition, changing either the DNA length or the type of sequence (nucleosome positioning sequences versus random DNA of the same size) used in the reconstitution alters the resulting fluorescence yield. The results are all consistent with the conclusion that a more rigid, less flexible nucleosome structure results in less fluorescence than a looser structure, presumably due to structural constraints on dye intercalation. This probe should be well suited to analyzing nucleosome dynamics and to following factor-mediated assembly and remodeling of nucleosomes in real time, particularly at the single-molecule level.     

Strategies for crystallizing a chromatin protein in complex with the nucleosome core particle

The molecular details of how chromatin factors and enzymes interact with the nucleosome are critical to understanding fundamental genetic processes including cell division and gene regulation. A structural understanding of such processes has been hindered by the difficulty in producing diffraction-quality crystals of chromatin proteins in complex with the nucleosome. We describe here the steps used to grow crystals of the 300-kDa RCC1 chromatin factor/nucleosome core particle complex that diffract to 2.9-Å resolution. These steps include both pre- and postcrystallization strategies potentially useful to other complexes. We screened multiple variant RCC1/nucleosome core particle complexes assembled using different RCC1 homologs and deletion variants, and nucleosomes containing nucleosomal DNA with different sequences and lengths, as well as histone deletion variants. We found that using RCC1 from different species produced different crystal forms of the RCC1/nucleosome complex consistent with key crystal packing interactions mediated by RCC1. Optimization of postcrystallization soaks to dehydrate the crystals dramatically improved the diffraction quality of the RCC1/nucleosome crystal from 5.0- to 2.9-Å resolution.     

Effects of histone acetylation by Piccolo NuA4 on the structure of a nucleosome and the interactions between two nucleosomes

We characterized the effect of histone acetylation on the structure of a nucleosome and the interactions between two nucleosomes. In this study, nucleosomes reconstituted with the Selex "Widom 601" sequence were acetylated with the Piccolo NuA4 complex, which acetylates mainly H4 N-terminal tail lysine residues and some H2A/H3 N-terminal tail lysine residues. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies topology change of the DNA. Interactions between two nucleosomes in solution were also monitored to discover multiple transient dinucleosomal states that can be categorized to short-lived and long-lived (～1 s) states. The formation of dinucleosomes is strongly Mg(2+)-dependent, and unacetylated nucleosomes favor the formation of long-lived dinucleosomes 4-fold as much as the acetylated ones. These results suggest that the acetylation of histones by Piccolo NuA4 disturbs not only the structure of a nucleosome but also the interactions between two nucleosomes. Lastly, we suggest a structural model for a stable dinucleosomal state where the two nucleosomes are separated by ～2 nm face-to-face and rotated by 34° with respect to each other.     

Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.