DNA-protein interactions in nucleosomes and in chromatin. Structural studies of chromatin stabilized by ultraviolet-light induced crosslinking.

Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.     

My favourite molecule: Polyamines,chromatin structure and transcription

Nucleosomes are the basic elements of chromatin structure. Polyamines, such as spermine and spermidine, are small ubiquitous molecules absolutely required for cell growth. Photoaffinity polyamines bind to specific locations in nucleosomes and can change the helical twist of DNA in nucleosomes. Acetylation of polyamines reduces their affinity for DNA and nucleosomes, thus the helical twist of DNA in nucleosomes could be regulated by cells through acetylation. I suggest that histone and polyamine acetylation act synergistically to modulate chromatin structure. On naked DNA, the photoaffinity spermine bound preferentially to a specific ‘TATA’ sequence element, suggesting that polyamines may be involved in the unusual chromatin structure in this region. Further work is needed to test whether the specificities shown by photoaffinity polyamines are also shown by cellular polyamines; such experiments are now feasible.     

Transgenic mouse models for studies of the role of polyamines in normal,hypertrophic and neoplastic growth

Transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner have considerable promise for evaluation of the roles of polyamines in normal, hypertrophic and neoplastic growth. This short review summarizes the available transgenic models. Mice with large increases in ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase or antizyme, a protein regulating polyamine synthesis by reducing polyamine transport and ODC in the heart, have been produced using constructs in which the protein is expressed from the alpha -myosin heavy-chain promoter. These mice are useful in studies of the role of polyamines in hypertrophic growth. Expression from keratin promoters has been used to target increased synthesis of ODC, spermidine/spermine-N(1)-acetyltransferase (SSAT) and antizyme in the skin. Such expression of ODC leads to an increased sensitivity to chemical and UV carcinogenesis. Expression of antizyme inhibits carcinogenesis in skin and forestomach. Expression of SSAT increases the incidence of skin papillomas and their progression to carcinomas in response to a two-stage carcinogenesis protocol. These results establish the importance of polyamines in carcinogenesis and neoplastic growth and these transgenic mice will be valuable experimental tools to evaluate the importance of polyamines in mediating responses to oncogenes and studies of cancer chemoprevention.     

Analysis of epigenetic modifications of chromatin at specific gene loci by native chromatin immunoprecipitation of nucleosomes isolated using hydroxyapatite chromatography

Chromatin immunoprecipitation (ChIP) is routinely used to examine epigenetic modification of histones at specific genomic locations. However, covalent modifications of histone tails can serve as docking sites for chromatin regulatory factors. As such, association of these regulatory factors with chromatin could cause steric hindrance for antibody recognition, resulting in an underestimation of the relative enrichment of a given histone modification at specific loci. To overcome this problem, we have developed a native ChIP protocol to study covalent modification of histones that takes advantage of hydroxyapatite (HAP) chromatography to wash away chromatin-associated proteins before the immunoprecipitation of nucleosomes. This fast and simple procedure consists of five steps: nuclei isolation from cultured cells; fragmentation of chromatin using MNase; purification of nucleosomes using HAP; immunoprecipitation of modified nucleosomes; and qPCR analysis of DNA associated with modified histones. Nucleosomes prepared in this manner are free of contaminating proteins and permit an accurate evaluation of relative abundance of different covalent histone modifications at specific genomic loci. Completion of this protocol requires approximately 1.5 d.     

Comparative study of nucleosome particles in chromatin from normal and tumor cells. II. Reconstitution, compaction and association induced by ionic strength of a solution

The method of circular dichroism (CD) has been used to investigate the reconstitution of mononucleosomes from C3HA mice liver and ascitic hepatoma 22A cells chromatin. It has been revealed that the more unfolding state of DNA in ascitic nucleosomes (discovered earlier) is determined by the peculiarities of the interactions between DNA and the dimers H2A-H2B, as well as by the linker histones of the H1 group. The investigation of the DNA folding in the oligonucleosome chains with increasing ionic strength has shown complete invariability of the DNA compactness in the ascitic chromatin up to 100 mM NaCl, while in liver nucleosomes an additional folding of the linker portion of the DNA was observed within the range of 20-40 mM NaCl. Oligonucleosomes from ascitic chromatin are less inclined to association upon increasing ionic strength, as compared with those from liver chromatin.     

DNA sequence plays a major role in determining nucleosome positions in yeast CUP1 chromatin.

The role of DNA sequence in determining nucleosome positions in vivo was investigated by comparing the positions adopted by nucleosomes reconstituted on a yeast plasmid in vitro using purified core histones with those in native chromatin containing the same DNA, described previously. Nucleosomes were reconstituted on a 2.5 kilobase pair DNA sequence containing the yeast TRP1ARS1 plasmid with CUP1 as an insert (TAC-DNA). Multiple, alternative, overlapping nucleosome positions were mapped on TAC-DNA. For the 58 positioned nucleosomes identified, the relative positioning strengths and the stabilities to salt and temperature were determined. These positions were, with a few exceptions, identical to those observed in native, remodeled TAC chromatin containing an activated CUP1 gene. Only some of these positions are utilized in native, unremodeled chromatin. These observations suggest that DNA sequence is likely to play a very important role in positioning nucleosomes in vivo. We suggest that events occurring in yeast CUP1 chromatin determine which positions are occupied in vivo and when they are occupied.     

Remodeling of sperm chromatin after fertilization involves nucleosomes formed by sperm histones H2A and H2B and two CS histone variants

The composition of nucleosomes at an intermediate stage of male pronucleus formation was determined in sea urchins. Nucleosomes were isolated from zygotes harvested 10 min post-insemination, whole nucleoprotein particles were obtained from nucleus by nuclease digestion, and nucleosomes were subsequently purified by a sucrose gradient fractionation. The nucleosomes derived from male pronucleus were separated from those derived from female pronucleus by immunoadsorption to antibodies against sperm specific histones (anti-SpH) covalently bound to Sepharose 4B (anti-SpH-Sepharose). The immunoadsorbed nucleosomes were eluted, and the histones were analyzed by Western blots. Sperm histones (SpH) or alternatively, the histones from unfertilized eggs (CS histone variants), were identified with antibodies directed against each set of histones. It was found that these nucleosomes are organized by a core formed by sperm histones H2A and H2B combined with two major CS histone variants. Such a hybrid histone core interacts with DNA fragments of approximately 100 bp. It was also found that these atypical nucleosome cores are subsequently organized in a chromatin fiber that exhibits periodic nuclease hypersensitive sites determined by DNA fragments of 500 bp of DNA. It was found that these nucleoprotein particles were organized primarily by the hybrid nucleosomes described above. We postulate that this unique chromatin organization defines an intermediate stage of male chromatin remodeling after fertilization.     

Electron microscopic and biochemical evidence that chromatin structure is a repeating unit

Electron microscopic and biochemical studies demonstrate that the fundamental structure of chromatin depleted of lysine-rich histones is composed of a flexible chain of spherical particles (nucleosomes), about 125 Å in diameter, connected by DNA filaments. Such a chromatin preparation can be separated by centrifugation into two fractions which differ in the spacing of the nucleosomes. In one fraction almost all of the DNA is condensed in nucleosomes, while the other fraction contains long stretches of free DNA connecting regions where the nucleosomes are closely packed. The isolated nucleosomes contain about 200 base pairs of DNA and the four histones F2a₁, F2a₂, and F2b, and F3 in an overall histone/DNA ratio of 0.97. In such a structure the DNA is compacted slightly more than five times from its extended length. The same basic structure can be visualized in chromatin spilling out of lysed nuclei. However, in this latter case the nucleosomes are very closely packed, suggesting that histone F1 is involved in the superpacking of DNA in chromosomes and nuclei. The chromatin fiber appears to be a self-assembling structure, since the nucleosomal arrangement can be reconstituted in vitro from DNA and the four histones F2a₁, F2a₂, F2b and F3 only, irrespective of their cellular origin.     

Histones of terminally differentiated cells undergo continuous turnover

In contrast to the widely accepted idea of the nearly absolute metabolic stability of histones, our experiments support the view that the histones of nonproliferating, terminally differentiated cells undergo continuous replacement. This conclusion is based on the incorporation of labeled amino acids into the histones of mouse kidney and liver cells after their intraperitoneal introduction. We have found that the intranuclear uptake of the histones made in the absence of replicative synthesis and their integration into chromatin proceed with striking delay. The metabolic rates of individual histones measured by calculating their half-lives suggest that each histone turns over at a specific rate. With regard to the basic chromatin structure, the nucleosome, such unequal turnover should mean that the histone core does not participate in this process as a single unit but rather as a protein mosaic in which each partner follows its own rate of removal. Additional experiments suggested that intact nucleosomes take part in the replacement, but the relative proportion of the nucleosomes involved should be limited. The nonnucleosomal H1A and H1 degree histones have been found to undergo faster replacement than the core histones. Moreover, in comparison to each other, these two histone subfractions are also replaced at a different rate. The results of autoradiography of isolated kidney and liver nuclei after continuous labeling with [3H]-thymidine suggest that the histone replacement is not associated with the repair of DNA.     

Differential association of linker histones H1 and H5 with telomeric nucleosomes in chicken erythrocytes.

Rat liver telomeric DNA is organised into nucleosomes characterised by a shorter and more homogeneous average nucleosomal repeat than bulk chromatin as shown by Makarov et al. (1). The latter authors were unable to detect the association of any linker histone with the telomeric DNA. We have confirmed these observations but show that in sharp contrast chicken erythrocyte telomeric DNA is organised into nucleosomes whose spacing length and heterogeneity are indistinguishable from those of bulk chromatin. We further show that chicken erythrocyte telomeric chromatin contains chromatosomes which are preferentially associated with histone H1 relative to histone H5. This contrasts with bulk chromatin where histone H5 is the more abundant species. This observation strongly suggests that telomeric DNA condensed into nucleosome core particles has a higher affinity for H1 than H5. We discuss the origin of the discrimination of the lysine rich histones in terms of DNA sequence preferences, telomere nucleosome preferences and particular constraints of the higher order chromatin structure of telomeres.     

Interactions between core histones and chromatin at physiological ionic strength

Addition of core histones to chromatin or chromatin core particles at physiological ionic strength results in soluble nucleohistone complexes when polyglutamic acid is included in the sample. The interaction between nucleosomes and added core histones is strong enough to inhibit nucleosome formation on a closed circular DNA in the same solution. Complexes consisting of core particles and core histones run as discrete nucleoprotein particles on polyacrylamide gels. Consistent with the electrophoretic properties of these particles, protein cross-linking with dimethyl suberimidate indicates that added core histones are bound as excess octamers. Histones in the excess octamers do not exchange with nucleosomal core histones at an ionic strength of 0.1 M and can be selectively removed from core particles by incubating the complexes in a solution containing sufficient DNA. Under conditions where added histones are confined to the surface of chromatin, the excess histones are mobile and can migrate onto a contiguous extension of naked DNA and form nucleosomes.     

Chromatin assembly during DNA replication in somatic cells.

Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.     

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites

Elevated expression of ornithine decarboxylase (ODC), the regulatory enzyme in polyamine biosynthesis, targeted to the epidermis is sufficient to promote skin tumor development following a single subthreshold dose of dimethylbenz(a)anthracene (DMBA). Since skin tumor promotion involves recruitment of hair follicle bulge stem cells harboring genetic lesions, we assessed the effect of increased epidermal ODC on recruitment of bulge stem cells in ODC-ER transgenic mice in which ODC activity is induced de novo in adult skin with 4-hydroxytamoxifen (4OHT). Bromodeoxyuridine-pulse labeling and use of K15.CrePR1;R26R;ODC-ER triple transgenic mice demonstrated that induction of ODC activity is sufficient to recruit bulge stem cells in quiescent skin. Because increased ODC activity not only stimulates proliferation but also increases reactive oxygen species (ROS) generation via subsequent induction of polyamine catabolic oxidases, we used an inhibitor of polyamine catabolic oxidase activity, MDL72527, to investigate whether ROS generation by polyamine catabolic oxidases contributes to skin tumorigenesis in DMBA-initiated ODC-ER transgenic skin. Newborn ODC-ER transgenic mice and their normal littermates were initiated with a single topical dose of DMBA. To assess tumor development originating from dormant bulge stem cells that possess DMBA-initiated mutations, epidermal ODC activity was induced in ODC-ER mice with 4OHT 5 weeks after DMBA initiation followed by MDL72527 treatment. MDL72527 treatment resulted in a shorter tumor latency time, increased tumor burden, increased conversion to carcinomas, and lower tumor levels of p53. Thus, elevated epidermal ODC activity promotes tumorigenesis by stimulating the recruitment of bulge stem cells but not via ROS generation by polyamine catabolic oxidases.     

Laser Raman spectra of calf thymus chromatin and its constituents.

Extensive Raman measurements have been made on calf thymus chromatin, core chromatin, the (H3,H4)/DNA complex, and isolated DNA. The results indicate that the alpha-helical content of the nucleosomal histones gradually increases as they form the heterocomplexes that lead to the formation of the octameric nucleosome core. The secondary structure of the latter is not modified as it binds to DNA. The spectra indicate that the DNA essentially retains its B conformation in nucleosomes, although slight changes probably occur in the ribose-phosphate backbone. No specific interactions between the nucleosomal histones and DNA can be established from the spectra, but histone H1 possibly interacts selectively with the thymine bases.     

Nucleosome-interacting proteins regulated by DNA and histone methylation

Modifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify "crosstalk" between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification binding "profile" for proteins regulated by DNA and histone methylation.