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In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.

In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequential ordered binding of the core and linker histone proteins. The acidic proteins termed histone chaperones are known to bind to core histones to neutralize their positive charges, thereby facilitating their proper deposition onto DNA to assemble the core of nucleosomes. For linker histones, however, little has been known about the regulatory mechanism for deposition of linker histones onto the linker DNA. Here we report that, in Xenopus eggs, the linker histone is associated with the Xenopus homologue of nucleosome assembly protein-1 (NAP-1), which is known to be a chaperone for the core histones H2A and H2B in Drosophila and mammalian cells [Ito, T., Bulger, M., Kobayashi, R. & Kadonaga, J. T. (1996) Mol. Cell Biol. 16, 3112-3124; Chang, L., Loranger, S. S., Mizzen, C., Ernst, S. G., Allis, C. D. & Annunziato, A. T. (1997) Biochemistry 36, 469-480]. We show that NAP-1 acts as the chaperone for the linker histone in both sperm chromatin remodeling into nucleosomes and linker histone binding to nucleosome core dimers. In the presence of NAP-1, the linker histone is properly deposited onto linker DNA at physiological ionic strength, without formation of nonspecific aggregates. These results strongly suggest that NAP-1 functions as a chaperone for the linker histone in Xenopus eggs.

A DNA fragment of 163 bp containing 11 GGA repeats formed two-end positioned mononucleosomes as efficiently as that of CTG repeats. However, the rotational positioning of the GGA fragment was weak because clear DNase I cleavage patterns with 10-base periodicity were not seen near the center of the GGA fragment but were detected in the entire region of the CTG fragment. Incubation of the GGA mononucleosomes with the same fragment provided the DNA-DNA complex, which had been shown by using naked DNA fragments. DNase I digestion of the complex exhibited protection in the GGA repeats and in flanking sequences of about 30 bp at both sides, suggesting that both the repeat and flanking regions were involved in the association. Interestingly, histone H1, which enhanced DNA-DNA association on naked DNA, did not affect the complex formation on mononucleosomes. These results imply that GGA microsatellites in genomes could associate with one another at multiple sites and that the association may play a role in functional organization of higher order chromatin architecture., ((c)2002 Elsevier Science.)

To investigate the relationship between chromatin dynamics and nucleotide excision repair (NER), we have examined the effect of chromatin structure on the formation of two major classes of UV-induced DNA lesions in reconstituted dinucleosomes. Furthermore, we have developed a model chromatin-NER system consisting of purified human NER factors and dinucleosome substrates that contain pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) either at the center of the nucleosome or in the linker DNA. We have found that the two classes of UV-induced DNA lesions are formed efficiently at every location on dinucleosomes in a manner similar to that of naked DNA, even in the presence of histone H1. On the other hand, excision of 6-4PPs is strongly inhibited by dinucleosome assembly, even within the linker DNA region. These results provide direct evidence that the human NER machinery requires a space greater than the size of the linker DNA to excise UV lesions efficiently. Interestingly, NER dual incision in dinucleosomes is facilitated by recombinant ACF, an ATP-dependent chromatin remodeling factor. Our results indicate that there is a functional connection between chromatin remodeling and the initiation step of NER.

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a 'beads on a string' structure with each nucleosome trapping 158 +/- 27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.

Nucleosomes and the chromatin structures they assemble provide the architectural foundation for the transcription process. We describe the use of reconstituted chromatin templates assembled using purified histones and HMGs to investigate the significance of chromatin structure for transcription. Our structural assays include sucrose gradient fractionation, nucleoprotein gel analysis, micrococcal nuclease digestion, DNase I and hydroxyl radical cleavage mapping of nucleosome position, and determination of nucleosome mobility. We determine the consequences of incorporation of linker histones or HMG1 on the properties of the nucleosome and the transcription process. Our results indicate that linker histones and HMG1 can direct the positioning of core histone-DNA interactions, restrict nucleosome mobility, and repress transcription.

We demonstrate using a dinucleosome template that acetylation of the core histones enhances transcription by RNA polymerase III. This effect is not dependent on an increased mobility of the core histone octamer with respect to DNA sequence. When linker histone is subsequently bound, we find both a reduction in nucleosome mobility and a repression of transcription. These effects of linker histone binding are independent of core histone acetylation, indicating that core histone acetylation does not prevent linker histone binding and the concomitant transcriptional repression. These studies are complemented by the use of a Xenopus egg extract competent both for chromatin assembly on replicating DNA and for RNA polymerase III transcription. Incorporation of acetylated histones and lack of linker histones together facilitate transcription by >10-fold in this system; however, they have little independent effect on transcription. Thus core histone acetylation significantly facilitates transcription, but this effect is inhibited by the assembly of linker histones into chromatin.

A fundamental step in the assembly of native chromatin is the specific recognition and binding of linker histones to the nucleoprotein subunit known as the nucleosome. A first step in defining this important interaction is the determination of residues within linker histones that are important for the structure-specific recognition of the nucleosome core. By combining in vitro assays for the native binding activity of linker histones and site-directed mutagenesis, we have examined a cluster of basic residues within the globular domain of H1(0), a somatic linker histone variant from Xenopus laevis. We show that these residues, which comprise a putative DNA binding surface within the globular domain, do not play an essential role in the structure-specific binding of a linker histone to the nucleosome.

We examined the structural and functional consequences of incorporating either histone H1, histone B4 or HMG1 into a synthetic dinucleosome containing two 5S rRNA genes. We found that all three proteins bind to linker DNA, stabilizing an additional 20 bp from micrococcal nuclease digestion and restrict nucleosome mobility. Histone H1 has the highest-affinity interaction with the dinucleosome; histone B4 and HMG1 associate with significantly reduced affinities. We found that histone H1 binds to the dinucleosome template with a dissociation constant (KD) of 7.4 nM, whereas the KD is 45 nM for histone B4 and 300 nM for HMG1. The KDs for the interaction of these proteins with naked DNA are 18 nM for H1, 80 nM for B4 and 300 nM for HMG1. The differences in association of these proteins with the dinucleosome are reflected in the efficiency with which the different proteins repress transcription from the 5S rRNA genes. Thus, although all three proteins can contribute to the organization of chromatin, the stability of the structures they assemble will vary. Our results provide a molecular explanation for the transcriptional promiscuity of Xenopus early embryonic chromatin, which is enriched in HMG1 and linker histone B4, but deficient in histone H1.

Nucleosome mobility facilitates the transcription of chromatin templates containing only histone octamers. Inclusion of linker histones in chromatin inhibits nucleosome mobility, directs nucleosome positioning and represses transcription. Transcriptional repression by linker histone occurs preferentially on templates associated with histone octamers relative to naked DNA. Mobile nucleosomes and the restriction of mobility by linker histones might be expected to exert a major influence on the accessibility of chromatin to regulatory molecules.

We demonstrate that acetylation of core histone tails does not significantly impair the ability of linker histones to bind preferentially and asymmetrically to a defined sequence mononucleosome core reconstituted in vitro. Thus a simple inhibition mechanism cannot explain models whereby acetylation is causal for the observed reduction in the linker histone content in chromatin. Other models to explain this correlation are discussed.