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Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II- and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation ∼3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a 'reader' of higher-order chromatin structure leading to gene silencing through DNA methylation.

The process of development can be viewed as a series of linker histone replacements which take place throughout spermatogenesis and oogenesis, as well as following fertilization or somatic nuclear transfer (SNT). Although few of the histone H1 variants in question have been shown to be essential for viability, the timing of their appearance as well as the affinity with which they are able to bind to chromatin seem to be important factors in their developmental role. A looser binding of linker histones to chromatin seems to correlate with the meiotic phases of gametogenesis and the establishment of a totipotent, as well as the maintenance of a pluripotent, state in early embryos, while tighter binding of linker histones to chromatin appears to be associated with the mitotic phases, as well as the increased levels of condensation that are required for the packaging of DNA into sperm. This latter process also involves the binding of certain basic non-histone proteins to DNA. While all proteins involved in chromatin compaction during development are highly basic in nature, in general they can be seen to change from lysine-rich variants to arginine-rich ones, and back again. The fact that linker histone transitions are conserved across diverse metazoan species speaks of their importance in packaging DNA in a variety of ways during this crucial period.

Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency.

The organization of DNA within eukaryotic cell nuclei poses special problems and opportunities for the cell. For example, assembly of DNA into chromatin is thought to be a principle mechanism by which adventitious general transcription is repressed. However, access to genomic DNA for events such as DNA repair must be facilitated by energy-intensive processes that either directly alter chromatin structure or impart post-translational modifications, leading to increased DNA accessibility. The assembly of DNA into chromatin affects both the incidence of damage to DNA and repair of that damage. Correction of most damage to DNA caused by UV irradiation occurs via the nucleotide excision repair (NER) process. NER requires extensive involvement of large multiprotein complexes with relatively large stretches of DNA. Here, we review recent evidence suggesting that at least some steps of NER require ATP-dependent chromatin remodeling activities while perhaps others do not.

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.

It is now generally believed that DNA methylation is responsible for genomic imprinting in mammals (1). Recent experimental evidence has provided an elegant mechanism for repression of gene expression by DNA methylation. This evidence suggests that proteins that recognize specifically methylated CpGs may contribute to the formation of inactive chromatin (2-5). Nucleosomes are the basic unit of chromatin, consisting of a core of 146 bp of DNA wrapped around a histone octamer (two molecules of each of H2A, H2B, H3, and H4) and a stretch of linker DNA between adjacent nucleosomes. The binding of a fifth histone, known as a linker histone or H1, promotes the packaging of strings of nucleosomes into a 30 nm chromatin fiber (6). Packaging of DNA into nucleosomes and the chromatin fiber greatly restricts the availability of the DNA for nuclear processes such as transcription.

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.

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.

Introduction: Type I interferons (IFNs) activate the JAK/STAT pathway and stimulate transcription from many IFN stimulated genes (ISGs). This process likely involves destabilization and reorganization of chromatin. However, chromatin events associated with ISG induction is poorly understood. To gain insight into a link between transcription and chromatin regulation, we studied whether IFN stimulation causes exchange of histones relevant to epigenetic regulation. Methods: Because the histone H3.3 is implicated in transcription coupled chromatin change, we constructed NIH3T3 cells expressing GFP- H3.3 and examined H3.3 incorporation into ISGs by ChIP in parallel with ISG transcription and transcription factor recruitment. Results: IFN stimulation led to rapid recruitment of RNA polymerase II and BRD4, an acetyl-histone binding factor to the ISG. This was followed by recruitment of the elongation factor P-TEFb, the pausing complex NELF/DSIF and SPT6. Along with these events, IFN stimulation caused rapid H3.3 accumulation in the ISGs. H3.3 accumulation was greater in the coding region and the gene end than in the promoter region where virtually no H3.3 incorporation was detected. Analysis with a BRD4 specific inhibitorJQ1 showed that H3.3 incorporation depended on BRD4 recruitment and ISG elongation. However, H3.3 incorporation into ISGs continued past ISG elongation, leaving the H3.3 mark on ISGs for at least two cycles of cell division. The mutant GFP-H3.3K36R was not incorporated into ISGs, indicating that methylation of K36 is requreid for H3.3 incorporation. Finally, H3.3 incorporation was also observed in another activation model, indicating the generality of transcription-induced histone exchange. Conclusion: We show that IFN stimulation triggers rapid and extensive H3.3 incorporation into ISGs, which is presumably associated with expulsion of the preexisting H3 (H3.1/H3.2). This event required active ISG elongation, suggesting that passage of RNA polymerase II through the ISG gene body destabilizes the architecture of the RNA-DNA-nucleosome, necessitating reconstruction of nucleosomes composed of H3.3. Based on the remarkable persistence of the H3.3 mark left on the ISGs long after transcription, we suggest that transcription-induced H3.3 deposition represents an epigenetic mark linked to transcriptional memory. [Copyright &y& Elsevier]