HMG-D and histone H1 alter the local accessibility of nucleosomal DNA

There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.     

Characterization of the interaction between HMGB1 and H3—a possible means of positioning HMGB1 in chromatin

High mobility group protein B1 (HMGB1) binds to the internucleosomal linker DNA in chromatin and abuts the nucleosome. Bending and untwisting of the linker DNA results in transmission of strain to the nucleosome core, disrupting histone/DNA contacts. An interaction between H3 and HMGB1 has been reported. Here we confirm and characterize the interaction of HMGB1 with H3, which lies close to the DNA entry/exit points around the nucleosome dyad, and may be responsible for positioning of HMGB1 on the linker DNA. We show that the interaction is between the N-terminal unstructured tail of H3 and the C-terminal unstructured acidic tail of HMGB1, which are presumably displaced from DNA and the HMG boxes, respectively, in the HMGB1-nucleosome complex. We have characterized the interaction by nuclear magnetic resonance spectroscopy and show that it is extensive for both peptides, and appears not to result in the acquisition of significant secondary structure by either partner.     

The interaction of HMGB1 and linker histones occurs through their acidic and basic tails

H1 and HMGB1 bind to linker DNA in chromatin, in the vicinity of the nucleosome dyad. They appear to have opposing effects on the nucleosome, H1 stabilising it by "sealing" two turns of DNA around the octamer, and HMGB1 destabilising it, probably by bending the adjacent DNA. Their presence in chromatin might be mutually exclusive. Displacement/replacement of one by the other as a result of their highly dynamic binding in vivo might, in principle, involve interactions between them. Chemical cross-linking and gel-filtration show that a 1:1 linker histone/HMGB1 complex is formed, which persists at physiological ionic strength, and that complex formation requires the acidic tail of HMGB1. NMR spectroscopy shows that the linker histone binds, predominantly through its basic C-terminal domain, to the acidic tail of HMGB1, thereby disrupting the interaction of the tail with the DNA-binding faces of the HMG boxes. A potential consequence of this interaction is enhanced DNA binding by HMGB1, and concomitantly lowered affinity of H1 for DNA. In a chromatin context, this might facilitate displacement of H1 by HMGB1.     

Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays.

We describe the cloning and analysis of Drosophila nucleosome assembly protein 1 (dNAP-1), a core histone-binding protein that functions with other chromatin assembly activities in a Drosophila chromatin assembly factor 1-containing fraction (dCAF-1 fraction) in the ATP-facilitated assembly of regularly spaced nucleosomal arrays from purified core histones and DNA. Purified, recombinant dNAP-1 acts cooperatively with a factor(s) in the dCAF-1 fraction in the efficient and DNA replication-independent assembly of chromatin. In the presence of histone H1, the repeat length of the chromatin is similar to that of native chromatin from Drosophila embryos. By coimmunoprecipitation analysis, dNAP-1 was found to be associated with histones H2A and H2B in a crude whole-embryo extract, which suggests that dNAP-1 is bound to the histones in vivo. Studies of the localization of dNAP-1 in the Drosophila embryo revealed that the factor is present in the nucleus during S phase and is predominantly cytoplasmic during G2 phase. These data suggest that NAP-1 acts as a core histone shuttle which delivers the histones from the cytoplasm to the chromatin assembly machinery in the nucleus. Thus, NAP-1 appears to be one component of a multifactor chromatin assembly machinery that mediates the ATP-facilitated assembly of regularly spaced nucleosomal arrays.     

Histone hyperacetylation facilitates chromatin remodelling in a Drosophila embryo cell-free system

We found that Drosophila embryo extract contains a protein activity (or activities) that can destabilize nucleosomes, resulting in increased sensitivity to DNase I, release of nucleosomal supercoiling, high levels of conformational flexibility of DNA and more diffuse micrococcal nuclease digestion patterns. Incorporation of histone H1 did not significantly affect this nucleosome remodelling. Remodelling occurs more efficiently in hyperacetylated chromatin. It was shown previously that hyperacetylated chromatin, reconstituted in a Drosophila embryo cell-free system, exhibits increased DNase I sensitivity and a high degree of conformational flexibility of DNA. The present data suggest that the more diffuse structure of acetylated chromatin is a result of chromatin remodelling by protein activities in the Drosophila embryo extract. Received: 4 November 1998 / Accepted: 10 May 1999     

Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H1(0) linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

Effect of salt on the binding of the linker histone H1 to DNA and nucleosomes

The linker histones are involved in the salt-dependent folding of the nucleosomes into higher-order chromatin structures. To better understand the mechanism of action of these histones in chromatin, we studied the interactions of the linker histone H1 with DNA at various histone/DNA ratios and at different ionic strengths. In direct competition experiments, we have confirmed the binding of H1 to superhelical DNA in preference to linear or nicked circular DNA forms. We show that the electrophoretic mobility of the H1/supercoiled DNA complex decreases with increasing H1 concentrations and increases with ionic strengths. These results indicate that the interaction of the linker histone H1 with supercoiled DNA results in a soluble binding of H1 with DNA at low H1 or salt concentrations and aggregation at higher H1 concentrations. Moreover, we show that H1 dissociates from the DNA or nucleosomes at high salt concentrations. By the immobilized template pull-down assay, we confirm these data using the physiologically relevant nucleosome array template.     

Evidence for a shared structural role for HMG1 and linker histones B4 and H1 in organizing chromatin.

The high mobility group proteins 1 and 2 (HMG1/2) and histone B4 are major components of chromatin within the nuclei assembled during the incubation of Xenopus sperm chromatin in Xenopus egg extract. To investigate their potential structural and functional roles, we have cloned and expressed Xenopus HMG1 and histone B4. Purified histone B4 and HMG1 form stable complexes with nucleosomes including Xenopus 5S DNA. Both proteins associate with linker DNA and stabilize it against digestion with micrococcal nuclease, in a similar manner to histone H1. However, neither histone B4 nor HMG1 influence the DNase I or hydroxyl radical digestion of DNA within the nucleosome core. We suggest that HMG1/2 and histone B4 have a shared structural role in organizing linker DNA in the nucleosome.     

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.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Prothymosin alpha modulates the interaction of histone H1 with chromatin.

Prothymosin alpha (ProTalpha) is an abundant acidic nuclear protein that may be involved in cell proliferation. In our search for its cellular partners, we have recently found that ProTalpha binds to linker histone H1. We now provide further evidence for the physiological relevance of this interaction by immunoisolation of a histone H1-ProTalpha complex from NIH 3T3 cell extracts. A detailed analysis of the interaction between the two proteins suggests contacts between the acidic region of ProTalpha and histone H1. In the context of a physiological chromatin reconstitution reaction, the presence of ProTalpha does not affect incorporation of an amount of histone H1 sufficient to increase the nucleosome repeat length by 20 bp, but prevents association of all further H1. Consistent with this finding, a fraction of histone H1 is released when H1-containing chromatin is challenged with ProTalpha. These results imply at least two different interaction modes of H1 with chromatin, which can be distinguished by their sensitivity to ProTalpha. The properties of ProTalpha suggest a role in fine tuning the stoichiometry and/or mode of interaction of H1 with chromatin.     

Inner nuclear membrane protein LBR preferentially interacts with DNA secondary structures and nucleosomal linker

The lamin B receptor (LBR) is an integral protein of inner nuclear membrane whose nucleoplasmic amino-terminal domain contributes to the attachment of the membrane to chromatin. Here we analyzed the interactions of a recombinant GST protein containing the amino-terminal domain of the protein with in vitro reconstituted nucleosomes and short DNA fragments. Data show that the LBR amino-terminal domain (AT) binds linker DNA but does not interact with the nucleosome core. Titration and competition studies revealed that the interaction between LBR AT and DNA is saturable, of high affinity (K(D) approximately 4 nM), independent of DNA sequence, and enhanced by DNA curvature and supercoiling. In this respect, LBR amino-terminal domain binding to nucleosomes is similar to that of histone H1 and non histone proteins HMG1/2 which both bind preferentially to linker DNA and present a significant affinity for DNA secondary structures.     

The two Plasmodium falciparum nucleosome assembly proteins play distinct roles in histone transport and chromatin assembly

The malarial parasite Plasmodium falciparum has two nucleosome assembly proteins, PfNapS and PfNapL (Chandra, B. R., Olivieri, A., Silvestrini, F., Alano, P., and Sharma, A. (2005) Mol. Biochem. Parasitol. 142, 237-247). We show that both PfNapS and PfNapL interact with histone oligomers but only PfNapS is able to deposit histones onto DNA. This property of PfNapS is divalent cation-dependent and ATP-independent. Deletion of the terminal subdomains of PfNapS abolishes its nucleosome assembly capabilities, but the truncated protein retains its ability to bind histones. Both PfNapS and PfNapL show binding to the linker histone H1 suggesting their probable role in extraction of H1 from chromatin fibers. Our data suggests distinct sites of interaction for H1 versus H3/H4 on PfNapS. We show that PfNapS and PfNapL are phosphorylated both in vivo and in vitro by casein kinase-II, and this modification is specifically inhibited by heparin. Circular dichroism, fluorescence spectroscopy, and chymotrypsin fingerprinting data together suggest that PfNapL may undergo very small and subtle structural changes upon phosphorylation. Specifically, phosphorylation of PfNapL increases its affinity 3-fold for core histones H3, H4, and for the linker histone H1. Finally, we demonstrate that PfNapS is able to extract histones from both phosphorylated and unphosphorylated PfNapL, potentially for histone deposition onto DNA. Based on these results, we suggest that the P. falciparum NapL is involved in the nucleocytoplasmic relay of histones, whereas PfNapS is likely to be an integral part of the chromatin assembly motors in the parasite nucleus.     

Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions.

BACKGROUND: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. METHODS: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. RESULTS: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. CONCLUSIONS: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.     

Base excision repair of 8-oxoG in dinucleosomes

In this work we have studied the effect of chromatin structure on the base excision repair (BER) efficiency of 8-oxoG. As a model system we have used precisely positioned dinucleosomes assembled with linker histone H1. A single 8-oxoG was inserted either in the linker or the core particle DNA within the dinucleosomal template. We found that in the absence of histone H1 the glycosylase OGG1 removed 8-oxoG from the linker DNA and cleaved DNA with identical efficiency as in the naked DNA. In contrast, the presence of histone H1 resulted in close to 10-fold decrease in the efficiency of 8-oxoG initiation of repair in linker DNA independently of linker DNA length. The repair of 8-oxoG in nucleosomal DNA was very highly impeded in both absence and presence of histone H1. Chaperone-induced uptake of H1 restored the efficiency of the glycosylase induced removal of 8-oxoG from linker DNA, but not from the nucleosomal DNA. We show, however, that removal of histone H1 and nucleosome remodelling are both necessary and sufficient for an efficient removal of 8-oxoG in nucleosomal DNA. Finally, a model for BER of 8-oxoG in chromatin templates is suggested.