DNA instructed displacement of histones H2A and H2B at an inducible promoter

Regulation of gene expression requires dynamic changes in chromatin, but the nature of these changes is not well understood. Here, we show that progesterone treatment of cultured cells leads to recruitment of progesterone receptor (PR) and SWI/SNF-related complexes to Mouse Mammary Tumor Virus (MMTV) promoter, accompanied by displacement of histones H2A and H2B from the nucleosome containing the receptor binding sites, but not from adjacent nucleosomes. PR recruits SWI/SNF to MMTV nucleosomes in vitro and facilitates synergistic binding of receptors and nuclear factor 1 to the promoter. In nucleosomes assembled on MMTV or mouse rDNA promoter sequences, SWI/SNF catalyzes ATP-dependent sliding of the histone octamer followed only on the MMTV promoter by displacement of histones H2A and H2B. In MMTV nucleosome arrays, SWI/SNF displaces H2A and H2B from nucleosome B and not from the adjacent nucleosome. Thus, the outcome of nucleosome remodeling by SWI/SNF depends on DNA sequence.     

Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

The core histone tail domains contribute to sequence-dependent nucleosome positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.     

Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.     

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.     

Heterogeneity of chromatin subunits in vitro and location of histone H1.

Chromatin subunits ("nucleosomes") which were purified by sucrose gradient centrifugation of a staphylococcal nuclease digest of chromatin have been studied. We found that such a preparation contains nucleosomes of two discrete types which can be separated from each other by polyacrylamide gel electrophoresis. Nucleosome of the first type contains all five histones and a DNA segment of approximately 200 base pairs long, whereas nucleosome of the second type lacks histone H1 and its DNA segment is approximately 170 base pairs long, i.e., about 30 base pairs shorter than the DNA segment of the nucleosome of the first type. Purified dimer of the nucleosome also can be fractionated by gel electrophoresis into three discrete bands which correspond to dinucleosomes containing two molecules of histone H1, one and no H1. These and related findings strongly suggest that the H1 molecule is bound to a short (approximately 30 base pairs) terminal stretch of the nucleosomal DNA segment which can be removed by nuclease (possibly in the form of H1-DNA complex) without any significant disturbance of main structural features of the nucleosome.     

Archaeal nucleosome positioning sequence from Methanothermus fervidus.

DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB. Here, we document the translational and rotational positioning of archaeal nucleosome assembly in vitro by a sequence from the 7S RNA encoding region of the M. fervidus genome. The minor groove of the DNA at the center of the DNA sequence, protected from micrococcal nuclease digestion by incorporation into a positioned archaeal nucleosome, faces away from the archaeal histone core.     

Interaction of maize chromatin-associated HMG proteins with mononucleosomes: role of core and linker histones

Two groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified. We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini. In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.     

Acetylated nucleosome assembly on telomeric DNAs

The role of histone N-terminal domains on the thermodynamic stability of nucleosomes assembled on several different telomeric DNAs as well as on 'average' sequence DNA and on strong nucleosome positioning sequences, has been studied by competitive reconstitution. We find that histone tails hyperacetylation favors nucleosome formation, in a similar extent for all the examined sequences. On the contrary, removal of histone terminal domains by selective trypsinization causes a decrease of nucleosome stability which is smaller for telomeres compared to the other sequences examined, suggesting that telomeric sequences have only minor interactions with histone tails. Micrococcal nuclease kinetics shows enhanced accessibility of acetylated nucleosomes formed both on telomeric and 'average' sequence DNAs. These results suggest a more complex role for histone acetylation than the decrease of electrostatic interactions between DNA and histones.     

A translational signature for nucleosome positioning in vivo

In vivo nucleosomes often occupy well-defined preferred positions on genomic DNA. An important question is to what extent these preferred positions are directly encoded by the DNA sequence itself. We derive here from in vivo positions, accurately mapped by partial micrococcal nuclease digestion, a translational positioning signal that identifies the approximate midpoint of DNA bound by a histone octamer. This midpoint is, on average, highly A/T rich (∼73%) and, in particular, the dinucleotide TpA occurs preferentially at this and other outward-facing minor grooves. We conclude that in this set of sequences the sequence code for DNA bending and nucleosome positioning differs from the other described sets and we suggest that the enrichment of AT-containing dinucleotides at the centre is required for local untwisting. We show that this signature is preferentially associated with nucleosomes flanking promoter regions and suggest that it contributes to the establishment of gene-specific nucleosome arrays.     

Unique translational positioning of nucleosomes on synthetic DNAs.

A computational study was previously carried out to analyze DNA sequences that are known to position histone octamers at single translational sites. A conserved pattern of intrinsic DNA curvature was uncovered that was proposed to direct the formation of nucleosomes to unique positions. The pattern consists of two regions of curved DNA separated by preferred lengths of non-curved DNA. In the present study, 11 synthetic DNAs were constructed which contain two regions of curved DNA of the form [(A5.T5)(G/C)5]4 separated by non-curved regions of variable length. Translational mapping experiments of in vitro reconstituted mononucleosomes using exonuclease III, micrococcal nuclease and restriction enzymes demonstrated that two of the fragments positioned nucleosomes at a single site while the remaining fragments positioned octamers at multiple sites spaced at 10 base intervals. The synthetic molecules that positioned nucleosomes at a single site contain non-curved central regions of the same lengths that were seen in natural nucleosome positioning sequences. Hydroxyl radical and DNase I digests of the synthetic DNAs in reconstituted nucleosomes showed that the synthetic curved element on one side of the nucleosomal dyad assumed a rotational orientation where narrow minor grooves of the A-tracts faced the histone surface with all molecules. In contrast, the curved element on the other side of the nucleosome displayed variable rotational orientations between molecules which appeared to be related to the positioning effect. These results suggest that asymmetry between the two halves of nucleosomal DNA may facilitate translational positioning.