Nucleosomal stability and dynamics vary significantly when viewed by internal versus terminal labels

Nucleosomes are a major impediment to regulatory factor activities and therefore to the operation of genomic processes in eukaryotes. One suggested mechanism for overcoming in vivo nucleosomal repression is factor-mediated removal of H2A/H2B from nucleosomes. Using nucleosomes labeled internally with FRET fluorophores, we previously observed significant, DNA sequence-dependent variation in stability and dynamics under conditions (subnanomolar concentrations) reported to produce H2A/H2B release from nucleosomes. Here, the same analytical approaches are repeated using 5S and MMTV-B nucleosomes containing FRET labels that monitor the terminal regions. The results show that stability and dynamics vary significantly within the nucleosome; terminally labeled constructs report significantly reduced stability and enhanced DNA dynamics compared to internally labeled constructs. The data also strongly support previous suggestions (1) that subnanomolar concentrations cause H2A/H2B release from nucleosomes, including the 5S, and (2) that stabilities in the internal regions of 5S and two promoter-derived nucleosomes (MMTV-B, GAL10) differ. Sequence-dependent nucleosome stability/dynamics differences could produce inherent variations in the accessibility of histone-associated DNA in vivo. Such intrinsic variation could also provide a mechanism for producing enhanced effects on specific nucleosomes by processes affecting large chromatin regions, thus facilitating the localized targeting of alterations to nucleosomes on crucial regulatory sequences. The results demonstrate clearly the importance of studying physiologically relevant nucleosomes.     

Structures of Native-like Nucleosomes: One Step Closer toward Understanding the Structure and Function of Chromatin

Genomic DNA in eukaryotes is organized into chromatin through association with core histone proteins to form nucleosomes. To understand the structure and function of chromatin, we must determine the structures of nucleosomes containing native DNA sequences. However, to date, our knowledge of nucleosome structures is mainly based on the crystallographic studies of the nucleosomes containing non-native DNA sequences. Here, we discuss the technical issues related to the determination of the nucleosome structures and review the few structural studies on native-like nucleosomes. We show how an antibody fragment-aided single-particle cryo-EM can be a useful method to determine the structures of nucleosomes containing genomic DNA. Finally, we provide a perspective for future structural studies of some native-like nucleosomes that play critical roles in chromatin functions.     

The nuclease sensitivity of active genes.

Brief micrococcal nuclease digestion of chick embryonic red blood cells results in preferential excision and solubilization of monomer nucleosomes associated with beta-globin sequences and also 5'-sequences flanking the beta-globin gene. Both regions are DNAse-I sensitive in nuclei. Such salt-soluble nucleosomes are enriched in all four major HMG proteins but HMG1 and 2 are only weakly associated. These nucleosomes appear to have lost much of the DNAse-I sensitivity of active genes. The HMG14 and 17-containing salt-soluble nucleosomes separated by electrophoresis are not DNAse-I sensitive and contain inactive gene sequences as well as active sequences. Reconstitution of HMG proteins onto bulk nucleosomes or chromatin failed to reveal an HMG-dependent sensitivity of active genes as assayed by dot-blot hybridization and it was found that the DNAse-I sensitivity of ASV proviral sequences as assayed by dot-blot hybridization was not HMG-dependent. These results indicate that higher order chromatin structures might be responsible for nuclease sensitivity of active genes.     

In vivo assembly of chromatin on pBR322 sequences cloned into yeast plasmids

In order to study the in vivo assembly of chromatin on prokaryotic DNA templates, we have transformed yeast cells with plasmids pAJ50 and pRB58, which contain pBR322 sequences. In both cases nucleosomes are assembled in vivo on pBR322 DNA, although the nucleosomes are not homogeneous in size. To explore whether there is any preference for nucleosome assembly along pBR322 sequences, we have used an indirect end labeling method. The results indicate that most nucleosomes are placed at random on pBR322, although the probability for histone octamers to interact with some short regions is somewhat reduced. These regions coincide with sequences in which the frequency distribution of nucleosomes reconstituted in vitro (E. Caffarelli et al. (1988) Eur. J. Biochem. 171, 497-501) is low. Sequence determinants that direct chromatin assembly in vitro seem thereby to act to some extent in vivo.     

Nucleotide sequence-directed mapping of the nucleosomes

The concept of sequence-dependent deformational anisotropy of DNA proposed earlier is further elaborated and a computational procedure is developed for the sequence-directed mapping of the nucleosomes along chromatin DNA nucleotide sequences. The deformational anisotropy is found to be nonuniform along the molecule of the nucleosomal DNA, suggesting that the DNA superhelix in the nucleosome is slightly oval rather than circular in projection. The number of superhelical turns in the nucleosome core particle is estimated to be 2.0 +/- 0.2. Preliminary mapping of the nucleosomes in various chromatin DNA sequences yields the distribution of linker lengths which shows several minima separated by about 10 base-pairs. This is explained by sterical exclusion effects due to overlapping of the nucleosomes in space when some specific linker lengths are chosen. The mapping procedure described is tested by comparing its results with all the most accurate experimental mapping data reported so far. The comparison demonstrates that the exact positions of all the nucleosomes appear to be determined exclusively by the nucleotide sequences.     

Nucleosomes are positioned with base pair precision adjacent to the alpha 2 operator in Saccharomyces cerevisiae.

Analysis of the chromatin structure of minichromosomes containing the binding site for the yeast alpha 2 repressor protein by indirect end-labeling has previously indicated that nucleosomes are stably positioned over sequences adjacent to the alpha 2 operator in the presence of the repressor. Development of a primer extension assay for nucleosome position now allows a more detailed examination of the location of these nucleosomes relative to the operator sequence, and indicates that nucleosomes are precisely and stably positioned both translationally and rotationally over sequences adjoining the operator. In addition, this assay enables analysis of the chromatin structure of single copy, genomic sequences. Chromatin structures determined for two genes regulated by alpha 2, STE6 and BAR1, are consistent with nucleosomes precisely positioned downstream of the operator sequence, incorporating promoter elements, in alpha cells but not in a-cells. The location of these nucleosomes relative to the operator sequence is highly analogous to that observed in the minichromosome. The stability of the nucleosomes adjacent to the operator together with the precision of their location suggests that they may play a role in repression of a specific gene expression by alpha 2. Further, the primer extension assay allows a comparison of the structure of these positioned nucleosomes formed in vivo to that previously described for core particles reconstituted in vitro.     

Maintenance DNA methylation of nucleosome core particles

The enzyme responsible for maintenance methylation of CpG dinucleotides in vertebrates is DNMT1. The presence of DNMT1 in DNA replication foci raises the issue of whether this enzyme needs to gain access to nascent DNA before its packaging into nucleosomes, which occurs very rapidly behind the replication fork. Using nucleosomes positioned along the 5 S rRNA gene, we find that DNMT1 is able to methylate a number of CpG sites even when the DNA major groove is oriented toward the histone surface. However, we also find that the ability of DNMT1 to methylate nucleosomal sites is highly dependent on the nature of the DNA substrate. Although nucleosomes containing the Air promoter are refractory to methylation irrespective of target cytosine location, nucleosomes reconstituted onto the H19 imprinting control region are more accessible. These results argue that although DNMT1 is intrinsically capable of methylating some DNA sequences even after their packaging into nucleosomes, this is not the case for at least a fraction of DNA sequences whose function is regulated by DNA methylation.     

Telomeric nucleosomes are intrinsically mobile

Nucleosomes are no longer considered only static basic units that package eukaryotic DNA but they emerge as dynamic players in all chromosomal processes. Regulatory proteins can gain access to recognition sequences hidden by the histone octamer through the action of ATP-dependent chromatin remodeling complexes that cause nucleosome sliding. In addition, it is known that nucleosomes are able to spontaneously reposition along the DNA due to intrinsic dynamic properties, but it is not clear yet to what extent sequence-dependent dynamic properties contribute to nucleosome repositioning. Here, we study mobility of nucleosomes formed on telomeric sequences as a function of temperature and ionic strength. We find that telomeric nucleosomes are highly intrinsically mobile under physiological conditions, whereas nucleosomes formed on an average DNA sequence mostly remain in the initial position. This indicates that DNA sequence affects not only the thermodynamic stability and the positioning of nucleosomes but also their dynamic properties. Moreover, our findings suggest that the high mobility of telomeric nucleosomes may be relevant to the dynamics of telomeric chromatin.     

Epigenetic nucleosomes: Alu sequences and CG as nucleosome positioning element

Alu sequences carry periodical pattern with CG dinucleotides (CpG) repeating every 31-32 bases. Similar distances are observed in distribution of DNA curvature in crystallized nucleosomes, at positions +/-1.5 and +/-4.5 periods of DNA from nucleosome DNA dyad. Since CG elements are also found to impart to nucleosomes higher stability when positioned at +/-1.5 sites, it suggests that CG dinucleotides may play a role in modulation of the nucleosome strength when the CG elements are methylated. Thus, Alu sequences may harbor special epigenetic nucleosomes with methylation-dependent regulatory functions. Nucleosome DNA sequence probe is suggested to detect locations of such regulatory nucleosomes in the sequences.     

Nucleosome positioning,nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae

The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.     

Sequence structure of Lowary/Widom clones forming strong nucleosomes

Lowary and Widom selected from random sequences those which form exceptionally stable nucleosomes, including clone 601, the current champion of strong nucleosome (SN) sequences. This unique sequence database (LW sequences) carries sequence elements which confer stability on the nucleosomes formed on the sequences, and, thus, may serve as source of information on the structure of “ideal” or close to ideal nucleosome DNA sequence. An important clue is also provided by crystallographic study of Vasudevan and coauthors on clone 601 nucleosomes. It demonstrated that YR·YR dinucleotide stacks (primarily TA·TA) follow one another at distances 10 or 11 bases or multiples thereof, such that they all are located on the interface between DNA and histone octamer. Combining this important information with alignment of the YR-containing 10-mers and 11-mers from LW sequences, the bendability matrices of the stable nucleosome DNA are derived. The matrices suggest that the periodically repeated TA (YR), RR, and YY dinucleotides are the main sequence features of the SNs. This consensus coincides with the one for recently discovered SNs with visibly periodic DNA sequences. Thus, the experimentally observed stable LW nucleosomes and SNs derived computationally appear to represent the same entity – exceptionally stable SNs.     

Sequence-dependent DNA torsional rigidity: a tetranucleotide code

Using fluorescence polarization anisotropy (FPA), we measured the torsional constant of various DNA oligomers in different sequences and calculated the value for each of the 136 unique tetranucleotides. From these values, we obtained a "rigidity profile" for every double-stranded DNA sequence. We tested the code in the analysis of DNA sequences able to form nucleosomes. More than 50% of the sequences studied showed a common 20 and/or 30 bp modulation of the torsional constant. Many other profiles of rigidity were observed in the remaining sequences and this variety in torsional constant modulation may be related to functional differences between nucleosomes.