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.     

Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites

We have previously shown that nucleosomes are conformationally dynamic: DNA sequences that in the time-average are buried inside nucleosomes are nevertheless transiently accessible, even to large proteins (or any other macromolecule). We refer to this dynamic behavior as "site exposure". Here we show that: (i) the equilibrium constants describing this dynamic site exposure decrease progressively from either end of the nucleosomal DNA in toward the middle; and (ii) these position-dependent equilibrium constants are strongly dependent on the nucleosomal DNA sequence. The progressive decrease in equilibrium constant with distance inside the nucleosome supports the hypothesis that access to sites internal to a nucleosome is provided by progressive (transient) release of DNA from the octamer surface, starting from one end of the nucleosomal DNA. The dependence on genomic DNA sequence implies that a specific genomic DNA sequence could be a major determinant of target site occupancies achieved by regulatory proteins in vivo, by either governing the time-averaged accessibility for a given nucleosome position, or biasing the time-averaged positioning (of mobile nucleosomes), which in turn is a major determinant of site accessibility.     

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.     

Nucleosomal locations of dominant DNA sequence motifs for histone-DNA interactions and nucleosome positioning

DNA sequence is an important determinant of the positioning, stability, and activity of nucleosomes, yet the molecular basis of these effects remains elusive. A "consensus DNA sequence" for nucleosome positioning has not been reported and, while certain DNA sequence preferences or motifs for nucleosome positioning have been discovered, how they function is not known. Here, we report that an unexpected observation concerning the reassembly of nucleosomes during salt gradient dialysis has allowed a breakthrough in our efforts to identify the nucleosomal locations of the DNA sequence motifs that dominate histone-DNA interactions and nucleosome positioning. We conclude that a previous selection experiment for high-affinity, nucleosome-forming DNA sequences exerted selective pressure chiefly on the central stretch of the nucleosomal DNA. This observation implies that algorithms for aligning the selected DNA sequences should seek to optimize the alignment over much less than the full 147 bp of nucleosomal DNA. A new alignment calculation implemented these ideas and successfully aligned 19 of the 41 sequences in a non-redundant database of selected high-affinity, nucleosome-positioning sequences. The resulting alignment reveals strong conservation of several stretches within a central 71 bp of the nucleosomal DNA. The alignment further reveals an inherent palindromic symmetry in the selected DNAs; it makes testable predictions of nucleosome positioning on the aligned sequences and for the creation of new positioning sequences, both of which are upheld experimentally; and it suggests new signals that may be important in translational nucleosome positioning.     

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.     

Functional role of extranucleosomal DNA and the entry site of the nucleosome in chromatin remodeling by ISW2

A minimal amount of extranucleosomal DNA was required for nucleosome mobilization by ISW2 as shown by using a photochemical histone mapping approach to analyze nucleosome movement on a set of nucleosomes with varied lengths of extranucleosomal DNA. ISW2 was ineffective in repositioning or mobilizing nucleosomes with 相似文献   

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.     

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.     

SWI/SNF- and RSC-catalyzed nucleosome mobilization requires internal DNA loop translocation within nucleosomes

The multisubunit SWI/SNF and RSC complexes utilize energy derived from ATP hydrolysis to mobilize nucleosomes and render the DNA accessible for various nuclear processes. Here we test the idea that remodeling involves intermediates with mobile DNA bulges or loops within the nucleosome by cross-linking the H2A N- or C-terminal tails together to generate protein "loops" that constrict separation of the DNA from the histone surface. Analyses indicate that this intranucleosomal cross-linking causes little or no change in remodeling-dependent exposure of DNA sequences within the nucleosome to restriction enzymes. However, cross-linking inhibits nucleosome mobilization and blocks complete movement of nucleosomes to extreme end positions on the DNA fragments. These results are consistent with evidence that nucleosome remodeling involves intermediates with DNA loops on the nucleosome surface but indicate that such loops do not freely diffuse about the surface of the histone octamer. We propose a threading model for movement of DNA loops around the perimeter of the nucleosome core.     

Long-range correlations between DNA bending sites: relation to the structure and dynamics of nucleosomes

It has been established that the precise positioning of nucleosomes on genomic DNA can be achieved, at least for a minority of them, through sequence-dependent processes. However, to what extent DNA sequences play a role in the positioning of the major part of nucleosomes is still debated. The aim of the present study is to examine to what extent long-range correlations (LRC) are related to the presence of nucleosomes. Using the wavelet transform technique, we perform a comparative analysis of the DNA text and of the corresponding bending profiles generated with curvature tables based on nucleosome positioning data. The exploration of a number of eukaryotic and bacterial genomes through the optics of the so-called "wavelet transform microscope" reveals a characteristic scale of 100-200 bp that separates two regimes of different LRC. Here, we focus on the existence of LRC in the small-scale regime (10-200 bp) which are actually observed in eukaryotic genomes, in contrast to their absence in eubacterial genomes. Analysis of viral DNA genomes shows that, like their host's genomes, eukaryotic viruses present LRC but eubacterial viruses do not. There is one exception for genomes of poxviruses (Vaccinia and Melamoplus sanguinipes) which do not replicate in the cell nucleus and do not exhibit LRC. No small-scale LRC are detected in the genomes of all examined RNA viruses, with the exception of retroviruses. These results together with the observation of LRC between particular sequence motifs known to participate in the formation of nucleosomes (e.g. AA dinucleotides) strongly suggest that the 10-200 bp LRC are a signature of the sequence-dependence of nucleosome positioning. Finally, we discuss possible interpretations of these LRC in terms of the physical mechanisms that might govern the positioning and the dynamics of the nucleosomes along the DNA chain through cooperative processes.     

Abnormal DNA methylation and deoxycytosine-deoxyguanine content in nucleosomes from lymphocytes undergoing apoptosis.

Systemic lupus erythematosus (SLE) is characterized by an accelerated apoptosis of peripheral lymphocytes and an impairment of the clearance of apoptotic cells. Since changes in DNA methylation and in deoxycytosine and deoxyguanine (GC) content have been shown to enhance the potential of DNA to activate murine and human B lymphocytes, we tested the capacity of lymphocytes undergoing apoptosis (under conditions that mimic the deletion of self-reactive cells after antigen receptor engagement) to generate nucleosomes with a particular base composition. Using two cell culture systems and four apoptosis triggers, we found an increase of deoxymethylcytosine in fragmented chromosomal DNA of apoptotic B and T lymphocytes. However, this increase was not associated with modulation of DNA (cytosine-5) methyltransferase, the enzyme that methylates eukaryotic DNA, which suggests that the changes in DNA methylation patterns are not linked to the process of de novo DNA methylation during cell death. In addition, we could not detect a unique methylation pattern in highly repetitive Alu sequences present in the human genome of SLE subjects, as compared with controls. However, the abnormal DNA methylation of apoptotic nucleosomes was associated with an unusual pattern of nuclease-resistant, GC-rich regions in these DNA fragments. We propose that the combination of an accelerated apoptosis with a defect in the clearance of apoptotic cells results in release of increased amounts of nucleosomes with abnormally methylated, GC-rich DNA and provides an autologous stimulation that could bypass tolerance to self in systemic autoimmune diseases. These findings support the concept that the structure and dynamics of nucleosomes are critical in determining their immunogenicity in SLE.     

Chromatin remodeling by ISW2 and SWI/SNF requires DNA translocation inside the nucleosome

Chromatin-remodeling complexes regulate access to nucleosomal DNA by mobilizing nucleosomes in an ATP-dependent manner. In this study, we find that chromatin remodeling by SWI/SNF and ISW2 involves DNA translocation inside nucleosomes two helical turns from the dyad axis at superhelical location-2. DNA translocation at this internal position does not require the propagation of a DNA twist from the site of translocation to the entry/exit sites for nucleosome movement. Nucleosomes are moved in 9- to 11- or approximately 50-base-pair increments by ISW2 or SWI/SNF, respectively, presumably through the formation of DNA loops on the nucleosome surface. Remodeling by ISW2 but not SWI/SNF requires DNA torsional strain near the site of translocation, which may work in conjunction with conformational changes of ISW2 to promote nucleosome movement on DNA. The difference in step size of nucleosome movement by SWI/SNF and ISW2 demonstrates how SWI/SNF may be more disruptive to nucleosome structure than ISW2.     

Stability of nucleosomes in native and reconstituted chromatins.

The stability of nucleosomes of SV40 minichromosomes extracted from infected cells or reconstituted by association of SV40 DNA and the four histones H2A, H2B, H3 and H4 was studied as a function of the ionic strength. As a measure of the stability of the nucleosome, we followed the disappearance of the nucleosomes from the original chromatin and their appearance on a "competing" DNA. We show here that the DNA and the histone components of the nucleosomes do not apprecially dissociate below 800 mM NaCl. At 800 mM and above, the histone moiety of the nucleosomes can dissociate from the DNA and efficiently participate to the formation of nucleosomes on a "competing" DNA.     

The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction

It was shown with the use of specific probes that mild micrococcal nuclease digestion releases from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A⁺) RNAs, however larger released fragments include some transcribed sequences, while the nuclease-resistant chromatin is considerably impoverished in coding sites. These evidences and the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5–2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.