Nucleosome Positioning Sequences Research Articles

DNA flexibility regulates transcription factor binding to nucleosomes.

Cell fate decisions are controlled by sequence-specific transcription factors (TFs), referred to as 'pioneer' factors, that bind their target sites within nucleosomes ('pioneer binding') and thus initiate chromatin opening. However, pioneers bind just a minority of their recognition sequences present in the genome, suggesting that local sequence context features may regulate pioneer binding. Here, we developed PIONEAR-seq, a highly parallel sequencing-based biochemical assay for high-throughput analysis of TF binding to nucleosomes on nucleosome positioning sequences. Using PIONEAR-seq, we characterized the pioneer binding of 7 human pioneer TFs. Comparison of TF binding to nucleosomes based on the synthetic Widom 601 (W601) model sequence versus three different genomic sequences revealed that the positional preferences of these TFs' binding to nucleosomes ( i.e. , dyad, periodic and end binding) is determined by the broader sequence context of the nucleosome, rather than being a property intrinsic to the TF. We propose a model where the flexibility and rigidity within nucleosomal DNA regulate where pioneers bind within nucleosomes. Our results suggest that the broader physical properties of nucleosomal DNA represent another layer of cis -regulatory information read out by TFs in eukaryotic genomes.

Bi-directional nucleosome sliding by the Chd1 chromatin remodeler integrates intrinsic sequence-dependent and ATP-dependent nucleosome positioning.

Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers exhibit a sequence-dependent bias in nucleosome positioning, which presumably reflects properties of the DNA duplex. Here, we show how nucleosome positioning by the Chd1 remodeler is influenced by local DNA perturbations throughout the nucleosome footprint. Using site-specific DNA cleavage coupled with next-generation sequencing, we show that nucleosomes shifted by Chd1 can preferentially localize DNA perturbations - poly(dA:dT) tracts, DNA mismatches, and single-nucleotide insertions - about a helical turn outside the Chd1 motor domain binding site, super helix location 2 (SHL2). This phenomenon occurs with both the Widom 601 positioning sequence and the natural+1 nucleosome sequence from the Saccharomycescerevisiae SWH1 gene. Our modeling indicates that localization of DNA perturbations about a helical turn outward from SHL2 results from back-and-forth sliding due to remodeler action on both sides of the nucleosome. Our results also show that barrier effects from DNA perturbations can be extended by the strong phasing of nucleosome positioning sequences.

High-yield ligation-free assembly of DNA constructs with nucleosome positioning sequence repeats for single-molecule manipulation assays

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70± 3h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.

Manipulating chromatin architecture in C. elegans

BackgroundNucleosome-mediated chromatin compaction has a direct effect on the accessibility of trans-acting activators and repressors to DNA targets and serves as a primary regulatory agent of genetic expression. Understanding the nature and dynamics of chromatin is fundamental to elucidating the mechanisms and factors that epigenetically regulate gene expression. Previous work has shown that there are three types of canonical sequences that strongly regulate nucleosome positioning and thus chromatin accessibility: putative nucleosome-positioning elements, putative nucleosome-repelling sequences, and homopolymeric runs of A/T. It is postulated that these elements can be used to remodel chromatin in C. elegans. Here we show the utility of such elements in vivo, and the extreme efficacy of a newly discovered repelling sequence, PRS-322.ResultsIn this work, we show that it is possible to manipulate nucleosome positioning in C. elegans solely using canonical and putative positioning sequences. We have not only tested previously described sequences such as the Widom 601, but also have tested additional nucleosome-positioning sequences: the Trifonov sequence, putative repelling sequence-322 (PRS-322), and various homopolymeric runs of A and T nucleotides.ConclusionsUsing each of these types of putative nucleosome-positioning sequences, we demonstrate their ability to alter the nucleosome profile in C. elegans as evidenced by altered nucleosome occupancy and positioning in vivo. Additionally, we show the effect that PRS-322 has on nucleosome-repelling and chromatin remodeling.

A giant virus genome is densely packaged by stable nucleosomes within virions

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome-positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than "beads on a string" with genic punctuation represents a distinct mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome.

Large‐scale analysis of Drosophila core promoter function using synthetic promoters

The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it “computes” expression remains poorly understood. To dissect its function, we carried out a comprehensive structure–function analysis in Drosophila. First, we performed a genome‐wide bioinformatic analysis, providing an improved picture of the sequence motifs architecture. We then measured synthetic promoters’ activities of ~3,000 mutational variants with and without an external stimulus (hormonal activation), at large scale and with high accuracy using robotics and a dual luciferase reporter assay. We observed a strong impact on activity of the different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, nucleosome positioning, and flanking sequences. A linear combination of the individual motif features largely accounts for the combinatorial effects on core promoter activity. These findings shed new light on the quantitative assessment of gene expression in metazoans.

Synthesis of Oriented Hexasomes and Asymmetric Nucleosomes Using a Template Editing Process.

Nucleosomes, the structural building blocks of chromatin, possess 2-fold pseudo symmetry which can be broken through differential modification or removal of one copy of a pair of sister histones. The resultant asymmetric nucleosomes and hexasomes have been implicated in gene regulation, yet the use of these noncanonical substrates in chromatin biochemistry is limited, owing to the lack of efficient methods for their preparation. Here, we report a strategy that allows the orientation of these asymmetric species to be tightly controlled relative to the underlying DNA sequence. Our approach is based on the use of truncated DNA templates to assemble oriented hexasomes followed by DNA ligation and, in the case of asymmetric nucleosomes, addition of the missing heterotypic histones. We show that this approach is compatible with multiple nucleosome positioning sequences, allowing the generation of desymmetrized mononucleosomes and oligonucleosomes with varied DNA overhangs and heterotypic histone H2A/H2B dimer compositions. Using this technology, we examine the functional consequences of asymmetry on BRG1/BRM associated factor (BAF) complex-mediated chromatin remodeling. Our results indicate that cancer-associated histone mutations can reprogram the inherent activity of BAF chromatin remodeling to induce aberrant chromatin structure.

Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the ‘601’ nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.

AID preferentially targets the top strand in nucleosome sequences

AID initiates both somatic hypermutation (SHM) and class switch recombination (CSR) in Ig genes. AID-induced mutations are linked with transcription initiation and elongation. Transcription occurs in the context of chromatin and thus RNA PolII and AID need to deal with nucleosomes. Both nucleosome stability and positioning significantly influence the accessibility of AID to Ig genes and the SHM pattern. Interestingly, in the nucleosome, SHM process seems to have a preference for the top strand. To know whether the preferential targeting of SHM to the top strand is due to a post-AID event, we expressed an inhibitor of Uracil DNA glycosylase (UNG), Ugi, into DT40 cells containing the nucleosome positioning sequence (MP2) and compared the SHM pattern. We observed a similar preference to the top strand for the high-affinity nucleosome positioning sequence in UNG inhibited cells. Furthermore, to understand whether the primary sequence of nucleosome sequence is influencing preferential targeting, we introduced two copies of MP2 sequence in the reverse orientation (MP2R) into a variable Ig gene. We observed that the MP2R cells also demonstrated preferential targeting of the non-transcribed strand in nucleosome as compared to the transcribed strand, confirming that in nucleosome sequences AID has better access to Cs on the top strand. The preferential targeting of AID on the top strand suggests that RNA Pol-II stalls while it transcribes the stable nucleosomes, thus giving ample opportunity for the transcribed strand to form R-loops with the nascent RNA, thereby gives limited access to AID on the bottom strand.

DNA sequence influences hexasome orientation to regulate DNA accessibility.

Nucleosomes, the fundamental organizing units of eukaryotic genomes, contain ∼146 base pairs of DNA wrapped around a histone H3–H4 tetramer and two histone H2A–H2B dimers. Converting nucleosomes into hexasomes by removal of a H2A–H2B dimer is an important regulatory event, but its regulation and functional consequences are not well-understood. To investigate the influence of hexasomes on DNA accessibility, we used the property of the Widom-601 Nucleosome Positioning Sequence (NPS) to form homogeneously oriented hexasomes in vitro. We find that DNA accessibility to transcription factors (TF) on the hexasome H2A–H2B distal side is identical to naked DNA, while the accessibility on the H2A–H2B proximal side is reduced by 2-fold, which is due to a 2-fold reduction in hexasome unwrapping probability. We then determined that a 23 bp region of the Widom-601 NPS is responsible for forming homogeneously oriented hexasomes. Analysis of published ChIP-exo data of hexasome containing genes identified two DNA sequence motifs that correlate with hexasome orientation in vivo, while ExoIII mapping studies of these sequences revealed they generate homogeneously oriented hexasomes in vitro. These results indicate that hexasome orientation, which is influenced by the underlying DNA sequence in vivo, is important for modulating DNA accessibility to regulate transcription.

Structure and Dynamics of the Telomeric Nucleosome and Chromatin

Eukaryotic genomic DNA is condensed into chromatin: 146bp DNA wraps around the histone octamer (HO) comprising two copies each of the core histones H2A, H2B, H3 and H4 forming the nucleosome core particle (NCP). A string of nucleosomes folds into the so-called called “30nm” chromatin fibre, which compacts into metaphase chromosomes. Human telomeres constitute the protective structure at the end of the chromosomes with repetitive TTAGGG sequences that are about 10kbp. Little is known about the consequences of this unique sequence for chromatin structural and dynamic properties. Although it is established that telomeric DNA is packaged in chromatin with an unusually short (157 bp) nucleosome repeat length (NRL) and can form nucleosomes and chromatin, almost nothing is known about the telomeric nucleosome core and telomeric chromatin structure at the detailed molecular level. We have characterised the telomeric human HO 145 bp DNA NCP (Telo-NCP), using biophysical methods and determined its structure at 2.1 Å resolution with X-ray crystallography. Although the structure is largely similar to the well-known published atomic resolution NCP structures, several differences can be identified, e.g. DNA stretching on the Telo-NCP is different from that of the “601” (high-affinity nucleosome positioning sequence) and alpha satellite NCPs of the same length. We have designed and prepared 20×157 bp telomeric DNA template repeats, reconstituted these with human HO and performed characterisation of the structural and dynamic properties of the telomeric chromatin fibres with EM and single molecule magnetic tweezer experiments. Telomeric chromatin fibres are considerably more homogeneous than corresponding 157 bp repeat fibres reconstituted from 601-DNA array templates. However, the structural and dynamic properties of individual telomeric arrays saturated with 20 nucleosomes, appear similar to equivalent 601 chromatin fibres under the influence of force in single molecule pulling experiment.

Kinky DNA in solution: Small-angle-scattering study of a nucleosome positioning sequence

DNA is a flexible molecule, but the degree of its flexibility is subject to debate. The commonly-accepted persistence length of $l_p \approx 500\,$\AA\ is inconsistent with recent studies on short-chain DNA that show much greater flexibility but do not probe its origin. We have performed X-ray and neutron small-angle scattering on a short DNA sequence containing a strong nucleosome positioning element, and analyzed the results using a modified Kratky-Porod model to determine possible conformations. Our results support a hypothesis from Crick and Klug in 1975 that some DNA sequences in solution can have sharp kinks, potentially resolving the discrepancy. Our conclusions are supported by measurements on a radiation-damaged sample, where single-strand breaks lead to increased flexibility and by an analysis of data from another sequence, which does not have kinks, but where our method can detect a locally enhanced flexibility due to an $AT$-domain.

CgDNAweb: a web interface to the cgDNA sequence-dependent coarse-grain model of double-stranded DNA.

The sequence-dependent statistical mechanical properties of fragments of double-stranded DNA is believed to be pertinent to its biological function at length scales from a few base pairs (or bp) to a few hundreds of bp, e.g. indirect read-out protein binding sites, nucleosome positioning sequences, phased A-tracts, etc. In turn, the equilibrium statistical mechanics behaviour of DNA depends upon its ground state configuration, or minimum free energy shape, as well as on its fluctuations as governed by its stiffness (in an appropriate sense). We here present cgDNAweb, which provides browser-based interactive visualization of the sequence-dependent ground states of double-stranded DNA molecules, as predicted by the underlying cgDNA coarse-grain rigid-base model of fragments with arbitrary sequence. The cgDNAweb interface is specifically designed to facilitate comparison between ground state shapes of different sequences. The server is freely available at cgDNAweb.epfl.ch with no login requirement.

Hypomethylated domain-enriched DNA motifs prepattern the accessible nucleosome organization in teleosts

BackgroundGene promoters in vertebrate genomes show distinct chromatin features such as stably positioned nucleosome array and DNA hypomethylation. The nucleosomes are known to have certain sequence preferences, and the prediction of nucleosome positioning from DNA sequence has been successful in some organisms such as yeast. However, at gene promoters where nucleosomes are much more stably positioned than in other regions, the sequence-based model has failed to work well, and sequence-independent mechanisms have been proposed.ResultsUsing DNase I-seq in medaka embryos, we demonstrated that hypomethylated domains (HMDs) specifically possess accessible nucleosome organization with longer linkers, and we reassessed the DNA sequence preference for nucleosome positioning in these specific regions. Remarkably, we found with a supervised machine learning algorithm, k-mer SVM, that nucleosome positioning in HMDs is accurately predictable from DNA sequence alone. Specific short sequences (6-mers) that contribute to the prediction are specifically enriched in HMDs and distribute periodically with approximately 200-bp intervals which prepattern the position of accessible linkers. Surprisingly, the sequence preference of the nucleosome and linker in HMDs is opposite from that reported previously. Furthermore, the periodicity of specific motifs at hypomethylated promoters was conserved in zebrafish.ConclusionThis study reveals strong link between nucleosome positioning and DNA sequence at vertebrate promoters, and we propose hypomethylated DNA-specific regulation of nucleosome positioning.

Evolutionary analysis of nucleosome positioning sequences based on New Symmetric Relative Entropy

New Symmetric Relative Entropy (NSRE) was applied innovatively to analyze the nucleosome sequences in S. cerevisiae, S. pombe and Drosophila. NSRE distributions could well reflect the characteristic differences of nucleosome sequences among three organisms, and the differences indicate a concerted evolution in the sequence usage of nucleosome. Further analysis about the nucleosomes around TSS shows that the constitutive property of +1/−1 nucleosomes in S. cerevisiae is different from that in S. pombe and Drosophila, which indicates that S. cerevisiae has a different transcription regulation mechanism based on nucleosome. However, in either case, the nucleosome dyad region is conserved and always has a higher NSRE. Base composition analysis shows that this conservative property in nucleosome dyad region is mainly determined by base A and T, and the dependence degrees on base A and T are consistent in three organisms.

Solid-phase synthesis of highly repetitive chromatin assembly templates

DNA templates for assembling chromatin model systems typically consist of numerous repeats of nucleosome positioning sequences, making their synthesis challenging. Here we describe a solid-phase strategy for generating such templates using sequential enzymatic ligation of DNA monomers. Using single nucleosome site monomers, we can either generate a twelve-nucleosome site target, or systematically access intermediate-sized templates. Using twelve nucleosome positioning site monomers, longer templates can be generated. Our synthesized templates assemble into well-defined chromatin model systems, demonstrating the utility of our solid-phase approach. Moreover, our strategy should be more widely applicable to generating other DNAs containing highly repetitive DNA sequences.

Application of Synthetic Tumor-Specific Promoters Responsive to the Tumor Microenvironment.

Activity of endogenous promoters can be altered by including additional responsive elements (REs). These elements can be responsive to features of the tumor environment or alternatively to signaling pathways specifically activated in cancer cells. These REs incorporated into tumor-specific promoters can improve cancer targeting, the replicative capacity, and lytic activity of conditionally replicative adenovirus. Here we outline an approach to incorporate hypoxia and inflammation REs into a specific fragment of the SPARC promoter and the steps to clone a nucleosome positioning sequence (NPS ) identified in the osteocalcin promoter that contains a Wnt RE upstream of a heterologous synthetic promoter.

Drosophila H2A and H2A.Z Nucleosome Sequences Reveal Different Nucleosome Positioning Sequence Patterns.

Nucleosomes are implicated in transcriptional regulation as well as in packing and stabilizing the DNA. Nucleosome positions affect the transcription by impeding or facilitating the binding of transcription factors. The DNA sequence, especially the periodic occurrences of dinucleotides, is a major factor that affects the nucleosome positioning. We analyzed the Drosophila DNA sequences bound by H2A and H2A.Z nucleosomes. Periodic patterns of dinucleotides (weak-weak/strong-strong or purine-purine/pyrimidine-pyrimidine) were identified as WW/SS and RR/YY nucleosome positioning sequence (NPS) patterns. The WW/SS NPS pattern of the H2A nucleosome has a 10-bp period of weak-weak/strong-strong (W = A or T; S = G or C) dinucleotides. The 10-bp periodicity, however, is disrupted in the middle of the sequence. At the dyad, the SS dinucleotide is preferred. On the other hand, the RR/YY NPS pattern has an 18-bp periodicity of purine-purine/pyrimidine-pyrimidine (R = A or G; Y = T or C) dinucleotides. The NPS patterns from H2A.Z nucleosomes differ from the NPS patterns from H2A nucleosomes. The RR/YY pattern of H2A.Z nucleosomes has major peaks shifted by 10 bp deviated from the H2A nucleosome pattern. The H2A and H2A.Z nucleosomes have different sequence preferences. The shifted peaks coincide with DNA regions interacting with the histone loops.

Nucleosome assembly and disassembly activity of GRWD1, a novel Cdt1-binding protein that promotes pre-replication complex formation

GRWD1 was previously identified as a novel Cdt1-binding protein that possesses histone-binding and nucleosome assembly activities and promotes MCM loading, probably by maintaining chromatin openness at replication origins. However, the molecular mechanisms underlying these activities remain unknown. We prepared reconstituted mononucleosomes from recombinant histones and a DNA fragment containing a nucleosome positioning sequence, and investigated the effects of GRWD1 on them. GRWD1 could disassemble these preformed mononucleosomes in vitro in an ATP-independent manner. Thus, our data suggest that GRWD1 facilitates removal of H2A-H2B dimers from nucleosomes, resulting in formation of hexasomes. The activity was compromised by deletion of the acidic domain, which is required for efficient histone binding. In contrast, nucleosome assembly activity of GRWD1 was not affected by deletion of the acidic domain. In HeLa cells, the acidic domain of GRWD1 was necessary to maintain chromatin openness and promote MCM loading at replication origins. Taken together, our results suggest that GRWD1 promotes chromatin fluidity by influencing nucleosome structures, e.g., by transient eviction of H2A-H2B, and thereby promotes efficient MCM loading at replication origins.

TA-periodic (“601”-like) centromeric nucleosomes of A. thaliana

The bulk of strong nucleosomes (SNs, with visibly periodic DNA sequences) is described by consensus pattern of 5 or 6 base runs of purines alternating with similar runs of pyrimidines – RR/YY SNs. Yet, the strongest known nucleosome positioning sequence, the 601 clone of Lowary and Widom, is rather periodic repetition of TA dinucleotides following one another every 10 bases. We located “601”-like TA-periodic sequences in the genome of A. thaliana. Several families of such sequences are discovered repeating almost exclusively in centromeres. Thus, while A. thaliana SNs of RR/YY type have strong affinity to pericentromeric regions, as it has been previously found, the SNs of TA periodic type concentrate rather in centromeres.