Thus, the suppression of transcription from cryptic TATA-like sequences in transcribed regions requires both the restoration of nucleosomes behind elongating RNA polymerase II, and their deacetylation to generate a repressive chromatin environment. First, Eaf3 was originally identified as a component of the NuA4 promoter-targeted histone acetyltransferase complex Eisen et al.
Does it continue to function through multiple rounds of transcription, perhaps eventually diluted away by nucleosome loss and replacement? Alternatively, it might be targeted for removal by JmjC domain-containing histone H3K36 demethylase Tsukada et al. Finally, at very highly transcribed genes where nucleosome replacement apparently cannot keep up and partial nucleosome depletion in the ORF occurs Lee et al.
One possibility is that high levels of accurate transcription prevent transcription complex formation at cryptic TATA-like sequences in the ORF. This type of mechanism has been described by Winston and colleagues Martens et al. In summary, nucleosome displacement appears to be a widespread event in the process of transcription.
A combination of transcription factor binding, histone acetylation, nucleosome remodeling complexes, and perhaps the histone variant H2AZ target promoter nucleosomes for displacement upon gene induction to clear space for assembling the transcription machinery.
In addition, nucleosomes are displaced during transcription elongation, where it is necessary to restore them to block TATA-like sequences in the transcribed region to prevent cryptic sites of initiation. Given the prevalence of nucleosome displacement in transcription, it seems likely that they will prove equally dynamic during other processes acting on the genome. I thank Susan Abmayr and the reviewers of the manuscript for many helpful suggestions. View all Nucleosome displacement in transcription Jerry L.
Previous Section Next Section. Figure 1. Previous Section. Adams C. Cell 72 : — Adkins M. Cell 14 : — Ahmad K. Cell 9 : — Akey C. Angelov D. Avolio-Hunter T. Nucleic Acids Res. Babiarz J. Z in Saccharomyces cerevisiae.
Barton M. Oncogene 20 : — Bednar J. Cell 4 : — Belotserkovskaya R. Science : — Bernstein B. Genome Biol. CrossRef Medline Google Scholar. Boeger H. Cell 11 : — Boeger, H. Removal of promoter nucleosomes by disassembly rather than sliding in vivo.
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The left figure exhibit perfect-positioning region, where the nucleosome center is located at the same basepair all over population cells; the other two showed partial-positioning and no-positioning region.
Early in , the 10—11 bp periodicities were reported [ 17 ]. In addition to 3-bp periodicity, which is due to the fact that three consecutive bases encode one type of amino acids, the genomic DNA exhibits 10—11 bp periodicities. The 10—11 bp periodicities in complete genomes reflect protein structure and DNA folding [ 17 ]. On the other hand, the 10—11 bp periodicities have an intimate association with nucleosome positioning.
To sharply bent and tightly wrapped around a histone protein octamer, DNA sequence has intrinsic bias. The position of certain dinucleotides, such as AA, TA, and TT in minor grooves facing toward every 10 bp and GG in minor grooves facing away from the histone octamer favors these Figure 5 distortions [ 15 ]. For the naked DNA, which is entirely devoid of nucleosomes, the oscillatory pattern in cleavage profile was disappeared in digesting [ 18 ].
All of these strongly suggested the role of the 10—11 bp periodicities of the specific dinucleotides in positioning nucleosomes. Based on the features of DNA sequences, many models are developed to predict nucleosomes Table 2. Illustration of nucleosome sequence preferences [ 16 ].
We assessed the roles of the 10—11 bp periodicities for different kinds of dinucleotides [ 20 ]. Near the transcription start site, the signals reveal a similar feature that the nucleosome organization exhibits Figure 6. But, it seems that the species do not share the same dinucleotides patterns. Furthermore, the dinucleotides patterns are dominant at the specific region of genome, indicating their diverse roles in forming and organizing nucleosomes.
The 10—11 bp periodicities signals of the dinucleotides patterns around TSSs of eight species human, mouse, chicken, worm, fly, fugu, lancelet, and yeast [ 20 ]. In Table 2 , the models for both nucleosome prediction and nucleosome sequencing data processing are listed. Chromatin remodeling complex helps cell to establish the access of genomic DNA for transcription factors. The complexes have two major groups, namely covalent histone-modifying complexes and ATP-dependent chromatin remodeling complexes [ 53 ].
They work in a different way. Covalent histone-modifying complexes modify the histone including acetylation, methylation, and phosphorylation which can change the interaction between histone and DNA; for example, methylation of specific lysine residues in H3 and H4 causes further condensation of DNA around histones, making it hard to bind transcription factor or other proteins.
A typical nucleosome distribution around TSS is shown in Figure 7 [ 56 ]. Nucleosomes are depleted around TSSs, resulting in a nucleosome-free region NFR that is flanked by two well-positioned nucleosomes whereas the nucleosomes downstream of the TSS are equally spaced in a nucleosome array.
Z and H3. These may help to the nucleosome eviction when transcription is needed. Z and less methylation and acetylation. In a barrier model for nucleosome organization, the nucleosome distribution is largely a consequence of statistical packing principles. The consensus distribution of nucleosomes gray ovals around all yeast genes is shown, aligned by the beginning and end of every gene.
The resulting two plots were fused in the genic region. The peaks and valleys represent similar positioning relative to the transcription start site TSS. The green-blue shading in the plot represents the transitions observed in nucleosome composition and phasing green represents high H2A.
Z levels, acetylation, H3K4 methylation and phasing, whereas blue represents low levels of these modifications. Each of these components has different contribution in nucleosome positioning. Interestingly, these components can affect each other thus resulting in different positioning pattern in a more complex way. The DNA sequence is critical for rotational positioning along the DNA helix, and it is also an important determinant for nucleosome occupancy.
In particular, poly dA:dT and poly dG:dC tracts are intrinsically inhibitory to nucleosome formation, whereas non-homopolymeric GC-rich regions favor nucleosome formation. Determinants of nucleosome positioning. Gray circles indicate nucleosomes. Micrococcal nuclease MNase , one kind of glycolprotein of Staphylococcus aureus , has capacity of digesting the naked DNA. MNase, firstly, induces single-strand breaks, and then cleaves the complementary strand near the first break [ 58 , 59 ].
Taking this advantage, a high throughput sequencing technique MNase-seq is developed to probe nucleosome positions in a genome-wide manner. MNase cleavage favors AT-rich region in limiting enzyme concentrations. The opening chromatin region is mainly the regulatory sites in gene transcription. Thus, the opening region may alter in different cells types.
This can be reflected in DHSs. The change of DHSs often associates one or more nucleosomes loss or formation [ 60 ]. DNase I hypersensitive sites within chromatin [ 60 ]. DNase-seq has been widely used in probing cell-specific chromatin accessibility. The rotational localization of individual nucleosomes is based on the inherent preference of DNA enzyme I cleavage of DNA at about 10 bp per nucleosome [ 61 ].
By coupling bioinformatics analysis, DNase-seq can be used in studying TF occupancy at nucleotide resolution in a qualitative and quantitative manner [ 62 ]. In DNase-seq, many cells and many sample preparations and enzyme titration steps are required [ 63 ].
ATAC-seq is an assay for transposase-accessible chromatin with high throughput sequencing [ 64 ]. Moreover, its procedure only involves two steps.
Therefore, it is able to study multiple aspects of chromatin architecture simultaneously at high resolution, including nucleosomes, chromatin accessibility [ 64 ]. Chromatin immunoprecipitation followed by sequencing ChIP-seq sequences the interest DNA fragments that are separated and collected from the immunoprecipitation [ 66 ]. Figure 10 shows a general procedure of a ChIP experiment [ 66 ]. This procedure includes the DNA-protein crosslinking with formaldehyde, sonication, immunoprecipitation, reversed crosslinking, and sequencing [ 66 ].
Using antibody of the histones, such as histone H3, ChIP-seq is immediately able to determine nucleosome positions. In addition to the techniques mentioned above, there are other techniques often used, such as Formaldehyde-assisted isolation of regulatory elements FAIRE-seq and ChIP-exo.
Sequencing provides information for regions of DNA that are not occupied by histones [ 67 ]. The nucleotides of the exonuclease-treated ends are determined using DNA sequencing. At the present, nucleosome sequencing dataset are mainly from MNase-seq.
A general analysis workflow includes data quality control, mapping, making nucleosome profile, determining nucleosome position, comparing between cell types, and associating with other omics-data expression data to find biological meanings. Sequencing quality control QC is to check the reads quality fraction of mapped reads and depth of coverage. Tools BWA and Bowtie are widely used in reads alignments. During the alignment process, multiple-mapping reads and duplication reads are often filtered so as to remove overrepresented regions of the genome due to technical bias [ 60 ].
Reads filtering can be performed with SAMtools or Picard tools. Data visualization helps to observe the reads distribution at specific locus. In IGV, the multiple types of annotation data are integrated, including gene information, epigenetic and expression data, single-nucleotide polymorphisms SNPs , repeat elements and functional information from the ENCODE, and other research projects.
With respect to nucleosomes sequencing data, there are two basic tasks in analysis. One is to calculate the nucleosome profile reads coverage both along the genomic coordinate and near the regulatory sites for instance the TSSs.
The other task is to infer the precise nucleosomes positions dyad position using the nucleosome profile so as to identify the nucleosome alteration among different cell types. For single-end MNase-seq data, one method to make nucleosome profile is as follows [ 70 ]. The absolute nucleosome occupancy value of each genomic site was expressed as the number of reads covering the genomic sites.
Second, nucleosome occupancy was scaled by dividing the occupancy value by the average nucleosome occupancy of the whole genome; i. With paired-end sequencing, it is assumed that the nucleosome midpoint is consistent with the midpoint of the forward and reverse reads.
Unless the reads are from the on type cell single cell , nucleosome positions actually represent the average positions in cell population. Therefore, the overlapping reads have to be clustered over genomic regions [ 60 ]. Calling nucleosomes actually is to find the peak positions along the nucleosome profile. Also, it allows us to detect three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework using MNase-seq datasets.
Other tools can be found in Table 2. Therefore, for these datasets, peaking calling is one central task. Based on the position-adjusted reads, MACS slides a window of size 2d across the genome to identify regions that are significantly enriched relative to the genome background. The P-value is derived from Poisson distribution. When a control sample is available, MACS can also estimate an empirical false discovery rate FDR for every peak by exchanging the ChIP-seq and control samples and identifying peaks in the control sample using the same set of parameters used for the ChIP-seq sample.
The bias can dominate the signal of interest for analyses and leads to false positive. More seriously, the bias tends to be different among the samples; thus, there is no general method to remove it [ 72 ]. Two facts associate the variability. One is GC content which is heterogeneous among the genome. Chromatin Remodeling and DNase 1 Sensitivity. Chromatin Remodeling in Eukaryotes. RNA Functions.
Citation: Phillips, T. Nature Education 1 1 In eukaryotes, DNA is tightly wound into a complex called chromatin.
Thanks to the process of chromatin remodeling, this complex can be "opened" so that specific genes are expressed. Aa Aa Aa. Chromatin Remodeling at a Glance. Nucleosomes, Histones, and Gene Transcription. Figure 1: Dynamic properties of nucleosomes. A Remodelling complexes of the SWR1 family can remove the canonical H2A-H2B dimers and replace them with Htz1-H2B dimers indicated in green , forming a variant nucleosome with unique tails that might bind unique regulatory proteins Reg.
Histone Modification and the Histone Code. Nucleosome Positioning and Reorganization. Signaling Function of Remodeled Chromatin. DNA Methylation. Figure 3: DNA methylation and gene silencing.
In early embryogenesis, DNA is largely devoid of methylation top left. References and Recommended Reading Cosgrove, M. Translating the histone code. Science , Lall, S. Genetics: A Conceptual Approach , 2nd ed. New York, Freeman, Sadava, D.
Life: The Science of Biology , 8th ed. New York, Freeman, Saha, A. Cell 98 , 1—4 Turner, B. Article History Close. Share Cancel. Revoke Cancel. Keywords Keywords for this Article. Save Cancel. Flag Inappropriate The Content is: Objectionable. Flag Content Cancel. Email your Friend. Submit Cancel. This content is currently under construction.
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Green Screen. Green Science. Bio 2. After the MNase digestion, we purified the digested products using a spin column A, Promega to avoid organic solvent contamination. The free energy of nucleosome assembly along the DNA sequence was estimated using a Markov model described in Ref. We performed coarse-grained MD simulations to investigate nucleosome repositioning when a translocase partially unwraps nucleosomal DNA.
The base-pairs are numbered relative to the dyad of the sequence. Initially, the nucleosome forms on the sequence. A model DNA translocase initially loaded on the upstream DNA proceeds in the downstream direction, colliding with the nucleosome. In these simulations, for histones, we used the coarse-grained model in which one particle represents one amino acid A structure-based potential stabilizes the native structure of the histone octamer The interactions between the histones were adjusted so that the complex disassembles in the absence of DNA For DNA, we used the coarse-grained model in which three particles represent one nucleotide Histone—DNA interactions include electrostatic interactions, excluded volume effects, and hydrogen bonds.
These setups have been previously calibrated and already applied to investigate nucleosome dynamics We modeled the translocase as a torus-shaped excluded volume potential. The translocation was realized by applying pN force toward the upstream direction half of the stall force [28 pN] of the bacterial RNA polymerase 28 to the base-pair most proximal to the center of the torus. The force causes the next downstream base-pair to be pulled into the torus; when this base-pair becomes the closest to the torus center, the force is then applied to this new base-pair.
The repetition of this procedure leads to a processive DNA translocation. Overall, the translocase relatively moves toward the nucleosome. In the initial structure, the translocase was placed at 91 bp upstream from the dyad Figure 1C i , D top left panel, and Supplementary Figure S2. As the simulation proceeded, the translocase collided with the nucleosome, unwrapping the nucleosomal DNA.
Note that the position of the translocase the center of the torus was, on average, 7 bp behind that of the base-pair to be detached Figure 1D top left panel due to the excluded volume of the translocase. In the current work, the translocase can partially unwrap the Widom strong nucleosome positioning sequence wrapping around the histone octamer.
Thus, it is reasonable to assume that the partial unwrapping can take place for the nucleosomes with a variety of DNA sequences. We repeatedly observed the repositioning in the 80 simulations Figure 1D , bottom panels and Supplementary Figure S2. In these simulations, the dyad repositioning distance ranged from 78 to bp Figure 1D , and Supplementary Figure S2.
In the repositioned nucleosome, the entry-side half of the histone octamer was wrapped by the right half of the sequence and the exit-side half by the random downstream DNA Figure 1A , C iv , and Supplementary Figure S4. In some trajectories, we observed a second repositioning event, though the limited length of the downstream DNA precludes the complete nucleosome formation Figure 1A and D , bottom left panel. This sudden and long-distance 78— bp repositioning cannot be explained by the pushing model in which one base-pair unwrapping is coupled to one base-pair nucleosome repositioning.
In our simulations, the force was applied as soon as the nucleotide enters the torus. In reality, however, the force must be generated at a certain stage of the ATP hydrolysis cycle second time scale , which is much slower than our simulation microsecond timescale. Thus, the in silico translocation speed is much faster than reality in which the partially unwrapped nucleosome structure would relax every time the translocase moves one base-pair. To study this process, we performed a second set of simulations starting from a partially unwrapped nucleosome structure obtained in the above simulations.
In this case, instead of pulling, we fixed the base-pair at the center of the torus at its initial position. First, we performed 80 simulations using the nucleosome conformation in which the translocase was at the —18th base-pair as the initial structure Figure 2A , top panel , which corresponds to just before the repositioning.
As the simulation proceeds, the structure relaxed and significantly changed from the initial structure Figure 2A and Movie S2. This result suggests that indeed the structure did not relax enough in the pulling simulations. Relaxation simulations of the partially unwrapped nucleosome. A Representative structures in the simulation trajectories. DNA, the histones, and the translocase are colored according to the color scheme in Figure 1C.
B Contact maps showing contacts between a histone octamer and DNA. The maps were averaged over the cluster members. C Cartoons explaining the lane-switch mechanism. The numbers next to the arrows represent relative transition rates. Then, we sought to classify the structures in the 80 relaxation simulations. To achieve this, we calculated the contact maps of the histone octamer and DNA and performed a k -means clustering. The optimal numbers of clusters were decided to be seven based on the Calinski—Harabasz index Supplementary Figure S5 41 , which is proportional to a ratio of inter- and intra-clusters dispersion.
Visual inspection revealed that the seven clusters contain one initial state, one off-pathway intermediate state, two on-pathway intermediate states, and three final states. Figure 2B and Supplementary Figure S6. The main pathway contains four of the seven states: one initial state, two on-pathway intermediate states, and one final state Figure 2B , C and Supplementary Figure S6A. In the initial state, the —9th to 59th base-pairs contact with superhelical locations SHL —1. In the intermediate I state, the probability of contacts between the -9th to 3rd base-pairs and SHL —1.
This result indicates the competitive binding of the —9th to 3rd base-pairs and the 59th to 71st base-pairs to the same amino acids. In the intermediate II state, the probability of contacts between the 3rd to 34th base-pairs and SHL 0. In this state, the DNA originally in the exit side switched its lane the binding site on the histone octamer.
In the final state, the 18th to th base-pairs bind to SHL —6. This state corresponds to the state after repositioning. Based on the simulations, we computed the transition rates between the states along the repositioning pathway.
This result suggests that spontaneous DNA dissociation after partial unwrapping is the bottleneck transition of downstream repositioning.
Next, we used the nucleosome structure in which the translocase is at the —31st base-pair as an initial structure. Note that, when the translocase is at the —31st base-pair and at the —18th base-pairs, the next base-pairs to be detached are at the —19th base-pairs and at the -9th base-pairs, respectively.
Thus, the simulation results suggest that the detachment of the —19th to —9th base-pairs from the histone octamer is essential for downstream repositioning. Based on these simulations, we propose the lane-switch mechanism. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding region lane to that just vacated by the unwrapping, and then the downstream DNA rewraps, completing downstream repositioning.
In the simulations, the several base-pairs of the wrapped end DNA occasionally dissociate from and re-associate to the histone octamer. Such breathing dynamics have also been observed experimentally, though the end position was altered by the translocase.
These tails are disordered polypeptides that occasionally become compact. These conformational changes may also help the tails to pass under DNA.
Collectively, the breathing dynamics, together with the conformational flexibility of the tails, may be necessary to facilitate the lane switching. Next, we sought to validate the lane-switch mechanism experimentally.
As the positioning sequence, the Widom sequence was modified so that the template strand does not contain thymine from the entry nucleotide to one nucleotide before the stall site. The stall sites were chosen at the —54th, —29th and —14th base-pairs relative position from the center of the positioning sequence Figure 3A and Supplementary Table S1.
MNase-seq assay 42 confirmed that this modification does not significantly affect nucleosome positioning Supplementary Figure S8. Restriction enzyme digestion assay. A The DNA sequence map of substrates used in the assay. The T7 RNAP stall sites where the translocase first encounter thymine are marked by the black arrows. B Cartoons of the experimental setup. On the other hand, if the nucleosome repositions upon transcription according to the lane-switch mechanism, the EcoRI site, but not the BssSI site, is occluded by the histone octamer.
The DNA substrates were digested at the pre- and post-transcription stages. The designed sequences contain the BssSI restriction site in the modified sequence, which is supposed to be occluded by a histone octamer The sequences also contain the EcoRI restriction site, which is supposed to be occluded when the histone octamer repositions downstream upon transcription. Thus, the repositioning frequency can be quantified by the restriction enzyme digestion assay Figure 3B.
The digested and undigested fragments were separated on a gel, and the intensities of the undigested product were measured Figure 3C.
These results suggests that the nucleosome repositions downstream when the T7 RNAP proceeds to the —14th base-pair.
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