Linker DNA Regions Research Articles

Structural basis of RNA polymerase II transcription on the chromatosome containing linker histone H1

In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing.

Structural basis for p53 binding to its nucleosomal target DNA sequence.

The tumor suppressor p53 functions as a pioneer transcription factor that binds a nucleosomal target DNA sequence. However, the mechanism by which p53 binds to its target DNA in the nucleosome remains elusive. Here we report the cryo-electron microscopy structures of the p53 DNA-binding domain and the full-length p53 protein complexed with a nucleosome containing the 20 base-pair target DNA sequence of p53 (p53BS). In the p53-nucleosome structures, the p53 DNA-binding domain forms a tetramer and specifically binds to the p53BS DNA, located near the entry/exit region of the nucleosome. The nucleosomal position of the p53BS DNA is within the genomic p21 promoter region. The p53 binding peels the DNA from the histone surface, and drastically changes the DNA path around the p53BS on the nucleosome. The C-terminal domain of p53 also binds to the DNA around the center and linker DNA regions of the nucleosome, as revealed by hydroxyl radical footprinting. These results provide important structural information for understanding the mechanism by which p53 binds the nucleosome and changes the chromatin structure for gene activation.

Structure of mitotic chromosomes

Chromatin fibers must fold or coil in the process of chromosome condensation. Patterns of coiling have been demonstrated for reconstituted chromatin, but the actual trajectories of fibers in condensed states of chromosomes could not be visualized because of the high density of the material. We have exploited partial decondensation of mitotic chromosomes to reveal their internal structure at sub-nucleosomal resolution by cryo-electron tomography, without the use of stains, fixatives, milling, or sectioning. DNA gyres around nucleosomes were visible, allowing the nucleosomes to be identified and their orientations to be determined. Linker DNA regions were traced, revealing the trajectories of the chromatin fibers. The trajectories were irregular, with almost no evidence of coiling and no short- or long-range order of the chromosomal material. The 146-bp core particle, long known as a product of nuclease digestion, is identified as the native state of the nucleosome, with no regular spacing along the chromatin fibers.

Oocyte-specific linker histone H1foo interacts with Esrrb to induce chromatin decondensation at specific gene loci

Linker histone H1 is mainly localized in the linker DNA region, between two nucleosome cores, and regulates chromatin structures linking gene expression. Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Although, from various previous studies, evidence related to H1foo function in chromatin structures is being accumulated, the distribution of H1foo at the target gene loci in a genome-wide manner and the molecular mechanism of H1foo-dependent chromatin architecture remain unclear. In this study, we aimed to identify the target loci and the physiological factor bound to H1foo at the loci. Chromatin immunoprecipitation sequencing analysis of H1foo-overexpressing mouse embryonic stem cells showed that H1foo is enriched around the transcriptional start sites of genes such as oocyte-specific genes and that the chromatin structures at these regions were relaxed. We demonstrated that H1foo was physiologically bound to the nuclear receptor estrogen-related receptor beta (Esrrb), and Esrrb was necessary for H1foo activity of chromatin decondensation at the target loci. The specific localization and interaction with Esrrb were validated in endogenous H1foo of oocytes. Overall, H1foo induces chromatin decondensation in a locus-specific manner and this function is achieved by interacting with Esrrb.

Distinct roles for RSC and SWI/SNF chromatin remodelers in genomic excision repair.

Nucleosomes are a significant barrier to the repair of UV damage because they impede damage recognition by nucleotide excision repair (NER). The RSC and SWI/SNF chromatin remodelers function in cells to promote DNA access by moving or evicting nucleosomes, and both have been linked to NER in yeast. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking RSC or SWI/SNF activity. Our data indicate that SWI/SNF is not generally required for NER but instead promotes repair of CPD lesions at specific yeast genes. In contrast, mutation or depletion of RSC subunits causes a general defect in NER across the yeast genome. Our data indicate that RSC is required for repair not only in nucleosomal DNA but also in neighboring linker DNA and nucleosome-free regions (NFRs). Although depletion of the RSC catalytic subunit also affects base excision repair (BER) of N-methylpurine (NMP) lesions, RSC activity is less important for BER in linker DNA and NFRs. Furthermore, our data indicate that RSC plays a direct role in transcription-coupled NER (TC-NER) of transcribed DNA. These findings help to define the specific genomic and chromatin contexts in which each chromatin remodeler functions in DNA repair, and indicate that RSC plays a unique function in facilitating repair by both NER subpathways.

Jagged Ends of Urinary Cell-Free DNA: Characterization and Feasibility Assessment in Bladder Cancer Detection.

Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends. Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing. The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83. Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.

Linker histone defines structure and self-association behaviour of the 177\xa0bp human chromatosome

Linker histones play essential roles in the regulation and maintenance of the dynamic chromatin structure of higher eukaryotes. The influence of human histone H1.0 on the nucleosome structure and biophysical properties of the resulting chromatosome were investigated and compared with the 177-bp nucleosome using Cryo-EM and SAXS. The 4.5 Å Cryo-EM chromatosome structure showed that the linker histone binds at the nucleosome dyad interacting with both linker DNA arms but in a tilted manner leaning towards one of the linker sides. The chromatosome is laterally compacted and rigid in the dyad and linker DNA area, in comparison with the nucleosome where linker DNA region is more flexible and displays structural variability. In solution, the chromatosomes appear slightly larger than the nucleosomes, with the volume increase compared to the bound linker histone, according to solution SAXS measurements. SAXS X-ray diffraction characterisation of Mg-precipitated samples showed that the different shapes of the 177 chromatosome enabled the formation of a highly ordered lamello-columnar phase when precipitated by Mg2+, indicating the influence of linker histone on the nucleosome stacking. The biological significance of linker histone, therefore, may be affected by the change in the polyelectrolyte and DNA conformation properties of the chromatosomes, in comparison to nucleosomes.

Linker histone variant H1T functions as a chromatin de-condenser on genic regions

Linker histone H1 is mainly localized in the linker DNA region, between two nucleosome cores, and regulates chromatin structures linking gene expression. There are 11 variants in histone H1, and each variant has unique functions. Our previous study demonstrates that one of the H1 variants, H1T is mainly localized in the nucleolus and targets the rDNA repeat region. Moreover, H1T condenses the chromatin structures on rDNA to repress pre-rRNA expression. Although H1T is partially localized in the nucleoplasm area, the functions of H1T in the non-repeat genic region are unclear. In this study, we aimed to identify the target loci and the role of H1T in the genic region. Chromatin immunoprecipitation sequencing analysis showed that H1T is localized around the transcriptional start site and the chromatin structures of the region were relaxed. H1T knockdown and overexpression experiments revealed that H1T induced chromatin de-condensation and was associated with the increased expression of target genes. Moreover, we observed H1T co-localization with transcriptional factor SPZ1 on the genic region. Collectively, H1T has opposing roles in the genic region and in rDNA repeats; H1T functions to facilitate chromatin relaxation linked gene activation.

UVA irradiation of BrU-substituted DNA in the presence of Hoechst 33258

Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil (BrU)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to BrU caused DNA cleavage preferentially at self-complementary 5′-AABrUBrU-3′ sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA.

N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena.

DNA N6-methyladenine (6mA) is newly rediscovered as a potential epigenetic mark across a more diverse range of eukaryotes than previously realized. As a unicellular model organism, Tetrahymena thermophila is among the first eukaryotes reported to contain 6mA modification. However, lack of comprehensive information about 6mA distribution hinders further investigations into its function and regulatory mechanism. In this study, we provide the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing. We provide evidence that 6mA occurs mostly in the AT motif of the linker DNA regions. More strikingly, these linker DNA regions with 6mA are usually flanked by well-positioned nucleosomes and/or H2A.Z-containing nucleosomes. We also find that 6mA is exclusively associated with RNA polymerase II (Pol II)-transcribed genes, but is not an unambiguous mark for active transcription. These results support that 6mA is an integral part of the chromatin landscape shaped by adenosine triphosphate (ATP)-dependent chromatin remodeling and transcription.

Post-translational modifications of the intrinsically disordered terminal domains of histone H1: effects on secondary structure and chromatin dynamics.

H1 linker histones are involved both in the maintenance of chromatin higher-order structure and in gene regulation. H1 binds to linker DNA regions on the surface of the nucleosome. In higher eukaryotes, H1 contains three distinct domains: a short N-terminal domain (NTD), a central globular domain, and a long C-terminal domain (CTD). Terminal domains determine subtype specificity and to a large extent the linker DNA binding and chromatin condensing properties of histone H1. This review is focused on the recent numerous studies that have provided insights in the role of H1 terminal domains in chromatin dynamics. The N- and C-terminal domains behave as intrinsically disordered proteins with coupled binding and folding. We examine the potential kinetic advantages of intrinsic disorder in the recognition of the specific H1 binding sites in chromatin. As typical intrinsically disordered regions, H1 terminal domains are post-translationally modified. Post-translational modifications in the NTD determine the interaction of histone H1 with other proteins involved in heterochromatin formation and transcriptional regulation, while phosphorylation by cyclin-dependent kinases modulates the secondary structure of the CTD and chromatin condensation. We review the arguments in favor of the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of partial phosphorylation in interphase chromatin relaxation. In addition, the interplay of histone H1 and other chromatin architectural proteins, such as proteins of the high-mobility group, protamines, and MeCP2, is associated with changes in chromatin structure.

A quantitative investigation of linker histone interactions with nucleosomes and chromatin.

Linker histones such as H1 are abundant basic proteins that bind tightly to nucleosomes, thereby acting as key organizers of chromatin structure. The molecular details of linker histone interactions with the nucleosome, and in particular the contributions of linker DNA and of the basic C-terminal tail of H1, are controversial. Here we combine rigorous solution-state binding assays with native gel electrophoresis and Atomic Force Microscopy, to quantify the interaction of H1 with chromatin. We find that H1 binds nucleosomes and nucleosomal arrays with very tight affinity by recognizing a specific DNA geometry minimally consisting of a solitary nucleosome with a single ~18 base pair DNA linker arm. The association of H1 alters the conformation of trinucleosomes so that only one H1 can bind to the two available linker DNA regions. Neither incorporation of the histone variant H2A.Z, nor the presence of neighboring nucleosomes affects H1 affinity. Our data provide a comprehensive thermodynamic framework for this ubiquitous chromatin architectural protein.

The shades of gray of the chromatin fiber: recent literature provides new insights into the structure of chromatin.

The chromatin fiber consists of a string of nucleosomes connected by linker DNA regions. The hierarchy of folding of this fiber within the cell has long been controversial, and the existence of an originally described 30 nm fiber has been debated and reviewed extensively. This review contextualizes two recent papers on this topic that suggest the 30 nm fiber to be an over-simplification. The idealized model from the first study provides good insight into the constraints and histone participation in the maintenance of the fiber structure. The second paper provides a theoretical description of a more realistic view of the highly heterogeneous and dynamic chromatin organization in the in vivo setting. It is now time to abandon the highly regular "one start" solenoidal 30 nm structure and replace it with a more realistic highly dynamic, polymorphic fiber.

In vitro determination of the 30 nm chromatin fiber structure (564.1)

To be able to fit 6 ft. of human DNA into a cell’s nucleus, an elaborate organizational system is necessary to compact the genomic DNA first into nucleosomes, and then into a 30 nm chromatin fiber. The ability to figure out the spatial conformation between these nucleosomes is vital in understanding gene regulation of underlying DNA. Literature data support two different models of the chromatin fiber that differ in their geometry and possible impact on the accessibility of the underlying DNA sequence. The “zig‐zag” model uses a straight linker DNA region, while the “solenoid” model illustrates a bent DNA linker, greatly influencing which nucleosomes are in close space in relation to one another. However, there is debate about which of these models actually represents the true structure. High resolution in vivo nucleosome maps show that the length of DNA between two nucleosomes is not constant across a genome. Computational studies have shown that changing this linker‐length can drastically change the available compacted structures accessible to an array of nucleosomes. To determine how much the properties of the linker DNA affect chromatin structure, we are developing a quantitative crosslinking assay. In our studies, we use this method to investigate the linker‐length dependence of chromatin structure in synthetic nucleosome arrays. Initial results have shown evidence for both zig‐zag and solenoid models depending on solution conditions.

An analysis and prediction of nucleosome positioning based on information content

Nucleosome positioning plays a key role in the regulation of many biological processes. In this study, the statistical difference of information content was investigated in nucleosome and linker DNA regions across eukaryotic organisms. By analyzing the information redundancy, D k , in Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans genomes, the short-range dominance of nucleotide correlation in nucleosome and linker DNA regions was confirmed. Significant difference of the D k value between the nucleosome and linker DNA regions was also found. The underlying reason for many successful oligonucleotide-based predictions of nucleosome positioning in eukaryotic model organisms may be attributed to the short-range dominance of nucleotide correlation in the nucleosome and linker DNA regions. When applying power spectrum analysis to the nucleosome and linker DNA regions, some obvious differences in sequence periodic signals were observed. The parameter F k was introduced to describe particular base correlation. Furthermore, the support vector machine combining F k was used to classify nucleosome and linker DNA regions in Homo sapiens, Oryzias latipes, C. elegans, Candida albicans, and S. cerevisiae. Independent test demonstrated that a good performance can be achieved by using this algorithm. This result further revealed that base correlation information has an important role in nucleosome positioning.

Interactive Chromatin Folding, Nucleosome (Mis)Positioning and Chromatin Structure

In the absence of the myriad influences that exist in a cell nucleus, the positioning and stability of nucleosomes on a short segment of DNA are governed by the material properties of the DNA. In the simplest view sequences of DNA that are intrinsically flexible or that are intrinsically curved lead to nucleosome positioning. Nucleosome positioning is thus an intra-nucleosome based effect. In vivo or for sequences of DNA that support multiple nucleosomes, nucleosome-nucleosome interactions and extra-nucleosomal (e.g. chromatin remodeling factors) interactions must also be considered.In case of intra-nucleosomal based positioning, DNA material defects (defined as variations in flexibility or conformation) are hidden because the histones force the DNA to assume a well defined super helical fold. Except for specifically constructed sequences of DNA, chromatin structures determined by intra-nucleosome interactions are irregular because the nucleosome positions will be irregular.In case inter- or extra- nucleosomal interactions determine the nucleosome positions some DNA defects will be exposed in linker DNA regions. Exposure of the defects impacts the global structure of chromatin, e.g. a bent linker yields bent chromatin.We propose that chromatin structure is biased toward one method of positioning or the other depending on environmental conditions. Thus for a given sequence of DNA both irregular and regular structures can be obtained. Our Interactive Chromatin Modeling Web Server http://www.latech.edu/∼bishop captures these ideas. Here we present a case study of how ICM Web can be used to study the folding of the mouse mammary tumor virus promoter complex(MMTV). ICM properly predicts the locations of six positioned nucleosomes in the MMTV and shows how mispositioning reveals sequence specific material defects that bend chromatin.

Correction to ‘Characteristics of nucleosomes and linker DNA regions on the genome of the basidiomycete Mixia osmundae revealed by mono- and dinucleosome mapping’

Correction to ‘Characteristics of nucleosomes and linker DNA regions on the genome of the basidiomycete <i>Mixia osmundae</i> revealed by mono- and dinucleosome mapping’

Nucleosome Positioning.

Nucleosome positioning is not only related to genomic DNA compaction but also to other biological functions. After the chromatin is digested by micrococcal nuclease, nucleosomal (nucleosome-bound) DNA fragments can be sequenced and mapped on the genomic DNA sequence. Due to the development of modern DNA sequencing technology, genome-wide nucleosome mapping has been performed in a wide range of eukaryotic species. Comparative analyses of the nucleosome positions have revealed that the nucleosome is more frequently formed in exonic than intronic regions, and that most of transcription start and translation (or transcription) end sites are located in nucleosome linker DNA regions, indicating that nucleosome positioning influences transcription initiation, transcription termination, and gene splicing. In addition, nucleosomal DNA contains guanine and cytosine (G + C)-rich sequences and a high level of cytosine methylation. Thus, the nucleosome positioning system has been conserved during eukaryotic evolution.

Characteristics of nucleosomes and linker DNA regions on the genome of the basidiomycete Mixia osmundae revealed by mono- and dinucleosome mapping

We present findings on the nucleosomal arrangement in the genome of the basidiomycete Mixia osmundae, focusing on nucleosomal linker DNA regions. We have assembled the genomic sequences of M. osmundae, annotated genes and transcription start sites (TSSs) on the genome, and created a detailed nucleosome map based on sequencing mono- and dinucleosomal DNA fragments. The nucleosomal DNA length distribution of M. osmundae is similar to that of the filamentous ascomycete Aspergillus fumigatus, but differs from that of ascomycetous yeasts, strongly suggesting that nucleosome positioning has evolved primarily through neutral drift in fungal species. We found clear association between dinucleotide frequencies and linker DNA regions mapped as the midpoints of dinucleosomes. We also describe a unique pattern found in the nucleosome-depleted region upstream of the TSS observed in the dinucleosome map and the precursor status of dinucleosomes prior to the digestion into mononucleosomes by comparing the mono- and dinucleosome maps. We demonstrate that observation of dinucleosomes as well as of mononucleosomes is valuable in investigating nucleosomal organization of the genome.

Linker Histone Subtypes Differ in Their Effect on Nucleosomal Spacing In Vivo

Linker histone H1 is located on the surface of the nucleosome where it interacts with the linker DNA region and stabilizes the 30-nm chromatin fiber. Vertebrates have several different, relatively conserved subtypes of H1; however, the functional reason for this is unclear. We have previously shown that H1 can be reconstituted in Xenopus oocytes, cells that lack somatic H1, by cytosolic mRNA injection and incorporated into in vivo assembled chromatin. Using this assay, we have expressed individual H1 subtypes in the oocytes to study their effect on chromatin structure using nucleosomal repeat length (NRL) as readout. We have compared chicken differentiation-specific histone H5, Xenopus differentiation-specific xH10 and the somatic variant xH1A as well as the ubiquitously expressed human somatic subtypes hH1.2, hH1.3, hH1.4 and hH1.5. This shows that all subtypes, except for human H1.5, result in a saturable increase in NRL. hH1.4 results in an increase of approximately 13–20 bp as does xH10 and xH1A. chH5 gives rise to the same or slightly longer increase compared to hH1.4. Interestingly, both hH1.2 and hH1.3 show a less extensive increase of only 4.5–7 bp in the NRL, thus yielding the shortest increase of the studied subtypes. We show for the first time in an in vivo system lacking H1 background that ubiquitously expressed and redundant H1 subtypes that coexist in most types of cells of higher eukaryotes differ in their effects on the nucleosomal spacing in vivo. This suggests that H1 subtypes have different roles in the organization and functioning of the chromatin fiber.