Presence Of Linker Histones Research Articles

HMGB1 restores a dynamic chromatin environment in the presence of linker histone by deforming nucleosomal DNA.

The essential architectural protein HMGB1 increases accessibility of nucleosomal DNA and counteracts the effects of linker histone H1. However, HMGB1 is less abundant than H1 and binds nucleosomes more weakly raising the question of how HMGB1 effectively competes with H1. Here, we show that HMGB1 rescues H1's inhibition of nucleosomal DNA accessibility without displacing H1. HMGB1 also increases the dynamics of condensed, H1-bound chromatin. Cryo-EM shows that HMGB1 binds at internal locations on a nucleosome and locally distorts the DNA. These sites, which are away from the binding site of H1, explain how HMGB1 and H1 co-occupy a nucleosome. Our findings lead to a model where HMGB1 counteracts the activity of H1 by distorting nucleosomal DNA and by contacting the H1 C-terminal tail. Compared to direct competition, nucleosome co-occupancy by HMGB1 and H1 allows a greater diversity of dynamic chromatin states and may be generalizable to other chromatin regulators.

Explicit ion modeling predicts physicochemical interactions for chromatin organization

Molecular mechanisms that dictate chromatin organization in vivo are under active investigation, and the extent to which intrinsic interactions contribute to this process remains debatable. A central quantity for evaluating their contribution is the strength of nucleosome-nucleosome binding, which previous experiments have estimated to range from 2 to 14 kBT. We introduce an explicit ion model to dramatically enhance the accuracy of residue-level coarse-grained modeling approaches across a wide range of ionic concentrations. This model allows for de novo predictions of chromatin organization and remains computationally efficient, enabling large-scale conformational sampling for free energy calculations. It reproduces the energetics of protein-DNA binding and unwinding of single nucleosomal DNA, and resolves the differential impact of mono- and divalent ions on chromatin conformations. Moreover, we showed that the model can reconcile various experiments on quantifying nucleosomal interactions, providing an explanation for the large discrepancy between existing estimations. We predict the interaction strength at physiological conditions to be 9 kBT, a value that is nonetheless sensitive to DNA linker length and the presence of linker histones. Our study strongly supports the contribution of physicochemical interactions to the phase behavior of chromatin aggregates and chromatin organization inside the nucleus.

Explicit ion modeling predicts physicochemical interactions for chromatin organization.

Molecular mechanisms that dictate chromatin organization in vivo are under active investigation, and the extent to which intrinsic interactions contribute to this process remains debatable. A central quantity for evaluating their contribution is the strength of nucleosome-nucleosome binding, which previous experiments have estimated to range from 2 to 14 kBT. We introduce an explicit ion model to dramatically enhance the accuracy of residue-level coarse-grained modeling approaches across a wide range of ionic concentrations. This model allows for de novo predictions of chromatin organization and remains computationally efficient, enabling large-scale conformational sampling for free energy calculations. It reproduces the energetics of protein-DNA binding and unwinding of single nucleosomal DNA, and resolves the differential impact of mono- and divalent ions on chromatin conformations. Moreover, we showed that the model can reconcile various experiments on quantifying nucleosomal interactions, providing an explanation for the large discrepancy between existing estimations. We predict the interaction strength at physiological conditions to be 9 kBT, a value that is nonetheless sensitive to DNA linker length and the presence of linker histones. Our study strongly supports the contribution of physicochemical interactions to the phase behavior of chromatin aggregates and chromatin organization inside the nucleus.

Regulation of chromatin architecture by transcription factor binding.

Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:Max binds to E-box regulatory DNA elements throughout the genome to control the transcription of a large group of specific genes. We introduce an implicit modeling protocol for Myc:Max binding to mesoscale chromatin fibers at nucleosome resolution to determine TF effect on chromatin architecture and shed light into its mechanism of gene regulation. We first bind Myc:Max to different chromatin locations and show how it can direct fiber folding and formation of microdomains, and how this depends on the linker DNA length. Second, by simulating increasing concentrations of Myc:Max binding to fibers that differ in the DNA linker length, linker histone density, and acetylation levels, we assess the interplay between Myc:Max and other chromatin internal parameters. Third, we study the mechanism of gene silencing by Myc:Max binding to the Eed gene loci. Overall, our results show how chromatin architecture can be regulated by TF binding. The position of TF binding dictates the formation of microdomains that appear visible only at the ensemble level. At the same time, the level of linker histone and tail acetylation, or different linker DNA lengths, regulates the concentration-dependent effect of TF binding. Furthermore, we show how TF binding can repress gene expression by increasing fiber folding motifs that help compact and occlude the promoter region. Importantly, this effect can be reversed by increasing linker histone density. Overall, these results shed light on the epigenetic control of the genome dictated by TF binding.

Determining the interaction behavior of calf thymus DNA with berberine hydrochloride in the presence of linker histone: a biophysical study

The binding of small molecules with histone-DNA complexes can cause an interference in vital cellular processes such as cell division and the growth of cancerous cells that results in apoptosis. It is significant to study the interaction of small molecules with histone-DNA complex for the purpose of better understanding their mechanism of action, as well as designing novel and more effective drug compounds. The fluorescence quenching of ct-DNA upon interaction with Berberine has determined the binding of Berberine to ct-DNA with Ksv = 9.46 × 107 M−1. Ksv value of ct-DNA-Berberine in the presence of H1 has been observed to be 3.10 × 107 M−1, indicating that the H1 has caused a reduction in the binding affinity of Berberine to ct-DNA. In the competitive emission spectrum, ethidium bromide (EB) and acridine orange (AO) have been examined as intercalators through the addition of Berberine to ct-DNA complexes, which includes ctDNA-EB and ctDNA-AO. Although in the presence of histone H1 , we have observed signs of competition through the induced changes within the emission spectra, yet there has been apparently no competition between the ligands and probes. The viscosity results have confirmed the different behaviors of interaction between ctDNA and Berberine throughout the binary and ternary systems. We have figured out the IC50 and viability percent values at three different time durations of interaction between Berberine and MCF7 cell line. The molecular experiments have been completed by achieving the results of MTT assay, which have been confirmed to be in good agreement with molecular modeling studies.Communicated by Ramaswamy H. Sarma

Forced unraveling of chromatin fibers with nonuniform linker DNA lengths

The chromatin fiber undergoes significant structural changes during the cell's life cycle to modulate DNA accessibility. Detailed mechanisms of such structural transformations of chromatin fibers as affected by various internal and external conditions such as the ionic conditions of the medium, the linker DNA length, and the presence of linker histones, constitute an open challenge. Here we utilize Monte Carlo (MC) simulations of a coarse grained model of chromatin with nonuniform linker DNA lengths as found in vivo to help explain some aspects of this challenge. We investigate the unfolding mechanisms of chromatin fibers with alternating linker lengths of 26–62 bp and 44–79 bp using a series of end-to-end stretching trajectories with and without linker histones and compare results to uniform-linker-length fibers. We find that linker histones increase overall resistance of nonuniform fibers and lead to fiber unfolding with superbeads-on-a-string cluster transitions. Chromatin fibers with nonuniform linker DNA lengths display a more complex, multi-step yet smoother process of unfolding compared to their uniform counterparts, likely due to the existence of a more continuous range of nucleosome-nucleosome interactions. This finding echoes the theme that some heterogeneity in fiber component is biologically advantageous.

Development of a “Realistic” Model of the Dynamic Chromatin Fiber using an Integrated Computational Approach

DNA in eukaryotic cells is highly compacted into a hierarchical chromatin structure to fit inside the nucleus. Linker histones play an important role in this packing. Their interaction with the nucleosomes and intervening DNA linkers, in combination with linker length, are believed to affect chromatin folding and long-range interactions. The details of chromatin structure at this level of compaction are still an open question but have profound implications on biological processes, such as gene expression. Two major challenges are that linker histones contain a long intrinsically disordered C-terminal domain (CTD) and determination of high-resolution structures of chromatin has not been possible due to the large size of the system and its dynamic nature.We are taking advantage of the recently published 11-A resolution electron microscopy maps of two chromatin fibers (Song et al., 2014), to construct a complete low-resolution model of chromatin. High-resolution crystal structures of nucleosome core particles are fitted to the electron density maps and the intervening segments of linker DNA are modeled based on a sampling protocol that utilizes new DNA optimization methods. Situs is used to score all models against the density maps. Linker histones are modeled on the densities extracted after accounting for the nucleosomes and linker DNA. The modeled paths of the DNA linkers and the sites of the nucleosomes provide spatial constraints for positioning the globular core and modeling the folding of the disordered CTD of the linker histone. The pathways of the CTD are selected from ensembles of conformations obtained using ab initio structure prediction tools like Rosetta. We are using the model in coarse-grained Monte Carlo simulations of long-range interactions along arrays of precisely positioned nucleosomes in the presence of linker histones.

The draft assembly of the radically organized Stylonychia lemnae macronuclear genome.

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia’s macronucleus has a very unusual architecture, comprised variably and highly amplified “nanochromosomes,” each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

Linker Histone Phosphorylation Regulates Global Timing of Replication Origin Firing

Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing.

Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding, but It Facilitates Its Association with the Nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.

DNA methylation-dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone.

Dynamic alterations in chromatin structure mediated by postsynthetic histone modifications and DNA methylation constitute a major regulatory mechanism in DNA functioning. DNA methylation has been implicated in transcriptional silencing, in part by inducing chromatin condensation. To understand the methylation-dependent chromatin structure, we performed atomic force microscope (AFM) studies of fibers isolated from cultured cells containing normal or elevated levels of m5C. Chromatin fibers were reconstituted on control or methylated DNA templates in the presence or absence of linker histone. Visual inspection of AFM images, combined with quantitative analysis of fiber structural parameters, suggested that DNA methylation induced fiber compaction only in the presence of linker histones. This conclusion was further substantiated by biochemical results.

DNA Accessibility to Minor Groove Ligands in Core Nucleosome and Chromatosome

Using the circular dichroism spectra induced in the visible by the binding to the minor groove of DNA, we found that Hoechst 33258 is able to occupy its specific sites even when these are located inside the nucleosome structure. This high accessibility of the DNA in the nucleosome is not modified by the removal of the amino-terminal domains of the octamer histones and is not reduced by the presence of linker histone. Interesting and reasonable differences were found in the association constants.

A signal encoded in vertebrate DNA that influences nucleosome positioning and alignment.

Evidence is provided that the nucleotide triplet con-sensus non-T(A/T)G (abbreviated to VWG) influences nucleosome positioning and nucleosome alignment into regular arrays. This triplet consensus has been recently found to exhibit a fairly strong 10 bp periodicity in human DNA, implicating it in anisotropic DNA bendability. It is demonstrated that the experimentally determined preferences for nucleosome positioning in native SV40 chromatin can, to a large extent, be pre-dicted simply by counting the occurrences of the period-10 VWG consensus. Nucleosomes tend to form in regions of the SV40 genome that contain high counts of period-10 VWG and/or avoid regions with low counts. In contrast, periodic occurrences of the dinucleotides AA/TT, implicated in the rotational positioning of DNA in nucleosomes, did not correlate with the preferred nucleosome locations in SV40 chromatin. Periodic occurrences of AA did correlate with preferred nucleosome locations in a region of SV40 DNA where VWG occurrences are low. Regular oscillations in period-10 VWG counts with a dinucleosome period were found in vertebrate DNA regions that aligned nucleosomes into regular arrays in vitro in the presence of linker histone. Escherichia coli and plasmid DNA, which fail to align nucleosomes in vitro, lacked these regular VWG oscillations.

Asymmetric linker histone association directs the asymmetric rearrangement of core histone interactions in a positioned nucleosome containing a thyroid hormone response element.

We describe histone-DNA cross-linking in a positioned nucleosome containing a thyroid hormone response element (TRE) from the Xenopus laevis thyroid hormone receptor betaA gene (TRbetaA). Histones H3 and H4 are cross-linked to DNA in the nucleosome core within 30 base pairs to either side of the dyad axis. Histone H2A cross-links to DNA in the core at the dyad axis, and histones H2A and H2B have extensive interactions with DNA 40-80 bp away from the dyad axis. Linker histone H5 and the globular domain of Xenopus H1(0) associate asymmetrically with DNA at one edge of the TRbetaA nucleosome. Nevertheless, the asymmetric association of H5 leads to a significant rearrangement of core histone-DNA contacts at the dyad axis of the nucleosome. In the presence of linker histone, cross-linkings of H4 within 15 bp to one side of the dyad axis, of histone H2A at the dyad axis, and of H2A and H2B 40-80 bp to one side of the dyad axis are all reduced. This reduction in cross-linking occurs preferentially on the side of the nucleosome to which H5 is bound. Our results indicate that core histone contacts within mononucleosomes are conformationally dynamic and that linker histone incorporation at the edge of the nucleosome can influence core histone-DNA interactions in an asymmetric way including contacts at the dyad axis.