Brownian dynamics simulation of the cross-talking effect among modified histones on conformations of nucleosomes

Using Brownian dynamics simulation, we studied the effect of histone modifications On conformations of an array of nucleosomes in a segment of chromatin. The simulation demonstrated that the segment of chromatin shows the dynamic behaviour that its conformation can switch between a state with nearly all of the histones being wrapped by DNA and a state with nearly all of the histones being unwrapped by DNA, thus involving the ＂cross-talking＂ interactions among the histones. Each state can stay for a sufficiently long time. These conformational states are essential for gene expression or gene silence. The simulation also shows that these conformational states can be inherited by the daughter DNAs during DNA replication, giving a theoretical explanation of the epigenetic phenomenon.

Statistical analysis of conformational properties of periodic dinucleotide steps in nucleosomes

Deformability of DNA is important for its superhelical folding in the nucleosome and has long been thought to be facilitated by periodic occurrences of certain dinucleotides along the sequences, with the period close to 10.5 bases. This study statistically examines the conformational properties of dinucleotides containing the 10.5 - base periodicity and those without that periodicity through scanning all nucleosome structures provided in PDB. By categorizing performances on the distribution of step parameter values, averaged net values, standard deviations and deformability based on step conformational energies, we give a detailed description as to the deformation preferences correlated with the periodicity for the 10 unique types of dinucleotides and summarize the possible roles of various steps in how they facilitate DNA bending. The results show that the structural properties of dinucleotide steps are influenced to various extents by the periodicity in nucleosomes and some periodic steps have shown a clear tendency to take specific bending or shearing patterns.

The engagement of histone lysine methyltransferases with nucleosomes: structural basis, regulatory mechanisms, and therapeutic implications

Histone lysine methyltransferases(HKMTs)deposit methyl groups onto lysine residues on histones and play important roles in regulating chromatin structure and gene expression.The structures and functions of HKMTs have been extensively investigated in recent decades,significantly advancing our understanding of the dynamic regulation of histone methylation.Here,we review the recent progress in structural studies of representative HKMTs in complex with nucleosomes(H3K4,H3K27,H3K36,H3K79,and H4K20 methyltransferases),with emphasis on the molecular mechanisms of nucleosome recognition and trans-histone crosstalk by these HKMTs.These structural studies inform HKMTs'roles in tumorigenesis and provide the foundations for developing new therapeutic approachestargetingHKMTs incancers.

Transcriptional Regulation of the Ambient Temperature Response by H2A.Z Nucleosomes and HSF1 Transcription Factors in Arabidopsis

Temperature influences the distribution, range, and phenology of plants. The key transcriptional activators of heat shock response in eukaryotes, the heat shock factors （HSFs）, have undergone large-scale gene amplification in plants. While HSFs are central in heat stress responses, their role in the response to ambient temperature changes is less well understood. We show here that the warm ambient temperature transcriptome is dependent upon the HSFA1 clade ofArabidopsis HSFs, which cause a rapid and dynamic eviction of H2A.Z nucleosomes at target genes. A transcriptional cascade results in the activation of multiple downstream stress-responsive transcription factors, triggering large-scale changes to the transcriptome in response to elevated temperature. H2A.Z nucleosomes are enriched at temperature-responsive genes at non-inducible temperature, and thus likely confer inducibility of gene expression and higher responsive dynamics. We propose that the antagonistic effects of H2A.Z and HSF1 provide a mechanism to activate gene expression rapidly and precisely in response to temperature, while preventing leaky transcription in the absence of an activation signal.

Roles of histones and nucleosomes in gene transcription

This article reviews the latest research developments in the field of eukaryotic gene regulation by the structural alterations of chromatin and nucleosomes. The following issues are briefly addressed: (i) nucleosome and histone modifications by both the ATP-dependent remodeling complexes and the histone acetyltransferases and their roles in gene activation; (ii) competitive binding of histones and transcription factors on gene promoters, and transcription repression by nucleosomes; and (iii) influences of linker histone HI on gene regulation. Meanwhile, the significance and impact of these new research progresses, as well as issues worthwhile for further study are commented.

DNA damage in nucleosomes

Eukaryotic genomic DNA is packed into chromatin, whose fundamental structural unit is the nucleosome. As DNA-histone protein complexes, nucleosomes show different properties toward exogenous and endogenous DNA-damaging agents. This review summarizes nucleosome DNA damage due to different sources, including alkylating agents, radicals, UV radiation and reactive DNA damage intermediates. In most cases, the histone core protects the associated DNA against damage via its structure and/or scavenging of damaging agents. In contrast, histones react with damaged DNA and, in some instances, catalyze DNA damage in the nucleosome. The biological consequence of nucleosome DNA damage and future prospects in this field are briefly discussed.

High mobility group protein 1: A collaborator in nucleosome dynamics and estrogen-responsive gene expression

High mobility group protein 1(HMGB1) is a multifunctional protein that interacts with DNA and chromatin to influence the regulation of transcription, DNA replication and repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome(N) in a nonenzymatic,adenosine triphosphate-independent manner. As a result, the canonical nucleosome is converted to two stable, physically distinct nucleosome conformers. Although estrogen receptor(ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes(N' and N'') remain stable and exhibit a number of characteristics that are distinctly different from the canonical nucleosome. These findings complement previous studies that showed(1) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and(2) knock down of HMGB1 expression by siR NA precipitously reduced transcriptional activation. The findings indicate that a major facet of the mechanism of HMGB1 action involves a restructuring of aspects of the nucleosome that appear to relax structural constraints within the nucleosome. The findings are extended to reveal the differences between ER and the other steroid hormone receptors. A working proposal outlines mechanisms that highlight the multiple facets that HMGB1 may utilize in restructuring the nucleosome.

Nucleosome Positions and Differential Methylation Status of Various Regions within MLH1 CpG Island

Objective： To determine the relationship between nucleosome positions and formation of differential methylation of the reported region A, B, C, and D within the MLH1 CpG island. Methods： Methylation of the MLH1 promoter was analyzed by combined of bisulfite restriction assay. Chromatin of RKO and MGC803 cells were extracted and digested by MNase. Mononucleosomal DNA fragment was isolated and used as templates for detection of nucleosomal distribution by a battery of quantitative PCRs covering the full MLH1 promoter region. Results： The MLH1 was methylated in RKO and unmethylated in MGC803. At the region B, where methylation of CpG sites did not correlated with transcription of this gene well, qPCR product of the M-3 （-599nt ～ -475nt） fragment was amplified in both RKO and MGC803 cells. However, at the region C and D within the core promoter, where methylation of CpG sites correlated with loss of MLH1 transcription well, the M-7 （-257nt ～ -153nt） and M-8 （-189nt ～ -71nt） fragments were amplified remarkably only in RKO cells. Conclusion： Nucleosome may be the basic unit for both CpG methylation and methylation-related regulation of gene transcription. Methylation status of CpG sites within the same nucleosome may be homogeneous; between different nucleosomes, homogeneous or heterogeneous.

A Simple Method to Produce Sub-Nucleosome Complexes of High Purity In Vitro

With the identification of increasing number of chromatin modifiers, histone variants, histone post-translational modifications and their cross-talk, it is essential to validate these findings and interactions in vitro for which pure histone complexes are required. Although, the production of such complexes has been described earlier but still it remains a challenge for a non-specialist lab. Here we describe a protocol to quickly obtain large quantities of highly pure histones using bacterial expression system for GST pull-down and reconstitution experiments. In addition, we describe methods to quickly reconstitute and purify H2A/H2B dimers, H3/H4 tetramers and histone octamers for in vitro experiments. We demonstrate that these sub-complexes are properly folded and are hence, true representatives of the actual substrates in vivo. We also show that histones have a propensity to be non-specifically cleaved by proteases. Our results suggest that TEV protease is the most suitable protease while working with histones. The methodology described here should allow researchers to purify histone complexes in three days enabling functional and structural analyses of histone variants, mutants and post-translational modifications.

Large-scale long terminal repeat insertions produced a significant set of novel transcripts in cotton

Genomic analysis has revealed that the 1,637-Mb Gossypium arboreum genome contains approximately 81%transposable elements(TEs),while only 57%of the 735-Mb G.raimondii genome is occupied by TEs.In this study,we investigated whether there were unknown transcripts associated with TE or TE fragments and,if so,how these new transcripts were evolved and regulated.As sequence depths increased from 4 to 100 G,a total of 10,284 novel intergenic transcripts(intergenic genes)were discovered.On average,approximately 84%of these intergenic transcripts possibly overlapped with the long terminal repeat(LTR)insertions in the otherwise untranscribed intergenic regions and were expressed at relatively low levels.Most of these intergenic transcripts possessed no transcription activation markers,while the majority of the regular genic genes possessed at least one such marker.Genes without transcription activation markers formed their+1 and-1 nucleosomes more closely(only(117±1.4)bp apart),while twice as big spaces(approximately(403.5±46.0)bp apart)were detected for genes with the activation markers.The analysis of 183 previously assembled genomes across three different kingdoms demonstrated systematically that intergenic transcript numbers in a given genome correlated positively with its LTR content.Evolutionary analysis revealed that genic genes originated during one of the whole-genome duplication events around 137.7million years ago(MYA)for all eudicot genomes or 13.7 MYA for the Gossypium family,respectively,while the intergenic transcripts evolved around 1.6 MYA,resultant of the last LTR insertion.The characterization of these low-transcribed intergenic transcripts can facilitate our understanding of the potential biological roles played by LTRs during speciation and diversifications.

AtMCM10 promotes DNA replication-coupled nucleosome assembly in Arabidopsis

Minichromosome Maintenance protein 10(MCM10)is essential for DNA replication initiation and DNA elongation in yeasts and animals.Although the functions of MCM10 in DNA replication and repair have been well documented,the detailed mechanisms for MCM10 in these processes are not well known.Here,we identified AtMCM10 gene through a forward genetic screening for releasing a silenced marker gene.Although plant MCM10 possesses a similar crystal structure as animal MCM10,AtMCM10 is not essential for plant growth or development in Arabidopsis.AtMCM10 can directly bind to histone H3-H4 and promotes nucleosome assembly in vitro.The nucleosome density is decreased in Atmcm10,and most of the nucleosome density decreased regions in Atmcm10 are also regulated by newly synthesized histone chaperone Chromatin Assembly Factor-1(CAF-1).Loss of both AtMCM10 and CAF-1 is embryo lethal,indicating that AtM CM10 and CAF-1 are indispensable for replication-coupled nucleosome assembly.AtMCM10 interacts with both new and parental histones.Atmcm10 mutants have lower H3.1abundance and reduced H3K27me1/3 levels with releasing some silenced transposons.We propose that AtM CM10 deposits new and parental histones during nucleosome assembly,maintaining proper epigenetic modifications and genome stability during DNA replication.

DPB3 and DPB4 proteins regulate Medicago flowering and leaf anthocyanin biosynthesis

As important subunits of the leading-strand DNA polymerase epsilon,chromatin remodeling,and histone acetylation complexes,the H2A/H2B-like histone-fold domain-containing proteins DNA PO-LYMERASE II SUBUNIT B3(DPB3)and DPB4 play key roles in nucleosome assembly and heterochromatin maintenance during DNA replication in yeast,Drosophila,and mammals(He et al.,2017;Bellelli et al.,2018;Yu et al.,2018;Casari et al.,2021).

Heparin resistance in severe thermal injury:a prospective cohort study

Background:Low molecular-weight heparin(LMWH)is routinely administered to burn patients for thromboprophylaxis.Some studies have reported heparin resistance,yet the mechanism(s)and prevalence have not been systematically studied.We hypothesized that nucleosomes,composed of histone structures with associated DNA released from injured tissue and activated immune cells in the form of neutrophil extracellular traps(NETs or NETosis),neutralize LMWH resulting in suboptimal anticoagulation,assessed by reduction in anti-factor Xa activity.Methods:Blood was sampled from>15%total body surface area(TBSA)burn patients receiving LMWH on days 5,10 and 14.Peak anti-factor Xa(AFXa)activity,anti-thrombin(ATIII)activity,cellfree DNA(cfDNA)levels and nucleosome levels were measured.Mixed effects regression was adjusted for multiple confounders,including injury severity and ATIII activity,and was used to test the association between nucleosomes and AFXa.Results:A total of 30 patients with severe burns were included.Mean TBSA 43%(SD 17).Twentythree(77%)patients were affected by heparin resistance(defined by AFXa activity<0.2 IU/mL).Mean peak AFXa activity across samples was 0.18 IU/mL(SD 0.11).Mean ATIII was 81.9%activity(SD 20.4).Samples taken at higher LWMH doses were found to have significantly increased AFXa activity,though the effect was not observed at all doses,at 8000 IU no samples were heparin resistant.Nucleosome levels were negatively correlated with AFXa(r=−0.29,p=0.050)consistent with the hypothesis.The final model,with peak AFXa as the response variable,was adjusted for nucleosome levels(p=0.0453),ATIII activity(p=0.0053),LMWH dose pre-sample(p=0.0049),drug given(enoxaparin or tinzaparin)(p=0.03),and other confounders including severity of injury,age,gender,time point of sample.Conclusions:Heparin resistance is a prevalent issue in severe burns.Nucleosome levels were increased post-burn,and showed an inverse association with AFXa consistent with the hypothesis that they may interfere with the anticoagulant effect of heparin in vivo and contribute to heparin resistance.Accurate monitoring of AFXa activity with appropriate therapy escalation plans are recommended with dose adjustment following severe burn injury.

Nuclear actin switch of the IN080 remodeler

Eukaryotic DNA is wrapped in nucleosomes, which impede the access of transcription factors and regulatory proteins to template DNA. Chromatin remodelers utilize the energy from ATP hydrolysis to drive histone movement relative to nucleosomal DNA and nucleosome editing. Thus, they play critical roles in transcription, DNA replication, and damage repair, and their dysfunctions are often associated with diseases including cancers (Klages-Mundt et al., 2018). Chromatin remodelers can be generally categorized into IN080, SWI/SNF, CHD, and ISWI subfamilies, which share conserved catalytic ATPasetranslocase motors. However, how other auxiliary components of these multi-subunit machinery control their genomic recruitment and actions of DNA translocation remains as a major challenge of the field.

H2A.Z Represses Gene Expression by Modulating Promoter Nucleosome Structure and Enhancer Histone Modifications in Arabidopsis

Deposition of the histone variant H2A.Z at gene bodies regulates transcription by modifying chromatin accessibility in plants. However, the role of H2A.Z enrichment at the promoter and enhancer regions is unclear, and how H2A.Z interacts with other mechanisms of chromatin modification to regulate gene expression remains obscure. Here, we mapped genome-wide H2A.Z, H3K4me3, H3K27me3, Pol II, and nucleosome occupancy in Arabidopsis inflorescence. We showed that H2A.Z preferentially associated with H3K4me3 at promoters, while it was found with H3K27me3 at enhancers, and that H2A.Z deposition negatively correlated with gene expression. In addition, we demonstrated that H2A.Z represses gene expression by establishing low gene accessibility at ＋1 nucleosome and maintaining high gene accessibility at -1 nucleosome. We further showed that the high measures of gene responsiveness correlate with the H2A.Z-associated closed ＋1 nucleosome structure. Moreover, we found that H2A.Z represses enhancer activity by promoting H3K27me3 and preventing H3K4me3 histone modifications. This study provides a framework for future studies of H2A.Z functions and opens up new aspects for decoding the interplay between chromatin modification and histone variants in transcrip- tional control.

Sequence Signatures of Nucleosome Positioning in Caenorhabditis elegans

Our recent investigation in the protist Trichomonas vaginalis suggested a DNA sequence periodicity with a unit length of 120.9 nt, which represents a sequence signature for nucleosome positioning. We now extended our observation in higher eukaryotes and identified a similar periodicity of 175 nt in length in Caenorhabditis elegans. In the process of defining the sequence compositional characteristics, we found that the 10.5-nt periodicity, the sequence signature of DNA double helix, may not be sufficient for cross-nucleosome positioning but provides essential guiding rails to facilitate positioning. We further dissected nucleosome-protected sequences and identified a strong positive purine （AG） gradient from the 5＇-end to the 3＂-end, and also learnt that the nucleosome-enriched regions are GC-rich as compared to the nucleosome-free sequences as purine content is positively correlated with GC content. Sequence characterization allowed us to develop a hidden Markov model （HMM） algorithm for decoding nucleosome positioning computationally, and based on a set of training data from the fifth chromosome of C. elegans, our algorithm predicted 60%-70% of the well-positioned nucleosomes, which is 15%-20% higher than random positioning. We concluded that nucleosomes are not randomly positioned on DNA sequences and yet bind to different genome regions with variable stability, well-positioned nucleosomes leave sequence signatures on DNA, and statistical positioning of nucleosomes across genome can be decoded computationally based on these sequence signatures.

H3K27me3 Signal in the Cis Regulatory Elements Reveals the Differentiation Potential of Progenitors During Drosophila Neuroglial Development

Drosophila neural development undergoes extensive chromatin remodeling and precise epigenetic regulation.However,the roles of chromatin remodeling in establishment and maintenance of cell identity during cell fate transition remain enigmatic.Here,we compared the changes in gene expression,as well as the dynamics of nucleosome positioning and key histone modifications between the four major neural cell types during Drosophila neural development.We find that the neural progenitors can be separated from the terminally differentiated cells based on their gene expression profiles,whereas nucleosome distribution in the flanking regions of transcription start sites fails to identify the relationships between the progenitors and the differentiated cells.H3K27me3 signal in promoters and enhancers can not only distinguish the progenitors from the differentiated cells but also identify the differentiation path of the neural stem cells(NSCs)to the intermediate progenitor cells to the glial cells.In contrast,H3K9ac signal fails to identify the differentiation path,although it activates distinct sets of genes with neuron-specific and glia-related functions during the differentiation of the NSCs into neurons and glia,respectively.Together,our study provides novel insights into the crucial roles of chromatin remodeling in determining cell type during Drosophila neural development.

Distinct Role of Core Promoter Architecture in Regulation of Light-Mediated Responses in Plant Genes

In the present study, we selected four distinct classes of light-regulated promoters. The light-regulated promoters can be distinctly grouped into either TATA-box-containing or TATA-less （initiator-containing） promoters. Further, using either native promoters or their swapped versions of core promoter elements, we established that TATA-box and Inr （Initiator） elements have distinct mechanisms which are involved in light-mediated regulation, and these elements are not swappable. We identified that mutations in either functional TATA-box or Inr elements lead to the formation of nucleosomal structure. The nucleotide diversity in either the TATA-box or Inr element in Arabidopsis ecotypes proposes that the nucleotide variation in core promoters can alter the gene expression. We show that motif overrepresentation in light-activated promoters encompasses different specific regulatory motifs present downstream of TSS （transcription start site）, and this might serve as a key factor in regulating light promoters which are parallel with these elements. Finally, we conclude that the TATA-box or Inr element does not act in isolation, but our results clearly suggests the probable involvement of other distinct core promoter elements in concurrence with the TATA-box or Inr element to impart selectivity to light-mediated transcription.

Nuclear reassembly in vitro is independent of nucleosome/chromatin assembly

It was shown that nuclear reassembly was induced by small pieces of DNA fragments in cell free extracts of Xenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM Cellulose are used to eliminate the capacity of the egg extract S 150 to assemble chromatin, while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S 150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell free system of Xenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin.

Genome-wide Nucleosome Occupancy and Organization Modulates the Plasticity of Gene Transcriptional Status in Maize

Nucleosomes are fundamental units of chromatin that play critical roles in gene regulation by modulating DNA accessibility. However, their roles in regulating tissue-specific gene transcription are poorly understood. Here, we present genome-wide nucleosome maps of maize shoot and endosperm generated by sequencing the micrococcal nuclease digested nucleosomal DNA. The changes of gene transcriptional status between shoot and endosperm were accompanied by preferential nucleosome loss from the promoters and shifts in the first nucleosome downstream of the transcriptional start sites （＋1 nucleosome） and upstream of transcriptional termination sites （-1 nucleosome）. Intrinsically DNA-encoded nucleosome orga- nization was largely associated with the capacity of a gene to alter its transcriptional status among different tissues. Compared with tissue-specific genes, constitutively expressed genes showed more pronounced 5＇ and 3＇ nucleosome-depleted regions as well as further ＋1 nucleosome to transcriptional start sites and -1 nucleosome to transcriptional termination sites. Moreover, nucleosome organization was more highly correlated with the plasticity of gene transcriptional status than with its expression level when examined using in vivo and predicted nucleosome data. In addition, the translational efficiencies of tissue-specific genes appeared to be greater than those of constitutively expressed genes. Taken together, our results indicate that intrinsically DNA-encoded nucleosome organization is important, beyond its role in regulating gene expression levels, in determining the plasticity of gene transcriptional status.