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 ...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.展开更多
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 ...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.展开更多
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 a...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.展开更多
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-damagin...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.展开更多
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 al...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.展开更多
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 vitr...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.展开更多
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 investi...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.展开更多
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,...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.展开更多
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...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).展开更多
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 systematica...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.展开更多
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...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.展开更多
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 tra...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.展开更多
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 assem...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.展开更多
A nucleosome contains two copies of each histone H2A,H2B,H3 and H4.Histone H3 K4me0 and K36me3are two key chromatin marks for de novo DNA methylation catalyzed by DNA methyltransferases in mammals.However,it remains u...A nucleosome contains two copies of each histone H2A,H2B,H3 and H4.Histone H3 K4me0 and K36me3are two key chromatin marks for de novo DNA methylation catalyzed by DNA methyltransferases in mammals.However,it remains unclear whether K4me0 and K36me3 marks on both sister histone H3s regulate de novo DNA methylation independently or cooperatively.Here,taking advantage of the bivalent histone H3 system in yeast,we examined the contributions of K4 and K36 on sister histone H3s to genomic DNA methylation catalyzed by ectopically co-expressed murine Dnmt3a and Dnmt3L.The results show that lack of both K4me0 and K36me3 on one sister H3 tail,or lack of K4me0 and K36me3 on respective sister H3s results in a dramatic reduction of 5mC,revealing a synergy of two sister H3s in DNA methylation regulation.Accordingly,the Dnmt3a or Dnmt3L mutation that disrupts the interaction of Dnmt3aADD domain-H3K4me0,Dnmt3LADD domain-H3K4me0,orDnmt3aPWWP domain-H3K36me3 causes a significant reduction of DNA methylation.These results support the model that each heterodimeric Dnmt3a-Dnmt3L reads both K4me0 and K36me3 marks on one tail of sister H3s,and the dimer of heterodimeric Dnmt3a-Dnmt3L recognizes two tails of sister histone H3s to efficiently execute de novo DNA methylation.展开更多
In eukaryotic cells,histones are packaged into octameric core particles with DNA wrapping around to form nucleosomes,which are the basic units of chromatin(Kornberg and Thomas,1974).Multicellular organisms utilise chr...In eukaryotic cells,histones are packaged into octameric core particles with DNA wrapping around to form nucleosomes,which are the basic units of chromatin(Kornberg and Thomas,1974).Multicellular organisms utilise chromatin marks to translate one single genome into hundreds of epigenomes for their corresponding cell types.Inheritance of epigenetic status is critical for the maintenance of gene expression profile during mitotic cell divisions(Allis et al.,2006).During S phase,canonical histones are deposited onto DNA in a replication-coupled manner(Allis et al.,2006).To understand how dividing cells overcome the dilution of epigenetic marks after chromatin duplication,DNA replication coupled(RC)nucleosome assembly has been of great interest.In this review,we focus on the potential influence of RC nucleosome assembly processes on the maintenance of epigenetic status.展开更多
Epigenetic research focuses on heritable changes beyond the DNA sequence, which has led to a revolution in biologicalstudies and benefits in many other fields. The well-known model ciliate, Tetrahymena thermophila off...Epigenetic research focuses on heritable changes beyond the DNA sequence, which has led to a revolution in biologicalstudies and benefits in many other fields. The well-known model ciliate, Tetrahymena thermophila offers a unique system forepigenetic studies due to its nuclear dimorphism and special mode of sexual reproduction (conjugation), as well as abundantgenomic resources and genetic tools. In this paper, we summarize recent progress made by our research team and collaboratorsin understanding epigenetic mechanisms using Tetrahymena. This includes: (1) providing the first genome-wide basepair-resolution map of DNA N6-methyladenine (6mA) and revealed it as an integral part of the chromatin landscape;(2)dissecting the relative contribution of cis・ and trans- elements to nucleosome distribution by exploring the unique nucleardimorphism of Tetrahymena, (3) demonstrating the epigenetic controls of RNAi-dependent Polycomb repression pathwayson transposable elements, and (4) identifying a new histone monomethyltransferase, TXR1 (Tetrahymena Trithorax 1), thatfacilitates replication elongation through 让s substrate histone H3 lysine 27 monomethylation (H3K27mel).展开更多
High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the prese...High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the human e-globin gene promoter (e-promo-ter, -177-+1 bp) has been examined by using both the in vitro nucleosome reconstitution and the electrophoresis mobility shift assay (EMSA). We found that HMG1/2 proteins could bind to the naked e-promoter DNA, however, HMG14/17 could not. Using the in vitro nucleosome reconstitution, we revealed that HMG14/17 could bind to the mononucleosome reconstituted in vitro with E-promoter, while HMG1/2 could not. Those results indicate that the binding of HMG proteins to e-promoter is dynamic as the nucleosome assembling and disassembling. We speculated that this selective binding of HMG proteins to e-promoter might play a critical role in the regulation of e-globin gene expression.展开更多
In the budding yeast Saccharomyces cerevisiae,heterochromatin structure is found at three chromosome regions,which are homothallic mating-type loci,rDNA regions and telomeres.To address how telomere heterochromatin is...In the budding yeast Saccharomyces cerevisiae,heterochromatin structure is found at three chromosome regions,which are homothallic mating-type loci,rDNA regions and telomeres.To address how telomere heterochromatin is assembled under physiological conditions,we employed a de novo telomere addition system,and analyzed the dynamic chromatin changes of the TRP1 reporter gene during telomere elongation.We found that integrating a 255-bp,but not an 81-bp telomeric sequence near the TRP1 promoter could trigger Sir2 recruitment,active chromatin mark(s)' removal,chromatin compaction and TRP1 gene silencing,indicating that the length of the telomeric sequence inserted in the internal region of a chromosome is critical for determining the chromatin state at the proximal region.Interestingly,Rif1 but not Rif2 or yKu is indispensable for the formation of intra-chromosomal silent chromatin initiated by telomeric sequence.When an internal short telomeric sequence(e.g.,81 bp) gets exposed to become a de novo telomere,the herterochromatin features,such as Sir recruitment,active chromatin mark(s)' removal and chromatin compaction,are detected within a few hours before the de novo telomere reaches a stable length.Our results recapitulate the molecular dynamics and reveal a coherent picture of telomere heterochromatin formation.展开更多
It has been demonstrated in the last ten years that the nuclear reassembly may occur in the cell-free systems from frog egg extracts added with exogenous naked DNA. However, there remains an open question : is the cel...It has been demonstrated in the last ten years that the nuclear reassembly may occur in the cell-free systems from frog egg extracts added with exogenous naked DNA. However, there remains an open question : is the cell-free reassembled nucleus structurally similar to the nucleus in the intact cell ? That is, does the cell-free reassembled nucleus contain nucleosomes and chromatin? For this issue, we have designed experiments for identifying the internal structures of the cell-free reassembled nucleus. These experiments show that the nucleus reassembled in vitro also contains chromatin which is composed of typical 10 nm nucleosome fibers of "beads-on-a-string", 30 nm filaments and the next higher-order structures. The digestion experiment with the enzyme micrococcal nuclease has demonstrated that the DNA in the nucleosome of the reconstituted chromatin is about 200 base pairs (bp) in length, of which 165 bp may be in the nucleosome particle, and 35 bp may be in the linker between two particles.展开更多
In eukaryotic cells,the smallest subunit of chromatin is the nucleosome,which consists of a segment of DNA wound on histone protein cores. Despite many years of effort,the process of nucleosome assembly and disassembl...In eukaryotic cells,the smallest subunit of chromatin is the nucleosome,which consists of a segment of DNA wound on histone protein cores. Despite many years of effort,the process of nucleosome assembly and disassembly is still not very clear. Here,we present a convenient method to investigate the process of nucleosome assembly at the single molecule level. We invented a novel system derived from the yeast nucleoplasmic extracts(YNPE),and demonstrated that the YNPE supports the nucleosome assembly under physiological condition. By combining the total internal reflection fluorescence microscopy with microfluidic flow-cell technique,the dynamic process of nucleosome assembly in YNPE was visualized at single-molecule level. Our system provides a novel in vitro single-molecule tool to investigate the dynamics of nucleosome assembly under physiological conditions.展开更多
文摘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.
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB37010303 to Y.C.)the National Natural Science Foundation of China(31670748,31970576 to Y.C.,and 32071195 and 31900934 to Y.L.)+1 种基金the Young Elite Scientist Sponsorship Program by Chinese Association for Science and Technology(YESS20170198 to Y.L.)the National Postdoctoral Program for Innovative Talents(Bx201700263 to Y.L.).
文摘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.
基金Part of the work in this article was supported by the National Natural Science Foundation of China (Grant No. 3997038) and the National "973" Basic Research Project (Grant No. G19I99053902).
文摘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.
基金supported by the National Natural Science Foundation of China(21572109,21877064,21740002)the National Key R&D Program of China(2017YFD0200501)
文摘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.
文摘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.
文摘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.
基金supported by the National Key Research and Development Program of China(2022YFF1001400)the National Natural Science Foundation of China(31690090,32200286,32070207)+2 种基金Foundation of Hubei Hongshan Laboratory(2021hszd014)Hubei Provincial Natural Science Foundation of China(2021CFA055,2021BBA102)the China Postdoctoral Science Foundation(2022TQ0240,2022M722470)。
文摘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.
基金supported by National Natural Science Foundation of China (31921001)。
文摘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.
基金supported by grants from Ministry of Agriculture and Rural Affairs of the People’s Republic of China(2022ZD04011)the National Natural Science Foundation of China(32070554,32271759)+1 种基金Hainan Yazhou Bay Seed Laboratory(B21HJ0215)Hohhot Innovation Consortium Project(RC2022-1)。
文摘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).
基金funded by the Scar Free Foundation and National Institute for Health Research Surgical Reconstruction and Microbiology Research Centre,British Society of Haematology and Royal College of Surgeons.
文摘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.
基金the National Key R&D Program of China(2018YFC1004500)the National Natural Science Foundation of China(31872817 to B.L).
文摘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.
基金supported by the National Key R&D Program of China(Grant No.2016YFA0100400)the National Natural Science Foundation of China(Grant Nos.31771419,31721003,and 31800858)China Postdoctoral Science Foundation(Grant No.2017M621526)
文摘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.
文摘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.
基金We thank members of Zhou lab for the help,discussions and suggestions for this project.This work was supported by grants from the Ministry of Science and Technology(2016YFA0500701)the National Natural Science Foundation of China(NSFC 31521061)the Chinese Academy of Sciences(XDB19000000)to J.QZ.
文摘A nucleosome contains two copies of each histone H2A,H2B,H3 and H4.Histone H3 K4me0 and K36me3are two key chromatin marks for de novo DNA methylation catalyzed by DNA methyltransferases in mammals.However,it remains unclear whether K4me0 and K36me3 marks on both sister histone H3s regulate de novo DNA methylation independently or cooperatively.Here,taking advantage of the bivalent histone H3 system in yeast,we examined the contributions of K4 and K36 on sister histone H3s to genomic DNA methylation catalyzed by ectopically co-expressed murine Dnmt3a and Dnmt3L.The results show that lack of both K4me0 and K36me3 on one sister H3 tail,or lack of K4me0 and K36me3 on respective sister H3s results in a dramatic reduction of 5mC,revealing a synergy of two sister H3s in DNA methylation regulation.Accordingly,the Dnmt3a or Dnmt3L mutation that disrupts the interaction of Dnmt3aADD domain-H3K4me0,Dnmt3LADD domain-H3K4me0,orDnmt3aPWWP domain-H3K36me3 causes a significant reduction of DNA methylation.These results support the model that each heterodimeric Dnmt3a-Dnmt3L reads both K4me0 and K36me3 marks on one tail of sister H3s,and the dimer of heterodimeric Dnmt3a-Dnmt3L recognizes two tails of sister histone H3s to efficiently execute de novo DNA methylation.
基金supported by the National Programs for High Technology Research and Development Program(863 Program)(Grant No.2007AA02Z1A6,to B.Z.).
文摘In eukaryotic cells,histones are packaged into octameric core particles with DNA wrapping around to form nucleosomes,which are the basic units of chromatin(Kornberg and Thomas,1974).Multicellular organisms utilise chromatin marks to translate one single genome into hundreds of epigenomes for their corresponding cell types.Inheritance of epigenetic status is critical for the maintenance of gene expression profile during mitotic cell divisions(Allis et al.,2006).During S phase,canonical histones are deposited onto DNA in a replication-coupled manner(Allis et al.,2006).To understand how dividing cells overcome the dilution of epigenetic marks after chromatin duplication,DNA replication coupled(RC)nucleosome assembly has been of great interest.In this review,we focus on the potential influence of RC nucleosome assembly processes on the maintenance of epigenetic status.
基金supported by Natural Science Foundation ofShandong Province (JQ201706)The Marine S&T Fund of ShandongProvince for Pilot National Laboratory for Marine Science and Technology(Qingdao) (2018SDKJ0406-2)+1 种基金Fundamental Research Fundsfor the Central Universities (201841005)the Blue Life BreakthroughProgram of LMBB of Qingdao National Laboratory for MarineScience and Technology (MS2018N004).
文摘Epigenetic research focuses on heritable changes beyond the DNA sequence, which has led to a revolution in biologicalstudies and benefits in many other fields. The well-known model ciliate, Tetrahymena thermophila offers a unique system forepigenetic studies due to its nuclear dimorphism and special mode of sexual reproduction (conjugation), as well as abundantgenomic resources and genetic tools. In this paper, we summarize recent progress made by our research team and collaboratorsin understanding epigenetic mechanisms using Tetrahymena. This includes: (1) providing the first genome-wide basepair-resolution map of DNA N6-methyladenine (6mA) and revealed it as an integral part of the chromatin landscape;(2)dissecting the relative contribution of cis・ and trans- elements to nucleosome distribution by exploring the unique nucleardimorphism of Tetrahymena, (3) demonstrating the epigenetic controls of RNAi-dependent Polycomb repression pathwayson transposable elements, and (4) identifying a new histone monomethyltransferase, TXR1 (Tetrahymena Trithorax 1), thatfacilitates replication elongation through 让s substrate histone H3 lysine 27 monomethylation (H3K27mel).
基金This work was supported by the National Natural Science Foundation of China (Grant No. 39893320)the Foundation of Chinese Academy of Sciences (Grant No. KJ 951 -A 1 -603 (2)).
文摘High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the human e-globin gene promoter (e-promo-ter, -177-+1 bp) has been examined by using both the in vitro nucleosome reconstitution and the electrophoresis mobility shift assay (EMSA). We found that HMG1/2 proteins could bind to the naked e-promoter DNA, however, HMG14/17 could not. Using the in vitro nucleosome reconstitution, we revealed that HMG14/17 could bind to the mononucleosome reconstituted in vitro with E-promoter, while HMG1/2 could not. Those results indicate that the binding of HMG proteins to e-promoter is dynamic as the nucleosome assembling and disassembling. We speculated that this selective binding of HMG proteins to e-promoter might play a critical role in the regulation of e-globin gene expression.
基金supported by the National Natural Science Foundation of China (Nos.31230040,31461143003 and 31521061 to J.Q.Z.)Ministry of Science and Technology of the People's Republic of China(No. 2013CB910403 toJ.Q.Z.)
文摘In the budding yeast Saccharomyces cerevisiae,heterochromatin structure is found at three chromosome regions,which are homothallic mating-type loci,rDNA regions and telomeres.To address how telomere heterochromatin is assembled under physiological conditions,we employed a de novo telomere addition system,and analyzed the dynamic chromatin changes of the TRP1 reporter gene during telomere elongation.We found that integrating a 255-bp,but not an 81-bp telomeric sequence near the TRP1 promoter could trigger Sir2 recruitment,active chromatin mark(s)' removal,chromatin compaction and TRP1 gene silencing,indicating that the length of the telomeric sequence inserted in the internal region of a chromosome is critical for determining the chromatin state at the proximal region.Interestingly,Rif1 but not Rif2 or yKu is indispensable for the formation of intra-chromosomal silent chromatin initiated by telomeric sequence.When an internal short telomeric sequence(e.g.,81 bp) gets exposed to become a de novo telomere,the herterochromatin features,such as Sir recruitment,active chromatin mark(s)' removal and chromatin compaction,are detected within a few hours before the de novo telomere reaches a stable length.Our results recapitulate the molecular dynamics and reveal a coherent picture of telomere heterochromatin formation.
基金Project supported by the National Natural Science Foundation of China.
文摘It has been demonstrated in the last ten years that the nuclear reassembly may occur in the cell-free systems from frog egg extracts added with exogenous naked DNA. However, there remains an open question : is the cell-free reassembled nucleus structurally similar to the nucleus in the intact cell ? That is, does the cell-free reassembled nucleus contain nucleosomes and chromatin? For this issue, we have designed experiments for identifying the internal structures of the cell-free reassembled nucleus. These experiments show that the nucleus reassembled in vitro also contains chromatin which is composed of typical 10 nm nucleosome fibers of "beads-on-a-string", 30 nm filaments and the next higher-order structures. The digestion experiment with the enzyme micrococcal nuclease has demonstrated that the DNA in the nucleosome of the reconstituted chromatin is about 200 base pairs (bp) in length, of which 165 bp may be in the nucleosome particle, and 35 bp may be in the linker between two particles.
基金supported by the National Natural Science Foundation of China (31371264)CAS Interdisciplinary Innovation Team and the Newton Advanced Fellowship (NA140085) from the Royal Society
文摘In eukaryotic cells,the smallest subunit of chromatin is the nucleosome,which consists of a segment of DNA wound on histone protein cores. Despite many years of effort,the process of nucleosome assembly and disassembly is still not very clear. Here,we present a convenient method to investigate the process of nucleosome assembly at the single molecule level. We invented a novel system derived from the yeast nucleoplasmic extracts(YNPE),and demonstrated that the YNPE supports the nucleosome assembly under physiological condition. By combining the total internal reflection fluorescence microscopy with microfluidic flow-cell technique,the dynamic process of nucleosome assembly in YNPE was visualized at single-molecule level. Our system provides a novel in vitro single-molecule tool to investigate the dynamics of nucleosome assembly under physiological conditions.
