Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction

Background Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development.In some mammals,like pigs,ejaculates are emitted in three separate fractions:pre-sperm,sperm-rich(SRF)and post sperm-rich(PSRF).These fractions are known to vary in volume,sperm concentration and quality,as well as in the origin and composition of seminal plasma(SP),with differences being also observed within the SRF one.Yet,whether disparities in the DNA integrity and chromatin condensation and pro-tamination of their sperm exist has not been interrogated.Results This study determined chromatin protamination(Chromomycin A3 test,CMA_(3)),condensation(Dibromobi-mane test,DBB),and DNA integrity(Comet assay)in the pig sperm contained in the first 10 m L of the SRF(SRF-P1),the remaining portion of the sperm-rich fraction(SRF-P2),and the post sperm-rich fraction(PSRF).While chromatin protamination was found to be similar between the different ejaculate fractions(P>0.05),chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF(P=0.018 and P=0.004,respectively).Regarding DNA integrity,no differences between fractions were observed(P>0.05).As the SRF-P1 has the highest sperm concentra-tion and ejaculate fractions are known to differ in antioxidant composition,the oxidative stress index(OSi)in SP,calcu-lated as total oxidant activity divided by total antioxidant capacity,was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF(0.42±0.06 vs.0.23±0.09 and 0.08±0.00,respectively;P<0.01);this index,in addition,was observed to be correlated to the sperm concentration of each fraction(Rs=0.973;P<0.001).Conclusion While sperm DNA integrity was not found to differ between ejaculate fractions,SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF.This could be related to the OSi of each fraction.

Targeting the chromatin structural changes of antitumor immunity

Epigenomic imbalance drives abnormal transcriptional processes,promoting the onset and progression of cancer.Although defective gene regulation generally affects carcinogenesis and tumor suppression networks,tumor immunogenicity and immune cells involved in antitumor responses may also be affected by epigenomic changes,which may have significant implications for the development and application of epigenetic therapy,cancer immunotherapy,and their combinations.Herein,we focus on the impact of epigenetic regulation on tumor immune cell function and the role of key abnormal epigenetic processes,DNA methylation,histone post-translational modification,and chromatin structure in tumor immunogenicity,and introduce these epigenetic research methods.We emphasize the value of small-molecule inhibitors of epigenetic modulators in enhancing antitumor immune responses and discuss the challenges of developing treatment plans that combine epigenetic therapy and immuno-therapy through the complex interaction between cancer epigenetics and cancer immunology.

Unveiling structural and dynamical features of chromatin using NMR spectroscopy This article is part of the virtual special issue“Solid-state NMR studies on polymers and biological solids”

Eukaryotic deoxyribonucleic acid(DNA)is wrapped around histone octamers(HOs)to form nucleosome core particles(NCPs),which in turn interact with linker DNA and linker histones to assemble chromatin fibers with more complex,high-order structures.The molecular properties of chromatin are dynamically regulated by several factors,such as post-translational modifications and effector proteins,to maintain genome stability.In the past two decades,high-resolution techniques have led to many breakthroughs in understanding the molecular mechanisms that govern chromatin regulation.Nuclear magnetic resonance(NMR)has emerged as one of the major techniques in this field,providing new insights into the nucleosomes and nucleosome-protein complexes in different states ranging from soluble form to condensed states.Solution-state NMR has proven valuable in elucidating the conformational dynamics and molecular interactions for histone N-terminal tails,histone core regions and DNA with the combination of specific isotopic labeling.Solid-state NMR,which is not constrained by the high molecular weights of complexes like nucleosomes,has been applied to capture the structural and dynamical characteristics of both flexible tails and rigid histone core regions in nucleosomes and their complexes with effector proteins.Furthermore,the combination of the two techniques allows tracking molecular properties of nucleosomes during phase separation processes,which potentially play essential roles in chromatin regulation.This review summarizes recent advances in NMR studies of chromatin structure and dynamics.It highlighted that NMR revealed unique molecular characteristics for nucleosomes that are often invisible experimentally by other techniques like cryogenic electron microscopy(cryo-EM)and X-ray diffraction(XRD).I envision that,with future ef-forts such as the development of NMR methods and optimization of sample production protocols,solution-state NMR and solid-state NMR will provide invaluable information to expand our understanding of chromatin activity and its regulatory processes.

SWI/SNF染色质重塑复合物在肿瘤耐药中的作用

肿瘤耐药是化疗和靶向药物治疗面临的重要问题,它是一个涉及染色质重塑的复杂过程。SWI/SNF是肿瘤发生中研究最多的ATP依赖性染色质重塑复合物之一,在染色质结构稳定性、基因表达和翻译后修饰的协调过程中起着重要作用,但其调控肿瘤耐药的机制尚未被系统梳理。SWI/SNF按其亚基组成可分为BAF、PBAF和ncBAF 3种,这3种亚型均包含ATP酶催化亚基、核心亚基和调节亚基,可以通过调控染色质结构来控制基因表达。SWI/SNF复合物亚基的改变是调控肿瘤耐药和进展的重要因素之一,充分了解该复合物在肿瘤耐药中的分子机制可以改善癌症的治疗。本文总结了SWI/SNF复合物的组成、引起肿瘤耐药的突变或异常表达亚基类型、主要的耐药机制以及肿瘤耐药的克服方法等,为临床上SWI/SNF亚基突变或异常表达引起的肿瘤预后不良的诊断和治疗提供了思路。

Sperm chromatin structure assay （SCSA）： a tool in diagnosis and treatment of infertility

Diagnosis of male infertility has mainly been based on the World Health Organization （WHO） manual-based semen parameter＇s concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay （SCSA）, a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection （ICSI）.

Relationship between chromatin organization, mRNAs profile and human male gamete quality

Spermiogenesis is a complex process leading to the formation of motile spermatozoa characterized by a highly stable chromatin compaction that transfers the paternal genome into the oocyte. It is commonly held that these haploid cells are devoid of transcriptional and translational activities and that the transcripts represent remnants of stored mRNAs. Recently, the chromatin organization of mature spermatozoa has been revisited as a double nucleoprotamine-nucleohistone structure possessing less-condensed regions sensitive to nuclease activity, which could be implicated in the expression of genes involved in the early embryo development. The existence of a complex population of mRNAs in human sperm is well-documented, but their role is not yet elucidated. Evidence for a latent transcriptional capacity and/or a potential de novo translation in mature spermatozoa from fertile men are essential for understanding the last steps of sperm maturation, such as capacitation and acrosome reaction. As such, we have documented the relationship between sperm quality and the distribution of sperm RNAs by showing divergent levels of transcripts encoding for proteins involved in either nuclear condensation （protamines 1 and 2） or in capacitation （eNOS and nNOS, c-myc） or in motility and sperm survival （aromatase） between low and high motile sperm issued from the same sample. Therefore, analyzing the profile of mRNAs could be helpful either as a diagnostic tool for evaluating male fertility after spermatogenesis or for prognosis use for fertilization.

Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF

The aim of this study was to investigate whether the sperm chromatin structure assay （SCSA） results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitrofertilization （IVF）. A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and sperm chromatin structure assay results were collected. Fertilization rate, embryo quality and IVF success rates of different DNA fragmentation index （DFI） subgroups and high DNA stainability （HDS） subgroups were compared. There were no significant differences in fertilization rate, clinical pregnancy or delivery rates between the DFI and HDS subgroups. However, the group with abnormal DFI had a lower good embryo rate. So, we concluded that the SCSA variables, either DFI or HDS after swim-up preparation, were not valuable in predicting fertilization failure or pregnancy rate, but an abnormal DFI meant a lower good embryo rate following IVF.

Epigenetics and chromatin plasticity in embryonic stem cells

The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for selfrenewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson's disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells.

Bioinformatic analysis of chromatin organization and biased expression of duplicated genes between two poplars with a common whole-genome duplication

The nonrandom three-dimensional organization of chromatin plays an important role in the regulation of gene expression.However,it remains unclear whether this organization is conserved and whether it is involved in regulating gene expression during speciation after whole-genome duplication(WGD)in plants.In this study,high-resolution interaction maps were generated using high-throughput chromatin conformation capture(Hi-C)techniques for two poplar species,Populus euphratica and Populus alba var.pyramidalis,which diverged~14 Mya after a common WGD.We examined the similarities and differences in the hierarchical chromatin organization between the two species,including A/B compartment regions and topologically associating domains(TADs),as well as in their DNA methylation and gene expression patterns.We found that chromatin status was strongly associated with epigenetic modifications and gene transcriptional activity,yet the conservation of hierarchical chromatin organization across the two species was low.The divergence of gene expression between WGD-derived paralogs was associated with the strength of chromatin interactions,and colocalized paralogs exhibited strong similarities in epigenetic modifications and expression levels.Thus,the spatial localization of duplicated genes is highly correlated with biased expression during the diploidization process.This study provides new insights into the evolution of chromatin organization and transcriptional regulation during the speciation process of poplars after WGD.

Chromatin boundary elements organize genomic architecture and developmental gene regulation in Drosophila Hox clusters

The three-dimensional(3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements(CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and "delivering" remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development.

The landscape of chromatin accessibility in skeletal muscle during embryonic development in pigs

Background:The development of skeletal muscle in pigs during the embryonic stage is precisely regulated by transcriptional mechanisms,which depend on chromatin accessibility.However,how chromatin accessibility plays a regulatory role during embryonic skeletal muscle development in pigs has not been reported.To gain insight into the landscape of chromatin accessibility and the associated genome-wide transcriptome during embryonic muscle development,we performed ATAC-seq and RNA-seq analyses of skeletal muscle from pig embryos at 45,70 and 100 days post coitus(dpc).Results:In total,21,638,35,447 and 60,181 unique regions(or peaks)were found across the embryos at 45 dpc(LW45),70 dpc(LW70)and 100 dpc(LW100),respectively.More than 91%of the peaks were annotated within−1 kb to 100 bp of transcription start sites(TSSs).First,widespread increases in specific accessible chromatin regions(ACRs)from embryos at 45 to 100 dpc suggested that the regulatory mechanisms became increasingly complicated during embryonic development.Second,the findings from integrated ATAC-seq and RNA-seq analyses showed that not only the numbers but also the intensities of ACRs could control the expression of associated genes.Moreover,the motif screening of stage-specific ACRs revealed some transcription factors that regulate muscle developmentrelated genes,such as MyoG,Mef2c,and Mef2d.Several potential transcriptional repressors,including E2F6,OTX2 and CTCF,were identified among the genes that exhibited different regulation trends between the ATAC-seq and RNA-seq data.Conclusions:This work indicates that chromatin accessibility plays an important regulatory role in the embryonic muscle development of pigs and regulates the temporal and spatial expression patterns of key genes in muscle development by influencing the binding of transcription factors.Our results contribute to a better understanding of the regulatory dynamics of genes involved in pig embryonic skeletal muscle development.

Cancer bioinformatics:detection of chromatin states, SNP-containing motifs, and functional enrichment modules

In this editorial preface, I briefly r eview cancer bioinformatics and introduce the four articles in this special issue highlighting important applications of the field: detection of chromatin states; detection of SNP- containing motifs and association with transcription factor-binding sites; improvements in functional enrichment modules; and gene association studies on aging and cancer. We expect this issue to provide bioinformatics scientists, cancer biologists, and clinical doctors with a better understanding of how cancer bioinformatics can be used to identify candidate biomarkers and targets and to conduct functional analysis.

Clinical and molecular spectrum of Wiedemann-Steiner syndrome, an emerging member of the chromatinopathy family

Wiedemann-Steiner syndrome(OMIM#605130)is a rare congenital malformation syndrome characterized by hypertrichosis cubiti associated with short stature;consistent facial features,including long eyelashes,thick or arched eyebrows with a lateral flare,wide nasal bridge,and downslanting and vertically narrow palpebral fissures;mild to moderate intellectual disability;behavioral difficulties;and hypertrichosis on the back.It is caused by heterozygous pathogenic variants in KMT2A.This gene has an established role in histone methylation,which explains the overlap of Wiedemann-Steiner syndrome with other chromatinopathies,a heterogeneous group of syndromic conditions that share a common trigger:The disruption of one of the genes involved in chromatin modification,leading to dysfunction of the epigenetic machinery.

Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy (AFM)

The organization of the higher order structure of chromatin in chicken erythrocytes has been examined withtapping-mode scanning force microscopy under conditionsclose to their native environment. Reproducible highresolution AFM images of chromatin compaction at several levels can be demonstrated. An extended beads-on-astring (width of ～ 15-20nm, height of ～ 2-3nm for eachindividual nucleosome) can be consistently observed. Furthermore, superbeads (width of ～ 40nm, height of ～ 7nm)are demonstrated. Visualization of the solenoid conformation at the level of 30nm chromatin fiber is attained eitherby using AFM or by using electron microscopy. In addition, tightly coiled chromatin fibers (～ 50-60nm and ～90-110nm) can be revealed. Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning thefolding pattern of interphase chromatin fibers.

Copper interactions with DNA of chromatin and its role in neurodegenerative disorders

In this study, we have demonstrated the conformational changes to DNA induced by abnormal interactions of copper using circular dichroism, in combination with UV-absorbance and fluorescence spectroscopy. Results confirm that binding of copper to bases of DNA in chromatin is concentration dependent. Binding efficiency of Cu2~ ions to DNA is increased in proportion to the degree of unwinding of the double helix induced by denaturation. Altered B-DNA conformation will alter the integrity of DNA which may affect the normal process of DNA replication and transcription. Copper induced DNA damage in the brain may cause neurotoxicity and the neuronal cell death and is implicated in Alzheimer＇s disease and other neurological disorders.

Effects of tamoxifen citrate on gene expression during nuclear chromatin condensation in male rats

Aim： To evaluate the effects of tamoxifen citrate on gene expression during nuclear chromatin condensation in male rats. Methods： The effects of an oral dose of 0.4 mg/（kg·d） tamoxifen citrate on rates of in vitro chromatin decondensation, acridine orange （AO） dye uptake, concentration of thiol-groups, levels and/or expression of transition proteins 1, 2 （TP1, TP2）, protamine 1 （P1）, cyclic AMP response element modulator-τ （CREMτ）, androgenbinding protein （ABP） and cyclic adenosine 3＇, 5＇ monophosphate （cAMP） were evaluated after 60 days of exposure in adult male rats. Controls received the vehicle. Results： Tamoxifen citrate enhanced the rates of chromatin decondensation, increased AO dye uptake and reduced free thiols in caput epididymal sperms and reduced the levels of TP1, TP2, P1, and CREMτ in the testis, while cAMP was unaffected. P1 deposition was absent in the sperm. The transcripts of TP1, TP2 were increased, of P1 and ABP decreased, while those of CREMτ unaffected in the testis. Conclusion： Tamoxifen citrate reduced caput epididymal sperm chromatin compaction by reducing the testicular levels of proteins TP1, TP2 and P1 and the CREMτ involved in chromatin condensation during spermiogenesis. Tamoxifen citrate affects the expression of these genes at both the transcriptional and post-transcriptional levels. （Asian J Androl 2005 Sep; 7： 311-321）

ASSOCIATION OF PSL (p55),A S-PHASE-RELATED NUCLEAR ANTIGEN,WITH CHROMATIN IN HeLa CELLS

PSL is a S-phase-related 55 kDa antigen which has been previously located inchromatin areas of the nucleus by electron microscopic studies. In the present report, weshow that it is released from purified HeLa cell nuclei by micrococcal nuclease treatment,which relieves chromatin under the form of nucleosome. However, the antigen is not associ-ated with nucleosomes but is part of a structure that sediments through 5-20% sucrosegradient. Moreover, PSL is completely retained on single-stranded DNA (ss-DNA) affini-ty columns and binds more efficiently HeLa cell ss-DNA than double stranded DNA.Although no direct evidence could be obtained, these data suggest that PSL might bea ss-DNA-binding or a RNA-binding protein.

Involvement of chromatin and histone acetylation in the regulation of HIV-LTR by thyroid hormone receptor

The HIV-1 LTR controls the expression of HIV-1 viral genes and thus is critical for viral propagation and pathology. Numerous host factors have been shown to participate in the regulation of the LTR promoter. Among them is the thyroid hormone (T3) receptor (TR). TR has been shown to bind to the critical region of the promoter that contain the NFbB and Sp1 binding sites. Interestingly, earlier transient transfection studies in tissue culture cells have yielded contradicting conclusions on the role of TR in LTR regulation, likely due to the use of different cell types and/or lack of proper chromatin organization. Here, using the frog oocyte as a model system that allows replication-coupled chromatin assembly, mimicking that in somatic cells, we demonstrate that unliganded heterodimers of TR and RXR (9-cis retinoic acid receptor) repress LTR while the addition of T3 relieves the repression and further activates the promoter. More importantly, we show that chromatin and unliganded TR/RXR synergize to repress the promoter in a histone deacetylase-dependent manner.

Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain

Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.

Chromatin domain boundaries: insulators and beyond

The eukaryotic genome is organized into functionally and structurally distinct domains, representing regulatory unitsfor gene expression and chromosome behavior. DNA sequences that mark the border between adjacent domains are theinsulators or boundary elements, which are required in maintenance of the function of different domains. Some insula-tors need others enable to play insulation activity. Chromatin domains are defined by distinct sets of post-translationallymodified histones. Recent studies show that these histone modifications are also involved in establishment of sharpchromatin boundaries in order to prevent the spreading of distinct domains. Additionally, in some loci, the high-orderchromatin structures for long-range looping interactions also have boundary activities, suggesting a correlation betweeninsulators and chromatin loop domains. In this review, we will discuss recent progress in the field of chromatin domainboundaries.