Epigenetics： heterochromatin meets RNAi

The term epigenetics refers to heritable changes not encoded by DNA. The organization of DNA into chromatin fibers affects gene expression in a heritable manner and is therefore one mechanism of epigenetic inheritance. Large parts of eukaryotic genomes consist of constitutively highly condensed heterochromatin, important for maintaining genome integrity but also for silencing of genes within. Small RNA, together with factors typically associated with RNA interference （RNAi） targets homologous DNA sequences and recruits factors that modify the chromatin, com- monly resulting in formation of heterochromatin and silencing of target genes. The scope of this review is to provide an overview of the roles of small RNA and the RNAi components, Dicer, Argonaute and RNA dependent polymerases in epigenetic inheritance via heterochromatin formation, exemplified with pathways from unicellular eukaryotes, plants and animals.

Two H3K36 methyltransferases differentially associate with transcriptional activity and enrichment of facultative heterochromatin in rice blast fungus

Di-and tri-methylation of lysine 36 on histone H3(H3K36me2/3)is catalysed by histone methyltransferase Set2,which plays an essential role in transcriptional regulation.Although there is a single H3K36 methyltransferase in yeast and higher eukaryotes,two H3K36 methyltransferases,Ash1 and Set2,were present in many filamentous fungi.However,their roles in H3K36 methylation and transcriptional regulation remained unclear.Combined with methods of RNA-seq and ChIP-seq,we revealed that both Ash1 and Set2 are redundantly required for the full H3K36me2/3 activity in Magnaporthe oryzae,which causes the devastating worldwide rice blast disease.Ash1 and Set2 distinguish genomic H3K36me2/3-marked regions and are differentially associated with repressed and activated transcription,respectively.Furthermore,Ash1-catalysed H3K36me2 was co-localized with H3K27me3 at the chromatin,and Ash1 was required for the enrichment and transcriptional silencing of H3K27me3-occupied genes.With the different roles of Ash1 and Set2,in H3K36me2/3 enrichment and transcriptional regulation on the stress-responsive genes,they differentially respond to various stresses in M.oryzae.Overall,we reveal a novel mechanism by which two H3K36 methyltransferases catalyze H3K36me2/3 that differentially associate with transcriptional activities and contribute to enrichment of facultative heterochromatin in eukaryotes.

Protein O-GlcNAcylation homeostasis regulates facultative heterochromatin to fine-tune sog-Dpp signaling during Drosophila early embryogenesis

Protein O-GlcNAcylation is a monosaccharide post-translational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Mutations in human OGT have recently been associated with neurodevelopmental disorders, although the mechanisms linking O-GlcNAc homeostasis to neurodevelopment are not understood. Here, we investigate the effects of perturbing protein O-GlcNAcylation using transgenic Drosophila lines that overexpress a highly active OGA. We reveal that temporal reduction of protein O-GlcNAcylation in early embryos leads to reduced brain size and olfactory learning in adult Drosophila. Downregulation of O-GlcNAcylation induced by the exogenous OGA activity promotes nuclear foci formation of Polycomb-group protein Polyhomeotic and the accumulation of excess K27 trimethylation of histone H3 (H3K27me3) at the mid-blastula transition. These changes interfere with the zygotic expression of several neurodevelopmental genes, particularly short gastrulation (sog), a component of an evolutionarily conserved sog-Decapentaplegic (Dpp) signaling system required for neuroectoderm specification. Our findings highlight the importance of early embryonic O-GlcNAcylation homeostasis for the fidelity of facultative heterochromatin redeployment and initial cell fate commitment of neuronal lineages, suggesting a possible mechanism underpinning OGT-associated intellectual disability.

Effects of SWI/SNF complex on DNA damage repair in heterochromatin of embryonic fibroblast cells

Objective:To investigate the impact of SWI/SNF complex on heterochromatin DNA damage repair after exposure to X-ray irradiation,in order to explore the underlying mechanism.Methods:NIH3T3 and MRC5 cells were treated with 50 nmol/L siRNA targeting SWI/SNF complex subunits(BRM,ARID1A,BRG1 and SNF5),and YAP/TAZ.At 24 h after transfection,the cells were irradiated with 0.5 and 1 Gy of X-rays.At 20,60 and 240 min post-irradiation,γH2AX assay was performed to evaluate the radiation response in total or heterochromatin.Comet assay was used to determine the role of YAP/TAZ in DNA damage when the cells were irradiated with 4 Gy of X-rays.NIH3T3 were treated with 50 nmol/L siRNA targeting BRM/BRG1 and YAP/TAZ to determine their relationship on heterochromatin DNA damage repair.Results:In NIH3T3,SWI/SNF complex subunits(BRM,ARID1A and BRG1)knock-down increasedγH2AX in total and heterochromatin at 1 Gy 60 min post-irradiation(P<0.05),while SNF5 knock-down decreased heterochromatinγH2AX at 1 Gy 20 min post-irradiation(P<0.05).In MRC5,BRM and BRG1 knock-down increasedγH2AX in total and heterochromatin at 1 Gy 60 min post-irradiation(P<0.05).Inconsistently,ARID1A knockdown did not affect it,and SNF5 knock-down increased heterochromatinγH2AX at 1 Gy 60 min post-irradiation(P<0.05).Moreover,YAP/TAZ knock-down decreased heterochromatinγH2AX in NIH3T3 and MRC5(P<0.05).Meanwhile,YAP/TAZ knock-down decreased Tail Moment in comet assay at 4 Gy 60 min post-irradiation(P<0.05).BRM/BRG1 combining with YAP/TAZ knock-down significantly decreased heterochromatinγH2AX compared with single BRM/BRG1 knock-down at 0.5 Gy 60 min post-irradiation(P<0.05).Conclusions:The SWI/SNF complex subunits exhibited varying effects on DNA damage repair.BRM/BRG1 knockdown promotedγH2AX accumulation in heterochromatin through YAP/TAZ.This study provides a novel direction for DNA damage repair and sheds light on the role of SWI/SNF complex in response to DNA damage repair in heterochromatin.

Alteration of Terminal Heterochromatin and Chromosome Rearrangements in Derivatives of Wheat-Rye Hybrids

Wheat-rye addition and substitution lines and their self progenies revealed variations in telomeric heterochromatin and centromeres, Furthermore, a mitotically unstable dicentric chromosome and stable multicentric chromosomes were observed in the progeny of a Chinese Spring-lmperial rye 3R addition line. An unstable multicentric chromosome was found in the progeny of a 6R/6D substitution line. Drastic variation of terminal heterochromatin including movement and disappearance of terminal heterochromatin occurred in the progeny of wheat- rye addition line 3R, and the 5RS ditelosomic addition line. Highly stable minichromosomes were observed in the progeny ofa monosomic 4R addition line, a ditelosomic 5RS addition line and a 6R/6D substitution line. Minichromosomes, with and without the FISH signals for telomeric DNA （TTTAGGG）n, derived from a monosomic 4R addition line are stable and transmissible to the next generation. The results indicated that centromeres and terminal heterochromatin can be profoundly altered in wheat-rye hybrid derivatives.

RNA Polymerase V Functions in Arabidopsis Interphase Heterochromatin Organization Independently of the 24-nt siRNA-Directed DNA Methylation Pathway

In Arabidopsis, pericentromeric repeats, retroelements, and silenced rRNA genes are assembled into heterochromatin within nuclear structures known as chromocenters. The mechanisms governing higher-order heterochromatin organization are poorly understood but 24-nt small interfering RNAs （siRNAs） are known to play key roles in heterochromatin formation. Nuclear RNA polymerase IV （Pol IV）, RNA-DEPENDENT RNA POLYMERASE 2 （RDR2）, and DICER-LIKE 3 （DCL3） are required for biogenesis of 24-nt siRNAs that associate with ARGONAUTE 4 （AGO4）. Nuclear RNA polymerase V （Pol V） collaborates with DRD1 （DEFICIENT IN RNA-DEPENDENT DNA METHYLATION 1） to generate transcripts at heterochromatic loci that are hypothesized to bind to siRNA-AGO4 complexes and subsequently recruit the de-novo DNA methylation and/or histone modifying machinery. Here, we report that decondensation of the major pericentromeric repeats and depletion of the heterochromatic mark histone H3 lysine 9 dimethylation at chromocenters occurs specifically in pol V and drdl mutants. Disruption of pericentromeric repeats condensation is coincident with transcriptional reactivation of specific classes of pericentromeric 180-bp repeats. We further demonstrate that Pol V functions independently of Pol IV, RDR2, and DCL3-mediated siRNA production to affect interphase heterochromatin organization, possibly by involving RNAs that recruit structural or chromatin-modifying proteins.

Specifications of Targeting Heterochromatin Modifications in Plants

Plants encode a diverse repertoire of DNA methyltransferases that have specialized to target cytosines for methylation in specific sequence contexts. These include the de novo methyltransferase, DOMAINS REARRANGED METHYLTRANSFERASE 2 （DRM2）, which methylates cytosines in all sequence contexts through an RNA-guided process, the CHROMOMETHYLASES （CMTs）, which methylate CHH and CHG cytosines （where H is A, T, or C）, and METHYLTRANSFERASE 1 （MET1）, which maintains methylation of symmetrical CG contexts. In this review, we discuss the sequence specificities and targeting of each of these pathways. In particular, we highlight recent studies that indicate CMTs preferentially target CWG or CWA/CAW motifs （where W is A or T）, and discuss how self-reinforcing feedback loops between DNA methyltransferases and histone modifications characteristic of heterochromatin specify targeting. Finally, the initiating events that lead to gene body methylation are discussed as a model illustrating how interde- pendent targeting of different silencing pathways can potentiate the establishment of off-target epialleles.

Functions of chromatin remodeling factors in heterochromatin formation and maintenance

Heterochromatin is characteristically more compact than chromatin is mediated by special histone modifications, euchromatin in the eukaryotic genome. The establishment of hetero- recruitment and propagation of heterochromatin specific proteins, as well as formation of special primary and high order structures of chromatin. Chromatin remodeling factors are ATPases that can alter the conformation and/or positioning of nucleosomes along DNA in an ATP-dependent manner. There is increasing evidence implicating chromatin remodeling activities in heterochromatin in various organisms ranging from yeasts to humans. Chromatin remodeling factors play roles in the establishment, maintenance and epigenetic inheritance of heterochromatin, but the underlying molecular mechanisms have just begun to be investigated.

Molecular dynamics of de novo telomere heterochromatin formation in budding yeast

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 TRPI 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, Rill 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 het- erochromatin formation.

The Long-Sought-After Plant Heterochromatin Protein 1

Heterochromatin,being transcriptionally inactive,is the highly condensed form of chromatin that is associated with Histone H3 lysine 9 methylation (H3K9me)and DNA methylation in eukaryotic cells.In fission yeast and animals,Heterochromatin Protein 1 (HP1)plays a major role in forming and maintaining heterochromatin.

同源异型盒蛋白转录因子-2在前列腺癌中表达及临床意义

目的对前列腺癌中同源异型盒蛋白转录因子-2(EN2)表达状况进行检测,探讨EN2与前列腺癌临床、病理分期及五年生化复发和生存的关系。方法选取81例前列腺癌和45例前列腺增生症(BPH),采用免疫组织化学SP法检测组织中EN2表达。对实验结果结合前列腺癌临床病理资料进行K-M生存分析其五年生化复发和总体生存之间的关系。结果EN2在前列腺癌中增高表达,在前列腺癌中的表达阳性率为72.83%、在BPH中表达阳性率为11.11%,差异有统计学意义(P<0.05)。增高表达的EN2与前列腺癌Gleason分级和临床分期相关,对EN2表达与前列腺癌五年生化复发和总体生存之间的关系进行K-M分析,结果显示EN2表达与前列腺癌五年生化复发和总体生存相关(P<0.05)。结论EN2在前列腺癌增高表达可能参与了前列腺癌发生进展,联合检测有望提高前列腺癌的预后评估。

miR-433-3p靶向HP1BP3抑制胃癌细胞的增殖、迁移和侵袭能力

目的:探究微小RNA-433-3p(miR-433-3p)对胃癌细胞增殖、迁移和侵袭的影响,以及miR-433-3p靶向异染色质蛋白1结合蛋白3(HP1BP3)对胃癌细胞的调控作用。方法:将人胃癌细胞系HGC-27和AGS分为阴性对照(NC)mimic组、miR-433-3p mimic组、NC siRNA(si-NC)组和HP1BP3 siRNA(si-HP1BP3)组,分别转染NC mimic、miR-433-3p mimic、si-NC和si-HP1BP3。RT-qPCR检测miR-433-3p在HGC-27和AGS细胞中的表达;CCK-8法和细胞集落形成实验检测细胞增殖;划痕愈合实验检测细胞迁移;Traswell实验检测细胞侵袭;Western blot检测HP1BP3蛋白表达。结果:与NC mimic组相比,miR-433-3p mimic组HGC-27和AGS细胞中miR-433-3p表达升高。过表达miR-433-3p抑制HGC-27和AGS细胞的增殖、迁移和侵袭,抑制HP1BP3蛋白表达;敲减HP1BP3抑制HGC-27和AGS细胞的增殖和迁移。结论:miR-433-3p靶向调控HP1BP3进而抑制胃癌细胞的增殖、迁移和侵袭。

肿瘤干细胞异染色质对人结直肠癌放射敏感性的影响及探究

背景与目的:放射治疗在结直肠腺癌综合治疗中发挥着重要的作用,与非肿瘤干细胞(non-cancer stem cells,non-CSCs)相比,肿瘤干细胞(cancer stem cells,CSCs)更具放射抵抗性,并且是影响放射治疗效果的重要因素之一。本研究旨在探讨人结直肠腺癌CSCs特征性染色体结构及组蛋白修饰方式对肿瘤放射抵抗效应的作用。方法:收集人结直肠癌手术标本并建立裸鼠皮下移植瘤模型。免疫组化检测人手术标本和异种移植动物肿瘤的组织来源。流式细胞仪分选人结直肠腺癌标本与移植瘤模型中放射干预前后CSCs(CD133+)与non-CSCs(CD133-),并检测CSCs表面标志物CD133的表达,利用免疫荧光染色检测CD133+及CD133-细胞核中异染色质标记物(H3K9me3,HP1-α,H3K4me1)和常染色质标志物(H3K4me3)的表达情况。结果:人手术标本和异种移植动物肿瘤的组织来源相同。人结直肠腺癌中CSCs与non-CSCs染色质结构存在明显差异。CD133+细胞染色质为致密斑块状结构,CD133-细胞染色质结构则明显呈疏松片状或网格状,免疫荧光染色显示CSCs细胞核中存在的致密斑块状结构为异染色质成分。行10Gy单次大剂量放射干预1h和24h后发现,结直肠腺癌CSCs异染色质区域出现明显空泡状缺损现象,non-CSCs染色质除结构稍疏松外无其他明显改变;放射后96h和144hCSCs异染色质空泡状缺损普遍得到修复,并逐渐恢复至放射干预前的致密斑块状结构。结论:CSCs在人结直肠腺癌放射抵抗效应中发挥作用,其机制可能与异染色质形成及组蛋白的甲基化修饰紧密相关。

远缘杂种孤雌生殖系Giemsa C-带的遗传稳定性分析

利用ＧｉｅｍｓａＣ－显带方法发现在玉米自交系自３３０和二倍体多年生类玉米远缘杂交孤雌生殖后代中出现广泛的Ｃ－带结构异染色质变异，表现为Ｃ－带数目和分布位置的变化以及异配型Ｃ－带的出现。这种变异不仅存在于表型不稳定的早代孤雌系中，而且还存在于表型稳定的高代系中。造成这种变异的原因可能是因为远缘种质的引入影响到遗传平衡进而引起染色体发生结构重排并引起染色质的变化，这种变化与玉米染色体组中非同源染色体间存在的同源片段和因此造成的减数分裂过程中非同源染色体配对有着直接的关系。这些研究对于认识孤雌生殖育种的理论基础和应用价值具有重要意义。

节节麦5D染色体上随体多态性的一个证据

５Ｄ染色体是节节麦整个染色体组中唯一的１对随体染色体，其随体位于５Ｄ短臂的末端。对来源于中国的９个不同的节节麦居群的吉姆萨Ｃ带分析表明：同一份节节麦居群内不同植株或不同细胞，这个端部随体在整个有丝分裂前期、中期和间期都很稳定，表现为大小、强弱一致的端部特征带。但是不同节节麦居群间５Ｄ染色体上的这个随体Ｃ带存在多态性。５Ｄ染色体上的随体区域可以作为小麦遗传分析和遗传操作的标记性状。同时本文还对这种多态性与普通小麦５Ｄ染色体短臂上随体的消失的关系作了分析。

两种突颜蝗的染色体C带核型研究

采用染色体C分带技术初次对癞蝗科Pamphagidae中国特有的突颜蝗属EotmethisB . Bienko 2个种 ,即天祝突颜蝗E .tientsuensis (Chang)和景泰突颜蝗E .jintaiensisXietZheng的精原细胞有丝分裂中期和初级精母细胞减数分裂中期 (中期I)染色体C带核型进行了比较研究。结果表明 :2种突颜蝗的染色体基数、着丝粒位置及染色体分组形式相同 ,且与前人的研究结果基本吻合 ,反映了癞蝗科昆虫核型的稳定性。同时 ,2种突颜蝗染色体相对长度值较为接近 ;结构异染色质总含量没有明显差异 ;并且两者无论在精原细胞有丝分裂中期还是减数分裂中期Ⅰ细胞 ,其 3号、4号和X染色体上都显现深着色较大块的着丝粒C带 ,9号染色体上都出现大块深着色的端部C带 ,2号染色体上都发生清晰的近中部居间C带 ,以上反映了 2个种在系统发生上的亲缘关系。另外 ,2种突颜蝗在C带带型上的差异 ,在 1号、7号、8号染色体上有不同程度的反映 ,尤其是对应的 7号。

大Y染色体的细胞遗传学研究及其临床效应分析

目的探讨大Y染色体与不良妊娠之间的关系以及临床意义。方法常规外周血淋巴细胞培养制备染色体标本,G显带核型分析716例检测者。结果检出异常核型124例,男性异常核型56例,大Y染色体携带者38例,占男性异常核型的67.86%,可导致其配偶发生习惯性流产、生育智力低下儿、长期不育、有死胎史、有畸形儿生育史以及新生儿死亡并流产等。结论大Y染色体与不良妊娠有关,有一定的临床意义。

9号染色体异染色质区的变异分析

目的分析疑有染色体异常个体的9号染色体异染色质区的变异。方法采集疑有染色体异常的3075名个体静脉血,其中男性1653例,女性1422例,以1515名正常人作对照,培养其淋巴细胞进行染色体核型分析。结果疑有染色体异常个体的9号染色体异染色质区的变异率为5.56%,而正常对照组9号染色体异染色质区的变异率仅为1.32%,9号染色体异染色质区的变异包括9qh+、9qh-及inv(9)。结论疑有染色体异常个体的9号染色体异染色质区的变异率高于正常人4.2倍(P<0.01),这说明9号染色体异染色质区的变异可能参与一些染色体病的发生。

转录水平siRNA介导的基因沉默

siRNA(smallinterferenceRNA)在转录后水平有效地沉默基因表达的现象称为RNAi(RNAinterference,RNA干涉),传统认为它能在转录后水平上有效地沉默基因的表达(posttranscriptionalgenesilence,PTGS)。然而,最近实验研究表明,siRNA是一些甲基化转移酶活化的起始信号,在siRNA的作用下,甲基化转移酶使DNA区域包括胞嘧啶鸟嘌呤核苷酸连续区(称为CG岛),CNG(N:A/T/C/G),CHH(H:A/C/T)中的胞嘧啶核苷C和组蛋白H3亚基第9个赖氨酸K(lysineresidueK9inhistoneH3,H3K9)发生甲基化,基因表达为此而受到抑制,即siRNA能在转录水平调控基因的表达(transcriptionalgenesilence,TGS).同时,siRNA在异染色质的形成中也起着重要的作用,RNAi突变会激活原异染色质区域中沉默基因的表达。

热激蛋白Hsp90在调控裂殖酵母异染色质区基因沉默中的功能

鉴于高等生物中Hsp90与Argonaute蛋白的功能相关性,研究了裂殖酵母中Hsp90/Swo1与Ago1蛋白之间的相互关系以及对异染色质区基因沉默的影响。结果表明,裂殖酵母中Swo1蛋白通过与Ago1蛋白的相互作用,可以稳定Ago1蛋白,并且这种相互作用依赖于Swo1的N端和中央结构域以及Ago1的N端和PAZ结构域。在着丝粒的otr区和imr区,swo1+基因的突变会引起区域内基因沉默的解除,并且与RNAi组分双突变后(ago1Δ或dcr1Δ),基因沉默解除的效果加强。在交配型区,swo1+基因突变后也会引起显著的基因沉默解除现象。当swo1+基因突变后,依赖于Tas3的人工异染色质区基因沉默解除。研究发现了裂殖酵母中热激蛋白Hsp90的新功能,即参与异染色质区的基因沉默调控。