人NPTX1相关H3K27ac富集的非保守性增强子鉴定

目的探索和鉴定人NPTX1相关H3K27ac富集的非保守性的增强子,完善NPTX1相关调控区的注释,为NPTX1相关疾病及非编码区突变的研究提供相应的理论基础。方法从CistromeDB数据库下载和分析涉及7种组织的39个H3K27ac ChIP-seq数据,选择NPTX1两侧100 kb范围内的峰。通过ECR浏览器分析各片段在人、小鼠和斑马鱼之间的序列保守性。将相应DNA片段插入双荧光素酶验证载体中,在人神经源细胞系SH-SY5Y和人肾源细胞系293T行双荧光素酶实验,以检测增强子活性。结果分析获得5个H3K27ac富集片段,各片段在人、小鼠和斑马鱼之间DNA序列不保守。NPTX1-E1-P在SH-SY5Y中双荧光素酶活性比值明显高于阴性对照组,但在293T细胞中其增强子活性下降约40%。结论NPTX1附近的H3K27ac富集片段中,NPTX1-E1-P在人神经细胞系SH-SY5Y中具有增强子活性,且在物种间序列保守性低。相对于人神经源细胞系SH-SY5Y,其在人肾源细胞系293T中增强子活性下降。

胃黏膜肠上皮化生的H3K27ac增强子重编程特征及转录调控机制

目的描绘胃黏膜肠上皮化生(intestinal metaplasia,IM)(肠化)组织增强子标志H3K27ac修饰的全基因组分布图谱,探讨增强子调控肠化的作用机制。方法于本院收集41例肠化胃黏膜与21例正常胃黏膜进行H3K27ac染色质靶向切割和标签化(Cleavage Under Targets and Tagmentation,CUT&Tag)测序与RNA测序,比较两种组织的H3K27ac修饰数量、信号强度差异,分析增强子重编程特征;探讨增强子相关转录因子调控肠化基因表达的分子机制。结果相比正常胃黏膜,肠化组织的H3K27ac修饰数量、信号强度均显著增加,其重编程区域主要位于增强子;在肠化组织的高变异增强子基因座中,活性升高的增强子占92.4%,后者可能上调大量肠上皮细胞相关基因表达,改变肠化细胞表型和生物学特性;肠化增强子区域富集了CDX2等14个转录因子的结合基序,它们在肠化组织表达上调,组成转录因子调控网络,可能在增强子激活肠化相关基因表达中发挥重要作用。结论本研究采用表观组、转录组测序及整合分析,发现全基因组增强子重塑是肠化的一个重要表观修饰特征,增强子可能通过招募CDX2等转录因子形成调控网络,协同上调肠化相关基因表达。

H3K27ac促进lncRNA OIP5-AS1的表达进而诱导食管鳞癌放射抵抗的产生

目的本研究旨在探索放射相关H3组蛋白修饰调控OIP5-AS1的分子机制,从而为逆转食管鳞癌放射抵抗提供依据。方法本研究纳入137例西安医学院第一附属医院食管鳞癌患者标本。qRT-PCR及Western blotting检测目的基因的表达。划痕实验及CCK8实验检测细胞迁移及增殖能力。应用ChIP检测OIP5-AS1与H3K27ac的结合情况。结果OIP5-AS1在食管癌放疗抵抗患者中高表达;且促进食管癌抵抗细胞的增殖及转移能力。H3K27ac在食管癌放射抵抗细胞中高表达,且富集于OIP5-AS1的启动子区域。CBP在食管癌放射抵抗细胞中高表达;其抑制剂C646可明显抑制H3K27ac在OIP5-AS1启动子区域的富集。结论OIP5-AS1参与调控食管鳞癌放射抵抗。CBP通过促进H3K27ac与OIP5-AS1的结合,进而激活OIP5-AS1的表达,从而促进放射抵抗的产生。

The transcriptional activator Klf5 recruits p300-mediated H3K27ac for maintaining trophoblast stem cell pluripotency

The effective proliferation and differentiation of trophoblast stem cells(TSCs)is indispensable for the development of the placenta,which is the key to maintaining normal fetal growth during pregnancy.Kruppel-like factor 5(Klf5)is implicated in the activation of pluripotency gene expression in embryonic stem cells(ESCs),yet its function in TSCs is poorly understood.Here,we showed that Klf5 knockdown resulted in the downregulation of core TSC-specific genes,consequently causing rapid differentiation of TSCs.Consistently,Klf5-depleted embryos lost the ability to establish TSCs in vitro.At the molecular level,Klf5 preferentially occupied the proximal promoter regions and maintained an open chromatin architecture of key TSC-specific genes.Deprivation of Klf5 impaired the enrichment of p300,a major histone acetyl transferase of H3 lysine 27 acetylation(H3K27ac),and further reduced the occupancy of H3K27ac at promoter regions,leading to decreased transcriptional activity of TSC pluripotency genes.Thus,our findings highlight a novel mechanism of Klf5 in regulating the self-renewal and differentiation of TSCs and provide a reference for understanding placental development and improving pregnancy rates.

Loss of LBP triggers lipid metabolic disorder through H3K27 acetylation-mediated C/EBPβ-SCD activation in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease(NAFLD)is associated with mutations in lipopolysaccharide-binding protein(LBP),but the underlying epigenetic mechanisms remain understudied.Herein,LBP^(-/-)rats with NAFLD were established and used to conduct integrative targetingactive enhancer histone H3 lysine 27 acetylation(H3K27ac)chromatin immunoprecipitation coupled with high-throughput and transcriptomic sequencing analysis to explore the potential epigenetic pathomechanisms of active enhancers of NAFLD exacerbation upon LBP deficiency.Notably,LBP^(-/-)reduced the inflammatory response but markedly aggravated high-fat diet(HFD)-induced NAFLD in rats,with pronounced alterations in the histone acetylome and regulatory transcriptome.In total,1128 differential enhancer-target genes significantly enriched in cholesterol and fatty acid metabolism were identified between wild-type(WT)and LBP^(-/-)NAFLD rats.Based on integrative analysis,CCAAT/enhancer-binding proteinβ(C/EBPβ)was identified as a pivotal transcription factor(TF)and contributor to dysregulated histone acetylome H3K27ac,and the lipid metabolism gene SCD was identified as a downstream effector exacerbating NAFLD.This study not only broadens our understanding of the essential role of LBP in the pathogenesis of NAFLD from an epigenetics perspective but also identifies key TF C/EBPβand functional gene SCD as potential regulators and therapeutic targets.

增强子及相关调控组在肠型胃癌中的重塑特征及其预后预测模型

目的研究组蛋白H3K27ac修饰在肠型胃癌的全基因组分布情况,探讨其增强子及相关调控组重塑特征,建立临床预后评估模型。方法收集于2022年1-12月在我院消化内科就诊患者的15例肠型胃癌组织与18例正常胃黏膜组织进行靶向H3K27ac修饰的染色质靶向捕获(cleavage under targets and tagmentation,CUT&Tag)测序,采用生物信息学分析,比较两种组织H3K27ac修饰在基因组的分布差异;基于H3K27ac修饰分布特征,鉴定增强子元件,探讨增强子及相关调控组的重塑特征;采用单因素Cox和多因素Cox等回归分析方法,构建增强子相关靶基因的预后预测模型。结果两种组织的H3K27ac修饰的基因组分布特征无明显差异,主要位于增强子区域;与正常胃黏膜相比,肠型胃癌增强子H3K27ac修饰的整体水平更高;共鉴定出8847个在肠型胃癌中活性增加的增强子(获得性增强子),占所有增强子的8.3%,其可能促进胃癌细胞增殖、黏附等恶性行为;联合6个获得性增强子相关靶基因(BHLHE41、CEP97、CHI3L2、NR6A1、SUPT3H和TOMM20)构建的预测模型,能够较好地推测患者总体生存期。结论增强子重塑是肠型胃癌显著的表观遗传学特征之一,增强子可能通过上调MYC、E2F3等基因表达促进癌细胞恶性增殖与黏附行为,并基于增强子靶基因构建预后模型。

Reorganization of 3D genome architecture provides insights into pathogenesis of early fatty liver disease in laying hens

Background Fatty liver disease causes huge economic losses in the poultry industry due to its high occurrence and lethality rate.Three-dimensional(3D)chromatin architecture takes part in disease processing by regulating tran-scriptional reprogramming.The study is carried out to investigate the alterations of hepatic 3D genome and H3K27ac profiling in early fatty liver(FLS)and reveal their effect on hepatic transcriptional reprogramming in laying hens.Results Results show that FLS model is constructed with obvious phenotypes including hepatic visible lipid deposi-tion as well as higher total triglyceride and cholesterol in serum.A/B compartment switching,topologically associat-ing domain(TAD)and chromatin loop changes are identified by high-throughput/resolution chromosome conforma-tion capture(HiC)technology.Targeted genes of these alternations in hepatic 3D genome organization significantly enrich pathways related to lipid metabolism and hepatic damage.H3K27ac differential peaks and differential expres-sion genes(DEGs)identified through RNA-seq analysis are also enriched in these pathways.Notably,certain DEGs are found to correspond with changes in 3D chromatin structure and H3K27ac binding in their promoters.DNA motif analysis reveals that candidate transcription factors are implicated in regulating transcriptional reprogram-ming.Furthermore,disturbed folate metabolism is observed,as evidenced by lower folate levels and altered enzyme expression.Conclusion Our findings establish a link between transcriptional reprogramming changes and 3D chromatin struc-ture variations during early FLS formation,which provides candidate transcription factors and folate as targets for FLS prevention or treatment.

Mapping and analysis of a spatiotemporal H3K27ac and gene expression spectrum in pigs

The limited knowledge of genomic noncoding and regulatory regions has restricted our ability to decipher the genetic mechanisms underlying complex traits in pigs. In this study, we characterized the spatiotemporal landscape of putative enhancers and promoters and their target genes by combining H3K27ac-targeted Ch IP-Seq and RNA-Seq in fetal(prenatal days 74–75) and adult(postnatal days 132–150) tissues(brain, liver, heart, muscle and small intestine) sampled from Asian aboriginal Bama Xiang and European highly selected Large White pigs of both sexes. We identified 101,290 H3K27ac peaks, marking 18,521promoters and 82,769 enhancers, including peaks that were active across all tissues and developmental stages(which could indicate safe harbor locus for exogenous gene insertion) and tissue-and developmental stage-specific peaks(which regulate gene pathways matching tissue-and developmental stage-specific physiological functions). We found that H3K27ac and DNA methylation in the promoter region of the XIST gene may be involved in X chromosome inactivation and demonstrated the utility of the present resource for revealing the regulatory patterns of known causal genes and prioritizing candidate causal variants for complex traits in pigs. In addition, we identified an average of 1,124 super-enhancers per sample and found that they were more likely to show tissue-specific activity than ordinary peaks. We have developed a web browser to improve the accessibility of the results(http://segtp.jxau.edu.cn/pencode/?genome=sus Scr11).

ERα promotes transcription of tumor suppressor gene ApoA-I by establishing H3K27ac-enriched chromatin microenvironment in breast cancer cells

Apolipoprotein A-I(Apo A-I),the main protein component of high-density lipoprotein(HDL),plays a pivotal role in reverse cholesterol transport(RCT).Previous studies indicated a reduction of serum Apo A-I levels in various types of cancer,suggesting Apo A-I as a potential cancer biomarker.Herein,ectopically overexpressed Apo A-I in MDA-MB-231 breast cancer cells was observed to have antitumor effects,inhibiting cell proliferation and migration.Subsequent studies on the mechanism of expression regulation revealed that estradiol(E2)/estrogen receptorα(ERα)signaling activates Apo A-I gene transcription in breast cancer cells.Mechanistically,our Ch IP-seq data showed that ERαdirectly binds to the estrogen response element(ERE)site within the Apo A-I gene and establishes an acetylation of histone 3 lysine 27(H3 K27 ac)-enriched chromatin microenvironment.Conversely,Fulvestrant(ICI 182780)treatment blocked ERαbinding to ERE within the Apo A-I gene and downregulated the H3 K27 ac level on the Apo A-I gene.Treatment with p300 inhibitor also significantly decreased the Apo A-I messenger RNA(m RNA)level in MCF7 cells.Furthermore,the analysis of data from The Cancer Genome Atlas(TCGA)revealed a positive correlation between ERαand Apo A-I expression in breast cancer tissues.Taken together,our study not only revealed the antitumor potential of Apo A-I at the cellular level,but also found that ERαpromotes the transcription of Apo A-I gene through direct genomic effects,and p300 may act as a co-activator of ERαin this process.

Long-read genome assemblies reveal a cis-regulatory landscape associated with phenotypic divergence in two sister Siniperca fish species

Due to the difficulty in accurately identifying structural variants(SVs) across genomes,their impact on cisregulato ry diverge n ce of closely related species,especially fish,remains to be explored.Recently identified broad H3K4me3 domains are essential for the regulation of genes involved in several biological processes.However,the role of broad H3K4me3 domains in phenotypic divergence remains poorly understood.Siniperca chuatsi and S.scherzeri are closely related but divergent in several phenotypic traits,making them an ideal model to study cis-regulatory evolution in sister species.Here,we generated chromosome-level genomes of S.chuatsi and S.scherzeri,with assembled genome sizes of 716.35 and740.54 Mb,respectively.The evolutionary histories of S.chuatsi and S.scherzeri were studied by inferring dynamic changes in ancestral population sizes.To explore the genetic basis of adaptation in S.chuatsi and S.scherzeri,we performed gene family expansion and contraction analysis and identified positively selected genes(PSGs).To investigate the role of SVs in cis-regulatory divergence of closely related fish species,we identified high-quality SVs as well as divergent H3K27ac and H3K4me3 domains in the genomes of S.chuatsi and S.scherzeri.Integrated analysis revealed that cis-regulatory divergence caused by SVs played an essential role in phenotypic divergence between S.chuatsi and S.scherzeri.Additionally,divergent broad H3K4me3 domains were mostly associated with cancer-related genes in S.chuatsi and S.scherzeri and contributed to their phenotypic divergence.

靶向敲除β-珠蛋白基因座控制区增强子HS2对K562细胞转录组的影响

人类β-地中海贫血的发病机制与β-样珠蛋白基因异常表达息息相关。人类β-样珠蛋白基因以5′-ε-Gγ-Aγ-δ-β-3′的顺序排列于β-珠蛋白基因座,受5′LCR (locus control region)中5个超敏位点(hypersensitive site,HS)5′HS5~5′HS1和3′HS1调控。其中5′HS2是最重要的增强子,能产生增强子RNA(enhancerRNA)并调控ε-globin、γ-globin和β-globin的表达。为了进一步探究K562细胞中增强子5′HS2的功能,本研究首先通过染色质构象捕获技术在人慢性髓原白血病K562细胞中探测到5′HS2介导的染色质相互作用集中在以包含CTCF(CCCTC-bindingfactor)位点的3′HS1和5′HS5为边界的拓扑结构域中,5′HS2在三维空间上与HBE1、HBG2和HBG1启动子区域相互靠近。其次运用CRISPRDNA片段编辑技术在K562细胞系中删除了增强子5′HS2。最后通过RNA-seq和CUT&Tag (cleavage under target&tagmentation)实验分析两个5′HS2删除的单克隆细胞系的转录组和染色质H3K27ac组蛋白修饰,发现91个基因表达显著下调而且其启动子区的H3K27ac修饰程度显著降低。这些基因主要聚类于氧气运输、免疫应答、细胞粘附、抗氧化和维持血栓形成等与红细胞功能相关的生物过程中,表明K562细胞系中增强子5′HS2对红细胞功能相关基因的转录产生了广泛影响。

Acetylation of H3K4,H3K9,and H3K27 mediated by p300 regulates the expression of GATA4 in cardiocytes

GATA4 is a particularly important cardiogenic transcription factor and serves as a potent driver of cardiogenesis.Recent progress in the field has made it clear that histone acetylation can influence gene expression through changing the structure of chromatin.Our previous research had revealed that hypo-acetylation could repress gata4 expression in cardiocytes,however the underlying mechanism by which this occurred was still unclear.To reveal the mechanism of histone acetylation involved in the regulation of gata4 transcription,we concentrated on P300,one of the important histone acetyltransferase associated with cardiogenesis.We found that P300 participated in gata4 expression through regulating histone acetylation in embryonic mouse hearts.RNAi-mediated downregulation of P300 modulated the global acetylation of H3 and the acetylation of H3K4,H3K9,and H3K27 in gata4 and Tbx5 promoters.Interestingly,there was an obvious inhibition of gata4 transcription,whereas Tbx5 was not influenced.Furthermore,SGC-CBP30,the selective inhibitor of the bromodomain in CBP/P300,downregulated gata4 transcription by repressing the acetylation of H3K4,H3K9,and H3K27 in the gata4 promoters.Taken together,our results identified that acetylation of H3K4,H3K9,and H3K27 mediated by P300 plays an important role in regulation of gata4 expression in cardiogenesis.

食管鳞癌中异常增强子促进肿瘤发生

食道鳞状细胞癌(esophagus squamous cell carcinoma,ESCC)是侵袭性恶性肿瘤,预后较差。Diffbind用于H3K27ac ChIP-seq差异分析以鉴定异常增强子(aberrant enhancer loci,AEL)在食管鳞癌发生中的作用。ROSE、HOMER软件分别用于鉴定超级增强子、计算转录因子。ClusterProfiler程序包用于GO和KEGG富集分析。根据异常增强子相关基因的表达,采用LASSO Cox回归构建风险评分模型。本研究在ESCC中鉴定了3323个激活和955个沉默的AEL,多数位于超级增强子内,被转录因子AP-1占据。对GSE53625等数据集进行差异分析发现,相比于癌旁组织,活化AEL基因在食管鳞癌组织中表达上调,而沉默AEL基因被抑制。AEL基因与染色质结合等功能密切相关,参与细胞粘附、细胞骨架、Rap1等信号通路。DNA甲基化差异分析发现,相比于癌旁组织,活化AEL在食管鳞癌组织中被低甲基化,而沉默的AEL被高甲基化。EGFR基因上游存在AEL,为超级增强子,该AEL在肿瘤细胞中被特异性激活,细胞实验证实此AEL对EGFR启动子有显著的增强作用。与低风险组相比,高风险组的患者在训练集(P=3.331×10^(-16))和测试集(P=4.5×10^(-2))中的总生存率显着降低。训练集和测试集的多因素Cox回归分析证明AEL基因风险评分模型是影响患者总生存率的独立因素。ESCC恶化过程中存在高频率的增强子活性改变,这些异常增强子可能是ESCC肿瘤发生中基因表达失调的驱动因素。根据AEL基因表达构建的风险评估模型在训练集和测试集中表现优异,有望作为ESCC患者预后的预测工具。

MACMIC Reveals A Dual Role of CTCF in Epigenetic Regulation of Cell Identity Genes

Numerous studies of relationship between epigenomic features have focused on their strong correlation across the genome,likely because such relationship can be easily identified by many established methods for correlation analysis.However,two features with little correlation may still colocalize at many genomic sites to implement important functions.There is no bioinformatic tool for researchers to specifically identify such feature pairs.Here,we develop a method to identify feature pairs in which two features have maximal colocalization minimal correlation(MACMIC)across the genome.By MACMIC analysis of 3306 feature pairs in 16 human cell types,we reveal a dual role of CCCTC-binding factor(CTCF)in epigenetic regulation of cell identity genes.Although super-enhancers are associated with activation of target genes,only a subset of super-enhancers colocalized with CTCF regulate cell identity genes.At super-enhancers colocalized with CTCF,CTCF is required for the active marker H3 K27 ac in cell types requiring the activation,and also required for the repressive marker H3 K27 me3 in other cell types requiring repression.Our work demonstrates the biological utility of the MACMIC analysis and reveals a key role for CTCF in epigenetic regulation of cell identity.The code for MACMIC is available at https://github.com/bxia888/MACMIC.