Genome-Wide Quantitative Enhancer Activity Maps Identified by STARR-seq

New Insights into a Controversial Topic: The Methylation–Cancer Connection

Mammalian development occurs by the progressive determination of cells from a pluripotent undifferentiated state through successive states of gradually restricted developmental potential, until the full complement of mature terminally differentiated cells has been specified. Embryonic development is a complex and highly orchestrated process during which multiple cell movements and changes in gene expression must be spatially and temporally coordinated to ensure that embryogenesis proceeds correctly. Complex genetic regulatory networks receive input in the form of extracellular signals and output instructions on the regulated expression of specific genes. The linchpins of the regulatory networks are the cis-regulatory elements that directly control gene expression through interpretation of the tissue-specific transcription factors (trans-elements). Embryonic stem cells are orientated across the dorso-ventral and the anterior-posterior axis of the early embryo. The orientation of progenitor cells along these two axes is thought to influence their fate by defining the identity and concentration of inductive signals to which they are exposed.In an effort to develop cell-based therapies, (i.e. for diabetes) experimental protocols aim to mimic the biological procedures that take place during embryonic development in order to differentiate embryonic stem cells towards specific cell types. One of the foremost challenges towards the development of cell therapies for diabetic people is to achieve the directed differentiation of cells capable of producing insulin. Elucidation of the genetic networks involved in the endocrine pancreas specification are thought to be essential for devising rational protocols to efficiently differentiate embryonic stem cells or pancreas progenitor cells into fully differentiated endocrine subtypes. Computational approaches allow the unravelling of complex regulatory networks including genomic (cis-cis) or proteomic (trans-trans) interactions or a combination (cis-trans) of both. In this study the genomic regulatory regions (cis elements) of several genes known and putative targets of the transcription factor NGN3 were analyzed. The NGN3 transcription factor is the major regulator of “insulin-producing cell” formation. Taking into account data from microarray experiments from pancreas progenitor cells, in which NGN3 has been induced, genes shown to be co-regulated (upregulated or downregulated) by this transcription factor were selected for analysis. Using a combination of sophisticated computational tools for exploiting and analyzing genomic data and developing the suitable algorithms, an extensive in silico analysis of the regulatory regions of these genes was performed.Evolutionarily conserved regions are linked with experimentally identified regulatory elements. Comparative genomics are commonly used in order to identify transcription factor binding sites, which are functionally important regions that are thought to be well-conserved. Analysis of genomic regulatory regions included not only genes corregulated by NGN3, but also their orthologs in several species including the most phylogenetically distant species (fish), which have pancreas. In parallel, housekeeping genes, like B-ACTIN, and those not expressed in embryos and stem cells, like B-GLOBIN, were used as negative controls. Regulatory region analysis revealed the presence of a highly conserved regulatory element, where many transcription factors with established involvement in pancreas development bind, in all the orthologs of several genes co-regulated by NGN3. Furthermore, motif identification in separate clusters of the regulatory elements of either upregulated or downregulated genes revealed the presence of additional binding motifs for the factor AP4 only in downregulated genes. In parallel, the regulatory region analysis of the entire mouse genome and the statistical analysis of the upcoming results showed that both types of regulatory elements (with and without AP4) were non-randomly identified inside the regulatory regions of genes whose transcription is controlled by NGN3. Moreover the selective presence of the AP4 binding sequence into this region renders it a highly specific suppressor found in only a small number of genes downregulated by NGN3. Taking into account that both these regulatory elements were identified at considerable distances from each gene’s transcription start site, it was assumed that they represent enhancers, and those capable of binding AP4 were considered silencers. This conclusion was enforced by the compositional analysis of these regions showing low GC levels, similarly to the majority of the regulatory regions implicated in embryonic development, something that has not been reported for promoter sequences. Moreover, analysis of protein-protein interactions showed that some of the transcription factors, predicted to bind onto these elements, together with other non-specific transcription factors, constitute a core transcription control complex. This protein complex interacts with the remaining members of the predicted cluster of transcription regulators and works either as an inducer or a suppressor of transcription. This is determined by the presence of a HAT and/or an HDAC in this protein complex assumed to locally control chromatin acetylation. Based on these data, we constructed a model of the complex regulatory network that describes how through the transcriptional regulation of the analyzed genes mainly guided by ΝGN3 the gradual differentiation of cells capable of producing insulin takes place.

Cell-type diversity is governed in part by differential gene expression programs mediated by transcription factor (TF) binding. However, there are few systematic studies of the genomic binding of different types of TFs across a wide range of human cell types, especially in relation to gene expression. In the ENCODE Project, we have identified the genomic binding locations across 11 different human cell types of CTCF, RNA Pol II (RNAPII), and MYC, three TFs with diverse roles. Our data and analysis revealed how these factors bind in relation to genomic features and shape gene expression and cell-type specificity. CTCF bound predominantly in intergenic regions while RNAPII and MYC preferentially bound to core promoter regions. CTCF sites were relatively invariant across diverse cell types, while MYC showed the greatest cell-type specificity. MYC and RNAPII co-localized at many of their binding sites and putative target genes. Cell-type specific binding sites, in particular for MYC and RNAPII, were associated with cell-type specific functions. Patterns of binding in relation to gene features were generally conserved across different cell types. RNAPII occupancy was higher over exons than adjacent introns, likely reflecting a link between transcriptional elongation and splicing. TF binding was positively correlated with the expression levels of their putative target genes, but combinatorial binding, in particular of MYC and RNAPII, was even more strongly associated with higher gene expression. These data illuminate how combinatorial binding of transcription factors in diverse cell types is associated with gene expression and cell-type specific biology.

Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection. Previous gene expression studies of hibernation have mainly focused on changes at the mRNA level. Large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species. We constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression throughout the torpor-arousal cycle during hibernation in arctic ground squirrels (Urocitellus parryii). We identified more than 3,000 unique proteins from livers of arctic ground squirrels. Among them, 517 proteins showed significant differential expression comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1-2 months after hibernation had ended (non-hibernating). Consistent with changes at the mRNA level shown in a previous study on the same tissue samples, proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed at the protein level in both late torpid and early aroused animals compared with non-hibernating animals, whereas proteins involved in fatty acid catabolism were significantly overexpressed. On the other hand, when we compared late torpid and early aroused animals, there were discrepancies between mRNA and protein levels for a large number of genes. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals compared with late torpid animals, whereas no significant changes at the mRNA levels between these stages had been observed. Our results suggest that there is substantial post-transcriptional regulation of proteins during torpor-arousal cycles of hibernation.

Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function

Single-cell chromatin accessibility analysis reveals the intricate regulatory landscape of mouse testicular development, uncovering novel cell subpopulations and transcription factors, and offering valuable insights into the molecular mechanisms driving germ cell and somatic cell maturation.

PDF HTML阅读 XML下载 导出引用 引用提醒 吉富尼罗罗非鱼Ikaros基因5'调控区的克隆、序列分析及抗无乳链球菌相关SNP位点筛选 DOI: 作者: 作者单位: 1. 中国水产科学研究院 珠江水产研究所, 农业部热带亚热带水产资源利用与养殖重点实验室, 广东 广州 510380;2. 上海海洋大学 水产与生命学院, 上海 201306;3. 惠州市渔业研究推广中心, 广东 惠州 516002 作者简介: 陈昆平(1991-),男,硕士研究生,研究方向为水产健康养殖及遗传育种.E-mail:colinchenkunping@163.com 通讯作者: 中图分类号: S917 基金项目: 现代农业产业技术体系专项资金项目（CARS-46）；广东省自然科学基金项目（2016A030313146）；国家自然科学基金项目（31502205）. Molecular cloning analysis of the 5' regulatory region of Ikaros gene from Oreochromis niloticus and screening of its SNP markers for Streptococcus agalactiae resistance Author: Affiliation: 1. Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China;2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;3. Fisheries Research and Extension Center of Huizhou, Huizhou 516002, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为获得尼罗罗非鱼（）抗链球菌病相关的单核苷酸多态性（single nucleotide polymorphism，SNP）标记，本研究通过染色体步移法克隆了尼罗罗非鱼基因的启动子和转录调控元件进行预测和分析。利用PCR产物直接测序法从亲本（P0）尼罗罗非鱼基因5'调控区中筛查到5个SNPs，分别为SNP1（g.562，G>A）、SNP2（g.217，G>T）、SNP3（g.-53，C>T）、SNP4（g.-220，T>C）和SNP5（g.-579，T>C）。利用Snapshot技术对子一代（F1）尼罗罗非鱼易感群体和抗病群体进行相应SNPs的基因分型，分析两个群体遗传多样性参数，结果显示多态信息含量（polymorphism information content，PIC）值在0.0872~0.3747之间，表明基因5'调控区SNPs与抗链球菌病性状关联分析结果表明，SNP2、SNP3、SNP4和SNP5的基因型频率和等位基因频率在易感群体和抗病群体中存在显著差异（基因5'调控区SNPs可形成1个单倍块和5种单倍型，其中GGCTT单倍型与抗病性显著相关（<0.05）。此外，还发现SNP2和SNP5处于完全连锁状态（Ikaros基因5'调控区筛选到4个抗链球菌病相关SNPs和1个单倍型（GGCTT），均可作为尼罗罗非鱼分子育种的候选分子标记。 Abstract:Tilapia () is one of the most important fishery species in the world and has been introduced and cultured widespreadly because of its fast growth rate, strong reproductive capacity, good adaptability and omnivorous feeding habit. However, disease has become the biggest threat for tilapia breeding. Recently, the epidemic and outbreak of tilapia disease caused substantial economic losses to aquaculture industry. Breeding resistant variety in tilapia was unimportant way to solve the problem of disease. The molecular marker assisted selection (MAS) has become an efficient breeding method for selecting and breeding tilapia, and will accelerate genetic improvement and increase selection intensity for disease resistance. The single nucleotide polymorphism (SNP) markers are used in many genetic and breeding studies because they are abundant in genomes, and can be genotyped easily. Ikaros is a kind of transcription factor with zinc finger structure that is essential to the development of lymphocyte and to the maintenance of normal immune function. Therefore, to obtain large amount of effective SNP molecular genetic markers and to perform MAS for disease resistance in tilapia, it is essential to study immune related candidate and to examine whether the SNPs in the gene are associated with disease resistance. In this study, the 5' regulatory region sequence of , length of 4178 bp, were obtained through Genome Walking method from . Bioinformatics software was used to analyze the 5' regulatory region sequence of gene. The predicted transcriptional start site (TSS) was in the initiation codon (ATG) upstream of 931 bp, and the core promoter regions was located at -57 bp to 48 bp when the TSS was specified as 1. The predicted promoter regions of gene included basic start of substructure components:TATA box, CCAAT box and octamer. The analysis of transcription factor binding sites (TFBS) showed that abundant of TFBS were located at -2200 bp to 1200 bp in 5' regulatory region of gene, such as GATA-1, Homeobox, CDP CR3+HD and AP-1. The analysis of CpG islands showed that two CpG islands were in 5' regulatory region sequence of gene, one of which was located in promoter regions and the other of which was located in the first exon. Five SNPs in the 5' regulatory region of gene were detected by direct sequencing method from the parents (P0), which are named SNP1 (g.562, G>A), SNP2(g.217, G>T), SNP3(g.-53, C>T), SNP4(g.-220, T>C) and SNP5(g.-579, T>C). The five SNPs were sited in various regulatory elements in promoter regions which could have major implications for exact expression of gene. Based on Snapshot method analysis, the frequencies of alleles and genotypes were calculated in susceptible groups and resistant groups of ). The polymorphisms and genetic parameter of the SNPs in resistant groups and susceptible groups were calculated by software Popgen 32 and PIC. The result showed that the polymorphism information content (PIC) of the 5' regulatory region of SNPs was 0.0872~0.3747, suggesting that all the SNPs had moderate of intermediate polymorphism. The correlation between SNPs and resistance to was analyzed by software SPSS 17.0, the results showed that four of them (SNP2, SNP3, SNP4and SNP5) were significantly associated with the resistance to <0.05). Moreover, All of the SNPs in the 5' regulatory region of could formed one haplotype block and five haplotypes from the prediction of linkage disequilibrium analysis used software Haploview 4.2. The haplotypes (GGCTT) were significantly associated with the resistance to <0.05), and two of the haplotypes (GGTCT and GTCCC) were significantly associated with <0.05). Furthermore, the SNP2 and SNP5 were completely linked with each other ('=1), which could be selected as tag SNP for research of genetic breeding in . The results suggest that the four SNPs (SNP2, SNP3, SNP4 and SNP5) and the haplotypes (GGCTT) in the 5' regulatory region of could be potential genetic markers for future molecular selection of . 参考文献 相似文献 引证文献

Bioinformatics Methods and Biological Interpretation for Next-Generation Sequencing Data.

Differential Expression of Genes Associated with Oncogene-Induced Senescence and Senescence Associated Secretory Phenotype in the Absence of Differential Expression of High Molecular Risk Genes and Genes Associated with JAK-STAT Pathway in Sorted Cells of Patients with Polycythemia Vera and Primary Myelofibrosis

S-adenosylmethionine Levels Regulate the Schwann Cell DNA Methylome

Changes in chromatin structure, especially in histone modifications (HMs), linked with chromatin accessibility for transcription machinery, are considered to play significant roles in transcriptional regulation. Alveolar macrophages (AM) are important immune cells for protection against pulmonary pathogens, and must readily respond to bacteria and viruses that enter the airways. Mechanism(s) controlling AM innate response to different pathogen-associated molecular patterns (PAMPs) are not well defined in pigs. By combining RNA sequencing (RNA-seq) with chromatin immunoprecipitation and sequencing (ChIP-seq) for four histone marks (H3K4me3, H3K4me1, H3K27ac and H3K27me3), we established a chromatin state map for AM stimulated with two different PAMPs, lipopolysaccharide (LPS) and Poly(I:C), and investigated the potential effect of identified histone modifications on transcription factor binding motif (TFBM) prediction and RNA abundance changes in these AM. The integrative analysis suggests that the differential gene expression between non-stimulated and stimulated AM is significantly associated with changes in the H3K27ac level at active regulatory regions. Although global changes in chromatin states were minor after stimulation, we detected chromatin state changes for differentially expressed genes involved in the TLR4, TLR3 and RIG-I signaling pathways. We found that regions marked by H3K27ac genome-wide were enriched for TFBMs of TF that are involved in the inflammatory response. We further documented that TF whose expression was induced by these stimuli had TFBMs enriched within H3K27ac-marked regions whose chromatin state changed by these same stimuli. Given that the dramatic transcriptomic changes and minor chromatin state changes occurred in response to both stimuli, we conclude that regulatory elements (i.e. active promoters) that contain transcription factor binding motifs were already active/poised in AM for immediate inflammatory response to PAMPs. In summary, our data provides the first chromatin state map of porcine AM in response to bacterial and viral PAMPs, contributing to the Functional Annotation of Animal Genomes (FAANG) project, and demonstrates the role of HMs, especially H3K27ac, in regulating transcription in AM in response to LPS and Poly(I:C).

Peroxisome Proliferator-Activated Receptor γ Dances with Different Partners in Macrophage and Adipocytes

A statistical model for locating regulatory regions in genomic DNA

Calcitriol (1,25-dihydroxycholecalciferol), the most active form of vitamin D, has selective anti-proliferative effects on tumor-derived endothelial cells (TDEC) compared with Matrigel-derived endothelial cells (MDEC). Although both cell types have an intact vitamin D receptor-signaling axis, this study demonstrates that upon treatment with calcitriol, 24-hydroxylase (CYP24) mRNA, protein and enzymatic activity were markedly induced in MDEC in a time-dependent manner but not in TDEC. Furthermore, treatment of MDEC with a CYP24 small interfering RNA restored sensitivity to calcitriol. To investigate the lack of CYP24 induction in TDEC, we examined methylation patterns in the promoter regions of the CYP24 gene in these two cell types. We identified two putative CpG island regions located at the 5' end. Using methylation-specific PCR and bisulfite sequencing, we determined that these CpG islands were hypermethylated in TDEC but not in MDEC. These data may explain the recruitment of vitamin D receptor to the promoter region in MDEC but not TDEC, as revealed by chromatin immunoprecipitation analyses. Treatment of TDEC with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored calcitriol-mediated induction of CYP24, which led to loss of sensitivity to calcitriol growth inhibitory effects. CYP24 promoter hypermethylation was also observed in endothelial cells isolated from other tumors but not in endothelial cells isolated from normal mouse tissues. These observations indicate that the methylation status of the CYP24 promoter differs in endothelial cells isolated from different microenvironments (tumor versus normal) and that methylation silencing of CYP24 contributes to selective calcitriol-mediated growth inhibition in endothelial cells.

Decision letter: Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates