A Statistical Method for Finding Transcription Factor Binding Sites
基于SREBPs通路的益肾通络方改善脂质代谢异常作用机制研究
基于SREBPs 通路的益肾通络方改善脂质代谢异常作用机制研究Δ赵靓 1＊，张效威 1，谢治深 1，向世勰 1，段亚飞 1，高改 1，王潘 1，马慧芬 1，孙意冉 1，陈洁 2，徐江雁 1，张振强 1 #（1.河南中医药大学中医药科学院，郑州 450046；2.马来西亚管理与科技大学国际学术部，马来西亚 雪兰莪州 莎阿南市 47300）中图分类号 R 965 文献标志码 A 文章编号 1001-0408（2023）23-2835-06DOI 10.6039/j.issn.1001-0408.2023.23.04摘要 目的 基于固醇调节元件结合蛋白（SREBPs ）通路探究益肾通络方改善脂质代谢异常的作用机制。
方法 以C 57BLKS/J（db /db ）小鼠为模型动物、 C 57BLKS/J （db /m ）小鼠为正常对照，通过测定小鼠肝脏系数及血清中总胆固醇（TC ）、甘油三酯（TG ）、低密度脂蛋白（LDL ）、高密度脂蛋白（HDL ）含量，观察小鼠肝组织脂肪变性和脂质蓄积情况，测定小鼠肝组织中SREBP-1、SREBP-2蛋白和Srebp-1c 、Srebp-2及其下游脂质代谢相关靶基因（Fasn 、Acc 1、Scd 5、Fads 1、Hmgcr 、Dhcr 24、Insig-1、Fdps ）的mRNA 转录水平，来考察1、2.5、5 g/kg 益肾通络方改善db /db 小鼠脂质代谢异常的作用机制。
以低脂培养的人肝癌细胞HepG 2为体外脂质代谢异常细胞模型，以25-HC （SREBPs 抑制剂，10 μmol/L ）为抑制剂对照，考察125、250、500 μg/mL 益肾通络方干预24 h 后对细胞中SREBP-1、SREBP-2蛋白和SREBP-1c 、SREBP-2其下游脂质代谢相关靶基因mRNA 转录水平的影响，进行体外机制验证。
结果 1、2.5、5 g/kg 益肾通络方均可显著降低模型小鼠TC 、TG 、LDL 水平，显著降低肝组织脂滴阳性面积百分比、肝脏系数，显著下调肝组织中Pre-SREBP-1、n-SREBP-1、Pre-SREBP-2、n-SREBP-2蛋白以及Srebp-1c 、Srebp-2及其下游靶基因的mRNA 转录水平，并可显著升高HDL 水平，差异均有统计学意义（P ＜0.05或P ＜0.01）。
GeneOntologyAnalysis：基因本体论分析
17
GO tree example
GO tree: A child can have more than one parent
⎯ Standard assignment of genes into functional categories ⎯ Controlled vocabulary for describing biological meanings
u Gene Ontology or GO project at NCBI
2) Define controlled terms (ontologies) for description of gene products from 3 aspects:
u Biological process (DNA repair, mitosis) u Molecular function (protein serine/threonine kinase activity, transcription factor
Gene Ontology -Cellular Component
/GO_nature_genetics_2000.pdf
Any one gene can be a member of more than one GO classification
21
Temporal snapshots of Go terms and mappings are available in BioC (~700, April 2014)
markov-chains 马尔科夫链
L
L
where aBx1 represents the transition from the begin state
• This gives a probability distribution over sequences of length L
Estimating the Model Parameters
gccgcgcttg gcttggtggc tggccgttgc
0 1 P r(a | g ) 12 4 7 1 P r(c | g ) 12 4 3 1 P r(g | g ) 12 4 2 1 P r(t | g ) 12 4
0 1 Pr(a | c) 74
Markov Chains for Discrimination
• suppose we want to distinguish CpG islands from other sequence regions • given sequences from CpG islands, and sequences from other regions, we can construct – a model to represent CpG islands – a null model to represent the other regions • can then score a test sequence by:
6 P r(a ) 0.2 30 9 P r(c) 0.3 30
7 P r(g ) 0.233 30 8 P r(t ) 0.267 30
Maximum Likelihood Estimation
• suppose instead we saw the following sequences gccgcgcttg gcttggtggc tggccgttgc • then the maximum likelihood estimates are
蛋白质相互作用
并非外源非特异蛋白，单克隆抗体的使用有助于避免污染的 发生；
(4) 要确保抗体的特异性，即在不表达抗原的细胞溶解物中 添加抗体后不会引起共沉淀；
Column CoIP
Column CoIP
NdkB
6755399
Sam68
6671538
4串联亲和纯化（TAP)
• 该方法选用了两个连续的标签进行相互作 用蛋白的纯化。
• 标签分三部分：蛋白A，C B P(calmodulin binding peptide，钙调素结合多肽)和中间 连接的TEV 酶识别的酶切位点。
A:标签中的ProteinA 与固化的IgG 结合，缓冲液淋洗去除不能结合的杂蛋白， TEV 酶切分离ProteinA 和靶蛋白； B:利用CBP(Calmodulin binding peptide)-Calmodulin 的相互作用进一步纯化 复合物，缓冲液洗去杂蛋白。加入过量的螯合剂(EGTA) 螯合Ca2+，使纯化的复合物与层析柱分离。
Bait Protein
Activation Domain
Prey Protein
Binding Domain
Reporter Gene
• Interaction of bait and prey proteins localizes the activation domain to the reporter gene, thus activating transcription.
• Since the reporter gene typically codes for a survival factor, yeast colonies will grow only when an interaction occurs.
基于癌症基因图集数据库与免疫组织化学方法分析SMARCB1对肝细胞癌早期诊断及预后的作用
基于癌症基因图集数据库与免疫组织化学方法分析SMARCB1对肝细胞癌早期诊断及预后的作用王健;曹天生;张升敏;吴佳勉;卢卓才;杨建荣;巫泓生;陈浩;林波;许荣华【摘要】目的: 通过研究SMARCB1在肝细胞癌(hepatocellular carcinoma,HCC)组织的表达,阐明其对HCC的早期诊断及预后的作用.方法: 在癌症基因组图集(The Cancer Genome Atlas,TCGA)数据库中筛选出SMARCB1基因,运用免疫组织化学(immunohistochemistry,IHC)技术和TCGA分析SMARCB1在HCC组织和正常组织的表达情况,阐述其在HCC发生、发展进程中的作用. 结果: IHC结果证实,与正常肝组织相比,HCC中SMARCB1的蛋白表达量显著上升(P<0.01).IHC的结果显示SMARCB1的蛋白表达量与原发肿瘤分期呈正相关(P<0.05),即SMARCB1表达量越高,原发肿瘤分期越趋向晚期.TCGA的结果显示SMARCB1的高表达是HCC的独立预后因素(P<0.05). 结论: SMARCB1可能起着促癌基因的作用,临床上根据其在组织中的表达差异,可鉴别早期的HCC及良性组织,并可能有效地进行预后判断.%AIM: To illuminate the effect of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1 (SMARCB1) in early diagnosis and prognosis of hepatocellular carcinoma (HCC) by determining the clinical expression of SMARCB1 in HCC tissue and benign liver tissue.METHODS: The specific target gene SMARCB1 was selected from these genes by using The Cancer Genome Atlas (TCGA).SMARCB1 expression in HCC tissue and benign liver tissue was measured by immunohistochemistry.Further statistical analysis of TCGA was performed to illuminate the role of SMARCB1 on HCC occurrence and progression.RESULTS: Compared with the benign liver tissue,immunohistochemical staining showed that SMARCB1 expression was significantly up-regulated in the HCC tissue (P<0.01).In addition, SMARCB1 expression was significantly associated with advanced tumor stage(P<0.05).The relation between SMARCB1 expression at mRNA level and clinical prognosis was analyzed.The results indicated that high SMARCB1 expression was an independent prognostic factor for HCC(P<0.05).CONCLUSION: SMARCB1 may play a part as a carcinogenic gene in tumorigenesis.We can distinguish primary HCC samples from non-malignant samples according to its different clinical expression.High SMARCB1 expression probably predicts poor outcome in HCC patients.【期刊名称】《中国病理生理杂志》【年(卷),期】2017(000)004【总页数】4页(P754-757)【关键词】肝细胞癌;SMARCB1;诊断;预后【作者】王健;曹天生;张升敏;吴佳勉;卢卓才;杨建荣;巫泓生;陈浩;林波;许荣华【作者单位】南方医科大学附属花都人民医院普外科,广东广州 510800;南方医科大学附属花都人民医院普外科,广东广州 510800;南方医科大学附属花都人民医院普外科,广东广州 510800;南方医科大学附属花都人民医院普外科,广东广州510800;南方医科大学附属花都人民医院普外科,广东广州 510800;南方医科大学附属花都人民医院普外科,广东广州 510800;南方医科大学附属花都人民医院普外科,广东广州 510800;南方医科大学附属花都人民医院普外科,广东广州 510800;南方医科大学附属花都人民医院普外科,广东广州 510800;海南医学院附属医院肿瘤外科,海南海口 570102【正文语种】中文【中图分类】R730.23肝细胞癌(hepatocellular carcinoma，HCC)是世界上第五大恶性肿瘤，病死率高居第3位[1-2]。
Lecture1 乳糖操纵子
Ph.D.Quan Chen,2002‐2007, Ph.D. in Biochemistry and Molecular Biology, USTC,,y,,,2007‐2009, Research Fellow, Mayo Clinic, Scottsdale, AZ, USA2009‐2011, Research Associate, University of Southern California, Los Angeles, CA, USA 2011‐present, Associate Professor, USTCMolecular Biology第一章前言I 第二章基因和基因组第三章DNA复制第四章转录Undergraduate 第五章翻译第六章基因表达的调控第七章突变与修复第八章癌症的分子生物学GraduateII 第九章转座子第十章基因操作和基因组分析第十一章细胞核的分子生物学第十二章基因表达调控的分子机制III 第十三章疾病产生的分子基础第十四章基因诊断和基因治疗Molecular Biology II ¾Regulation of Gene Expression‐In Prokaryotes‐In Eukaryotes¾Mutation and RepairG ti f C¾Genetics of Cancer¾Transposable Elements¾Gene Manipulation & Genomic AnalysisReferences《Essential i Genetics》, Peter J Russell The Benjamin/Cummings Publishing Company, Inc 《Genetics》, 5th ed.，Peter J Russell The Publishing Company, ed Benjamin/Cummings Company Inc Genes IX, Benjamin Lewin Jan. 2008 ISBN: 9787030182982.Grading g30% ‐Attendance, homework and quizzes70% ‐Final examFi lWhy is Regulation necessary• the level of Not all genes are expressed continuously:gene expression differ according to‐‐cell types, stages of cell cycle•Organisms live in changing environments•Regulation allows organisms to grow and reproduce optimally in different environmentsti ll i diff t i tAre all genes’ activity regulated?•The housekeeping genes (constitutive genes)Genes that are essential for normal cell functionf fand are constitutionally expressedg g•The regulated genesTheir activity is controlled in response to the needs of a cell ororganism•Distinction between regulated and constitutivegenes is somewhat arbitraryRegulation of Gene ExpressionThe most direct control pointRegulate by changing sets of genes’ expression level in response to environment alterationi l iEfficiency and economicsRegulation of Gene Expression in Prokaryotes ¾The lac Operon of E.coli¾The trp Operon of E.coli¾Regulation of gene expression in Phage Lambdal f h bdThe Logic of Prokaryotic Gene RegulationEfficiency and economicsMetabolic enzymes are required to break down different carbon sources to yield energy.Carbon sources: lactose, glucose, galactose, xylose etc.Selective repression and activation mechanisms must fulfill two criteria:They must have the ability to reversibly turn on or switch off transcription of each specific gene or group of genes.They must be responsive to environmental conditions in which transcription of the relevant gene(s) should be activated or repressed.Prokaryotic transcriptionPromoter–recruit RNA PolymeraseOperator sites–bind activators/ repressors10¾‐Element¾‐35 Element¾Extended ‐10 Element¾UP elementKey ConceptsTwo types of DNA-p rotein interactions are required for regulated transcription. determines where transcription begins-Promoter-RNA Polymeraseyregulates whether or not promoter-driven transcription occursOperators-Operators-Activators/ repressorsgMessageGenes must contain two kinds of binding sites to permit regulated transcription. First, binding sites for RNA polymerase must be present. Second, binding sites for activator or proteins must be present in the vicinity of the promoter.repressorAllosteric effectorseffectors toggle switches to control the ability or Allosteric work like of activator repressor proteins to bind to their DNA target sites.Operons‐Fine Control of Prokaryotic TranscriptionThe genes, the operator, and the promoter constitute an operonOperon‐‐Genes grouped together; transcribed into single mRNA molecule containing coding sequences for more than one gene (polycistronic mRNA). Grouped together with one promoter.Structural gene–lacZ, Y, ARegulatory gene ‐lacIOperon. A cluster genes, their expression are coordinatelyof genesregulated by operator/regulator protein interactions, plus the operator and promoterregion the¾l lac operon¾trp operoncontrol–repressorNegative controlPositive control–activatorNegative and positive regulation are not mutually exclusiveLactose metabolism and the operonWhat led to the study of operon regulationNoDiauxiediauxieGlucose GalactoseMannose ArabinoseFructose XyloseMannitol RhamnoseSorbitolS bi lDulcitolMaltoseLactose•when cultures of E.coli were grown in mixtures of two different sugars. Depending on the particular sugarsinvolved, the resulting growth curve showed eitherinvolvedcontinuous or biphasic growthThey wanted to know•How bacteria know that lactose is present in the growth medium?•What role the inducer plays in enzyme synthesis?•How do the cells respond so rapidly to the presence of a new sugar?Bacterium’s adaptation to utilizing different nutrients •Glucose is the main source of carbon•Enzymes required for glucose metabolism are coded byconstitutive genesi i•In the absence of Glucose and presence of other carbonsources such as lactose,the genesfor utilizing these substance are ‘turned on”.•The related induciblegenes are:(regulated genes)•The alternate substance : inducer•the effectors that turn repressoroff these genes:The general structure of a inducible geneofMetabolism lactosesimplified lac operon model ‐galactosidase: cleave the lactose moleculeb l id l h l l l Permease: transport lactose into the cellTransacetylase: not required for lactose metabolismβ‐galactosidase function in two waysCarbon sourceInducerdRegulation of the lac operonPolycistronic mRNA: several genes are transcribed into one mRNA.To ensure: gene products required for the same event are produced equally.T d t i d f th t d d lllactose must be present in the environmentglucose cannot be present in the cell's environmentLac operon controlling regionNegative regulation of the lac operon•lac I gene encodes lac repressor•The repressor is an allosteric protein•Inducer: allolactose, IPTGand a low basal level•Repressor is leaky of the lac operon products is always present.•Only about 10 tetramers of repressor are present per cell.The repressor protein•tetramer•an allosteric protein with two domains:a domainDNA bindingan inducer binding domainInducer binding Æstructural change at DNA binding domain Ælose its DNA binding ability Ætranscription repression relieved.The lac operon repression model In absence of lactheIn the presence of lactoseinducer ‐allolactoseE i l id f h Experimental evidence for the regulation of lac genesMutation in the Protein‐Coding Genes‐‐‐‐polar effect on a polygenic mRNA•LacZ, LacY and Lac A mutation to map theirlocation•Missense mutation•Nonsense mutationIn sum, nonsense mutations in the cluster of three genes involved in lactose utilization have different effects, depending on where they are located within the cluster: The nonsense mutations are said to exhibit polar effects, and the phenomenon is called polarity. (nonsense mutations that show polar effects therefore are called polar mutation)Mutation affecting the Regulation of Gene ExpressionConstitutive Mutation: Mutation that cause its downstream genes constitutively C i i M i M i h i d i i lexpressed with or without inducerTwo groups of constitutive mutations:Operator Mutation‐‐‐‐‐‐mapped to a small region near lac Z(lac O)LacI Gene Regulatory Mutation‐‐‐‐‐‐mapped to a DNA region a small distance away (lac I)‐‐repressor geneThe Operator Mutations•Partial diploid: F’lacO+ lacZ‐lacY+‐lacO c lacZ+ lacYg•F’strains in which a few chromosomal genes on an extrachromosomal genetic element called F factor are introduced into a bacterial cell•Test for gene products both in the presence and the absence of inducer allolactoseb f i d ll lβ•in the absence of inducer: ‐galactosidase•in the presence of inducer: β‐gal and permeaseCis‐dominance•Partial diploid: F’lacO+ lacZ‐lacY+‐lacO c lacZ+ lacYp g q y •The phenomenon that a gene or DNA sequence only controlling structure genes on the same, contiguous of DNA‐piece cis dominance•generally speaking, a cis‐dominant element has no diffusible gene product (protein)diff ibl d(i)•lacO+/lacO c on different chromosomes do not interfere. And has no position effectlacI gene mutations•Partial diploid: F’lacI+lacO+lacZ‐lacY+‐++‐lacI lacO lacZ lacY•Test for structural gene expression in the presence and the absence of inducerIn no βgal and permease•the absence of inducer:‐•in the presence of inducer: both proteins were made •The lacI+ gene can overcome lacI‐defect. Even when they are located on different chromosomesTrans‐dominance•Partial diploid: F’lacI+lacO+lacZ‐lacY+lacI‐lacO+lacZ+lacY‐•A gene or DNA sequence that can control genes on different, noncontiguous piece of DNA,g p•A trans‐dominant element must produce a diffusible geneproduct(protein)•In one system, a lacI+ gene can overcome any lacI‐gene defect.defectmutations ‐‐‐‐‐‐Cis‐dominantlac promoter()g •Promoter mutations Plac‐): affect all three structural genes (none or few), even with inducer present•Plac‐only control genes that are on the same DNA strand‐‐‐Cis‐dominantEffect of MutationsOperator mutationsLacO c mutation: operator DNA sequence mutatesp qcould not be recognized by the repressorits downstream genes constitutively expressed(with or inducer)withoutPartial diploid in the absence of inducerNoexpressionConstitutiveexpressionPartial diploid in the presence of inducerInducibleexpressionconstitutiveexpressionHaploid in presence or absence of inducerconstitutiveexpressionu a ep esso cou d o b d o ope a oMutant repressor could not bind to operatorPartial diploid in the absence of inducerNoexpressionPartial diploid in the presence of inducerInducibleexpressionInducibleexpression。
高通量测序技术简介共31页文档
Reversible Terminator Chemistry 可逆终止反应
• All 4 labelled nucleotides in 1 reaction
O HN
ON
PPP
O
cleavage site fluor
O
X
5’
HN
DNA O N
O
O
3’
block
Incorporation Detection Deblock; fluor removal
Methy-seq（1）： 肿瘤和MCF7细胞系中 BRCA!启动子区域的甲基化差异
Methy-seq（2）：
Some highlights:
Correlation between ChIP-Seq and his prior SAGE-like method (called GMAT) has r=0.906
Digital expression profiling & microRNA re-sequencing：
hESC： human embryonic stem cells EB： embryoid bodies
ChIP-seq（1）： 人一号染色体DNA-蛋白相互作用
ChIP-seq（2）：
Cycle 2-n: Add sequencing reagents and repeat
1、每轮测序反应加入四种带有荧光标记的dNTP，末端带有可 以被去除的阻断基团 2、每轮反应只能整合一个核苷酸，仪器读取相应的荧光信号 3、信号读取结束，用化学方法去除阻断基团，进行下一轮测序 反应
Base calling from the raw data T G C TAC GAT …
Bioinformatics
Chien-Yu Chen Graduate school of biotechnology and bioinformatics, Yuan Ze University
Course Information
Web page
– .tw/~cychen/course/922BioInfo/922-BioInfo-CourseInformation.htm
12
Functional Genomics – Research Issues
Which genes are expressed in which tissues? How is the expression of a gene affected by extracellular influences? Which genes are expressed during the development of an organism? What is the effect of misregulated expression of a gene? What patterns of gene expression cause a disease or lead to disease progression? What patterns of gene expression influence response to treatmen Reading Frames
Consider the double-stranded DNA sequence below
5' CAATGGCTAGGTACTATGTATGAGATCATGATCTTTACAAATCCGAG 3' 3' GTTACCGATCCATGATACATACTCTAGTACTAGAAATGTTTAGGCTC 5'
第四章 生物信息学的主要研究方法
（2） 生物信息学软件和工具的 应用
(Software & application)
第二式 飞龙在天
利用成熟的生物信息学工具（专业网站、软件）解决生物信息学问题 ——基因组序列分析、序列比对软件（GCG、BLAST、CLUSTAL等） ——系统发育树构造软件（PHYLIP、PALM等……） ——基因芯片检测分析软件（商业软件ScanArray、Array-Pro等 ……） ——分子动力学模拟软件（GROMACS、NAMD等……）
受体位点（acceptor site）单碱基的权重矩阵（k=1）：
-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 A 9.0 8.4 7.5 6.8 7.6 8.0 9.7 9.2 7.6 7.8 23.7 4.2 100 0.0 23.9 C 31.0 31.0 30.7 29.3 32.6 33.0 37.3 38.5 41.0 35.2 30.9 70.8 0.0 0.0 13.8 G 12.5 11.5 10.6 10.4 11.0 11.3 11.3 8.5 6.6 6.4 21.2 0.3 0.0 100 52.0 U 42.3 44.0 47.0 49.4 49.4 46.3 40.8 42.9 44.5 50.4 24.0 24.6 0.0 0.0 10.4
Deciphering a secret message……
《The Gold-Bug》 A parchment written by pirate Captain Kidd:
53##+305))6*;4826)4#.)4#):806*;48+8$60))85; 1#(;:#*8+83(88)5*+;46(88*96*?;8)*#(;485);5* +2:*#(;4956*2(5*--4)8$8*;4069285);)6+8)4##; 1(#9;48081;8:8#1;48#85;4)485+528806*81(#9; 48;(48;(88;4(#?34;48)4#161;:188;#?;
第十一讲 Tiling array and exon array
Promoter tiling array
Whole-Genome Tiling Array Sets
• The GeneChip Human Tiling 1.0R Array Set is a 14-array set designed for transcript mapping or other analyses that benefit from both perfect match and mismatch probes. The GeneChip Human Tiling 2.0R Array Set is designed for ChIP experiments and features all of the perfect match probes from the Human Tiling 1.0R Array Set. Each array within the sets contains over 6.5 million probes to specifically interrogate genomic regions
• • •
•
Location Analysis ( ChIP-on-chip )
ChIP-on-chip Workflow
• 染色质免疫共沉淀芯片将染色质沉淀和芯 片技术结合在一起，简单的说，在体内 DNA和蛋白质在甲醛作用下交联，并且通 过与目标蛋白对应的抗体实现免疫沉淀。 通过超声波作用将DNA降解为0.2-2 kb 的 片段。需要对超声波参数进行设定，以获 得长度在一定范围内的DNA片段。Pulldown DNA和合适的对照用荧光标记，加在 载玻片上，用于芯片分析，可以找到特异 性蛋白在基因组中的结合位点。
PCR产物用于tiling的缺点