细胞分化的分子机制The genetic core of development: Differential gene expression细胞生长细胞代谢细胞增殖细胞分化细胞通讯细胞衰老细胞死亡……个体发育细胞分化（cell differentiation ）：经分裂形成在形态经分裂形成在形态、、结构和功能上不同的稳定的细胞类群的过程群的过程；；是个体发育的基础和核心是个体发育的基础和核心。
细胞表型Cell phenotype ：是细胞特定基因型在一定的环境条件下的表现一定的环境条件下的表现，，是细胞的特定性状是细胞的特定性状。
Totipotent cell:产生有机体全部细胞表型细胞 Pluripotent cell: 产生几种特定类型的细胞Differentiated cell:多潜能细胞通过分离和分化发育成的特殊细胞表型个体发育的过程个体发育的过程：：全能性细胞全能性细胞→→多潜能性细胞多潜能性细胞→→分化细胞细胞分化是基因差异性表达的结果细胞分化是个体行使正常功能的保证细胞分化是个体行使正常功能的保证。
本质本质：：细胞的基因组相同细胞的基因组相同，，但表达谱不同但表达谱不同；；使细胞能行使不同的功能使细胞能行使不同的功能（（分工分工）；）；核心核心：：基因是如何有序表达的基因是如何有序表达的？？细胞分化过程是基因差异表达的结果细胞分化过程是基因差异表达的结果。
每个基因的表达都必须每个基因的表达都必须：：在正确的细胞中在正确的细胞中；；在正确的时间在正确的时间；；对正确的信号产生正确的反应对正确的信号产生正确的反应；；产生正确的表达产物产生正确的表达产物，，表达水平表达水平。
问题问题：：细胞是如何协调细胞是如何协调（（coordinate ）这一过程的？Evidence for genomic equivalenceGenetic evidence-Polytenic chromosomes 多线染色体Embryology evidence-differentiated cells can retain their potential to produce other cell types.Transdifferentiation-metaplasiaMolecular evidence•DNA isolation techniques •Northern blotting•In situ hybridization•The exception to the rule: Immunoglobin genesGene deletionGene amplificationGene rearrangementDifferent cell types make different sets of proteins, even though their genomes are identical.Each cell use only small subset of these genes, different cell types use different subsets of these genes.Composition of genes1.Promoter2.Transcription initiation site3.Translation initiation site4.Exons5.Introns6.Translation termination site7.3’untranslated region (3’UTR):8.poly(A) addition site (usually AATAAA), transcription termination site1. Transcription•Synthesis of nuclear RNA from DNA matrix•Nuclear RNA contains both exonsand introns•Transcription machinery -protein complex•Proceeds in nucleus2. Processing•Synthesis of mRNA from nuclear RNA matrix•mRNA contains just exons•Proceeds in nucleus•mRNA is transported to the cytoplasm3. Translation•Synthesis of a protein from mRNA matrix•Takes place in the cytoplasm•Ribosomesare involved in translation machinery4.Posttranslational modifications•Folding the protein•Glycosylation•Phosphorylation•Lipidation•Adding prosthetic groups (non-amino acid component of a protein)The regulation of gene expression can be accomplished at several levels •Differential gene transcription•Selective nuclear RNA processing•Selective mRNA translation•Differential protein modification1) 1) 染色体水平的转录调控染色体水平的转录调控X chromosome inactivation Mammals barr bodyX chromosome inactivation 虽然是随机的虽然是随机的，，但某些情况下有一定的选择性和规律性1、转录水平的调控X chromosome inactivation in mammalsThe mechanism of X chromosome inactivation Xist基因引发X chromosome inactivation维持X chromosome silencemethylationRegulatory network from early sea urchin developmentLevine and Davidson, PNAS, 2005Transcription is the synthesis of RNA on a DNA template. Thanscription requires an enzyme, RNA polymerase, and a host of ubiquitous proteins that help to load the enzyme onto the promoter and establish the basal apparatus .Transcription factors bind near the promoter, or enhancers,influence gene expression either by interacting with the basal apparatus, many transcription factors are dominant, and can be activated by signal transductionpathways.2) 转录水平的调控•Promoters are the sites where RNA polymerase binds to the DNA to initiate transcription, most of these promoters contain the sequence TATA，where RNA Polymerase will be bound.•An Enhancer is a DNA sequence that can activate the utilization of a promoter, controlling the efficiency and rate of transcription from that particular promoter.•Like promoters, enhancers function by binding specific regulatory proteins-transcription factors.•Enhancers can regulate the temporal and tissue-specific expression of any differentially regulated gene, but different types of genes normally have different enhancers.The genetic elements regulating tissue-specific transcription can be identified by fusing reporter genes to suspected enhancer regions of the genes expressed in particular cell types•Silencer: repress gene expression (negative enhancer)•Ex) Neural restrictive silencer element (NRSE), which is recognized by NRSF, repress gene expression except in neural tissues •Insulator: provides barrier for enhancer’s actiondifferential gene expression1.Enhancers are the major determinant of differential gene expression2.One gene can have multiple enhancer elements3.Multiple transcription factors can bind together on one enhancer element4.Interaction between transcription factors on the enhancer and transcription initiation complex at the promoter5.Mix and match of transcription factors for thedifferential gene expression6.Enhancers are modulator of gene expression: thestrength of enhancer7.Enhancers remodel chromatin to activatepromoter or facilitate the binding of RNApolymerase to the promoter8.Enhancer also can repress gene expression9.Most genes require enhancers for their expressionTranscription factor •Transcription factors are proteins that bind to enhancer or promoter regions and interact to activate or repress the transcription of a particular gene.•Transcription factors have three major domain. DNA–binding domain(recognizes a particular DNA sequence); trans-activating domain(activate or suppress transcription); protein-protein interaction domain(allows modulated by TAF ir other transcription factors).Some major transcription factor families and subfamilies Muscle and nervespecification; drosophila sex determinationMyoD, achaete, daughterlessBasic helix-loop-helix (bHLH)Neural specification; eye developmentPax1, 2, 3 etc.Pax Head developmentLim-1, Forkhead Lim Pituitary formation; neural fatePit-1, Unc-86, Oct-2Pou Axis formationHoxa-1, Hoxb-2, etc.Hox Homeodomain Some functionsRepresentativetranscription factorsFamilyBend DNA;Mammalian primarysex determination; ectoderm differentiationSry, SoxD, Sox2Sry-soxSecondary sex determination; craniofacial; limb development Glucocorticoid receptor, estrogen receptor Nuclear homone receptors Kidney, gonad, and macrophage development; drosophila segmentation WT1, Kruppel,engrailedStandardZinc fingerExamples of transcription factors •MITF(microphthalmia) is necessary for the production of pigment cells and their pigments.•MITF has a protein-protein interaction domain that enables it to dimerize with another MITF protein, this homodimer can bind to DNA and activate the transcription of certain gene.•Without MITF, the pigment-cell-specific proteins of the tyrosinase family are not synthesized properly.•Pax6 is needed for mammalian eye, nervous system and pancreas development.•The use of Pax6 by different organs demonstrates the modular nature of transcriptional regulatory units.A. chick δcrystallin gene;B. rat somatostatinGriffin et al. 2002Enhancer (α-enhancer)Baumer et al. 2002EnhancerEnhancer Kleinjan et al. 2004Autoregulatory Enhancer Williams et al. 1998Key role of MyoD in muscle developmentFig. 21.93) DNA甲基化和转录的调控methylation and the control of transcriptionHow can cells undergo rounds of mitosis and still maintain their differentiated characteristics? DNA MethylationMethylation of globin genes in human embryonic blood cells•甲基化抑制基因转录的可能机制甲基化的DNA可以稳定核小体Acetylated histones are relatively unstable and cause nucleosomes to disperse;DNA methyation is linked to histone deacetylation2 Roles of differential RNA processing duringdevelopmentSome examples of alternative RNA splicingAlternative RNA splicing to form a family of rat-tropomyosin proteinsFGF8 splicing in xenopusFGF8b is the FGF8 spliceform affecting mesodermal developmentDevelopment 2006 133: 1703-17143 Control of Gene Expression at the Levelof Translation•mRNA longevity-Primarily depends on the poly(A) tails-Influenced by external factors (ex. Casein mRNAstays longer during lactation periods)•Selective inhibition of mRNA translation-Regulatory sequences: 5’cap and3’UTR-microRNADegradation of casein mRNA in the presence and absence of prolactinRNA also acts as a regulator of gene expression siRNA miRNA。
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