细胞分化的分子机制

1、一、绪论细胞分化的分子机制细胞分化的分子机制细胞分化 多细胞生物个体生长发育过程中，细胞在结构、形态、生理功能及生化特征等方面逐步产生稳定的差异，形成不同的细胞类型，形成不同的组织器官和系统。细胞类型分化细胞类型分化基因表达调控基因表达调控细胞类型形成细胞类型形成组织器官形成组织器官形成个体发育个体发育细胞分化：渐进过程；贯穿生命过程细胞决定：细胞在发育的某个时刻被定向 被定向的细胞最终发育成为成熟细胞细胞表型n全能性细胞（totipotent cell)n多潜能细胞（pluripotent cell)n分化的细胞（differentiated cell)能够产生全部细胞表型；具有分化形成全部

2、细胞类型的能力发育潜能一定的局限性 ; 具有分化形成大多数细胞类型的能力发育命运在一定程度上被限定；基因表达受到一定程度的限定由多潜能细胞分裂分化发育成的特殊细胞表型；有丝分裂频率明显降低甚至停止分裂；一般仅5%10%的基因表达细胞分化过程全能性细胞多潜能细胞分化细胞基因选择性表达基因选择性表达的结果的结果随着个体发育的进行，细胞核指导发育的潜能被限定，甚至丧失指导全部发育的能力豹蛙 囊胚期细胞核 激活的去核卵 60% 正常发育成囊胚 其中8085%形成正常蝌蚪 豹蛙 原肠胚早期内胚层细胞核 激活的去核卵 50% 的胚胎能正常发育 成正常蝌蚪 豹蛙 神经胚内胚层细胞核 激活的去核卵 10% 以

3、下的胚胎能正常发育 全能性及多能性的维持由外部信号和内部决定因素控制-外部信号-内部决定因素各种细胞因子各种转录调节因子细胞分化是基因差异表达的结果n细胞内环境影响差异表达 卵质不均匀分布n细胞外环境的影响 胚胎细胞处于不同区域，接受不同位置信息；邻近细胞相互关系；信号转导 细胞诱导 细胞诱导细胞生长因子邻近细胞表面分子细胞不对称分裂 不对称分裂受体及信号转导分子基因差异表达是最重要的调控机制差异表达基因 时间特异性： 只在发育的某个特定时期表达空间特异性： 组织、细胞特异性基因表达在时间和空间上的特异性n时间特异性n空间特异性在发育的某个或某些时期表达在发育的某个或某些时期表达检测方法：检测

4、方法：转录组测序；消减杂交；转录组测序；消减杂交；RT-PCR；real-time PCR基因表达的组织特异性、细胞特异性基因表达的组织特异性、细胞特异性检测方法：原位杂交检测方法：原位杂交胚胎诱导胚胎发育过程中一部分细胞影响相邻细胞向一定方向分化胚胎发育过程中一部分细胞影响相邻细胞向一定方向分化不对称分裂 mRNA不对称分配不对称分配视胞诱导外胚层形成晶体视胞诱导外胚层形成晶体晶体诱导外胚层形成角膜晶体诱导外胚层形成角膜细胞环境影响转录抑制剂（放线菌素转录抑制剂（放线菌素D D）处理受精卵，胚胎发育）处理受精卵，胚胎发育仍能进行至囊胚期仍能进行至囊胚期蛋白质翻译抑制剂处理受精卵（嘌呤霉素），

5、受蛋白质翻译抑制剂处理受精卵（嘌呤霉素），受精卵停止发育精卵停止发育Figure 21-18. Asymmetric divisions segregating P granules into the founder cell of the C. elegans germ line. The micrographs in the upper row show the pattern of cell divisions, with cell nuclei stained blue with a DNA-specific fluorescent dye; below are the same ce

6、lls stained with an antibody against P granules. These small granules (0.51 m in diameter) are distributed randomly throughout the cytoplasm in the unfertilized egg (not shown). After fertilization, at each cell division up to the 16-cell stage, both they and the intracellular machinery that localiz

7、es them asymmetrically are segregated into a single daughter cell. (Courtesy of Susan Strome.) the Par proteins serve to bring a set of ribonucleoprotein particles called to the posterior pole, so that the posterior daughter cell inherits P granules and the anterior daughter cell does not. 二、基因的差异表达

8、机制二、基因的差异表达机制细胞分化的分子机制细胞分化的分子机制真核生物基因表达受转录调节因子真核生物基因表达受转录调节因子的组合调控的组合调控启动子启动子 + DNA调控序列调控序列Figure 7-41. The gene control region of a typical eucaryotic gene. The promoter is the DNA sequence where the general transcription factors and the polymerase assemble (see Figure 6-16). The regulatory sequenc

9、es serve as binding sites for gene regulatory proteins, whose presence on the DNA affects the rate of transcription initiation. These sequences can be located adjacent to the promoter, far upstream of it, or even within introns or downstream of the gene. DNA looping is thought to allow gene regulato

10、ry proteins bound at any of these positions to interact with the proteins that assemble at the promoter. Whereas the general transcription factors that assemble at the promoter are similar for all polymerase II transcribed genes, the gene regulatory proteins and the locations of their binding sites

11、relative to the promoter are different for each gene. 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.基因组中有成千种不同转录调节因子基因组中有成千种不同转录调节因子 - vary from one gene control region to the next- usually present in very small amounts in a cell, often less tha

12、n 0.01% of the total protein.- most of them recognize their specific DNA sequences using one of the DNA-binding motifs , although some do not recognize DNA directly but instead assemble on other DNA-bound proteins.For example, of the roughly 30,000 human genes, an estimated 510% encode gene regulato

13、ry proteins. Name DNA Sequence Recognized * Bacteria lac repressor 5 AATTGTGAGCGGATAACAATT 3 TTAACACTCGCCTATTGTTAA CAP TGTGAGTTAGCTCACT ACACTCAATCGAGTGA lambda repressor TATCACCGCCAGAGGTA ATAGTGGCGGTCTCCAT Yeast Gal4 CGGAGGACTGTCCTCCG GCCTCCTGACAGGAGGC Mat2 CATGTAATT GTACATTAA Gcn4 ATGACTCAT TACTGAG

14、TA Drosophila Kruppel AACGGGTTAA TTGCCCAATT Bicoid GGGATTAGA CCCTAATCT Mammals Sp1 GGGCGG CCCGCC Oct-1 Pou domain ATGCAAAT TACGTTTA GATA-1 TGATAG ACTATC MyoD CAAATG GTTTAC p53 GGGCAAGTCT CCCGTTCAGA 基因表达调控的保守机制基因表达调控的保守机制蛋白蛋白- -核酸相互作用核酸相互作用通常搭建转录起始复合体时是低效的，需要转录激活远距离互作远距离互作顺式元件顺式元件增强子增强子(enhancer)In h

15、igher eucaryotes it is not unusual to find the regulatory sequences of a gene dotted over distances as great as 50,000 nucleotide pairs. Figure 9.26. Activators of eukaryotic transcription initiation. The blue activator is attached to a regulatory module upstream of a gene, and influences transcript

16、ion initiation only at that single gene. The green activator is attached to a site within an enhancer and is influencing transcription of all three genes. 2002 Garland Science时间特异增强子增强子能使基因转录频率增强子能使基因转录频率增加增加10200倍倍Figure 7-59. Model for the control of the human -globin gene. The diagram shows some

17、of the gene regulatory proteins thought to control expression of the gene during red blood cell development. Some of the gene regulatory proteins shown, such as CP1, are found in many types of cells, while . As indicated by the double-headed arrows, several of the binding sites for GATA-1 overlap th

18、ose of other gene regulatory proteins; it is thought that occupancy of these sites by GATA-1 excludes binding of other proteins. Once bound to DNA, the gene regulatory proteins recruit chromatin remodeling complexes, histone modifying enzymes, the general transcription factors and RNA polymerase to

19、the promoter. (Adapted from B. Emerson, in Gene Expression: General and Cell-Type Specific M. Karin, ed., pp. 116161. Boston: Birkhauser, 1993.) Figure 7-47. Transcriptional synergy. In this experiment, the rate of transcription produced by three experimentally constructed regulatory regions is comp

20、ared in a eucaryotic cell. Transcriptional synergy, the greater than additive effect of the activators, is observed when several molecules of gene activator protein are bound upstream of the promoter. Synergy is also typically observed between different gene activator proteins from the same organism

21、 and even between activator proteins from widely different eucaryotic species when they are experimentally introduced into the same cell. This last observation reflects the high degree of conservation of the transcription machinery协同作用的效应不是单个激活因子效应之和而是其乘积协同作用的效应不是单个激活因子效应之和而是其乘积不同的激活因子也具有这样的协同效应基因转录

22、活性在不同细胞中可由不同增强子控制silencer A regulatory sequence that reduces the rate of transcription of a gene or genes located some distance away in either direction.neural restrictive silencer element (NRSE)Figure 5.16. The importance of silencers in liver-specific gene transcription. (A) In the early digestive

23、 tube endoderm, most of the transcription factors are not bound to their sites on the enhancer for serum albumin. (B) As endoderm development proceeds, the sites on the enhancer become occupied by five proteins whose presence is essential for activating the gene, and one protein, bound to the silenc

24、er (site eY), that can inhibit transcription. (C) As the liver forms, the inhibitory protein is no longer found on the enhancer, and the serum albumin gene is transcribed. Interestingly, this change may take place shortly after the association of the pre-liver endodermal region with heart-forming ti

25、ssue. At this time, the chromatin in this region clumps together to form a nucleoprotein activation complex that spans 180 base pairs of DNA and activates the albumin promoter. (After Gualdi et al. 1996.) Figure 7-50. Eucaryotic gene regulatory proteins often assemble into complexes on DNA. Seven ge

26、ne regulatory proteins are shown in (A). The nature and function of the complex they form depends on the specific DNA sequence that seeds their assembly. In (B), some assembled complexes activate gene transcription, while another represses transcription. Note that the red protein is shared by both a

27、ctivating and repressing complexes Figure 7-57. Integration at a promoter. Multiple sets of gene regulatory proteins can work together to influence transcription initiation at a promoter, as they do in the eve stripe 2 module illustrated previously in Figure 7-55. It is not yet understood in detail

28、how the integration of multiple inputs is achieved, but it is likely that the final transcriptional activity of the gene results from a competition between activators and repressors that act by the mechanisms summarized in Figures 7-43, 7-44, 7-45, 7-46, and 7-49. eve 基因调控区基因调控区Figure 7-56. Distribu

29、tion of the gene regulatory proteins responsible for ensuring that eve is expressed in stripe 2. The distributions of these proteins were visualized by staining a developing Drosophila embryo with antibodies directed against each of the four proteins (see Figures 7-52 and 7-53). The expression of ev

30、e in stripe 2 occurs only at the position where the two activators (Bicoid and Hunchback) are present and the two repressors (Giant and Krppel) are absent. In fly embryos that lack Krppel, for example, stripe 2 expands posteriorly. Likewise, stripe 2 expands posteriorly if the DNA-binding sites for

31、Krppel in the stripe 2 module (see Figure 7-55) are inactivated by mutation and this regulatory region is reintroduced into the genome. The eve gene itself encodes a gene regulatory protein, which, after its pattern of expression is set up in seven stripes, regulates the expression of other Drosophi

32、la genes. As development proceeds, the embryo is thus subdivided into finer and finer regions that eventually give rise to the different body parts of the adult fly, as discussed in Chapter 21. This example from Drosophila embryos is unusual in that the nuclei are exposed directly to positional cues

33、 in the form of concentrations of gene regulatory proteins. In embryos of most other organisms, individual nuclei are in separate cells, and extracellular positional information must either pass across the plasma membrane or, more usually, generate signals in the cytosol in order to influence the ge

34、nome. 中胚层祖细胞成肌细胞多核肌管肌纤维externalsignaldeterminationdifferentiationmaturationmyoDMyf-5MRF-4myogeninGrowth regulatormuslespecific genesFigure 21-7. The standard test for cell determinationFigure 21-8. Prospective thigh tissue grafted into the tip of a chick wing bud forms toes. (After J.W. Saunders et

35、al., Dev. Biol. 1:281301, 1959.) n果蝇具有能分化上皮细胞或神经母细胞潜能的细胞n肌母细胞决定基因细胞记忆装置细胞记忆装置 Figure 7-68. Schematic diagram showing how a positive feedback loop can create cell memory. Protein A is a gene regulatory protein that activates its own transcription. All of the descendants of the original cell will ther

36、efore “remember” that the progenitor cell had experienced a transient signal that initiated the production of the protein. The ABC genes code for transcription factors that initiate a cascade of events leading to the actual production of floral partsMyoD, Myf5, myogenin, Mrf4中任何中任何一个在成纤维细胞中表达都能诱导肌一个

37、在成纤维细胞中表达都能诱导肌细胞分化细胞分化推测：推测： 成纤维细胞中可能已经积累一些调节蛋白，它们成纤维细胞中可能已经积累一些调节蛋白，它们可以与肌原性蛋白协作，开启可以与肌原性蛋白协作，开启 muscle-specific genes。 特定的调节蛋白组合决定肌肉分化特定的调节蛋白组合决定肌肉分化Ey基因编码的调节蛋白引发果蝇眼形成基因编码的调节蛋白引发果蝇眼形成The Ey protein is known to bind directly to numerous target genes for eye development, including those encoding len

38、s crystallins (see Figure 7-119), rhodopsins, and other photoreceptor proteins. (Adapted from T. Czerny et al., Mol. Cell 3:297307, 1999.) Figure 7-75. Gene regulatory proteins that specify eye development in Drosophila. toy (twin of eyeless) and ey (eyeless) encode similar gene regulatory proteins,

39、 Toy and Ey, either of which, when ectopically expressed, can trigger eye development. In normal eye development, expression of ey requires the toy gene. Once its transcription is activated by Toy, Ey activates transcription of so (sine oculis) and eya (eyes absent) which act together to express the

40、 dac (dachshund) gene. As indicated by the green arrows, some of the gene regulatory proteins form positive feedback loops which reinforce the initial commitment to eye development. The Ey protein is known to bind directly to numerous target genes for eye development, including those encoding lens c

41、rystallins (see Figure 7-119), rhodopsins, and other photoreceptor proteins. (Adapted from T. Czerny et al., Mol. Cell 3:297307, 1999.) 发育过程中经常出现基因表达协调调节一种类型细胞中表达多种细胞类型特异基因激素诱导几种基因表达（如类固醇受体激活的基因）饥饿、剧烈运动饥饿、剧烈运动 糖皮质素激素糖皮质素激素肝细胞中肝细胞中 氨基酸氨基酸 葡萄糖葡萄糖糖皮质素激素受体糖皮质素激素受体（转录调节因子）（转录调节因子） 多个基因表达多个基因表达 酶酶Figure 1

42、5-12. Some signaling molecules that bind to nuclear receptors. Note that all of them are small and hydrophobic. The active, hydroxylated form of vitamin D3 is shown. Estradiol and testosterone are steroid sex hormones. 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Robert

43、s, and Peter Walter.核受体的信号核受体的信号类固醇激素：甾醇、类固醇性激素、维生素类固醇激素：甾醇、类固醇性激素、维生素D D、蜕皮素；前、蜕皮素；前体为胆固醇体为胆固醇Figure 12.5. Gene activation by a steroid hormone. Estradiol is one of the estrogen steroid hormones. After entering the cell, estradiol attaches to its receptor protein and the complex enters the nucleus

44、where it binds to the 15-bp estrogen response element (abbreviation: N, any nucleotide), which is located upstream of those genes activated by estradiol and other estrogens. Other steroid hormone receptors recognize other response elements. For example, glucocorticoid hormones target the sequence 5-

45、AGAACANNNTGTTCT-3. Note that this sequence, and that of the estrogen response element, is an inverted palindrome. The response element for vitamin D3, which is a steroid derivative that activates transcription via a nuclear receptor (see the text), has the sequence 5-AGGTCANNNAGGTCA-3, which is a di

46、rect repeat rather than an inverted palindrome. 2002 Garland ScienceOnce inside the cell, each hormone binds to a specific steroid receptor protein, which is usually located in the cytoplasm After binding, the activated receptor migrates into the nucleus, where it attaches to a response element upst

47、ream of a target gene.雌二醇雌二醇雌二醇受体雌二醇受体Figure 15-14. Responses induced by the activation of a nuclear hormone receptor. (A) Early primary response and (B) delayed secondary response. The figure shows the responses to a steroid hormone, but the same principles apply for all ligands that activate this

48、family of receptor proteins. Some of the primary-response proteins turn on secondary-response genes, whereas others turn off the primary-response genes. The actual number of primary- and secondary-response genes is greater than shown. As expected, drugs that inhibit protein synthesis suppress the tr

49、anscription of secondary-response genes but not primary-response genes, allowing these two classes of gene transcription responses to be readily distinguished. 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.Responses induced by the activation of

50、a nuclear hormone receptor (A) early primary response (B) delayed secondary response. Figure 9.13. The steroid receptor zinc finger. The R groups of the amino acids involved in the interactions with the zinc atoms are shown as solid green lines. N and C indicate the N- and C-termini of the motif, re

51、spectively. Reprinted from DNA-Protein Interactions by Andrew Travers, published by Chapman & Hall, 1993. Reprinted with kind permission of A. Travers. 2002 Garland ScienceResponse elements for each receptor are located upstream of 50100 genes and, once bound, the receptor acts as a transcriptio

52、n activator. Figure 21-4. How regulatory DNA defines the succession of gene expression patterns in development. the rules for stepping from one state to the next, as the , switching on new sets of genes according to the activities of the proteins that they currently contain 1组合调控在发组合调控在发 育中的意义育中的意义小

53、结选择性基因转录与染色质变化Figure 4-27. The assembly of a histone octamer. The histone H3H4 dimer and the H2A-H2B dimer are formed from the handshake interaction. An H3-H4 tetramer forms the scaffold of the octamer onto which two H2A-H2B dimers are added, to complete the assembly. The histones are colored as in

54、Figure 4-26. Note that all eight N-terminal tails of the histones protrude from the disc-shaped core structure. In the x-ray crystal (Figure 4-25), most of the histone tails were unstructured (and therefore not visible in the structure), suggesting that their conformations are highly flexible. (Adap

55、ted from figures by J. Waterborg.) 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.Figure 4-35. Covalent modification of core histone tails. (A) Known modifications of the four histone core proteins are indicated: Me = methyl group, Ac = acetyl gr

56、oup, P = phosphate, u = ubiquitin. Note that some positions (e.g., lysine 9 of H3) can be modified in more than one way. Most of these modifications add a relatively small molecule onto the histone tails; the exception is ubiquitin, a 76 amino acid protein also used in other cellular processes (see

57、Figure 6-87). The function of ubiquitin in chromatin is not well understood: histone H2B can be modified by a single ubiquitin molecule; H2A can be modified by the addition of several ubiquitins. (B) A histone code hypothesis. Histone tails can be marked by different combinations of modifications. A

58、ccording to this hypothesis, each marking conveys a specific meaning to the stretch of chromatin on which it occurs. Only a few of the meanings of the modifications are known. In Chapter 7, we discuss the way a doubly-acetylated H4 tail is “read” by a protein required for gene expression. In another

59、 well-studied case, an H3 tail methylated at lysine 9 is recognized by a set of proteins that create an especially compact form of chromatin, which silences gene expression. The acetylation of lysine 14 of histone H3 and lysines 8 and 16 of histone H4usually associated with gene expressionis perform

60、ed by the type A histone acetylases (HATs) in the nucleus. In contrast, the acetylation of lysines 5 and 12 of histone H4 and a lysine of histone H3 takes place in the cytosol, after the histones have been synthesized but before they have been incorporated into nucleosomes; these modifications are catal