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肿瘤的表观遗传调控肿瘤的表观遗传调控(Epigenetic Regulation of Cancer)

主讲：罗志勇 教授

中南大学生物科学与技术学院

分子生物学系

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Chromatin packaging

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表观遗传学概念

表观遗传 (epigenetics) 的概念是在 1942 年由Waddington 提出。指 DNA 序列不发生变化但是基因表达却发生了可遗传的改变，也就是说基因型未变化而表型却发生了改变，这种变化是细胞内除了遗传信息以外的其他可遗传物质的改变，并且这种改变在发育和细胞增殖过程中能稳定地传递下去。

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Cancer epigenetics In addition to having genetic causes, cancer

can also be considered an epigenetic disease.

Regulation by genetics involves a change in

the DNA sequence, whereas epigenetic regulation involves alteration in chromatin structure and methylation of the promoter region

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表观遗传学的特点： 可遗传的，即这类改变通过有丝分裂或减数分裂，能在细胞或个体世代间遗传；

可逆性的基因表达调节，也有较少的学者描述为基因活性或功能的改变；

没有 DNA 序列的改变或不能用 DNA 序列变化来解释。

表观遗传修饰主要包括 DNA 以及一些与 DNA 密切相关的蛋白质 (例如组蛋白 )的化学修饰，另外某些非编码的 RNA 也在表观遗传修饰中起着重要的作用。

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Epigenetic Regulation of Cancer

Site SpecificHypermethylation

GlobalHypomethylation

Histone Modifications

Regulating FactorsDietary

HormonalGenetic

Verma M, Srivastava S. Lancet Oncol. (2002) 3:755-63.

DNA MethyltransferasesHistone MethyltransferasesHistone Acetylases/Deacetylases

Epigenetics Regulates:Cell Cycle ControlDNA DamageApoptosisInvasionX-Chromosome InactivationImprintingAging

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表观遗传修饰从多个水平调控基因表达

DNA: DNA 甲基化

蛋白质：组蛋白修饰

染色质：染色质重塑

RNA ：非编码 RNA

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DNA 甲基化 DNA 甲基化 (DNA methylation) 是研究得最清楚、 也是最重要的表观遗传修饰形式，通过将 S 一腺苷甲硫氨酸作为甲基供体，并在 DNA

甲基转移酶 (DNA methyltransferase ， DNMT)

的催化下， CpG 二核苷酸中的胞嘧啶环上 5′位置的氢被活性甲基所取代，从而转变成为 5

甲基胞嘧啶 (5-methylcytosine ， 5mC) 。

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Enzymatic methylation of the C–5 position of cytosine residues can effect epigenetic inheritance by altering the expression of genes and by transmission of DNA

methylation patterns through cell division. Thus, in addition to its well–known role in deamination mutational hotspots in human DNA, DNA methylation may contribute to gene inactivation in cancer.

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哺乳动物基因组中 5mC 占胞嘧啶总量的 2%-7%，约 70%的 5mC 存在于 CpG 二连核苷。

在结构基因的 5’端调控区域 , CpG 二连核苷常常以成簇串联形式排列，这种富含 CpG 二连核苷的区域称为 CpG 岛 (CpG islands) ，其大小为500-1000bp，约 56%的编码基因含该结构。

基因调控元件 (如启动子 )所含 CpG 岛中的 5mC会阻碍转录因子复合体与 DNA 的结合。 DNA 甲基化一般与基因沉默相关联； 非甲基化一般与基因的活化相关联； 而去甲基化往往与一个沉默基因的重新激活相关联。

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癌细胞的整个基因组水平处于低甲基化状态，比正常低 20% ～ 60%，这种低甲基化大多发生于编码区和内含子区域，以及约占人类基因组20% ～ 30%的重复序列区

抑癌基因启动子区域 CpG 岛高度甲基化，且与DNA 结合的组蛋白广泛去乙酰化

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Esteller, and J. G. Herman. Cancer as an epigenetic disease: DNA

methylation and chromatin alterations in human tumors. J Pathol, 2001.

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Gene silencingDNA methylation is a powerful mechanism for thesuppression of gene activity.There is reciprocal relationship between the density of methylated cytosine residues and the transcriptional activity of a gene.The methyl groups do not affect base pairing but can influence protein–DNA interactions by protruding into the major groove.

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●The strong effect of 5–methylcytosine (5mC) in mammalian promoter regions suggests that DNA methylation inhibits transcription by interfering with transcription initiation.

● DNA methylation reduces the binding affinity of sequence–specific transcription factors.

● Methylation–dependent, sequence–specific DNA–binding

proteins, such as MDBP may act as transcriptional repressors.

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组蛋白修饰组蛋白修饰 (histone modification) 是表观遗传研究的重要内容。

组蛋白的 N端是不稳定的、无一定组织的亚单位，其延伸至核小体以外，会受到不同的化学修饰，这种修饰往往与基因的表达调控密切相关。

被组蛋白覆盖的基因如果要表达，首先要改变组蛋白的修饰状态，使其与 DNA 的结合由紧变松，这样靶基因才能与转录复合物相互作用。因此，组蛋白是重要的染色体结构维持单元和基因表达的负控制因子。

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Histone modifications— including acetylation, methylation and phosphorylation — are import

ant in transcriptional regulation and many are stably maintained during cell division, although the mechanism for this epigenetic inheritance is not yet well understood.Proteins that mediate these modifications are often associated within the same complexes as those that regulate DNA methylation.

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Covalent modification of histones:

•Acetylation of lysines

•Methylation of lysines and arginines

•Phosphorylations of serines and threonines

Histone Modifications

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Histones•5 types: H2A, H2B (slightly lys rich), H3, H4 (arg rich) H1 (lys rich). All relatively small proteins.• Per 200 bp of DNA: 2 molecules each of

H2A, H2B, H3, H4 and one molecule of H1.

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Histone AcetylationAcetylation of the lysine residues at the N terminus of histone proteins removes positive charges, thereby reducing the affinity between Histones and DNA. This makes RNA polymerase and transcription factors easier to access the promoter region. Therefore, in most cases, histone acetylation enhances transcription while histone deacetylation represses transcription

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Transcription process and its regulation by histone modification

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Histone acetylation and cancer

In acute promyelocytic leukaemia:

The oncoprotein produced by the fusion of the PML (promyelocytic leu

kaemia) gene and the retinoic acid receptor a gene appears to suppres

s the transcription of specific genes through the recruitment of HDACs.

Thus the cancer cell is unable to undergo differentiation, leading to excess

ive proliferation.

Similar phenomena:

retinoic acid receptor a–PLZF (promyelocytic leukaemia zinc finger pr

otein)fusion, AML1 (acute myelocytic leukaemia protein 1)–ETO fusio

n, and also in theMyc/Mad/Max signalling pathway involved in solid m

alignancies.

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It is clear that HDAC enzymes seldom operate alone.

Many proteins, with various functions such as recruitment, co-repression or

chromatin remodelling, are involved in forming a complex that results in the repressor complex.

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There are two protein families with HDAC activity: the recently discovered

SIR2 family of NAD+-dependent HDACs and the classical HDAC family.

Members of the classical HDAC family fall into two different phylogenetic

classes - class I and class II The class I HDACs (HDAC1, 2, 3 and 8) are most closel

y related to the yeast(Saccharomyces cerevisiae) transcriptional regulator RPD3.

Class II HDACs (HDAC4, 5, 6, 7, 9 and 10) share domains with similarity to HDA1, another deacetylase found in yeast .

Recently a new member of the HDAC family has been identified, HDAC11

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A wide variety of processes are associated with the inh

ibition of HDACs,such as apoptosis, necrosis, differen

tiation, inhibition of proliferation and cytostasis.

Currently, many efforts are being made to expand

our knowledge of the HDACs and to develop potent

and stable HDACi.

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RESPONSIVE GENES

By studying the effects of HDACi on the expression of various genes and their regulatory pathways, a more detailed picture will emerge of how the inhibition of HDACs, combined with the HDAC expression profile of that cell, ultimately determines the fate of the cell.

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组蛋白修饰的种类

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染色质重塑

染色质重塑（ chromatin remodeling）是一个重要的表观遗传学机制。

染色质重塑是由染色质重塑复合物介导的一系列以染色质上核小体变化为基本特征的生物学过程。

组蛋白尾巴的化学修饰（乙酰化、甲基化及磷酸化等）可以改变染色质结构，从而影响邻近基因的活性。

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Johnstone 2002

Epigenetic Mechanisms

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Chromatin-Level Regulation of Gene Expression

Garfinkel and Ruden Nutrition:20:56-62, 2004

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染色质修饰与重塑（共价修饰型与 ATP 依赖型）

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MicroRNAmiRNA 是近年来生命科学领域的研究热点，是一组生物体基因组编码的内源性非编码小 RNA 。

miRNA 主要采用降解靶 mRNA 和抑制靶 mRNA 的翻译两种作用方式在转录后水平调控基因表达。

降解靶 mRNA 的方式与 siRNA 的作用方式相似，直接作用于靶 mRNA ，直接导致 mRNA 表达水平下降。

但绝大多数哺乳动物细胞中的 miRNAs 并不导致靶 mRNA 的降解，而是通过与靶 mRNA 的 3’端非翻译区（ UTR）不完全匹配结合，抑制 mRNA翻译成蛋白质，使靶基因的蛋白质表达水平下降。

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A primary transcript (pri-miRNA) is first processed into a stem-loop structure about 60–80 nt (pre-miRNA) by the RNase endonuclease Drosha .pre-miRNAs are exported to the cytoplasm by Exportin-5. Dicer cleaves the hairpin, releasing a miRNA:-miRNA duplex, which has a two base overhang at both 30 ends. The strands of this duplex separate and release a 21–25 nt mature miRNA.

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http://microrna.sanger.ac.uk/sequences/search.shtml

利用公共数据库寻找某个miRNA 的 target 基因

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http://cbio.mskcc.org/cgi-bin/mirnaviewer/mirnaviewer.pl

利用公共数据库还能找被几个miRNA 的共同调节的 target 基因

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miRNA 与肿瘤发生的关系存在两种可能的模式 [Caldas, et al. （ Nature Medicine 2005 ） ] ：1 、若 miRNA 表达上调，其对应抑癌基因表达下调时，则可能导致肿 瘤发生；2 、若 miRNA 表达下调，其对应癌基因表达上调时，同样可能导致肿 瘤发生。

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许多瘤基因和肿瘤抑制基因都受到 miRNAs 分子的调控，此时的 miRNAs 可能起到癌基因或是肿瘤抑制基因的作用。 miRNA 癌基因的活化和 miRNA 抑癌基因失活导致肿瘤抑瘤基因表达下调以及这些 miRNA 基因与蛋白编码癌基因、抑癌基因协同作用导致肿瘤发生 .

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miRNA 的甲基化修饰

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miRNA芯片是目前筛选和研究 miRNA 调控的高通量手段

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思考题：

1. 何谓表观遗传学？2. 表观遗传学的主要研究内容是什么？3. 如何运用表观遗传策略对肿瘤进行诊断

与治疗？

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T h a n k s