EZH2 | Ezh2 | 组蛋白甲基化酶/组蛋白甲基转移酶 | 关键调控因子

以Ezh2为例，彻底搞明白一个调控因子regulator的调控原理。

这个是一个非常典型的调控因子，广泛涉及了epigenome, regulatome and transcriptome，这里还会加入miRNA，一旦把这个搞清楚了将会受益无穷。

Polycomb-group proteins

Polycomb Group (PcG) Proteins - 这里强调的是一个蛋白家族

Polycomb repressive complexes (PRCs) - 这里强调的是一个复合体蛋白

Transcriptional regulation by Polycomb group proteins - nature review

Polycomb group (PcG) proteins are epigenetic regulators of transcription that have key roles in stem-cell identity, differentiation and disease. Mechanistically, they function within multiprotein complexes, called Polycomb repressive complexes (PRCs), which modify histones (and other proteins) and silence target genes.

Polycomb Repressive Complex 2 (PRC2) - YouTube

HOX family

Polycomb-group proteins are a family of protein complexes first discovered in fruit flies that can remodel chromatin such that epigenetic silencing of genes takes place. Polycomb-group proteins are well known for silencing Hox genes through modulation of chromatin structure during embryonic development in fruit flies (Drosophila melanogaster).

They derive their name from the fact that the first sign of a decrease in PcG function is often a homeotic transformation of posterior legs towards anterior legs, which have a characteristic comb-like set of bristles. 这个名字的来源很懵。

Polycomb-Dependent Chromatin Looping Contributes to Gene Silencing during Drosophila Development - YouTube

PRC还有chromatin looping的功能，折叠线性的染色质。

小结：PcG和PRC2是非常重要的表观调控因子，它们会化学修饰组蛋白，从而沉默基因表达，与胚胎发育和癌症密切相关。

EZH2/甲基转移酶

Ezh2， histone-lysine N-methyltransferase enzyme，组蛋白赖氨酸N-甲基转移酶

EZH2 catalyzes the addition of methyl groups to histone H3 at lysine 27 - H3K27

Methylation activity of EZH2 facilitates heterochromatin formation thereby silences gene function.

EZH2 is the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), which is responsible for healthy embryonic development through the epigenetic maintenance of genes responsible for regulating development and differentiation.

EZH2 is responsible for the methylation activity of PRC2, and the complex also contains proteins required for optimal function (EED, SUZ12, JARID2, AEBP2, RbAp46/48, and PCL).

Mutation or over-expression of EZH2 has been linked to many forms of cancer.[9] EZH2 inhibits genes responsible for suppressing tumor development, and blocking EZH2 activity may slow tumor growth.

As a histone methyltransferase (HMTase), EZH2's primary function is to methylate Lys-27 on histone 3 (H3K27me) by transferring a methyl group from the cofactor S-adenosyl-L-methionine (SAM).

PRC2/EZH2-EED-mediated H3K27 methylation and subsequent recruitment of PRC1 which facilitates condensation of chromatin and formation of heterochromatin.

甲基化酶：在组蛋白或者DNA上添加甲基CH3，指向底物引入甲基的过程，一般是以甲基取代氢原子。

甲基转移酶：把氨基、羟基、硫氢基（thiol）甲基化

甲基转移酶和甲基化酶区别：一个是促进甲基的生成,一个是促进甲基的重排

小结：

Ezh2是Polycomb Repressive Complex 2（PRC2）复合体的一个重要的催化酶，它负责将H3K27甲基化，形成H3K27me3的组蛋白修饰，这种修饰会沉默其他基因的表达。

目前已经发现Ezh2的高表达会引发癌症，原理就是Ezh2把一些抑癌基因（好基因）的promoter给甲基化了，导致其不表达，从而让坏的癌症出现。

EZH2、EED和SUZ12是PRC2复合体的三个核心subunits，可以设计一个几何平均模型来通过这三个基因表达来衡量PRC2的活性【已实现】。

Ezh2 DNA binding

另外Ezh2也被当做了转录因子，看这里：https://github.com/aertslab/SCENICprotocol/blob/master/example/allTFs_mm.txt

这说明它确实有DNA binding的属性。

EZH2 binding at the AR promoter may recruit additional transcriptional coactivators, such as SP1 or KLF5, to induce gene expression, which will be interesting lines for future investigation - 见下面cell report文章

These authors found that a subset of EZH2-bound genes did not bind the PRC2 subunit SUZ12 or display H3K27me3. Many of these genes were downregulated upon EZH2 knockdown, suggesting that the role of EZH2 as an activator was independent of the PRC2 complex.

我们的结果：通过SCENIC的共表达和motif binding分析，发现EZH2确实通过DNA binding激活了很多下游基因的表达。

小结：Ezh2的两面性，一面催化甲基化，沉默某些基因；一面作为TF，激活某些基因。

PRC2 DNA binding

先看看这些DNA binding的factor有没有被收录为TF：https://github.com/aertslab/SCENICprotocol/blob/master/example/allTFs_hg38.txt

What brings PRC2 to chromatin?

Choi et al. also identify a DNA-binding domain in PRC2 and a protein cofactor that increases this binding.

The Complexity of PRC2 Subcomplexes

Here, we elaborate on how the facultative PRC2 subunits regulate catalytic activity, locus-specific PRC2 binding, and propagation of H3K27me3, and how this affects chromatin structure, gene expression, and cell fate.

DNA binding by PHF1 prolongs PRC2 residence time on chromatin and thereby promotes H3K27 methylation

Moreover, they reveal the molecular basis for how Polycomblike proteins stabilize PRC2 on chromatin and stimulate its activity.

Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA

Instead, protein-free linker DNA dominates the PRC2–nucleosome interaction.

PRC2 preferentially binds methylated DNA regulated by its AEBP2 subunit, suggesting how DNA and histone methylation collaborate to repress chromatin.

小结：Ezh2甲基化时是不展现DNA binding能力的，这时是PRC2进行DNA binding，目前还无法确定PRC2是如何定位到指定DNA片段，进行组蛋白甲基化修饰的。

Ezh2和miR124

MicroRNA miR-124 controls the choice between neuronal and astrocyte differentiation by fine-tuning Ezh2 expression

On the other hand, naturally elevated expression of miR-124 in embryonic carcinoma cells undergoing neuronal differentiation correlated with down-regulation of Ezh2 levels.

Importantly, overexpression of Ezh2 mRNA with a 3'-untranslated region (3'-UTR) lacking a functional miR-124 binding site, but not with the wild-type Ezh2 3'-UTR, hampered neuronal and promoted astrocyte-specific differentiation in P19 and embryonic mouse neural stem cells.

MicroRNA-124 inhibits proliferation and induces apoptosis by directly repressing EZH2 in gastric cancer

Our study indicate that miR-124 can suppress gastric cancer cell growth by directly targeting the EZH2 gene and sensitize the treatment effect of 5-FU. Therefore, miR-124 shows tumor-suppressive activity and may be a new and useful approach of gastric cancer therapy.

小结：miR-124是Ezh2上游的一个调控因子，microRNA一般都是作用于UTR区域的，降解目标mRNA。

cell mitosis

Remodeling of chromosomal heterochromatin by EZH2 is also required during cell mitosis.

根据我们的数据，Ezh2的target genes基本都是cell cycle相关的基因。

neurodegeneration

EZH2-mediated H3K27 trimethylation mediates neurodegeneration in ataxia-telangiectasia

Enhancer of zeste homolog 2 (EZH2), a core catalytic component of PRC2, is a new ATM kinase target, and ATM-mediated S734 phosphorylation of EZH2 reduces protein stability.

The change of H3K27me3 chromatin-binding pattern is directly related to cell cycle re-entry and cell death of ATM-deficient neurons.

我的任务

如何揭示Ezh2在Kif7-caused GI defect中的作用？

能不能找到公共的甲基化的数据支持？

能不能根据co-expression把抑制和激活的下游基因给抓出来？在co-expression的基础上，根据Ezh2的DNA binding把activate的下游基因抓出来；根据PRC2的DNA binding区域，把甲基化的抑制的基因抓出来。【这部分可以用H3K27me3的ChIP-seq获得直接的证据】

根据DEG的数据再筛一遍，这些就是高度可信的基因集。【超级筛选策略，如果再加上表观组和三维基因组的数据，那这个模型基本就接近完美了】

这些都差不多是已知的，如何讲一个新的，让人能感觉到insight的故事呢？

能把Ezh2在ENS发育调控的关系讲清楚也算不错

Digital-LI