组蛋白变异体在上皮间质转化过程中的作用以及相关组蛋白变异体密码的研究

Epithelial-to-mesenchymal transition (EMT), which has been considered as an extreme example of cell plasticity, is important for malignant progression. Emerging evidence suggests widespread epigenetic reprogramming occur during malignant transformation. Like histone modifications, which are intensively investigated in epigenetic regulation, histone variants have a role of importance in chromatin structure maintenance and gene expression. But whether histone variants contribute to EMT related epigenetic reprogramming is still not known. Taking advantage of the oncomine cancer database, we found that histone H2A variant macroH2A2 (mH2A2), but not mH2A1, is profoundly associated with breast cancer differentiation, metastasis and patient survival. In addition, the expression level of mH2A2 negatively correlates with the invasive ability of breast cancer cells. mH2A2 depletion in MCF-7 cells promotes Twist and N-cadherin expression and down-regulates E-cadherin expression, while gain of function of mH2A2 in MDA-MB-231-cells results in decreased expression of Twist and N-cadherin and increased E-cadherin level. Twist has been reported as a master controller of EMT in breast cancer. N-cadherin and E-cadherin represent the mesenchymal and Epithelial marker, respectively. These results indicated that mH2A2 might be a novel EMT governor of breast cancer progression. Furthermore, we found that another major type of histone H2A variant, H2A.Z functions to maintain Twist and N-cadherin expression in MDA-MB-231 cells, implicating that H2A.Z could also be involved into breast cancer EMT process. This observation is consistent with the previous finding that H2A.Z is significantly associated with breast cancer lymph node metastasis and patient survival. Basing on the facts that mH2A2 negatively correlates with H2A.Z expression in breast cancer and they both govern twist expression as well as their antagonizing effect in gene transcription, we propose the interplay between mH2A2 and H2A.Z might composes a pair of histone variants code，similar to histone code that determines transcriptional output, to regulate EMT progression. In the future work, we will try to indentify whether mH2A2 and H2A.Z could influence on EMT in vitro and in vivo. Importantly, we will map the binding profiles of both mH2A2 and H2A.Z on chromatin, find out the pivotal downstream effectors of the two variants during EMT and eventually generate the potential EMT related histone variants code. Our findings support emerging links between chromatin structures and EMT, and, for the first time to our knowledge, demonstrate a direct role of histone variants in this process. We envision that the further understanding of the molecular mechanisms through which histone variants operate in EMT, in combination with the elucidation of histone variants interplay, will aid the understanding of the regulation of breast cancer metastasis and will benefit the therapeutic pursuits for human cancer.

上皮细胞间质转化(EMT)，作为一种典型的由表观遗传重编程机制决定的细胞可塑性现象，在肿瘤恶性转化过程中起着重要作用。与组蛋白修饰类似，组蛋白变异体在染色质结构维持和基因表达中也扮演着重要角色。但是组蛋白变异体是否在EMT重编程中起作用，尚且无人知晓。通过生物信息学分析我们发现组蛋白变异体mH2A2表达与乳腺癌的分化程度和转移能力呈负相关；初步研究表明，乳腺癌细胞中mH2A2维持了上皮标志物且抑制了间质标志物的表达。同时我们发现组蛋白变异体H2A.Z也调节着EMT相关分子的表达。今后，我们将在细胞和动物水平进一步探索二者在EMT中的作用。基于二者的表达在乳腺癌中呈负相关，且共同调节EMT相关分子Twist的表达，我们猜想它们很可能共同决定着EMT相关的组蛋白变异体密码，这一密码的破解将有助于我们从表观遗传学的角度更好的理解乳腺癌的EMT过程和分子机制，并为其治疗提供理论基础。

迄今为止，在该基金的资助下，申请者取得了一些进展并在较高水平的杂志上如PNAS, Oncogene和Biomaterials作为责任作者报道了我们的研究成果。关于macroH2A和乳腺转移的工作，简述如下：上皮细胞间质转化(EMT)，作为一种典型的由表观遗传重编程机制决定的细胞可塑性现象,在肿瘤恶性转化过程中起着重要作用。与组蛋白修饰类似，组蛋白变异体在染色质结构维持和基因表达中也扮演着重要角色。但是组蛋白变异体是否在EMT 重编程中起作用，尚且无人知晓。通过生物信息学分析我们发现组蛋白变异体mH2A2 表达与乳腺癌的分化程度和转移能力呈负相关；初步研究表明，乳腺癌细胞中mH2A2 维持了上皮标志物且抑制了间质标志物的表达。利用免疫亲和纯化和质谱技术，我们初步鉴定出可以调节macroH2A1.2/2在染色质上沉积的伴侣分子MDC1，并通过SNAP实验证明其参与macroH2A1.2/2靶基因的调节；通过RNA-seq分析，我们发现了受制于macroH2A/MDC1调节的参与乳腺癌转移的靶分子MAP1B，初步证明其与macroH2A2介导的抑制乳腺癌迁移密切相关，接下来将进一步明确其是否会在乳腺癌细胞EMT过程中发挥作用。