Functions of Histone Ubiquitination and Methylation

描述（由申请人提供）：组蛋白的翻译后修饰在染色质的组织和基因转录的调节中起着重要作用。我们的长期目标是阐明组蛋白泛素酶和组蛋白甲基转移酶如何调节基因表达和控制细胞生长。两个这样的酶，这是高度保守的，是组蛋白H2B泛素化酶Rad6和组蛋白H3赖氨酸4（K4）甲基转移酶Set1。我们最近证明，Rad6介导的组蛋白H2B K123泛素化是必不可少的组蛋白H3 K4甲基化在酵母中，从而连接这两种形式的组蛋白修饰基因激活。Rad6是一种E2泛素结合酶，其功能从酵母到人类都是进化保守的。重要的是，发现人RAD6同源物（hHR6B）在乳腺癌细胞系和乳腺癌中过表达。此外，外源hHRGB的过表达导致染色体不稳定和人上皮细胞的转化。H3 K4甲基转移酶SET 1的许多人类同源物（例如，MLL，混合谱系白血病）也在多种人类癌症中突变或破坏。总的来说，这些发现建立了组蛋白泛素化和甲基化与癌症之间的密切联系。鉴于Rad6在调节H2B泛素化和H3甲基化中的高度保守功能，因此了解Rad6在介导这些组蛋白修饰，并最终控制基因表达和细胞生长中的作用至关重要。我们的总体假设是，通过H2B泛素化和随后的H3 K4甲基化，Rad6调节对控制细胞生长重要的特定基因的转录。因此，有必要了解Rad6介导的H2B泛素化在建立组蛋白H3甲基化中的基本分子作用机制，从而深入了解Rad6在控制基因转录中的作用。使用芽殖酵母作为模式生物，我们计划使用生物化学和遗传学的结合来剖析Rad6在这些过程中的功能。为此，提出了三个具体目标。目标1。识别和表征介导Bre1和Rad6功能的因素。目标二。定义磷酸化和乙酰化对Rad6的作用。目标3。确定Rad6介导的H2B泛素化在建立组蛋白H3甲基化中的分子作用机制。

DESCRIPTION (provided by applicant): Post-translational modifications of histone proteins play a fundamental role in the organization of chromatin and in the regulation of gene transcription. Our long-term objective is to elucidate how histone ubiquitinases and histone methyltransferases regulate gene expression and control cell growth. Two such enzymes, which are highly conserved, are the histone H2B ubiquitinase Rad6 and the histone H3 lysine 4 (K4) methyltransferase Set1. We have recently demonstrated that Rad6-mediated histone H2B K123 ubiquitination is essential for histone H3 K4 methylation in yeast, thereby linking these two forms of histone modification to gene activation. Rad6 is an E2 ubiquitin-conjugating enzyme whose functions are evolutionary conserved from yeast to human. Importantly, the human RAD6 homolog (hHR6B) was found to be over-expressed in breast cancer cell lines and in breast carcinomas. Moreover, over-expression of exogenous hHRGB resulted in chromosome instability and transformation of human epithelial cells. Many human homologs of the H3 K4 methyltransferase SET1 (e.g., MLL, Mixed Lineage Leukemia) are also mutated or disrupted in a variety of human cancers. Collectively, these findings establish a strong link between histone ubiquitination and methylation with cancer. Given the highly conserved functions of Rad6 in regulating H2B ubiquitination and H3 methylation, it is therefore of paramount importance to understand the roles of Rad6 in mediating these histone modifications, and ultimately, controlling gene expression and cell growth. Our overall hypothesis is that through H2B ubiquitination and subsequent H3 K4 methylation, Rad6 regulates the transcription of specific genes that are important for controlling cell growth. Therefore, it is imperative to understand the basic molecular mechanisms of action of Rad6-mediated H2B ubiquitination in establishing histone H3 methylation, and thereby, gain insight into the role of Rad6 in controlling gene transcription. Using budding yeast as a model organism, we plan to use a combination of biochemistry and genetics to dissect the function of Rad6 in these processes. To achieve this, three Specific Aims are proposed. Aim 1. Identify and characterize factors that mediate the functions of Bre1 and Rad6. Aim 2. Define the roles of phosphorylation and acetylation on Rad6. Aim 3. Determine the molecular mechanisms of action of Rad6-mediated H2B ubiquitination in establishing histone H3 methylation.