Chromatin Regulation of Tissue Regeneration and Stem Cell Function

基因组调节因子如转录因子和染色质修饰酶共同作用， 在发育过程中指导细胞命运，包括在发育过程中发生的常规细胞更替， 体内平衡和主要的组织替换和再生过程中发生的重新图案化。一个具体 一组染色质修饰复合物的活性与基因表达密切相关， Set 1/MLL组蛋白甲基转移酶家族。这些酶复合物共有几个基本的核心， 亚基，但每个特定的酶（Set 1，MLL 1，MLL 2等）在多细胞中是功能上非冗余的 动物即，单个无效突变是致命的。这些数据和其他数据，包括我实验室的一些数据， 表明每个Set 1/MLL复合物调节其自身不同的基因组。然而，许多分子 驱动这种生物化学和功能特异性的机制细节仍然未知。 为了揭示这一过程的基本分子机制，我的实验室研究了 动物再生的真涡虫模型。Planarians是自由生活的扁形虫， 能力。它们也可以通过RNAi进行遗传干扰，很容易分离为单细胞 分析，并编码染色质修饰蛋白质与其他生物体具有很强的同源性。为 例如，涡虫Set 1和MLL 1/2酶显示出与它们的哺乳动物的强序列同源性， 我以前的研究表明，任何一种酶的缺失都会导致其保守的 组蛋白修饰活性，组蛋白H3在赖氨酸4处的三甲基化（H3 K4 me 3）。此外，我们还发现， 酶靶向一组特定的基因，这些基因与酶丧失后的表型相关。具体来说，损失 MLL 1/2通过其转录本的RNAi导致高度保守的纤毛基因处的H3 K4 me 3的丢失，以及 纤毛和纤毛细胞的丧失。然而，我们不知道MLL 1/2如何特异性靶向这些纤毛基因， vivo.为了开始确定这种靶向的分子和机制基础，我们首先需要表征 涡虫MLL 1/2复合体的生物化学组成和功能。 值得注意的是，在核心Set 1/MLL复合物亚基dpy-30， 可能提供了一个机会，揭示机制，区分Set 1从MLL 1/2功能在体内。到 为了验证这个想法，我招募了一名有前途的本科生来鉴定真涡虫DPY-30的相互作用， proteins.他将开始克隆带有表位标签的DPY-30的两个真涡虫版本。随后他将 在哺乳动物细胞中表达这些Planarian蛋白，并询问它们是否与哺乳动物MLL 1/2稳定相互作用 蛋白质并形成功能性组蛋白甲基转移酶复合物。这些研究将告诉我们， DPY-30需要MLL 1/2，而不是Set 1，组装和/或作为组蛋白甲基转移酶复合物发挥作用。 该项目还将使伊森获得建立生物医学职业所需的经验和技能 research.

Genomic regulators such as transcription factors and chromatin-modifying enzymes work together to instruct cell fate during developmental processes, including both the routine cell turnover that occurs during homeostasis and the major tissue replacement and repatterning that occurs during regeneration. One specific group of chromatin-modifying complexes whose activity is strongly associated with gene expression is the Set1/MLL family of histone methyltransferases. These enzyme complexes share a core of several essential subunits, yet each particular enzyme (Set1, MLL1, MLL2, etc.) is functionally non-redundant in multicellular animals i.e., individual null mutations are lethal. These data and others, including some from my laboratory, indicate that each Set1/MLL complex regulates its own distinct set of genes. However, many of the molecular and mechanistic details driving this biochemical and functional specificity remain unknown. To uncover the fundamental molecular mechanisms underlying this process, my laboratory studies the planarian model of animal regeneration. Planarians are free-living flatworms with incredible regenerative capacities. They are also amenable to genetic perturbation through RNAi, easily dissociated for single cell analyses, and encode chromatin modifying proteins with strong homology to those of other organisms. For example, planarian Set1 and MLL1/2 enzymes show strong sequence homology to their mammalian counterparts and my previous research has shown that loss of either enzyme leads to loss of their conserved histone modifying activity, trimethylation of histone H3 at lysine 4 (H3K4me3). Moreover, we find that each enzyme targets a specific set of genes that correlate with their phenotype after enzyme loss. Specifically, loss of MLL1/2 through RNAi of its transcript leads to loss of H3K4me3 at highly conserved cilia genes as well as loss of cilia and ciliated cells. However, we do not know how MLL1/2 targets these cilia genes specifically in vivo. To begin identifying the molecular and mechanistic basis of this targeting, we first need to characterize the planarian MLL1/2 complex in terms of its biochemical composition and function. Notably, the phenotype induced in planarians after RNAi of a core Set1/MLL complex subunit, dpy-30, may provide an opportunity to uncover the mechanism that distinguishes Set1 from MLL1/2 function in vivo. To test this idea, I have recruited a promising undergraduate student to identify planarian DPY-30 interacting proteins. He will begin by cloning the two planarian versions of DPY-30 with an epitope tag. He will then express these planarian proteins in mammalian cells and ask if they stably interact with mammalian MLL1/2 proteins and form a functional histone methyltransferase complex. These studies will inform whether planarian DPY-30 is required MLL1/2, but not Set1, assembly and/or function as a histone methyltransferase complex. This project will also allow Ethan to gain the experience and skills needed to build a career in biomedical research.