Function of chromatin modifiers in cranial neural crest development

 描述(由申请人提供)：我们对染色质重塑蛋白、甲基转移酶和乙酰转移酶缺陷如何导致人类颅面表型的理解存在根本差距。这是一个重要的问题，因为颅面部缺陷经常发生在人类群体中，在美国，每年每1000名活产儿中就有一名(CDC，2011)，其中许多与基因组的表观遗传调节有关。我们的长期目标是更好地了解染色质重构体在脑神经脊(CNCC)发育过程中的功能。本研究的目的是确定KAT2a赖氨酸乙酰转移酶和PRDM赖氨酸甲基转移酶这两个表观遗传调节因子家族在斑马鱼和小鼠CNCC发育中的作用机制。我们将使用两个优秀的发育模型系统，并将遗传工具与斑马鱼和小鼠CNCC行为的活细胞成像以及转录研究相结合，以解决为什么Kat2a和Prdms突变会导致头面部异常的问题。总的假设是，这些染色质修饰酶作为相反的转录调节因子，自主地发挥细胞功能，调节CNCC的增殖和迁移。这项研究的基本原理是，了解KAT2a和PRDM如何调控CNCC发育的机制将有可能转化为更好地理解由于表观遗传调节因子突变而导致的头面部缺陷的发病机制，包括唇腭裂以及影响人类群体的各种综合征，如歌舞伎和SBBYSS。根据较强的初步数据，我们设计了三个特异的目标：1)确定KAT2A和PRDMS在脑神经脊发育中的发育功能；2)检测KAT2A和PRDMS对基因靶标的遗传交互作用和调控；3)确定KAT2A和PRDMS靶基因的酶调节和染色质状态。在第一个目标下，我们已经确定Prdm1、Prdm3、Prdm16和Kat2a在小鼠和斑马鱼中都存在头面部缺陷。我们有工具和专业知识来确定特定的头面部缺陷，并确定cNCC增殖和迁移的异常。对于第二个目标，我们已经产生并获得了大部分斑马鱼和小鼠品系，并在斑马鱼和小鼠身上进行了转录图谱分析，证明了可行性。对于第三个目标，我们展示了组织和生物化学中的乙酰化和甲基化状态的分析。我们的方法在检验新假说方面具有概念上的创新性，在使用LIVE方面也具有技术创新性 细胞成像和模拟人类颅面发育的两个物种之间的相互作用。这项拟议的研究具有重要意义，因为它有望促进对cNCC如何形成颅面骨骼的理解，这有可能为神经脊相关出生缺陷和颅面综合征的治疗提供信息。

 DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how defects in chromatin remodeling proteins, methyltransferases and acetyltransferases are causative for human craniofacial phenotypes. This represents an important problem, because craniofacial defects occur frequently in the human population, 1 in every 1000 live births annually in the U.S. (CDC, 2011) and many are associated with epigenetic regulators of the genome. Our long-term goal is to better understand the function of chromatin remodelers during cranial neural crest (cNCC) development. The objective of this application is to determine the mechanism by which two families of epigenetic regulators, KAT2a lysine acetyltransferase and PRDM lysine methyltransferases that regulate each other and act to modify the same H3K9 residue on histone 3, function in zebrafish and mouse cNCC development. We will use two excellent developmental model systems and combine genetic tools with live cell imaging of zebrafish and mouse cNCC behaviors and transcriptional studies to tackle the question of why mutations in Kat2a and Prdms lead to craniofacial abnormalities. The overall hypothesis is that these chromatin modifying enzymes act as opposing transcriptional regulators and function cell autonomously to regulate cNCC proliferation and migration. The rationale for this research is that understanding the mechanism of how KAT2a and PRDMs regulate cNCC development will have the potential to translate into a better understanding of the pathogenesis of craniofacial defects due to mutations in epigenetic regulators, including cleft lip and palate and various syndromes such as Kabuki and SBBYSS that affect the human population. From strong preliminary data, we have designed 3 specific aims: 1) Determine the developmental function of KAT2A and PRDMs in cranial neural crest development, 2) Examine the genetic interaction and regulation of gene targets by KAT2A and PRDMs, and 3) Determine the enzymatic regulation and chromatin state of KAT2A and PRDMs target genes. Under the first aim, we have determined that Prdm1, Prdm3, Prdm16 and Kat2a have craniofacial defects in both mouse and zebrafish. We have the tools and expertise to determine the specific craniofacial defects and to define abnormalities in proliferation and migration of cNCCs. For the second aim, we have generated and obtained most of the zebrafish and mouse strains, and performed transcriptional profiling in both zebrafish and mouse, demonstrating feasibility. For aim three, we have shown analysis of acetylation and methylation states in both tissue and biochemically. Our approach is conceptually innovative in testing a novel hypothesis and technically innovative in the use of live cell imaging and the interplay between two species that model human craniofacial development. The proposed research is significant because it is expected to advance an understanding of how cNCCs form the craniofacial skeleton, which has the potential to inform the treatment of neural crest associated birth defects and craniofacial syndromes.