Epigenetic Markings in Developing and Diseased Prefrontal Neurons

描述(由申请人提供)：精神分裂症的定义是绝大多数受影响的个人缺乏直接的遗传原因。前额叶皮质(PFC)，以及其他大脑区域，被认为经常出现在精神分裂症患者身上，这反映在它的功能性活动不足和一组不同基因的异常表达上。潜在的分子机制尚不清楚，但有证据表明，PFC的长期成熟一直持续到甚至超过第二个十年，对正常的人类发育和精神分裂症的神经生物学起着至关重要的作用。最近，我们首次提出证据表明，表观遗传标记的一个子集，包括基因启动子上的三甲基化组蛋白H3-赖氨酸4，参与了PFC染色质在整个出生前和出生后发育过程中的动态调节，并在某些精神分裂症病例中可能发生改变。这项挑战拨款提案已经做好了准备，既创造了新的就业机会，又保留了现有的就业机会。具体地说，我们将结合神经科学领域中与表观遗传学相关的两种最具创新性的方法，通过选择性地对神经元染色质进行分类，然后对免疫沉淀物进行大规模并行测序(CHIP-SEQ)，以深入了解前额叶神经元的表观基因组图景。我们将描述PFC神经元中与活性启动子(三甲基-H3K4)或转录(三甲基-H3K36)相关的组蛋白甲基化标记的发育和疾病相关变化。此外，我们将通过在断奶的PFC中选择性地表达Cre重组酶，在条件突变小鼠中研究候选组蛋白赖氨酸甲基转移酶MLL1(混合血统白血病1)在正常和疾病前额叶发育中的作用，然后监测成年动物的行为和分子变化。这里提出的实验将在挑战拨款的24个月奖励期限内完成，将首次阐明人类PFC神经元的染色质是否在疾病中受到发育调节和改变。在全基因组水平上回答这个问题将是至关重要的，以便找出基于染色质的机制是否是导致大部分被诊断为精神分裂症的受试者前额叶功能障碍的最终常见病理生理学的一部分。 公共健康相关性：对于大多数被诊断为精神分裂症的患者来说，没有直接的遗传原因被确定。关于精神分裂症的一个重要理论表明，在大脑的某些区域，如“前额叶皮质”，许多基因在正常发育过程中没有被适当地激活，就像健康受试者一样。然而，到目前为止，还缺乏研究这些现象的分子技术。这项挑战拨款提案是“准备就绪的”，将创造新的就业机会并保留现有的就业机会。它还基于我们实验室最近开发的极其创新的技术。我们第一次能够有选择地从死后获得的人脑神经细胞中分离染色体材料和染色质，并在全基因组水平上研究“表观遗传标记”(基本上是调节基因表达和功能的化学修饰)。在这项提议中，我们计划检查正常前额叶皮质发育过程中的表观遗传基因激活模式，并在精神分裂症和旨在在分子水平上建立疾病模型的突变小鼠中寻找潜在的变化。这项研究应该澄清人类神经元染色质中的表观遗传标记是否受到动态调节，以及精神分裂症等主要精神疾病是否与神经元表观基因组的变化有关。

DESCRIPTION (provided by applicant): Schizophrenia is defined by a lack of a straightforward genetic cause for a very large majority of affected individuals. The prefrontal cortex (PFC), among other brain regions, is thought to be frequently in subjects in schizophrenia, as reflected by its functional hypoactivity and dysregulated expression for a diverse set of genes. The underlying molecular mechanisms remain unknown but there is evidence that the prolonged maturation of the PFC, extending into or even beyond the second decade of life, plays a crucial role for normal human development and the neurobiology of schizophrenia. Recently, we presented the first evidence that a subset of epigenetic markings, including trimethylated histone H3-lysine 4 at sites of gene promoters, is involved in the dynamic regulation of PFC chromatin during throughout pre- and postnatal development, and may be altered in some cases with schizophrenia. This challenge grant proposal is "shovel ready" and create new jobs as well as retain existing ones. Specifically, we will combine two of the most innovative approaches in the field of neurosciences as it pertains to epigenetics, by selectively sorting neuronal chromatin followed by massively parallel sequencing of immunprecipitates (ChIP-seq) to obtain insight into the epigenomic landscape of prefrontal neurons. We will profile developmental and disease-related changes in histone methylation markings associated with active promoters (trimethyl-H3K4) or transcription (trimethyl-H3K36) selectively in PFC neurons. Furthermore, we will study in conditional mutant mice the role of a candidate histone lysine methyltransferase, Mll1 (mixed-lineage leukemia 1) for normal and diseased prefrontal development, by expressing Cre recombinase selectively in weanling PFC and then monitor behavioral and molecular alterations in the adult animal. The experiments proposed here, which are designed to be accomplished within the challenge grant's award period of 24 months, will clarify, for the first time, whether or not chromatin of human PFC neurons is developmentally regulated and altered in disease. The answer to this question on a genome-wide level will be critical in order to find out whether or not chromatin-based mechanisms are part of a final common pathophysiology underlying prefrontal dysfunction in the substantial portion of subjects diagnosed with schizophrenia. PUBLICH HEALTH RELEVANCE: For the majority of patients diagnosed with schizophrenia, no straightforward genetic cause has been identified. One of the important theories about schizophrenia implies that in some brain regions, such as the "prefrontal cortex", a number of genes are not switched on properly during normal development, as they are in healthy subjects. However, until now the molecular techniques to study these phenomena have been lacking. This challenge grant proposal is "shovel ready", will create new jobs and retain existing ones. It is also based on extremely innovative techniques that were recently developed in our laboratories. We were able, for the first time, to selectively isolate chromosomal materials and chromatin from the nerve cells of the human brain obtained postmortem, and study "epigenetic markings" (basically, chemical modifications that regulate gene expression and function without altering the genetic code) on a genome-wide level. In this proposal, we plan to examine epigenetic gene activation patterns during normal prefrontal cortex development and search for potential alterations in schizophrenia and in mutant mice designed to model the disease on a molecular level. This research should clarify whether or not epigenetic markings are dynamically regulated in human neuronal chromatin , and if major psychiatric diseases such as schizophrenia are associated with changes in the neuronal epigenome.