Implication of the DotCom complex members AF9 and DOT1L in cerebral cortex development

神经干细胞的神经元特性主导着大脑皮层分层的胚胎发育。神经干细胞存在于两个细胞室中，并产生下层或上层神经元。后者是由来自脑室下区的干细胞产生的，这些干细胞在分子水平上没有很好的特征。然而，它们在高等认知任务的进化过程中起着关键作用。在这个项目中，我们的目标是进一步鉴定这些已确定的干细胞。我们的初步数据确定AF9/MLLT3(髓系/淋巴系或混合血统白血病(三胸同源，果蝇)；易位到，3)是抑制上层命运和影响脑室下祖细胞增殖的转录因子。我们现在正在解决AF9如何控制祖细胞增殖和分化的问题。AF9在不同的蛋白质复合体中发挥作用：一方面在Dotcom中含有组蛋白甲基转移酶DOT1L(DOT1样组蛋白H3甲基转移酶)，另一方面在与转录延长有关的超延长复合体(SEC)中发挥作用。我们将产生AF9和DOT1L缺陷小鼠，并在涉及各种转录因子的分级发育序列的背景下描述祖细胞和神经元的规范。我们将通过高通量的RNA测序进一步确定AF9和DOT1L的靶基因，特别是在脑室下祖细胞中。靶基因将根据DOT1L依赖和非依赖转录进行分类。利用染色质免疫沉淀，我们将阐明特定染色质修饰、RNA聚合酶II、AF9和DOT1L在全基因组的分布，以确定在脑室下祖细胞规范的背景下，在各自的AF9靶基因座位上是否存在Dotcom或SEC蛋白。因此，我们将提供有关在皮质发育过程中通过染色质修饰发挥作用的转录因子的转录调控数据。此外，我们将提供对特定干细胞群体的发展能力的见解，这些干细胞群体产生了与更高认知任务有关的神经元网络。

Neuronal specification of neural stem cells predominates embryonic development of the stratified cerebral cortex. Neural stem cells reside in two cellular compartments and produce either lower or upper layer neurons. The latter are produced by stem cells from the subventricular zone, which are not well characterised on a molecular level. However, they play a pivotal role during evolution of higher cognitive tasks. In this project we aim to further characterise these determined stem cells.Our preliminary data identified AF9/MLLT3 (myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 3) as transcription factor that suppresses upper layer fate and influences subventricular progenitor proliferation. We are now addressing the question how AF9 controls progenitor proliferation and differentiation. AF9 acts in different protein complexes: on one hand in the DotCom that contains the histone methyltransferase DOT1L (DOT1-like, histone H3 methyltransferase), and on the other hand in the super elongation complex (SEC) that is implicated in transcriptional elongation. We will generate AF9- and DOT1L-deficient mice and characterise progenitor and neuronal specification in a context of a hierarchical developmental sequence involving a variety of transcription factors. We will further identify target genes for AF9 and DOT1L specifically in subventricular progenitors using high throughput sequencing of RNA. Targets will be classified according to DOT1L-dependent and -independent transcription. Using chromatin-immunoprecipitation we will elucidate genome-wide distribution of specific chromatin modifications, RNA polymerase II, AF9 and DOT1L to define presence of DotCom or SEC proteins at the respective AF9 target gene loci in the context of subventricular progenitor specification. We will thus provide data about transcriptional control through transcription factors that act via chromatin modifications during cortical development. Furthermore we will provide insights into the developmental capacities of a specific stem cell population that gives rise to neuronal networks implicated in higher cognitive tasks.