Deregulation of 3D genome structure in models of memory and learning disability

在伴有神经损伤的发育障碍中，染色质调节因子经常发生突变。例如，智力障碍与具有不同功能的染色质蛋白的突变有关，如CTCF、ATRX和SATB2。在基于恐惧的实验中，使用条件性基因敲除(CKO)小鼠模型，在有丝分裂后锥体神经元中耗尽这两种因子中的任何一种，都会导致电生理功能障碍，并损害海马区相关的学习和长期记忆。然而，CTCF、SATB2和ATRX缺失对染色质调节的直接影响和受影响的基因仍有待研究。在这里，我们建议在体内直接在成熟小鼠海马区锥体谷氨酸能神经元中剖析CTCF、ATRX或SATB2缺失的后果。我们将使用最先进的方法，基因组结构映射(GAM)来绘制3D染色质结构图，并使用10x基因组多组体来分析单个细胞中的基因表达和染色质可及性。我们将提供所有海马细胞受CKO影响的调控区域的图集，以及染色质接触改变的图景。我们将在3D基因组组织的不同水平上绘制全基因组染色质结构的变化图，以及影响基因表达和3D基因组折叠的转录因子和染色质调控途径。我们将重点研究三个突变体共同的染色质结构和基因表达的变化，以确定参与学习和记忆功能障碍的候选途径。我们将验证所有cKO中受影响的基因，因为这些基因为干预改善学习障碍提供了潜在的候选靶点。最终，我们的目标也是促进我们对有丝分裂后特殊神经元染色质调节的了解。我们的假设是，学习障碍患者海马区功能障碍的开始植根于染色质功能障碍的机制，并有望开发出一个框架，可以用来加速对人类患者样本的评估。

Chromatin regulators are often mutated in developmental disorders accompanied by neurological impairments. For example, intellectual disability is associated with mutations in chromatin proteins with diverse functions, such as CTCF, ATRX and SATB2. Depletion of either factor in post-mitotic pyramidal neurons, using conditional knockout (cKO) mouse models, results in eletrophysiological dysfunction and impairs hippocampal-related learning and long-term memory in fear-based experiments. However, the direct effects of CTCF, SATB2 and ATRX loss on chromatin regulation and the affected genes remain to be investigated.Here, we propose to dissect the consequences CTCF, ATRX or SATB2 loss in vivo, directly in pyramidal glutamatergic neurons of the mature murine hippocampus. We will use state-of-the-art methodologies, Genome Architecture Mapping (GAM), to map 3D chromatin structure, and 10x Genomics multiome, to profile both gene expression and chromatin accessibility in single cells. We will deliver an atlas of cKO-affected regulatory regions for all hippocampal cells, and the landscape of altered chromatin contacts. We will map the changes in chromatin structure genome-wide at different levels of 3D genome organization, and the transcription factors and chromatin regulatory pathways underlying the altered gene expression and 3D genome folding. We will focus on changes in chromatin structure and gene expression common to the three mutants, to identify candidate pathways involved in the learning and memory dysfunctions. We will validate genes affected in all cKOs, as these provide candidate targets with potential for intervention to ameliorate learning disabilities. Ultimately, we also aim to advance our knowledge of chromatin regulation in specialized post-mitotic neurons. Our hypothesis is that the inception of hippocampal dysfunction in learning disabilities is rooted in chromatin dysfunction mechanisms, and expect to develop a framework that can be adopted to accelerate the evaluation human patient samples.