Control of human neurodevelopment by a group of hominoid-specific transposons

人类大脑的发育需要发育基因的及时激活。当细胞分化时，基因激活主要由非编码DNA元件调节，称为增强子，通常位于远离目标基因的地方。随着dna结合蛋白（转录因子）的输入，增强子与靶基因相互作用，引发它们的激活。转座因子（te）是寄生的基因组因子，利用宿主基因组进行跨代繁殖。尽管如此，一些te表现出对宿主基因组有用的特定特征。例如，te的DNA序列可能包括被特定转录因子识别的特定序列基序。在这种情况下，te可能会被基因组“增选”成为功能增强子。这种宿主-寄生虫相互作用通常是细胞类型特异性的，因为te可能在某些细胞类型中被增选为增强子，而在其他细胞类型中被抑制。在这种情况下，一个名为sin - vntr - alu （SVA）的年轻te家族可能在人类神经发育过程中发挥重要作用。类人猿（猩猩、大猩猩、黑猩猩和人类）不存在SVAs，人类基因组中约2700个拷贝中有近一半是我们这个物种所不存在的。有强有力的初步证据表明，人类特异性SVAs通过增强剂的作用来控制人类神经发育。在这里，我们将探讨SVAs控制人类神经发育的机制。我们想解决几个问题：1)人类神经发育的哪个阶段是由SVAs控制的？2) SVAs只在特定的脑细胞类型中起促进发育的作用，还是这种现象在大脑中是普遍存在的？3) sva衍生的脑增强子是否在整个人群中积累了序列上的遗传变异？这种变异是否与特定的人类神经发育特征有关？为了回答这些问题，我们将在体外利用诱导多能干细胞（iPSCs）产生脑类器官来模拟人类神经发育。多能干细胞是从人体细胞中提取的干细胞，在体外被改造成干细胞样细胞。iPSCs可以用特定的试剂处理，以触发被称为脑类器官的脑样组织的快速发育。只需要10天就能形成神经识别，20-30天就能形成明确的大脑区域。类器官在两个月内达到最大尺寸，但它们可以无限期地保存在培养中。我们将把这个系统与单细胞基因组学和基因组编辑结合起来。更具体地说，单细胞基因组技术（单细胞RNA-seq和单细胞ATAC-seq）将被用于评估在脑类器官生成过程中，哪些SVAs在每个细胞中都是活跃的，以及它们控制哪些基因。此外，我们将采用crispr干扰，这是CRISPR-Cas9技术的一种修改，该技术已被优化以招募抑制特定基因组位点的蛋白质。在这种情况下，crispr干扰将用于抑制类器官生成过程中不同时间点的所有人类SVAs，并评估其对不同脑细胞类型发育的影响。除了在类器官上进行的工作外，我们将利用公开的人类全基因组序列来描述sva衍生的神经发育增强子的遗传和结构变异，并将在基因组全关联研究（GWASs）的背景下解释这种变异，该研究已经由其他人进行，以预测与特定人类神经发育特征相关的遗传变异。总之，这些实验将为人类神经发育提供新的见解，特别是揭示在所有不同大脑成分的发育过程中基因开启和关闭的新机制。

Human brain development requires the timely activation of developmental genes. As cells differentiate, gene activation is modulated primarily by non-coding DNA elements, called enhancers, usually located far away from their target genes. Following the input of DNA-binding proteins (transcription factors), the enhancers interact with the target genes, eliciting their activation.Transposable Elements (TEs) are parasitic genomic elements that take advantage of the host genomes to propagate across generations. Nonetheless, some TEs present specific characteristics that are useful to the host genome. For instance, the DNA sequence of the TEs may include particular sequence motifs that are recognized by specific transcription factors. When this is the case, the TEs may be "co-opted" by the genome to become functional enhancers. This host-parasite mutualism is usually cell type specific, in that the TEs may be co-opted as enhancers in some cell-types and repressed in others. In this context, a young family of TEs, called SINE-VNTR-Alu (SVA), may play an important role during human neurodevelopment. SVAs are exclusive of the great apes (orangutan, gorilla, chimpanzee, humans), and nearly half of the ~2,700 copies present in the human genome are exclusive of our species.There is strong preliminary evidence suggesting that the human-specific SVAs control human neurodevelopment by acting as enhancers. Here, we will investigate the mechanisms by which SVAs control human neurodevelopment. There are several questions we would like to address: 1) which stages of human neurodevelopment are controlled by the SVAs? 2) Do SVAs function as developmental enhancers only in specific brain cell types, or is this phenomenon universal in the brain? 3) Have the SVA-derived brain enhancers accumulated genetic variation in their sequence across human populations? And is this variation associated with specific neurodevelopmental human traits?To answer these questions, we will model human neurodevelopment in vitro using cerebral organoid generation from induced pluripotent stem cells (iPSCs). The iPSCs are stem cells derived from human somatic cells that have been engineered in vitro to become stem-like cells. The iPSCs can be treated with specific reagents to trigger the rapid development of brain-like tissues, termed cerebral organoids. Only 10 days are required for the appearance of neural identity and 20-30 days for defined brain regions to form. The organoids reach the maximum size in two months, but they can be kept in culture indefinitely. We will couple this system with single cell genomics and genome editing. More specifically, single cell genomic techniques (single-cell RNA-seq and single-cell ATAC-seq) will be employed to assess which SVAs are active in every individual cell during brain organoid generation, and which genes they control. Additionally, we will employ CRISPR-interference, which is a modification of the CRISPR-Cas9 technology that has been optimized to recruit proteins that repress specific genomic sites. In this case, the CRISPR-interference will be used to repress ALL the human SVAs at different time-points during organoid generation and assess the consequences on the development of the different brain cell types. In addition to the work performed on organoids, we will harness publicly available human whole genome sequences to profile genetic and structural variation in SVA-derived neurodevelopmental enhancers and will interpret this variation in the context of Genome Wide Association Studies (GWASs) that have been performed by others to predict genetic variants associated with specific human neurodevelopmental traits.Together these experiments will provide novel insights into human neurodevelopment, specifically unveiling novel mechanisms by which genes are turned on and off during the development of all the different brain components.