Programmable DNA loops between PU.1 gene regulatory elements

空间染色体结构的破坏是人类疾病中一种新兴的机制，因此提供了一种尚未探索的潜在治疗选择。然而，大多数基因的空间染色体需求尚不清楚，特别是那些与疾病有关的基因。Ets结构域转录因子PU.1是产生髓系和淋巴系祖细胞所必需的，PU.1表达异常可导致白血病。我们和其他人之前的工作揭示了PU.1的表达受到谱系特异性转录因子与远端基因调控元件的多层相互作用的严格控制。此外，我们最近发布了高分辨率的染色体构象图，揭示了PU1基因在健康细胞和白血病细胞中的三维结构。我们的工作将PU.1自动调节与特定的长距离染色体内接触联系起来，并将动态染色质循环与PU.1表达联系起来。根据目前的建议，我们将通过一种基于CRISPR-dCas9的新方法在不同的PU.1调控元件之间设计可逆的染色质环，从而从功能上访问三维染色体结构在驱动谱系特异性和疾病相关的PU.1表达水平中的参与。我们的工作将提供空间染色质景观如何协调髓系分化和恶性转化的因果信息。

Disruption of spatial chromosomal architecture is an emerging mechanism in human disease, thus presenting a yet unexplored potential therapeutic option. However, the spatial chromosomal requirements of most genes are yet unknown, especially of those involved in diseases. The ETS-domain transcription factor PU.1 is essential for the production of myeloid and lymphoid progenitor cells and disturbed PU.1 expression can lead to leukemia. Previous work by us and others revealed that PU.1 expression is under tight control of a multilayered interplay of lineage-specific transcription factors with distal gene regulatory elements. Furthermore, we have recently published high-resolution chromosome conformation maps revealing the three-dimensional organization of the PU.1 locus in both healthy and leukemic cells. Our work linked PU.1 autoregulation with specific long-range intrachromosomal contacts, and correlated dynamic chromatin looping with PU.1 expression. With the present proposal, we will engineer reversible chromatin loops between different PU.1 regulatory elements by a new CRISPR-dCas9-based method, and thus will functionally access involvement of the three-dimensional chromosomal architecture in driving lineage-specific and disease-associated PU.1 expression levels. Our work will provide causal information on how the spatial chromatin landscape orchestrates myeloid differentiation and malignant transformation.