Bohaczuk Stephanie C, Amador Zachary J, Li Chang, Mallory Benjamin J, Swanson Elliott G, Ranchalis Jane, Vollger Mitchell R, Munson Katherine M, Walsh Tom, Hamm Morgan O, Mao Yizi, Lieber Andre, Stergachis Andrew B
Division of Medical Genetics, University of Washington School of Medicine, Seattle, Washington 98195, USA.
Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.
Genome Res. 2024 Dec 23;34(12):2269-2278. doi: 10.1101/gr.279747.124.
Accurately quantifying the functional consequences of noncoding mosaic variants requires the pairing of DNA sequences with both accessible and closed chromatin architectures along individual DNA molecules-a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic, and epigenomic profiling across targeted >100 kb loci with ∼10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated γ-globin (/) promoters in primary human hematopoietic cells induced toward an erythroblast lineage increases the accessibility of the promoter as well as neighboring regulatory elements. Overall, we find that these non-protein coding mosaic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant.
准确量化非编码镶嵌变异的功能后果需要将DNA序列与单个DNA分子上可及的和封闭的染色质结构进行配对——这种配对无法通过传统的基于片段化的染色质分析来实现。我们证明，靶向单分子染色质纤维测序（Fiber-seq）实现了这一点，它能够对靶向的>100 kb位点进行单分子、长读长的基因组和表观基因组分析，相对于非靶向测序有~10倍的富集。靶向Fiber-seq揭示，肌强直性营养不良1的致病CTG重复序列扩增具有体细胞不稳定性以及多个附近调控元件的破坏的特征，这两者都依赖于重复序列长度。此外，我们发现，在向成红细胞谱系诱导的原代人类造血细胞中，对片段重复的γ-珠蛋白（/）启动子进行治疗性腺嘌呤碱基编辑会增加启动子以及邻近调控元件的可及性。总体而言，我们发现这些非蛋白质编码镶嵌变异可对染色质结构产生复杂影响，包括超出携带变异的调控元件的范围。
