PO.BCS01.15 · 生物信息与计算
对15个尤因肉瘤细胞系的长读长测序揭示了广泛的GGAA微卫星变异，塑造了EWSR1::FLI1结合图景
Long-read sequencing of 15 Ewing sarcoma cell lines uncovers extensive GGAA microsatellite variation shaping the landscape of EWSR1::FLI1 binding
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
尤因肉瘤（EwS）是一种罕见、侵袭性的骨和软组织肿瘤，主要发生于青少年和青年。EwS由FET基因家族成员与ETS转录因子之间获得性的染色体易位所定义（例如超过85%的病例中的EWSR1::FLI1），这些融合蛋白结合GGAA重复序列并重连局部转录活性。GGAA微卫星等位基因长度和基序纯度的种系变异可改变EWSR1::FLI1的结合，并破坏其失调核心驱动基因的能力。对这些种系-体细胞相互作用背后分子特征的进一步研究，可能为尤因肉瘤的遗传病因提供新的洞见。
我们对从15个EWSR1::FLI1阳性EwS细胞系中提取的高分子量DNA进行了全基因组PacBio Revio HiFi测序，以在全基因组范围内表征GGAA微卫星的种系变异。我们实施了一个定制的TRGT流程，利用基因组重复文库和隐马尔可夫模型来调用至少含有3个GGAA基序的微卫星。为研究可能增强EWSR1::FLI1结合的基因组特征，我们利用线性混合模型，将微卫星等位基因调用和DNA甲基化的PacBio数据与来自尤因肉瘤细胞系图谱（ESCLA）的染色质可及性和EWSR1::FLI1结合ChIP-seq数据相结合。
总计表征了39,319个GGAA微卫星，在微卫星长度（中位数=16 bp，四分位距=13-51）和GGAA基序纯度（均值=93.4%，标准差=9.2%）方面观察到显著变异。细胞系之间存在等位基因变异，平均为4个等位基因，最多的情况下全部30个单倍型均表现出不同的等位基因。微卫星长度增加是EWSR1::FLI1结合最强的预测因子（beta=0.711，95% CI=0.703,0.719），其次是DNA甲基化降低（beta=-0.023，95% CI=-0.024,-0.022）和衡量开放染色质的H3K27ac增加（beta=0.154，95% CI=0.138-0.170）。对由EWSR1::FLI1失调的已确立EwS核心基因的分析正在进行中，以识别促进EWSR1::FLI1改变这些基因表达的特定微卫星和等位基因组合。
总之，来自长读长全基因组测序的种系数据与来自ESCLA的多组学谱系，使得整合分析成为可能，以厘清改变EWSR1::FLI1结合及下游转录调控的种系-体细胞相互作用。
查看英文原文 English abstract
Ewing sarcoma (EwS) is a rare, aggressive bone and soft tissue tumor occurring primarily in adolescents and young adults. EwS is defined by acquired chromosomal translocations between a member of the FET gene family and an ETS transcription factor (e.g., EWSR1::FLI1 in >85% of cases) that bind GGAA repetitive sequences and rewire local transcriptional activity. Germline variation in GGAA microsatellite allele length and motif purity can alter binding of EWSR1::FLI1 and disrupt the ability to dysregulate core driver genes. Further investigation into the molecular features underlying these germline-somatic interactions may yield new insights into the genetic etiology of Ewing sarcoma.
We performed whole-genome PacBio Revio HiFi sequencing on high molecular weight DNA extracted from 15 EwS cell lines positive for EWSR1::FLI1 to characterize germline variation in GGAA microsatellites genome-wide. We implemented a custom TRGT pipeline to call microsatellites containing at least 3 GGAA motifs utilizing a genomic repeat library and hidden Markov models. To investigate genomic features that could enhance EWSR1::FLI1 binding, we utilized linear mixed models incorporating PacBio data on microsatellite allele calls and DNA methylation with chromatin accessibility and ChIP-seq of EWSR1::FLI1 binding from the Ewing Sarcoma Cell Line Atlas (ESCLA).
In total, 39,319 GGAA microsatellites were characterized, with notable variation observed in microsatellite length (median=16 bp, interquartile range=13-51) and purity of GGAA motifs (mean=93.4%, standard deviation=9.2%). Allelic variation was present between cell lines with a mean of 4 alleles and a maximum of all 30 haplotypes exhibiting a different allele. Increased microsatellite length was the strongest predictor of EWSR1::FLI1 binding (beta=0.711, 95% CI=0.703,0.719), followed by reduced DNA methylation (beta=-0.023, 95% CI=-0.024,-0.022) and increased H3K27ac measures of open chromatin (beta=0.154, 95% CI=0.138-0.170). Analyses of established core EwS genes dysregulated by EWSR1::FLI1 are underway to identify specific microsatellites and allelic combinations that promote EWSR1::FLI1-altered expression of these genes.
Together, germline data from long-read whole-genome sequencing and multi-omic profiles from the ESCLA enable integrative analyses for disentangling germline-somatic interactions that alter EWSR1::FLI1 binding and downstream transcriptional regulation.
利益披露 Disclosure
A. Song, None..
X. Li, None.
E. Dolzhenko,
PacBio Employment.
H. Neyret Kahn, None..
K. Jones, None..
D. Wang, None..
K. Jain, None..
A. Hubbard, None..
O. Lee, None..
S. Chanock, None..
D. Dutta, None..
O. Delattre, None..
M. J. Machiela, None.