Introduction

Human papillomavirus (HPV) integration can induce gene expression dysregulation by destroying higher-order chromatin structure in cervical cancer.

Objectives

We established a 13q22 site-specific HPV16 gene knock-in cell model to interrogate the changes in chromatin structure at the initial stages of host cell malignant transformation.

Methods

We designed a CRISPR-Cas9 system with sgRNA targeting 13q22 site and constructed the HPV16 gene donor. Cells were cotransfected, screened, and fluorescence sorted. The whole genome sequencing (WGS) was used to confirm the precise HPV16 gene integration site. Western blot and qRT-PCR were used to measure gene expression. In vitro and in vivo analysis were performed to estimate the tumorigenic potential of the HPV16 knock-in cell model. Combined Hi-C, chromatin immunoprecipitation and RNA sequencing analyses revealed correlations between chromatin structure and gene expression. We performed a coimmunoprecipitation assay with anti-PIBF1 antibody to identify endogenous interacting proteins. In vivo analysis was used to determine the role of PIBF1 in the tumor growth of cervical cancer cells.

Results

We successfully established a 13q22 site specific HPV16 gene knock-in cell model. We found that HPV integration promoted cell proliferation, invasion and stratified growth in vitro, and monoclonal proliferation in vivo. HPV integration divided the affected topologically associated domain (TAD) into two smaller domains, and the progesterone-induced blocking factor 1 (PIBF1) gene near the integration site was upregulated, although PIBF1 was not enriched at the domain boundary by CUT-Tag signal analysis. Moreover, PIBF1 was found to interact with the cohesin complex off chromatin to reduce contact domain formation by disrupting the cohesin ring-shaped structure, causing dysregulation of tumorigenesis-related genes. Xenograft experiments determined the role of PIBF1 in the proliferation in cervical cancer cells.

Conclusion

We highlight that PIBF1, a potential chromatin structure regulatory protein, is activated by HPV integration, which provides new insights into HPV integration-driven cervical carcinogenesis.

中文翻译：

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PIBF1通过阻碍染色质长程相互作用调控多种基因表达驱动HPV16整合上皮细胞恶性转化

介绍

人乳头瘤病毒 (HPV) 整合可通过破坏宫颈癌中的高级染色质结构来诱导基因表达失调。

目标

我们建立了一个 13q22 位点特异性 HPV16 基因敲入细胞模型来研究宿主细胞恶性转化初期染色质结构的变化。

方法

我们设计了一个带有 sgRNA 靶向 13q22 位点的 CRISPR-Cas9 系统，并构建了 HPV16 基因供体。细胞被共转染、筛选和荧光分选。全基因组测序 (WGS) 用于确认精确的 HPV16 基因整合位点。Western blot 和 qRT-PCR 用于测量基因表达。进行体外和体内分析以估计 HPV16 敲入细胞模型的致瘤潜力。结合 Hi-C、染色质免疫沉淀和 RNA 测序分析揭示了染色质结构和基因表达之间的相关性。我们使用抗 PIBF1 抗体进行了免疫共沉淀测定，以鉴定内源性相互作用蛋白。体内分析用于确定 PIBF1 在宫颈癌细胞肿瘤生长中的作用。

结果

我们成功建立了 13q22 位点特异性 HPV16 基因敲入细胞模型。我们发现 HPV 整合在体外促进细胞增殖、侵袭和分层生长，在体内促进单克隆增殖。HPV 整合将受影响的拓扑相关结构域 (TAD) 分为两个较小的结构域，并且整合位点附近的孕激素诱导阻断因子 1 (PIBF1) 基因被上调，尽管通过 CUT-Tag 信号分析 PIBF1 未在结构域边界富集. 此外，发现 PIBF1 与染色质外的粘连蛋白复合物相互作用，通过破坏粘连蛋白环状结构来减少接触域的形成，从而导致肿瘤发生相关基因的失调。异种移植实验确定了 PIBF1 在宫颈癌细胞增殖中的作用。

结论

我们强调 PIBF1，一种潜在的染色质结构调节蛋白，被 HPV 整合激活，这为 HPV 整合驱动的宫颈癌发生提供了新的见解。