Table 1_Integrated promoter-capture Hi-C and Hi-C analysis reveals fine-tuned regulation of the 3D chromatin architecture in colorectal cancer.docx

Introduction:Hi-C is a widely used technique for mapping chromosomal interactions within a 3D genomic framework, however, its resolution is often constrained by sequencing depth, making it challenging to detect fine-scale interactions. To overcome this limitation, Promoter-Capture Hi-C (PCHi-C), as it selectively enriches for promoter-associated interactions, was employed. This study integrates PCHi-C and Hi-C datasets from colorectal cancer (CRC) models investigate chromosomal interaction dynamics across various regulatory levels, from cis-regulatory elements to topologically associated domains (TADs). The primary goal is to examine how genomic structural alterations shape the epigenomic landscape in CRC and to assess their potential role in colorectal cancer susceptibility.
Methods:PCHi-C and Hi-C datasets from multiple colorectal cancer (CRC) studies were integrated to enhance the resolution of chromatin interaction mapping. The analysis focused on identifying fine-scale interactions within topologically associated domains (TADs) while incorporating histone modification landscapes (H3K27ac, H3K4me3) and transcriptomic signatures from CRC cell lines and the TCGA database. For experimental validation, ChIP-quantitative PCR was performed at the promoters of target genes using the highly malignant colorectal cell line HT29 and compared it to an embryonic cell line NT2D1.
Results:Our integrated analysis revealed significant genomic structural instability in CRC cells, closely associated with tumor-suppressive transcriptional programs. We identified nine dysregulated genes, including long non-coding RNAs (MALAT1, NEAT1, FTX, and PVT1), small nucleolar RNAs (SNORA26 and SNORA71A), and protein-coding genes (TMPRSS11D, TSPEAR, and DSG4), all of which exhibited a substantial increase in expression in CRC cell lines compared to human embryonic stem cells (hESCs). Additionally, we observed enriched activation-associated histone modifications (H3K27ac and H3K4me3) at the potential enhancer regions of these genes, indicating possible transcriptional activation. ChIP-quantitative PCRs conducted using in the highly malignant CRC cell line HT29, compared to the embryonic cell line NT2D1, further validated these findings, reinforcing the link between altered chromosomal interactions and gene dysregulation in CRC.
Discussion:This study sheds light on the dynamic 3D genome organization in CRC, highlighting critical structural changes associated with disease-associated loci. The identification of nine dysregulated genes points to potential biomarkers for colorectal cancer, with implications for diagnostic and therapeutic strategies. The combination of Hi-C and PCHi-C offers a refined approach for detecting chromosomal interactions at a higher resolution, laying the foundation for future studies on cancer-associated chromatin architecture.

引言：高通量染色体构象捕获技术（Hi-C）是一种在三维基因组框架内绘制染色体相互作用的广泛应用技术，但其分辨率常受测序深度限制，难以检测精细尺度的染色质相互作用。为克服这一局限，研究者采用了启动子捕获Hi-C（PCHi-C），该技术可选择性富集启动子相关的相互作用。本研究整合了结直肠癌（CRC）模型中的PCHi-C与Hi-C数据集，以解析从顺式调控元件到拓扑关联结构域（TADs）等多种调控层级的染色体相互作用动态。本研究的核心目标是探究基因组结构改变如何塑造结直肠癌的表观基因组景观，并评估其在结直肠癌易感性中的潜在作用。
方法：本研究整合了多项结直肠癌（CRC）研究中的PCHi-C与Hi-C数据集，以提升染色质相互作用绘图的分辨率。分析聚焦于识别拓扑关联结构域（TADs）内的精细尺度相互作用，同时结合了结直肠癌细胞系及癌症基因组图谱（TCGA）数据库中的组蛋白修饰图谱（H3K27ac、H3K4me3）与转录组特征。为进行实验验证，本研究使用高恶性结直肠癌细胞系HT29，针对靶基因启动子开展染色质免疫共沉淀-定量聚合酶链反应（ChIP-quantitative PCR），并与胚胎细胞系NT2D1进行对比分析。
结果：本研究的整合分析揭示了结直肠癌细胞中存在显著的基因组结构不稳定性，该现象与肿瘤抑制性转录程序密切相关。我们共鉴定出9个失调基因，包括长链非编码RNA（long non-coding RNAs）MALAT1、NEAT1、FTX及PVT1，小核仁RNA（small nucleolar RNAs）SNORA26与SNORA71A，以及蛋白编码基因TMPRSS11D、TSPEAR与DSG4；相较于人类胚胎干细胞（hESCs），上述基因在结直肠癌细胞系中的表达水平均显著升高。此外，我们观察到这些基因的潜在增强子区域存在富集的激活相关组蛋白修饰（H3K27ac与H3K4me3），提示可能存在转录激活。针对高恶性结直肠癌细胞系HT29与胚胎细胞系NT2D1开展的ChIP-quantitative PCR实验进一步验证了上述发现，强化了结直肠癌中染色体相互作用改变与基因失调之间的关联。
讨论：本研究阐明了结直肠癌中动态变化的三维基因组组织模式，突出了与疾病相关基因座相关的关键结构改变。本次鉴定出的9个失调基因有望成为结直肠癌的潜在生物标志物，可为结直肠癌的诊断与治疗策略提供参考。Hi-C与PCHi-C的联合应用为高分辨率检测染色体相互作用提供了一种精细化方法，为未来开展癌症相关染色质结构研究奠定了基础。