Table_5_A CTCF-Binding Element and Histone Deacetylation Cooperatively Maintain Chromatin Loops, Linking to Long-Range Gene Regulation in Cancer Genomes.xlsx

BackgroundGenes spanning long chromosomal domains are coordinately regulated in human genome, which contribute to global gene dysregulation and carcinogenesis in cancer. It has been noticed that epigenetic modification and chromatin architecture may participate in the regulation process. However, the regulation patterns and functional elements of long-range gene regulation are unclear.
MethodsBased on the clinical transcriptome data from different tumor sets, a novel expressional correlation analysis pipeline was performed to classify the co-regulated regions and subsets of intercorrelated regions. The GLAM2 program was used to predict conserved DNA elements that enriched in regions. Two conserved elements were selected to delete in Ishikawa and HeLa cells by CRISPR-Cas9. SAHA treatment and HDAC knockdown were used to change the histone acetylation status. Using qPCR, MTT, and scratch healing assay, we evaluate the effect on gene expression and cancer cell phenotype. By DNA pull-down and ChIP, the element-binding proteins were testified. 3C and 3D-FISH were performed to depict the alteration in chromatin architecture.
ResultsIn multiple cancer genomes, we classified subsets of coordinately regulated regions (sub-CRRs) that possibly shared the same regulatory mechanisms and exhibited similar expression patterns. A new conserved DNA element (CRE30) was enriched in sub-CRRs and associated with cancer patient survival. CRE30 could restrict gene regulation in sub-CRRs and affect cancer cell phenotypes. DNA pull-down showed that multiple proteins including CTCF were recruited on the CRE30 locus, and ChIP assay confirmed the CTCF-binding signals. Subsequent results uncovered that as an essential element, CRE30 maintained chromatin loops and mediated a compact chromatin architecture. Moreover, we found that blocking global histone deacetylation induced chromatin loop disruption and CTCF dropping in the region containing CRE30, linked to promoted gene regulation. Additionally, similar effects were observed with CRE30 deletion in another locus of chromosome 8.
ConclusionsOur research clarified a new functional element that recruits CTCF and collaborates with histone deacetylation to maintain high-order chromatin organizations, linking to long-range gene regulation in cancer genomes. The findings highlight a close relationship among conserved DNA element, epigenetic modification, and chromatin architecture in long-range gene regulation process.

背景 人类基因组中，跨越长染色体结构域的基因会受到协同调控，这会促进癌症中全局基因表达失调与癌变发生。已有研究表明，表观遗传修饰与染色质高级结构可能参与该调控过程，但长距离基因调控的具体模式及功能元件仍不明确。
方法 基于不同肿瘤队列的临床转录组数据，本研究构建了全新的表达相关性分析流程，以分类协同调控区域及互相关联区域的子集。通过GLAM2程序预测在上述区域中富集的保守DNA元件。选取两个保守元件，利用CRISPR-Cas9技术在Ishikawa细胞与海拉（HeLa）细胞中进行敲除。采用SAHA处理与组蛋白去乙酰化酶（HDAC）敲低的方式改变细胞的组蛋白乙酰化状态。通过实时定量PCR（qPCR）、MTT实验及划痕愈合实验，评估其对基因表达与癌细胞表型的影响。借助DNA下拉实验与染色质免疫共沉淀（ChIP）验证元件结合蛋白。通过染色质构象捕获（3C）与三维荧光原位杂交（3D-FISH）分析染色质结构的改变。
结果 在多种癌症基因组中，我们鉴定出协同调控区域的子集（sub-CRRs），这类区域可能共享相同的调控机制并呈现相似的表达模式。一种新的保守DNA元件（CRE30）在sub-CRRs中富集，且与癌症患者的生存率相关。CRE30可抑制sub-CRRs内的基因调控，并影响癌细胞表型。DNA下拉实验显示，包括CCCTC结合因子（CTCF）在内的多种蛋白被招募至CRE30基因座，ChIP实验证实了CTCF的结合信号。后续研究发现，作为关键功能元件，CRE30可维持染色质环并介导紧密的染色质高级结构。此外，我们发现阻断全局组蛋白去乙酰化会导致包含CRE30的区域发生染色质环破坏与CTCF丢失，这与基因调控的增强相关。另外，在8号染色体的另一基因座敲除CRE30也观察到了相似的效应。
结论 本研究明确了一种全新的功能元件，该元件可招募CTCF并协同组蛋白去乙酰化，以维持高级染色质结构，进而参与癌症基因组中的长距离基因调控。本研究结果揭示了长距离基因调控过程中，保守DNA元件、表观遗传修饰与染色质高级结构三者之间的紧密关联。