Disruption of a DNA loop as a complementary mechanism of H3.3K27M mutations

DNA 环的破坏作为 H3.3K27M 突变的补充机制

• 批准号: 10762308
• 负责人: Lukas Chavez
• 金额: $ 2.2万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-07 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10762308
• 关键词: Disruption; DNA; loop; complementary; mechanism

Somatic histone mutations are a hallmark of pediatric high-grade gliomas (pHGGs). Recurrent mutations in the genes encoding for histone variants H3.3 (H3F3A) and H3.1 (HIST1H3B, HIST1H3C) lead to amino acid substitutions at two key residues in the histone tail: lysine to methionine at position 27 (K27M) and glycine to arginine or valine at position 34 (G34R/V). H3.3K27M mutations are associated with distinct clinicopathological characteristics, such as anatomical distribution of tumors carrying these mutations, histological features, age at presentation and survival time. Although great progress has been made in understanding the inhibition of methyltransferases by K27M mutations, additional molecular mechanisms that contribute to the overall poor survival of H3.3K27M pHGG patients may have been overlooked. By DNA sequence analysis, we have recently predicted that the H3.3K27M mutation simultaneously disrupts the K27 codon of the H3F3A gene and the core unit of a DNA binding motif, which is recognized by the DNA-binding protein CCCTC-binding factor (CTCF). Preliminary chromatin immunoprecipitation data in patient derived pHGG cell lines and primary pHGG tumors indicate that CTCF indeed binds the H3F3A wild type, but not the H3.3K27M mutant allele. Based on our compelling preliminary results, we now propose the central hypothesis that the exonic CTCF binding site plays a critical role in the regulation of DNA loops and gene expression, and the disruption of the CTCF binding site by H3.3K27M mutations contributes to gliomagenesis. Toward this objective, we propose: (i) To validate the effects of the mutated CTCF binding site on the disruption of DNA loops and enhancer-mediated misregulation nearby genes in primary pHGG tumors and patient derived pHGGs cell lines (Aim 1); (ii) To validate the relevance of the disrupted exonic CTCF binding site for early transformation leading to pHGGs. We aim to generate targeted induced pluripotent stem cells (iPSCs) by replacing the endogenous H3F3A allele with a wildtype H3.3 sequence (H3.3-CTCF+) fused to an inducible H3.3 version with synonymous nucleotide substitutions (H3.3-CTCF-). Synonymous H3.3-CTCF- substitutions disrupt the CTCF binding site while retaining the wild-type H3F3A amino acid sequence. Targeted isogenic iPSCs will be differentiated into different cell types of the neural lineage, including oligodendroglial precursor cells (OPCs), and assessed for relative changes in proliferation, apoptosis, and differentiation in induced H3.3-CTCF- compared to uninduced H3.3-CTCF+ cells (Aim 2). Upon conclusion, we will have functionally tested the influence of the mutated exonic CTCF binding site on chromosome conformation, gene regulation, and impaired differentiation into OPCs as an additional molecular mechanism of H3.3K27M gliomagenesis.

体细胞组蛋白突变是儿童高级别胶质瘤（pHGG）的标志。基因组中的复发性突变 编码组蛋白变体H3.3（H3 F3 A）和H3.1（HIST 1H 3B，HIST 1H 3C）的基因导致氨基酸 在组蛋白尾部的两个关键残基处的取代：在位置27处的赖氨酸至甲硫氨酸（K27 M）和在位置27处的甘氨酸至甲硫氨酸（K27 M）。 在位置34处的精氨酸或缬氨酸（G34 R/V）。H3.3K27M突变与不同的临床病理学特征相关。 特征，如携带这些突变的肿瘤的解剖分布，组织学特征，年龄， 介绍和生存时间。虽然在理解抑制的作用方面已经取得了很大的进展， 甲基转移酶的K27 M突变，其他分子机制，有助于整体穷人 H3.3K27M pHGG患者的存活率可能被忽略。 通过DNA序列分析，我们最近预测H3.3K27M突变同时破坏了 H3 F3 A基因的K27密码子和DNA结合基序的核心单元，其被DNA结合基序识别。 蛋白CCCTC结合因子（CTCF）。患者来源的pHGG中的初步染色质免疫沉淀数据 细胞系和原发性pHGG肿瘤表明CTCF确实结合H3 F3 A野生型，但不结合H3.3K27M 突变等位基因 基于我们令人信服的初步结果，我们现在提出一个中心假设，即外显子CTCF 结合位点在DNA环和基因表达的调节中起着关键作用，并且结合位点的破坏在DNA环和基因表达的调节中起着关键作用。 H3.3K27M突变的CTCF结合位点有助于胶质瘤的发生。为实现这一目标，我们建议： 为了验证突变的CTCF结合位点对DNA环的破坏和增强子介导的细胞凋亡的影响， 在原发性pHGG肿瘤和患者来源的pHGG细胞系中附近基因的误调节（Aim 1）;（ii）为了验证 破坏的外显子CTCF结合位点与导致pHGG的早期转化的相关性。我们的目标是 通过将内源性H3 F3 A等位基因替换为靶向的诱导多能干细胞（iPSC）， 野生型H3.3序列（H3.3-CTCF+）与具有同义核苷酸的诱导型H3.3形式融合 3-CTCF-）。同义H3.3-CTCF-取代破坏CTCF结合位点，同时保留 野生型H3 F3 A氨基酸序列。靶向的同基因iPSC将分化成不同的细胞类型 包括少突胶质前体细胞（OPCs），并评估 与未诱导的H3.3-CTCF+细胞相比，诱导的H3.3-CTCF-细胞的增殖、凋亡和分化 (Aim 2）。在结论中，我们将在功能上测试突变的外显子CTCF结合位点的影响， 对染色体构象、基因调控和向OPCs分化受损的影响， H3.3K27M胶质瘤发生的分子机制。

项目成果

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

• DOI: 10.1038/s41467-023-38044-0
• 发表时间: 2023-04-21
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Okonechnikov, Konstantin;Camgoez, Aylin;Chapman, Owen;Wani, Sameena;Park, Donglim Esther;Huebner, Jens-Martin;Chakraborty, Abhijit;Pagadala, Meghana;Bump, Rosalind;Chandran, Sahaana;Kraft, Katerina;Acuna-Hidalgo, Rocio;Reid, Derek;Sikkink, Kristin;Mauermann, Monika;Juarez, Edwin F.;Jenseit, Anne;Robinson, James T.;Pajtler, Kristian W.;Milde, Till;Jaeger, Natalie;Fiesel, Petra;Morgan, Ling;Sridhar, Sunita;Coufal, Nicole G.;Levy, Michael;Malicki, Denise;Hobbs, Charlotte;Kingsmore, Stephen;Nahas, Shareef;Snuderl, Matija;Crawford, John;Wechsler-Reya, Robert J.;Davidson, Tom Belle;Cotter, Jennifer;Michaiel, George;Fleischhack, Gudrun;Mundlos, Stefan;Schmitt, Anthony;Carter, Hannah;Michealraj, Kulandaimanuvel Antony;Kumar, Sachin A.;Taylor, Michael D.;Rich, Jeremy;Buchholz, Frank;Mesirov, Jill P.;Pfister, Stefan M.;Ay, Ferhat;Dixon, Jesse R.;Kool, Marcel;Chavez, Lukas
• 通讯作者: Chavez, Lukas