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.

体细胞蛋白突变是小儿高级神经胶质瘤（PHGGS）的标志。复发突变 编码组蛋白变异的基因H3.3（H3F3A）和H3.1（Hist1h3b，Hist1h3c）导致氨基酸 在组蛋白尾部的两个关键残基上取代：赖氨酸至27（k27m）的蛋氨酸，甘氨酸和甘氨酸 位置34（G34R/V）处的精氨酸或瓣膜。 H3.3K27M突变与不同的临床病理学有关 特征，例如携带这些突变的肿瘤的解剖学分布，组织学特征，年龄 表现和生存时间。尽管在理解抑制方面取得了巨大进展 通过K27M突变，甲基转移酶，促成整体较差的其他分子机制 H3.3K27M PHGG患者的生存率可能被忽略了。 通过DNA序列分析，我们最近预测，H3.3K27M突变同时破坏了 H3F3A基因的K27密码子和DNA结合基序的核心单位，该基序是由DNA结合所识别的 蛋白CCCTC结合因子（CTCF）。患者衍生的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确实结合了H3F3A野生型，但不结合H3.3K27M 突变等位基因。 基于我们引人注目的初步结果，我们现在提出了一个中心假设，即外显子CTCF 结合位点在DNA环和基因表达的调节中起着至关重要的作用，并且破坏了 H3.3K27M突变的CTCF结合位点有助于神经胶质作用。朝向这个目标，我们提出：（i） 验证突变的CTCF结合位点对DNA环和增强子介导的破坏的影响 原发性PHGG肿瘤和患者衍生的PHGGS细胞系中附近基因附近的正调（AIM 1）； （ii）验证 破坏的外显子CTCF结合位点的相关性对于早期转化导致PHGG的相关性。我们的目标 通过用A替换内源性H3F3A等位基因，生成靶向诱导的多能干细胞（IPSC） WildType H3.3序列（H3.3-CTCF+）融合到具有同义核苷酸的诱导H3.3版本 取代（H3.3-CTCF-）。保留时，同义H3.3-CTCF-替换破坏了CTCF绑定位点 野生型H3F3A氨基酸序列。靶向的ISEGENIC IPSC将分为不同的细胞类型 神经谱系，包括寡头前体细胞（OPC），并评估了相对变化 与未诱导的H3.3-CTCF+细胞相比，诱导H3.3-CTCF-的增殖，凋亡和分化 （目标2）。总结一下，我们将在功能上测试突变的外显子CTCF结合位点的影响 关于染色体构象，基因调节和分化为OPC的损害 H3.3K27M神经胶质作用的分子机制。

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