Investigations of possible epigenetic mechanisms of ID3 in DNA repair and genomic stability - Potential implications for cancer therapy

ID3 在 DNA 修复和基因组稳定性中可能的表观遗传机制的研究 - 对癌症治疗的潜在影响

• 批准号: 429192355
• 负责人: Dr. Ali Bakr, Ph.D.
• 金额: --
• 依托单位: Abteilung Epigenomik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/429192355?language=en
• 关键词: Investigations; possible; epigenetic; mechanisms; ID3

The inhibitor of DNA-binding 3 (ID3) is a transcriptional repressor protein that limits the binding of basic helix-loop-helix (bHLH) transcription factors to DNA. ID3 expression is frequently deregulated in human cancers. We recently reported (Bakr et al., 2021) that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR), and that its loss confers sensitivity to therapeutically available PARP inhibitor. Mechanistically, ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Further unpublished data of our lab demonstrate possible protein interactions between ID3 and important chromatin modifiers as well as downregulation of key epigenetic players in ID3-depleted cells. In addition, preliminary ChIP-qPCR analyses showed a reduced enrichment of H4K16ac, H3K27ac and H3K36me3 upon cellular ID3 knockdown, in particular at HR-prone DSBs. We also observed that the loss of ID3 leads to sensitivity of tumor cells to HDAC inhibition which might offer a new therapeutic opportunity to treat ID3-deficient tumors. Based on these data we hypothesize (i) that ID3 has the potential to epigenetically affect DNA repair and genome stability by changing the chromatin environment of genomic regions flanking DSBs, and (ii) that epigenetic drugs could be effective tools to kill ID3-deficient cancer cells. In this application, it is our aim to elucidate the mechanisms how ID3 deficiency affects both chromatin accessibility and histone post-translational modifications around DSBs. For this, ATAC-seq (assay for transposase-accessible chromatin using sequencing), ACT-seq (antibody‐guided chromatin tagmentation using sequencing) and ChIP-seq (chromatin immunoprecipitation followed by sequencing) technologies will be used to monitor the dynamics of chromatin accessibility and histone modifications both genome-wide and at specific DSB sites in wild-type and ID3-depleted cells. In addition, we will perform a comprehensive drug screening with an epigenetic drug library in ID3-deficent cells to identify potential therapeutic candidates. The efficacy of the most promising candidates will subsequently be validated in vivo using ID3-WT and ID3-KO xenograft models. The resulting knowledge of this research project should allow to elucidate the epigenetic consequences of ID3 deficiency and provide evidence how ID3-deficient cancer cells can be actively treated.

DNA结合3抑制剂（ID3）是一种转录抑制蛋白，限制碱性螺旋-环-螺旋（bHLH）转录因子与DNA的结合。在人类癌症中，ID3的表达经常不受控制。我们最近报道（Bakr et al., 2021）， ID3在促进DNA双链断裂（DSB）修复，特别是同源重组（HR）方面具有双重作用，并且其缺失使其对治疗可用的PARP抑制剂敏感。在机制上，ID3与MRN复合体和RECQL解旋酶相互作用，激活DSB修复，并促进RAD51的加载和HR的下游步骤。此外，ID3通过调节染色质可及性和转录因子E2F1的活性，促进HR基因在电离辐射下的表达。我们实验室进一步未发表的数据表明，ID3与重要的染色质修饰因子之间可能存在蛋白质相互作用，以及ID3缺失细胞中关键表观遗传因子的下调。此外，初步ChIP-qPCR分析显示，细胞ID3敲除后，H4K16ac、H3K27ac和H3K36me3的富集减少，尤其是在hr易发的dsb中。我们还观察到ID3的缺失导致肿瘤细胞对HDAC抑制的敏感性，这可能为治疗ID3缺陷肿瘤提供新的治疗机会。基于这些数据，我们假设(i) ID3有可能通过改变dsb两侧基因组区域的染色质环境，从表观遗传学上影响DNA修复和基因组稳定性；（ii）表观遗传药物可能是杀死缺乏ID3的癌细胞的有效工具。在这项应用中，我们的目的是阐明ID3缺陷如何影响dsb周围染色质可及性和组蛋白翻译后修饰的机制。为此，ATAC-seq（利用测序技术测定转座酶可及的染色质）、ACT-seq（利用测序技术测定抗体引导的染色质标记）和ChIP-seq（利用测序技术测定染色质免疫沉淀）技术将用于监测野生型和id3缺失细胞中全基因组和特定DSB位点的染色质可及性和组蛋白修饰的动态。此外，我们将在id3缺陷细胞中使用表观遗传药物文库进行全面的药物筛选，以确定潜在的治疗候选药物。最有希望的候选药物的有效性随后将在体内使用ID3-WT和ID3-KO异种移植模型进行验证。本研究项目的结果将有助于阐明ID3缺乏的表观遗传后果，并为如何积极治疗ID3缺乏的癌细胞提供证据。