. Methylation and silencing of human p15INK4b in acute myeloid leukemia

。

• 批准号: 7733433
• 负责人: Linda Wolff
• 金额: $ 34.01万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733433
• 关键词: Acetylation; Acute; Acute Myelocytic Leukemia; Binding; Bone Marrow; CD34 gene; CDKN2A gene; Cell Cycle; Cell Cycle Arrest; Cell Line; Cells; Characteristics; Chromatin; Chromatin Structure; Chromosomes, Human, Pair 9; Clinic; Combined Modality Therapy; CpG Islands; Cultured Cells; DNA; DNA Methylation; DNA Methyltransferase; DNA Methyltransferase Inhibitor; DNA Modification Methylases; Data; Decitabine; Diagnosis; Dysmyelopoietic Syndromes; EZH2 gene; Epigenetic Process; Exons; Gene Activation; Gene Expression; Gene Silencing; Genes; Goals; HL60; Histone Deacetylase Inhibitor; Histones; Human; Hypermethylation; Ink; Laboratories; Laboratory Study; Localized; Lysine; Maintenance; Methylation; Modification; Molecular Profiling; Mutate; Myelogenous; Myosin Heavy Chains; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Polycomb; Promoter Regions; Protein Binding; Proteins; Publishing; Purpose; RUNX1 gene; Reporting; Research; Resolution; Sampling; Smooth Muscle Myosins; Stem cells; Subgroup; Transcriptional Activation; Trichostatin A; Tumor Suppressor Genes; Up-Regulation; base; chromatin immunoprecipitation; design; embryonic stem cell; gene repression; histone methyltransferase; human CBFB protein; inhibitor/antagonist; interest; leukemia; mRNA Expression; progenitor; promoter; research study; response; transcription factor

A recently published study from the laboratory has shown that acute myeloid leukemia patient samples have undergone different degrees of methylation throughout the CpG island of the gene. We observe three types, those that have a high degree of hypermethylation, those that have essentially no methylation and those that have an intermediate degree of methylation. In all cases the gene was not expressed. When there is no hypermethylation in patient samples at the p15INK4b locus CpG island we presume that there are other mechanisms that inhibit expression of the gene and for some acute myeloid cases we have determined an alternate pathway. In acute myeloid leukemia with inv(16) we have found that the INK4b silencing is caused by another mechanism. In these leukemias the inv(16)-encoded core binding factor beta-smooth muscle myosin heavy chain (CBFbeta-SMMHC) protein targets the promoter and displaces the transcription factor RUNX1 causing transcriptional repression. For the inv(16) acute myeloid leukemia patients, re-expression from the INK4b locus would not be predicted to occur using hypmethylating drugs. In our onging experiments we are determining repressive histone modifications that accompany the three different methylation states of p15INK4b DNA . Chromatin immunoprecipitation and DNA tiling microarrays (ChIP-on-chip) with 20bp resolution are being used to assess the distribution of histone modifications over a 1.2 megabase region of human chromosome 9 including p15INK4b and adjacent tumor suppressor genes p14ARF and p16INK4a. We found that acute myeloid leukemia cell lines with p15INK4b high and intermediate degrees of hypermethylation (Kasumi-1, KG-1, and KG-1a) have high levels of the repressive histone modification, trimethylation of lysine 27 of histone H3 (H3K27me3). Remarkably, this modification was found to span the entire INK4b-ARF-INK4a region while little binding was observed in adjacent regions of chromosome 9. Binding of EZH2, the polycomb associated H3K27 histone methyltransferase, co-localized with H3K27me3 distribution over the INK locus. H3K27me3 was not identified at this region in acute myeloid leukemia cell lines without p15INK4b DNA methylation (U937 and HL-60). Rather, histone modifications associated gene activation, trimethylation of lysine 4 of H3 (H3K4me3) and acetylation of lysine 9 (H3K9Ac), were found at the p15INK4b promoter in the latter cells. Enrichment of another repressive histone modification, trimethylation of histone H3 on lysine 9 (H3K9me3), did not correlate with the DNA methylation status of p15INK4b and appeared highest in exons 2 and 3 of p16INK4a in most cell lines. Since p15INK4b reactivation has been described as a component of a patients response to epigenetic therapies in acute myeloid leukemia treatment, we sought to determine the dynamics of histone modifications following treatment with the DNA methyltransferase (DNMT) inhibitor 5-aza-2-deoxycytidine and histone deacetylase (HDAC) inhibitor tricostatin A. So far only KG-1 cells with intermediate hypermethylation, have been examined. A reduction in p15INK4b DNA methylation and an increase in cell cycle arrest were observed following treatment with DNMT inhibitors. In contrast to other reports, treatment with HDAC inhibitors alone was capable of reducing p15INK4b DNA methylation, increasing mRNA expression and inducing cell cycle arrest suggesting that a repressive chromatin structure contributes to the DNA methylation in this cell line. Loss of DNA methylation was not sufficient for reactivation of p15INK4b expression as detectible expression was only observed following the combined treatment of DNMT and HDAC inhibitors. Reactivation was associated with an increase in the activation-associated histone modifications H3K4me3 and H3K9Ac at the promoter region and, unexpectedly, maintenance of the repressive modification H3K27me3. This bivalent histone modification pattern is characteristic of many developmentally poised genes in embryonic stem cells and correlates with the histone methylation status of p15INK4b that we found in CD34+ bone marrow progenitor/stem cells. This data supports the further use of HDAC inhibitors in addition to DNA demethylating agents for the treatment of AML.

该实验室最近发表的一项研究表明，急性髓细胞性白血病患者样品在整个基因的CpG岛中都经历了不同程度的甲基化。我们观察到三种类型，即高度甲基化的类型，基本无甲基化的甲基化和具有中等程度的甲基化的类型。在所有情况下，该基因均未表达。当P15INK4B基因座CPG岛的患者样品中没有高甲基化，我们认为还有其他机制抑制基因表达，并且对于某些急性髓样病例，我们已经确定了另一种途径。在具有INV的急性髓样白血病中（16），我们发现Ink4b沉默是由另一种机制引起的。在这些白血病中，INV（16）编码的核心结合因子β-平滑肌肌球蛋白重链（CBFBETA-SMMHC）蛋白靶向启动子并取代转录因子RUNX1引起转录抑制作用。对于INV（16）急性髓样白血病患者，不会预测使用甲基化药物的INK4B基因座的重新表达。在我们的启用实验中，我们确定伴随p15INK4B DNA的三种不同甲基化状态的抑制性组蛋白修饰。 具有20BP分辨率的染色质免疫沉淀和DNA瓷砖微阵列（芯片）用于评估人类9的1.2兆碱基区域的组蛋白修饰的分布，包括P15INK4B和包括邻接的肿瘤抑制剂基因P14ARF和P16INK4A。我们发现，具有p15INK4B高和中等程度的高甲基化（Kasumi-1，kg-1和kg-1a）的急性髓样白血病细胞系具有高水平的抑制性组蛋白修饰，三甲基化组蛋白H3的赖氨酸27（H3K27ME3）。值得注意的是，这种修饰被发现跨越了整个Ink4b-arf-ink4a区域，而在染色体9的相邻区域中几乎没有结合。EZH2的结合，polycomb相关的H3K27组蛋白甲基转移酶，与H3K27ME3分布在Ink Locus上共同定位。 H3K27me3在没有P15INK4B DNA甲基化（U937和HL-60）的急性髓细胞性白血病细胞系中未鉴定在该区域。相反，在后一种细胞的P15INK4B启动子上发现了组蛋白修饰，H3（H3K4ME3）的赖氨酸4（H3K4ME3）的三甲基化和赖氨酸9（H3K9AC）的乙酰化。在大多数细胞系中，在赖氨酸9（H3K9ME3）上的另一种抑制性组蛋白修饰，在赖氨酸9（H3K9ME3）上的三甲基化（H3K9ME3）在赖氨酸9（H3K9me3）上的富集与p15ink4b的DNA甲基化状态无关，在大多数细胞系中的p16ink4a的外显子2和3中最高。 Since p15INK4b reactivation has been described as a component of a patients response to epigenetic therapies in acute myeloid leukemia treatment, we sought to determine the dynamics of histone modifications following treatment with the DNA methyltransferase (DNMT) inhibitor 5-aza-2-deoxycytidine and histone deacetylase (HDAC) inhibitor tricostatin A. So far only KG-1已经检查了具有中间高甲基化的细胞。在用DNMT抑制剂处理后，观察到P15INK4B DNA甲基化的降低和细胞周期停滞的增加。与其他报告相反，单独使用HDAC抑制剂的治疗能够降低P15INK4B DNA甲基化，增加mRNA表达并诱导细胞周期停滞，这表明抑制性染色质结构有助于该细胞系中的DNA甲基化。 DNA甲基化的丧失不足以重新激活p15INK4B表达，因为仅在对DNMT和HDAC抑制剂的联合处理后才能观察到可检测的表达。重新激活与启动子区域的激活相关组蛋白修饰H3K4ME3和H3K9AC的重新激活有关，并且出乎意料地维持了抑制性修饰H3K27me3。这种二价组蛋白修饰模式是胚胎干细胞中许多发育固定基因的特征，并且与我们在CD34+骨髓祖细胞/干细胞中发现的P15INK4B的组蛋白甲基化状态相关。该数据支持除DNA脱甲基剂以治疗AML外，还支持HDAC抑制剂的进一步使用。