Long distant regulation of c-myb in normal cells and acute myeloid leukemia

正常细胞和急性髓系白血病中 c-myb 的长距离调控

• 批准号: 8552950
• 负责人: Linda Wolff
• 金额: $ 41.84万
• 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/8552950
• 关键词: 3-Dimensional; Acute Myelocytic Leukemia; Affect; Animals; Apoptosis; Binding; Biological Assay; Blood Platelets; Boundary Elements; Breast Carcinoma; Cell Line; Cell physiology; Cells; ChIP-on-chip; Chromatin; Chromatin Loop; Chromatin Structure; Chromosomes; Code; Colon Carcinoma; Complex; Computer Simulation; DNA; DNA-Binding Proteins; Data; Development; Disease; Distal; Distant; Enhancers; Erythrocytes; Erythroid; Eukaryota; Event; Fetal Hemoglobin; Firefly Luciferases; Future; Gene Activation; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genomics; Hematopoiesis; Hematopoietic; Histones; Human; Insertional Mutagenesis; Intercistronic Region; Interleukin-6; Investigation; Laboratories; Lead; Leukemic Cell; Location; Luciferases; MYB gene; Malignant Neoplasms; Mediator of activation protein; Modeling; Modification; Molecular Conformation; Mus; Mutate; Mutation; Myeloid Leukemia; Names; Normal Cell; Nuclear Matrix; Nucleic Acid Regulatory Sequences; Oncogenes; Oncogenic; Play; Positioning Attribute; Post-Translational Protein Processing; Pristane; Proto-Oncogene Proteins c-myb; Proviruses; Regulation; Regulatory Element; Reporter Genes; Reporting; Retroviridae; Role; Single Nucleotide Polymorphism; Site; Staging; Structure; T-Cell Leukemia; T-Lymphocyte; Time; Tissue Differentiation; Transcriptional Activation; Transgenes; Virus Integration; Work; base; cell type; cohesin; gene repression; genome sequencing; histone modification; leukemia; leukemia virus; monocyte; mouse model; neoplastic cell; novel; overexpression; progenitor; programs; promoter; transcription factor; 3-Dimensional; Acute Myelocytic Leukemia; Affect; Animals; Apoptosis; Binding; Biological Assay; Blood Platelets; Boundary Elements; Breast Carcinoma; Cell Line; Cell physiology; Cells; ChIP-on-chip; Chromatin; Chromatin Loop; Chromatin Structure; Chromosomes; Code; Colon Carcinoma; Complex; Computer Simulation; DNA; DNA-Binding Proteins; Data; Development; Disease; Distal; Distant; Enhancers; Erythrocytes; Erythroid; Eukaryota; Event; Fetal Hemoglobin; Firefly Luciferases; Future; Gene Activation; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genomics; Hematopoiesis; Hematopoietic; Histones; Human; Insertional Mutagenesis; Intercistronic Region; Interleukin-6; Investigation; Laboratories; Lead; Leukemic Cell; Location; Luciferases; MYB gene; Malignant Neoplasms; Mediator of activation protein; Modeling; Modification; Molecular Conformation; Mus; Mutate; Mutation; Myeloid Leukemia; Names; Normal Cell; Nuclear Matrix; Nucleic Acid Regulatory Sequences; Oncogenes; Oncogenic; Play; Positioning Attribute; Post-Translational Protein Processing; Pristane; Proto-Oncogene Proteins c-myb; Proviruses; Regulation; Regulatory Element; Reporter Genes; Reporting; Retroviridae; Role; Single Nucleotide Polymorphism; Site; Staging; Structure; T-Cell Leukemia; T-Lymphocyte; Time; Tissue Differentiation; Transcriptional Activation; Transgenes; Virus Integration; Work; base; cell type; cohesin; gene repression; genome sequencing; histone modification; leukemia; leukemia virus; monocyte; mouse model; neoplastic cell; novel; overexpression; progenitor; programs; promoter; transcription factor

We have begun to look for distal c-myb transcriptional regulators in the region 25-100 kb upstream region. Since MuLV-induced myeloid leukemias have integrated proviruses upstream of c-myb in three regions named Mml1, 2, and 3 (25, 50 and 70kb upstream, respectively), we hypothesized that the retrovirus accesses regulatory elements. Accordingly, the provirus locations might be able to assist us in identifying regulatory regions that are important to normal cells as well as leukemic cells that have dysregulated c-myb expression. We first looked by ChIP-on-chip for histone modifications implicating gene activation in normal cells, because this might help in locating regulatory sites. H3K4me3, H3K4me1 and H3K9/14ac, which are known to be enriched at promoters that are activated, were enriched at Mml1 and/ or Mml2 in the c-myb-expressing myeloblastic cell line, M1. The enrichment of all of these histone marks decreased with IL-6 induced differentiation and down-regulation of the gene in M1 cells. However, these marks were increased and spread in tumor cells containing integrated provirus. To examine the enhancer activity of sequences representing three peaks of H3Kme4 in Mml1, 3 fragments were cloned upstream of the c-myb promoter controlling a firefly luciferase reporter gene. We were able to show in Luciferase assays that sequences within one of the regions increased luciferase activity, indicating the presence of an enhancer element.Importantly, using Chromosome Conformation Capture (3C) q-PCR assays, interactions between the 5 prime region including the promoter and all Mml sites (Mml1, 2 and 3) were detected due to DNA looping in M1 cells and tumor cells with provirus in Mml1, 2 or 3. The Mml1 region contains 2 loops giving a total of 4 loops and provirus was found to be inserted in each of these loops. Interestingly, M1 cells, that are induced to differentiate with IL-6 and do not express appreciable levels of c-myb, still maintain the same chromatin looping structure observed in cells with active transcription. Therefore, this chromatin looping structure is insufficient by itself for c-myb expression and may require addition of transcription factors to result in expression. Examination of the chromatin conformation in a non-hematopoietic cell line, NIH3T3, which does not express c-myb, revealed that differences in the looping structure at the c-myb locus exist in different cell types. Apparently, a novel interaction peak, not found in the hematopoietic cells, was present very close the promoter at approximately -11 kb. We can conclude that our study provides a new mechanism of retrovirus insertional mutagenesis whereby spatial chromatin organization allows distally located provirus, with its own enhancer elements, to access the 5 prime regulatory region of the gene. Although such a model could be predicted based upon recent studies that show that transcriptional activation in higher eukaryotes frequently involves the long range action by regulatory elements, this is the first time it has actually been shown that the provirus is in a position to interact with the promoter and/or control region in intron1 through a 3-dimensional chromatin structure. Our study has, in addition, delineated some important regulatory regions that are distal to the c-myb coding region and can know be studied in more detail in future studies. Our investigation also showed that CTCF binds in the vicinity of the loop structures, which is not surprising because it is known that CTCF contributes to looping associated with gene activation. Although we discovered 4 dominant promoter interacting loops, only 3 were associated with CTCF. The most proximal loop was not associated with this DNA binding protein. Interestingly, others have shown that enhancer and promoter sites are often bound by cohesin and mediator complexes in the absence of CTCF. The MARWIZ online program was used to predict, within 100 kb region upstream of the c-myb in M1 cells, potential MARs which are evolutionarily conserved genome sequences that anchor DNA to the nuclear matrix and are reported to act as boundary elements for chromatin functional domains. In silico data showed that potential MARs are located at the boundaries of the loops identified in the 3C assay, thus assisting in our development of a hypothetical chromatin structure of the c-myb extended locus. In summary, retrovirus insertional mutagenesis has assisted in the identification of several potential regulatory regions in the far upstream region of c-myb that likely to be important in normal hematopoietic cell development and could be mutated or altered epigenetically in leukemia.

我们已经开始寻找25-100 kb上游区域的远端C-MYB转录调节器。由于MULV诱导的髓样白血病已在名为MML1、2和3（分别上游25、50和70KB上游）的三个区域的C-MYB上游集成了Proviruse，因此我们假设逆转录病毒访问调节元件。 因此，原病毒位置可能能够帮助我们识别对正常细胞以及患有C-MYB表达失调的白血病细胞重要的调节区域。我们首先通过芯片片查看了涉及正常细胞基因激活的组蛋白修饰，因为这可能有助于定位调节位点。 H3K4ME3，H3K4ME1和H3K9/ 14AC已知在被激活的启动子中富集，在表达C-MYB的髓生成细胞系中富含MML1和/或MML2，M1。所有这些组蛋白标记的富集随着IL-6的诱导分化和M1细胞中基因下调而降低。但是，这些标记在含有综合病毒的肿瘤细胞中增加并扩散。为了检查代表MML1中H3KME4峰的序列的增强剂活性，将3个片段克隆为C-MYB启动子的上游，这些启动子控制着萤火虫荧光素酶报告基因。我们能够在荧光素酶测定中表明，其中一个区域内的序列增加了荧光素酶的活性，表明增强子元件的存在。发生染色体构象捕获（3C）Q-PCR测定，5个质量区域之间的相互作用，包括启动子和所有MML位点（MML1，2和3）的5个单元格（MML1，2和3）在MML1，2和3）中（MML1，2和3）都在MML中（MML1和3）。 2或3。MML1区域包含2个环，总共有4个环，并且在这些环中的每个环中都插入了预告症。有趣的是，被诱导的M1细胞与IL-6区分开并且不表达明显的C-MYB，但仍保持相同的染色质循环结构，在具有活性转录的细胞中观察到。因此，这种染色质循环结构本身不足以用于C-MYB表达，可能需要添加转录因子才能导致表达。对未表达c-Myb的非脊髓质细胞系NIH3T3中染色质构象的检查表明，C-MYB基因座的循环结构的差异存在于不同的细胞类型中。显然，在造血细胞中未发现的一种新型相互作用峰值在大约-11 kb的启动子非常接近。我们可以得出结论，我们的研究提供了逆转录病毒插入诱变的新机制，从而使空间染色质组织允许远端放置的病毒具有其自身的增强剂元素，以访问该基因的5个主要调节区域。尽管可以基于最近的研究来预测这种模型，这些研究表明，高等真核生物中的转录激活经常涉及调节元素的远距离作用，但这实际上是第一次证明该病毒可以通过三维染色质结构与内含子中的启动子和/或控制区相互作用。此外，我们的研究还描述了一些重要的调节区域，这些调节区域是C-MYB编码区域远端的，并且可以在未来的研究中进行更详细的研究。 我们的研究还表明，CTCF在环结构附近结合，这并不奇怪，因为众所周知，CTCF有助于与基因激活相关的环。尽管我们发现了4个主要启动子相互作用循环，但只有3个与CTCF相关。最近端的环与该DNA结合蛋白无关。有趣的是，其他人表明，在没有CTCF的情况下，增强子和启动子位点通常受到粘蛋白和介体复合物的约束。 Marwiz在线计划用于预测M1细胞中C-MYB上游的100 Kb区域内的潜在火星，这些火星是进化保守的基因组序列，可将DNA锚定在核基质上，并据报道充当染色质功能域的边界元素。在硅数据中显示，潜在的火星位于3C分析中鉴定的环的边界，从而有助于我们发展C-Myb扩展基因座的假设染色质结构。总而言之，逆转录病毒插入诱变有助于鉴定C-MYB上游遥远地区的几个潜在调节区域，这些区域可能在正常造血细胞发育中很重要，并且可以在白血病中突变或表观遗传变化。