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: 8157597
• 负责人: Linda Wolff
• 金额: $ 38.55万
• 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/8157597
• 关键词: Acute Myelocytic Leukemia; Distant; MYB gene; Normal Cell; Proto-Oncogene Proteins c-myb; Regulation

We have begun to look for distant c-myb transcriptional regulators in the region 25-100 kb upstream region. Because of recent developments in the epigenetics field we can now begin to predict the function of regions of chromatin based upon the presence of histones with specific modifications or specific DNA-binding proteins. Promoter regions and enhancers of activated genes are enriched in histone 3, mono-and tri-methylated on lysine 4 (H3K4me1, H3K4me3) and have acetylated histone 3, for example, H3K9Ac. In an attempt to find regulatory sequences in the upstream region of c-myb, chromatin immunopreciptation analyzed by microchip (ChIP-on-chip) was carried out using antibodies specific for these marks and a tiling microarray representing the 600kb region surrounding the c-myb gene (40bp spacing). When an antibody specific for trimethyl-H3K4 was used in an experiment performed in myeloid blast cells, M1, it was found that this mark was strongly enriched in the region corresponding to the Mml1 integration site (25kb upstream of c-myb) as well as at the c-myb gene. Subsequent experiment with anti- H3K9,14Ac antibodies revealed that these regions were also enriched in acetylated histones, confirming that both of these peaks are associated with transcriptional activity or are poised for activity. Interestingly, abundance of H3K4me3 at both of these regions was decreased in M1 cells following IL-6-induced differentiation, which suggests that activity of the region corresponding to Mml1 integration is correlated with the transcriptional state of c-myb gene. Abundance of the H3K4me3 on the control genes was not affected. Changes in H3K27me3 and H3K9me, reported to be associated with transcriptional repression states were not found to largely differ. Next, ChIP-on-Chip with anti-H3K4me3 was performed in the cells lines carrying retrovirus in the Mml1 region. It was shown previously that the steady-state level of c-Myb mRNA and protein did not differ from M1 cells in these cells (Koller et al., 1996). Interestingly, ChIP-on-chip experiment revealed that the H3K4me3 has a different pattern of distribution at c-myb in 3 out of 4 analyzed Mml1 cell lines, compared to control M. The H3K4me3 signal in these cells spread dramatically toward the 3 end of c-myb and the signal was stronger. This data indicated that virus integration at the Mml results in predominant spreading of the active transcription-related histone mark 25 kb down-stream at the c-myb locus. Since the above observations together suggest that the -25 to -35kb region is associated with the transcriptional activity of the c-myb promoter, we decided to look for evidence of physical interaction of this and other regions upstream of c-myb with the promoter of the gene. Using a Chromatin Conformation Capture (3C, chromatin looping) assay we have been able to show that several regions upstream of c-myb, including the region in proximity of -25 to -35kb H3K4me3 peak, interact with the c-myb promoter in myb-expressing cells. All these interactions disappeared if c-myb was down-regulated by IL-6, suggesting that these interactions are important for the activity of c-myb promoter. In addition there was no interaction detected in NIH/3T3 cells, in which c-myb is not expressed. Interactions observed in M1 cells seem to be preserved in cells carrying retrovirus in Mml1. Interestingly, even larger upstream regions seem to participate in the interactions with the c-myb promoter in the latter cell lines. Since CTCF has been shown by many others to be involved in facilitating looping, we carried out ChIP-on-Chip to look for locations of CTCF binding in M1 cell DNA. Prominent CTCF binding sites were discovered at each of the three retrovirus integration sites, MmL1, MmL2 and Mml3. Studies are being performed to determine if these bound CTCFs are indeed involved in looping. In addition, since our experiments so far suggest that there are c-myb transcriptional regulator regions upstream of the gene, future experiments will also aim to determine whether the DNA sequences in regions of acetylation and H3K4 trimethylation actually function as transcriptional enhancers.

我们已经开始寻找25-100 kb上游区域的区域中遥远的C-MYB转录调节器。由于表观遗传学领域的最新发展，我们现在可以根据具有特定修饰或特定DNA结合蛋白的组蛋白的存在来预测染色质区域的功能。活化基因的启动子区域和增强子富含组蛋白3，单甲基化的赖氨酸4（H3K4ME1，H3K4ME3），例如，乙酰化组蛋白3，例如H3K9AC。为了尝试在C-MYB上游区域找到调节序列，使用微芯片（Chip-On-Chip）分析的染色质免疫沉淀使用这些标记的抗体和代表C-Myb基因（40bp space）的600KB区域的平铺微阵列进行了特定的抗体。当在M1中进行的实验中使用了对三甲基-H3K4的抗体时，发现该标记在与MML1积分位点（C-MYB上游25KB）以及C-MYB基因的区域中非常富集。随后对抗H3K9,14Ac抗体的实验表明，这些区域也富含乙酰化组蛋白，证实这两个峰都与转录活性相关或有助于活性。有趣的是，在IL-6诱导的分化后，M1细胞中H3K4ME3的丰度降低了，这表明与MML1整合相对应的区域的活性与C-MYB基因的转录状态相关。对照基因上的H3K4me3的丰度不受影响。 H3K27ME3和H3K9ME的变化据报道与转录抑制态有关，没有发现很大不同。接下来，用抗H3K4me3芯片芯片在MML1区域携带逆转录病毒的细胞系中进行。先前表明，在这些细胞中，C-MYB mRNA和蛋白质的稳态水平与M1细胞没有差异（Koller等，1996）。有趣的是，与对照M相比，Chip-on-Chip实验表明，在4个分析的MML1细胞系中，H3K4ME3在C-MYB的3个分布模式不同。这些细胞中的H3K4ME3信号急剧向C-MYB的3端扩散，信号较强。该数据表明，在MML处的病毒整合会导致在C-MYB基因座处有活性转录相关组蛋白的主要扩散。由于上述观察结果共同表明-25至-35KB区域与C -MYB启动子的转录活性有关，因此我们决定寻找C -MYB上游该基因启动子上游该区域的物理相互作用的证据。使用染色质构象捕获（3C，染色质环）测定，我们能够证明C-MYB上游的几个区域，包括-25至-35KB H3K4ME3峰的接近区域，与表达myb表达细胞中的C-MYB启动子相互作用。如果C-MYB被IL-6下调，所有这些相互作用都消失了，这表明这些相互作用对于C-MYB启动子的活性很重要。另外，在NIH/3T3细胞中未检测到C-MYB的相互作用。在M1细胞中观察到的相互作用似乎保存在MML1中携带逆转录病毒的细胞中。有趣的是，甚至更大的上游区域似乎参与了后一种细胞系中与C-MYB启动子的相互作用。由于许多其他人已显示CTCF参与促进循环，因此我们进行了芯片芯片，以寻找M1细胞DNA中CTCF结合的位置。在三个逆转录病毒整合位点MML1，MML2和MML3中发现了突出的CTCF结合位点。正在进行研究以确定这些结合的CTCF是否确实参与了循环。此外，由于到目前为止我们的实验表明该基因上游有C-MYB转录调节剂区域，因此未来的实验还将旨在确定乙酰化和H3K4三甲基化区域中的DNA序列是否实际上是转录增强子。