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The Function and Pathogenesis of A Novel Fusion Gene TBLR1-RARα in Acute Promyelocytic Leukemia The Binding Sites and Transcriptional Regulation of Transcriptional Factor AML1

Author: ChenYiRui
Tutor: WangJianXiang
School: Beijing Union Medical College
Course: Internal Medicine
Keywords: acute promyelocytic leukemia RARα fusion gene transcriptional regulation ATRA treatment differentiationAML1 ChIP Sequencing Transcriptional regulation signal transductionpathway
CLC: R733.71
Type: PhD thesis
Year: 2013
Downloads: 47
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Abstract


Objective:In the study of an intractable case encountered in our daily clinical work, we discovered a new t(3;17)(q26;q21) chromosomal translocation. A novel RARa fusion gene TBLRl-RARa which has never been reported was identified by5’RACE technique. In this study, the structure, pathogenesis and response to drug therapy of the new fusion gene was investigated to illustrate the characteristics, pathogenesis and the therapeutic effect in this novel APL. Thus, this study may consummate the knowledge of APL.Methods:To identify and amplificate the new chimeric fusion transcript,5’RACE experiment and RT-PCR was performed. The TBLR1-RARa expression vector was constructed and was transfected into293T cell line by lipofectamine2000reagent. When the transfected293T cell line was treated with or without ATRA, the expression level and the subcellular localization of TBLR1-RARa were investigated by Western blot and immunofluorescence analysis, and then coimmunoprecipitation and immunofluorescence analysis were executed to study the formation of homodimer and the recruitment of the corepressors by TBLRl-RARa. Dual-luciferase assay was used to clarify the transcriptional activity of TBLR1-RARa. Then, a lentiviral vector of TBLRl-RARa was constructed and infected the HL-60cell line. The HL-60cell which highly expressed TBLR1-RARa was sorted by flow cytometer. Colony formation assay and flow cytometry analysis were performed to detect the differentiation status in the TBLR1-RARa highly expressed HL-60cells.Results:In our study of an intractable case with a new t(3;17)(q26;q21) chromosomal translocation, a novel RARa fusion gene TBLR1-RARa was discovered. There have been nine RARa fusion genes reported so far. Like other RARa fusion genes, TBLRl-RARa contains the exon3and its3’sequence of RARa. TBLR1-RARa oncoprotein contains the LisH domain from TBLR1and the B-F domains from RARa. TBLR1-RARa diffusely locates in nuleus and cytoplasm. Like other RARa fusion protein, TBLRl-RARa can form homodimer and recruit corepressors to inhibit the transcription of the RARa target gene. TBLR1-RARa inhibits the RARa transcriptional activation in a dominant-negative manner and the transcriptional inhibition can be rescued by overexpressed RARa. When treated with ATRA, TBLR1-RARa can be down regulated and cannot form homodimer anymore. Besides, corepressors can be dismissed and degraded through TBLR1-RARa when treated with ATRA. On the other hand, TBLR1-RARa can also partially stabilize the binding activity of corepressors to RARE when expose to ATRA. As a result, TBLR1-RARa can inhibit the transcriptional activity of RARa target genes, which can be reduced by ATRA, and finally leads to cell differentiation.Conclusions:In this study, we discovered a novel RARa fusion gene TBLR1-RARa and investigated its function, pathogenesis and response to durg treatment. Unlike wild-type RARa, TBLRl-RARa can form homodimer, recruit more corepressors to inhibit the transcription of RARa target gene and cause APL. The transcriptional inhibition of TBLRl-RARa can be rescued by overexpression of RARa and ATRA treatment and finally leads to cell differentiation. Objective:To find new targets of AML1, to explore the interaction between AML1and other transcriptional factors, to understand the role of AML1in the entire signal transduction pathway, and ultimately to consummate the role of AML1in the process of hematopoiesis and leukemogenesis.Methods:Semi-quantitative RT-PCR and Western-blot were used to screen out the high endogenous AML1b expression cell line NB4as research object. After fixed processing, NB4was sent to the Genomics Institute (BGI) to conduct the following ChIP-seq experiments and bioinformatics analysis. The new target genes of AMLlb were validated by ChIP.Results:The data acquired from ChIP-seq were analyzed by bioinformatics and showed that AML1mainly bind to the intergenic region, intron and upstream20k region of genes acting as a transcriptional factor. By GO analysis, peak related genes were connected with cell proliferation, death, growth, development, localization, signaling, etc. A total of63353peak related genes were obtained after analyzing peak related genes by softwares and the most important one was pig7(LITAF) which could be bound by AML1in the upstream20k region, which was later validated by ChIP. Two tumor related transcriptional factors Fos and Klf4were found by motif analysis. They may interact with AML1and transcriptionally regulated downstream genes. Conclusion:AML1is one of the most important transcriptional factors in hematopoietic regulation. It can regulate many hematopoiesis related genes. In this research, a new target gene pig7(LITAF) of AML1was found and consequent biological experiment in our lab showed that AML1could up-regulate the expression of pig7(LITAF), which leads to differentiation and apoptosis. On the other hand, two motifs Fos and Klf4were found and they might bind to AML1. By interaction between AML1and the motifs, they might participate in some important cell signal transduction pathways, such as cell cycle, P53signalling pathways, mTOR signalling pathways, Wnt signalling pathways, apoptosis, and regulate some important genes, such as PUMA, PI3K, Wnt, TRAIL, c-kit, ERK, STAT5, etc.

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Leukemia > Acute leukemia
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