RESUMO
This study was purposed to characterize the genomic distribution of the binding sites for AML1-ETO fusion protein on chromosome 2, 9 and 19, and to further gain insights into the characteristics of transcriptional regulation by AML1-ETO in acute myeloid leukemia so as to provide theoretical basis for the development of targeted therapy and optimization for treatment. Chromatin immunoprecipitation (ChIP) coupled with high density tiling arrays (chip), also known as ChIP-chip, was utilized in this study. ChIP-DNA enriched by an anti-ETO antibody and total genomic DNA of Kasumi cells were hybridized to tiling arrays, tiled through chromosome 2, 9 and 19. The ChIP enriched regions were identified using a model based analytical tool (MAT). Genomic distribution of the ChIP regions was analyzed using publicly available CEAS web server. The Gene Ontology (GO) enrichment analysis was performed to excavated the biological significance. The results indicated that a total of 588 enriched regions were identified on chromosome 2, 9 and 19 by the anti-ETO antibody. A number of the identified regions were located within enhancers (48.86%) or introns (37.35%), much smaller fractions were within proximal promoters (5.96%) or exons (5.49%). Functional enrichment analysis showed that cell proliferation and signal transduction biological pathways were enriched in potential genes of AML-ETO. It is concluded that half of the AML1-ETO binding sites are located within known transcriptional regulatory regions (promoter, 5' UTR and enhancer), while almost another half were within the sequences which were not previously reported as regulatory regions. The potential target molecular network of AML1-ETO is involved in several essential biological processes.
Assuntos
Humanos , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Subunidade alfa 2 de Fator de Ligação ao Core , Genética , Metabolismo , Proteínas de Ligação a DNA , Metabolismo , Genoma Humano , Leucemia Mieloide Aguda , Genética , Proteínas de Fusão Oncogênica , Genética , Metabolismo , Regiões Promotoras Genéticas , Proteína 1 Parceira de Translocação de RUNX1 , Translocação GenéticaRESUMO
This study was purposed to investigate the effect of AML1-ETO fusion protein resulted from hematopoietic transcription factor (AML1) and acute myeloid leukemia M(2b)(AML-M(2b)) on transcription activity of nucleobindin 2 (nucb2) promoter, and to explore the role of AML1-ETO in molecular pathogenesis of AML-M(2b). The real-time RT-PCR was used to study the regulation of AML1-ETO on nucb2 at transcription level in AML1-ETO inducible leukemia cell line, the chromatin immunoprecipitation (ChIP)-based qPCR was used to investigate the direct in vivo interaction between the AML1, AML1-ETO and nucb2 promoter in AML1-ETO positive leukemia cell line, the luciferase report gene assay was used to detect the regulation of AML1, AML1-ETO on the transcription activity of nucb2 promoter. The results showed that the expression level of nucb2 was reduced with the increase of AML1-ETO. The promoter of nucb2 could be bound by both AML1 and AML1-ETO. The promoter of nucb2 was trans-repressed by AML1 and AML1-ETO respectively. It is concluded that the nucb2 is the direct target gene of AML1 and AML1-ETO, the transcription regulation of AML1, AML1-ETO on nucb2 is carried out via repressing its promoter activity.