Study On Spatial Organization And Functionality Of BCL11B Locus In Nuclei

Leukemia is a high-grade malignant tumor of the hematopoietic system and the most common type of malignancy in pediatric patients.Rapamycin has a certain effect on the treatment of leukemia.The BCL11 B gene belongs to the BCL family and is an identical important transcriptional regulator.BCL11 B gene involves in various biological processes such as thermogenesis,T cell proliferation and differentiation,and also regulates lymphatic hematopoietic system development and proliferation.Its absence is beneficial to derivation of radiation-induced leukemia.Chromatin conformation capture(3C)technology examines the chromatin spatial organization within the nucleus and can also explore long-range interactions between DNA molecules.Chromatin immunoprecipitation(ChIP)is a powerful tool for analyzing DNA-protein interactions nowadays.When combined with 3C,ChIP,FAIRE technology,and histone modification technology,the understanding of the internal structure of the nucleus can be enhanced.Interactions between nuclear chromatin are sophisticated and diverse,and these technologies can help us better understanding the interaction between chromatin.In this study,3C,ChIP,and formaldehyde-assisted separation control(FAIRE)techniques were used to investigate the effect of rapamycin on the dynamic spatial organization of BCL11 B locus in T-cell acute leukemia cells from a three-dimensional level to expedite the digging process of molecular mechanism of intracellular regulation of T cell acute leukemia governed by BCL11 B gene.Objective: Study the effect of rapamycin on the dynamic spatial organization of BCL11 B locus in T-cell acute leukemia cells,to further investigate the molecularmechanism of BCL11 B gene expression and regulation in T-cell acute leukemia cells.Methods: 1.CD4+ T cells(Jurkat cells),with normal culture conditions and in logarithmic growth phase,were treated with appropriate concentrations of rapamycin,and then examined with MTT assay to determine cell viability.2.Real-time quantitative PCR(qPCR)and Western Blot were used to detect the transcription level and expression level of related genes in Jurkat cells treated with rapamycin for 24 h.3.The 3C technique was used to study the interactions between the sites in the BCL11 B locus before and after treatment with rapamycin in Jurkat cells.4.The FAIRE technique was used to study the changes between the open site loci of the BCL11 B locus before and after treatment with rapamycin in Jurkat cells.5.Changes in the interaction between the BCL11 B locus and CTCF protein related sites before and after treatment with rapamycin by Jurkat cells by ChIP technology.6.Changes in the level of histone modification at the BCL11 B locus before and after treatment of rapamycin by Jurkat cells by ChIP technology.Result:1.MTT assay showed that rapamycin could inhibit the proliferation of Jurkat cells.The degree of inhibition was positively correlated with the drug concentration.The transcription and expression levels of BCL11 B and CTCF,detected in Jurkat cells treated with rapamycin,are up-regulated in different degrees.2.3C study demonstrated that there is an interaction between the BCL11 B gene locus and the rapamycin stimulated the dynamic changes of the spatial organization of BCL11 B in Jurkat cells.3.The enrichment efficiency of FAIRE on BCL11 B locus was different at each site,and rapamycin stimulated the BCL11 B locus activity of Jurkat cells.4.Rapamycin affects the frequency of binding of CTCF to BCL11 B locus in Jurkat cells.5.Rapamycin affects the level of histone modification at the BCL11 B locus in Jurkat cells.Conclusions: Rapamycin treatment altered the epigenetic environment in Jurkat cells,which in turn stimulated the spatial organization of the BCL11 B locus.This change in spatial organization of the BCL11 B locus functionally regulates gene expression.