The Expression Levels And Regulatory Mechanism Of BCL11A In Chinese With Acute Myeloid Leukemia

Purpose:5 acute myeloid leukemia (AML)-related genes were chosen for comparison of difference in mutation frequency between newly-diagnosed Chinese AML (ChAML) and persons of predominately European descent with AML (PPED-AML). We measured the BCL11A expression levels in Chinese with AML and focused on in-depth research about its regulatory mechanism, including determination of transcription start site, analysis of promoter activity, identification of potential binding sites within BCL11A promoter for key regulatory factors, such as CEBPa, involved in myeloid differentiation, and examination of the transcriptional regulation of BCL11A by CEBPa, and further exploration of the role of BCL11A as an oncogene in the progression of leukemia.Approach:(1) 269 bone marrow samples were collected from patients with AML and amplified for 5 AML-related genes, including NF1, TP53, NRAS, FLT3 and DNMT3A, by PCR and then analyzed for mutations by sequencing.(2) The TaqMan probe real-time fluorescence quantitative PCR was used for accurate measurement of the BCL11A expression levels in AML patients.12 bone marrow samples from healthy donors were compared and analyzed whether the expression of BCL11A was dysregulated.(3) The transcriptional start site of BCL11A was identified by bioinformatic analysis and 5’-RACE.(4) The BCL11A promoter and its serial deletion plasmids were constructed. The dual luciferase reporter system was used to measure promoter activity and to narrow down possible binding sites of transcription factor.(5) A series of experiments, including construction of CEBPA expression vectors, co-transfection with BCL11A promoter, generation of point mutation, evaluation of such mutations, were used to determine the influence of CEBPa on the promoter activity of BCL11 A.(6) CEBPA over-expression experiments were used to verify the effect of over-expression of CEBPA on the expression of endogenous BCL11A.(7) HL-60 cell differentiation model was established and used to study the role of BCL11A in ATRA-induced myeloid differentiation.Results:(1) NF1, TP53, DNMT3A, NRAS and FLT3 mutation frequency in Chinese AML patients were 0,<1%,6%,7% and 23%, respectively. The five mutations were insignificantly associated with clinical characteristics including age, gender, FAB-type and cytogenetics. Except for FLT3-ITD, mutational frequencies were substantially lower in Chinese with AML than in persons of predominately European descent with AML.(2) In Chinese AML, the levels of BCL11A expression were abnormally increased.(3) Our 5’-RACE results showed that the transcription initiation site of human BCL11A gene extends to the upstream 142 bp compared with that released by the NCBI.(4) The 500-bp fragment upstream of the new transcription initiation site of BCL11A displayed significantly high promoter activity.(5) The transcription factor CEBPa suppressed BCL11A promoter activity, in a manner dependent on the incubation time and the amount of expression vectors used. However, the inhibition was lost upon site-directed mutation of a CEBPa binding site in BCL11A promoter, and accordingly, BCL11A promoter activity was recovered by about 40%.(6) CEBPA over-expression suppressed endogenous BCL11A expression as well.(7) During HL-60 cell differentiation, the expression levels of CEBPA were increased and, on the contrary, those of BCL11A reduced, indicating for a negative correlation between CEBPA and BCL11A. Morever, BCL11A over-expression inhibited HL-60 cell differentiation.Conclusions:This study found that the mutation frequency of AML-related genes was generally low in Chinese patients with AML compared with Western AML patients, indicating that there may be unknown genes yet to be discovered which are involved in the development of Chinese AML. The dysregulated BCL11A expression was for the first time discovered in Chinese AML patients. Subsequently, we identified a new BCL11A transcription initiation site. We also first found that the regulatory factor essential for myeloid differentiation, CEBPa, might be an important inhibitor of BCL11A expression, with a dependence on the incubation time and the amount of expression vectors used. During HL-60 cell differentiation, the expression levels of CEBPA were increased and, on the contrary, those of BCL11A reduced, indicating for a negative correlation between CEBPA and BCL11A. Morever, BCL11A over-expression inhibited HL-60 cell differentiation. Together, these findings suggest that downregulation of BCL11A may mediate CEBPa induction of myeloid differentiation, and lay a solid foundation for further confirmation of the role of BCL11A as an oncogene in leukemogenesis.