The Mechanism Study Of ETV6-RUNX1 And RUNX1 Directly Regulating RAG1 Expression

Background and Objectives:ETV6-RUNX1 B cell precursor acute lymphoblastic leukemia（BCP-ALL）is a common malignant tumor in children and adolescents,which emerges from a multi-hit leukemogenesis process.Schematically,two sequential genetic events occur.The first one consists in the formation of the ETV6-RUNX1 fusion gene by the t（12;21）（p13;q22）chromosomal translocation and occurs in utero.The cell harboring ETV6-RUNX1 are considered leukemia precursor cell,which are prone to transform into acute leukemia.The second event implicates aberrant activity of RAG recombinase,which occurs several years later of the baby birth.Abnormal activity of RAG recombinase leads to acquired genetic abnormalities,namely second hit or multiple hits.The accumulation of acquired genetic abnormalities ultimately drives the transformation of ETV6-RUNX1 positive precursor cells into acute leukemia.Previous studies have demonstrated that dysregulated immune response occurs during infections or inflammation in children were the cause of the abnormal activity of RAG recombinase.However,whether there is a causal link between the first hit,ETV6-RUNX1,and the second hit,dysregulated RAG recombinase activity,is still unknown.Therefore,the purpose of this study is to explore whether there is a causal link between the first hit and second hit in ETV6-RUNX acute lymphoblastic leukemia,that is to say,the ETV6-RUNX1 fusion gene and the abnormal activity of RAG recombinase.If so,clarify the regulatory mechanism.The study of us tends to be of great significance to reveal the leukemogenesis mechanism in pediatric acute lymphoblastic leukemia,and guiding the prevention and treatment of leukemia.Methods:1.We constructed RUNX1 or ETV6-RUNX1 stable expressed cell lines,Nalm6^RUNX1 and Nalm6^ETV6-RUNX1through lentivirus transduction and antibiotic selection.RT-q PCR and Western blot were performed to verify whether the expression of RUNX1 or ETV6-RUNX1 was positively correlated with the expression of RAG1 both at m RNA and protein levels.The Nalm6^{truncated Runx1} cell line with deletion of RUNT DNA binding domain of RUNX1 was constructed,the changes of RAG1 transcript induced by RUNX1 inactivation were examined.2.Bone marrow mononuclear cells from BCP-ALL patients and precursor B cell lines （Nalm6 and REH）were used to perform Ch IP-Seq,anti-ETV6 antibody and anti-RUNX1 antibody were used to bind ETV6-RUNX1 and RUNX1.The aim of this experiment is to reveal whether the transcription factors ETV6-RUNX1 and RUNX1 can specifically bind to the transcriptional regulation region of RAG1 gene,if so,read the specific binding sequence.3.Five Nalm6 cell lines,Nalm6^{RUNX1-Halotag},Nalm6^RUNX1-HA,Nalm6^{RUNX1-HA+ETV6-} RUNX1,Nalm6ETV6-RUNX1-Halotag,and Nalm6ETV6-RUNX1-Halotag+RUNX1 were constructed through lentivirus transduction and antibiotic selection.Ch IP-q PCR using tag antibodies（anti-Halotag and anti-HA）was performed to verify the specific binding of ETV6-RUNX1 and RUNX1 to the transcriptional regulatory region of RAG1, and the competition between these two transcription factors when they bind to the RAG1 transcriptional regulatory region.4.Luciferase assay with the sequence of RAG1-80bp promoter or-1200bp enhancer cloned into luciferase reporter plasmid was performed to examine the expression of luciferase in presence of ETV6-RUNX1 or RUNX1.Jaspar software was used to predict the potential binding site of RUNX1 on RAG1 upstream regulatory region,highly conserved binding sites were enrolled in the following luciferase assay.Luciferase reporter plasmids with the highly conserved binding sites deletion were constructed,and the difference of luciferase activity between the normal cis- acting elements and the elements with highly conserved binding sites deletion was examined.5.CRISPR-d Cas9 mediated transcriptional activation system was constructed,and several sg RNAs targeting RAG1-80bp promoter and-1200bp enhancer were designed.RT-q PCR and Western blot were performed to verify whether these two cis-acting elements were involved in the physiological regulation of RAG1 expression.6.The RAG recombinase activity reporting plasmid GFPi was expressed in Nalm6 cells through retrovirus transduction.The recombinase activity in presence of RUNX1 or ETV6-RUNX1 was quantitative detected by flow cytometry.Results:1.The expression of RUNX1 and ETV6-RUNX1 were positively correlated with the expression of RAG1 both at m RNA and protein levels,that is,when the expression of RUNX1 or ETV6-RUNX1 increased,the expression of RAG1 increased too.However,when the RUNT DNA binding domain of RUNX1 was truncated,the inactivated RUNX1 can no longer upregulate the expression of RAG1.2.Chromatin immunoprecipitation-sequencing analysis showed that the specific binding signals of ETV6-RUNX1 and RUNX1 with the distal-1200bp sequence and proximal-80bp sequence within the upstream transcriptional regulation region of RAG1 were observed in the precursor B blasts of human BCP-ALL patients and precursor B cell lines（Nalm6 and REH）.The histone binding peak at proximal-80bp showed that H3k4me3 was positive,suggesting the-80bp sequence was an activated promoter.The H3k4me1 peak can be detected at distal-1200bp region, which is considered to be an activated enhancer.3.Chromatin immunoprecipitation-q PCR results suggest that the transcription factors ETV6-RUNX1 and RUNX1 can bind to the-1200bp enhancer and-80bp promoter sequences of RAG1 competitively.ETV6-RUNX1 preferentially binds the- 1200bp enhancer whereas RUNX1 binds predominantly the-80bp promoter.4.Luciferase assay showed that RUNX1 and ETV6-RUNX1 could induce specific luciferase expression of RAG1-80bp promoter and-1200bp enhancer,respectively.When the potential binding site of RUNX1 predicted by Jaspar software was deleted,the luciferase expression induced by the two transcription factors on RAG1-80bp promoter and-1200bp enhancer disappeared,which suggest that the specific sequence predicted by Jaspar is crucial for the binding of RUNX1 transcriptional factor to RAG1.5.CRISPR-d Cas9 mediated transcriptional activation showed that under the physiological conditions of precursor B lymphoblasts,the expression of RAG1 can be significantly upregulated by the sg RNA targeting-80bp promoter,and the RAG1 protein can be significantly downregulated by the sg RNA targeting-1200bp enhancer,which indicates that the-80bp promoter and-1200bp enhancer play physiological regulatory roles on RAG1 expression.6.RAG recombinase activity reporter assay showed that both ETV6-RUNX1 and RUNX1 could directly induce the increase of RAG recombinase activity in precursor B lymphoblasts.Conclusions:In conclusion,the study of us reveals that ETV6-RUNX1 and RUNX1 can directly upregulate the expression of RAG1 by binding to the-1200bp distal enhancer and-80bp proximal promoter of RAG1 in precursor B lymphoblasts.Meanwhile,ETV6-RUNX1 and RUNX1 can also directly induce the increase of RAG recombinase activity.The overexpression and aberrant increased recombinase activity of RAG induce inappropriate genomic rearrangements,that is,the second hit or multi-hit,and finally convert the ETV6-RUNX1 preleukemic clone into overt leukemia.