Landscapes of TET1 and STAT5B binding on DNA across the entire genome in PDX2 B-ALL cells. Landscapes of TET1 and STAT5B binding on DNA across the entire genome in PDX2 B-ALL cells

Here we report both TET1-WT and catalytically dead mutant TET1 (TET1-MUT) directly interact with STAT5B in PDX2 B-ALL cells. To study the landscape of TET1 and STAT5B binding in whole genomic DNA and test if the STAT5B binding signal is affected by TET1, we conduct the ChIP-seq assay with flag antibody (TET1-WT, TET1-MUT) and STAT5B antibody (sgNS or sgTET1) respectively. The results showed that TET1 bound peaks overlap with STAT5B very well and the tag density of STAT5B on target genes decreases in TET1-impeded cells. We conclude that TET1 recruit STAT5B to the promoters of its target genes and further promote the target genes’ transcriptions. Overall design: We generate the stable PDX2 cells with forced expression of flag-tagged TET1-CD-WT and TET1-CD-MUT. Meanwhile we also conduct the ChIP in wild type or TET1 knockout PDX2 cells(sgNS and sgTET1).

本研究证实，野生型TET1（TET1-WT）与催化失活突变体TET1（TET1-MUT）均可在PDX2 B细胞急性淋巴细胞白血病（B-ALL）细胞中与STAT5B发生直接相互作用。为探究全基因组DNA中TET1与STAT5B的结合图谱，并验证STAT5B的结合信号是否受TET1调控，我们分别采用Flag标签抗体（针对TET1-WT、TET1-MUT实验组）及STAT5B抗体（针对sgNS、sgTET1实验组）进行染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）实验。实验结果显示，TET1结合峰与STAT5B结合峰的重合度极高，且在TET1功能受抑制的细胞中，STAT5B在靶基因上的标签密度显著降低。综上，TET1可将STAT5B招募至靶基因的启动子区域，进而促进靶基因的转录。总体实验设计：本研究构建了稳定过表达带Flag标签的TET1-CD-WT与TET1-CD-MUT的PDX2细胞；同时，我们还在野生型及TET1基因敲除的PDX2细胞（分别转染sgNS与sgTET1）中开展了染色质免疫共沉淀实验。