高精度解析TAL1+51KB增强子在红系发育分化中的调控机制

转录因子TAL1的特异性表达对红系发育分化至关重要，而特异性转录的调控机制尚不清晰。申请人发现TAL1基因座上增强子-启动子的染色质构象互动参与介导基因的特异性表达，成果已发表于《核酸研究》。后续实验明确+51Enh增强子决定TAL1在红系细胞中的转录激活，其核苷酸序列或转录因子结合情况的改变可特异性下调K562细胞的TAL1表达。据此提出：+51Enh通过精细调控TAL1红系特异性地转录而促成红系发育正确进行。项目拟利用人CD36+细胞和红系细胞株，采用反向ChIP-质谱和RNA干扰技术以明确+51Enh所结合的转录因子；通过基因编辑，整合STARR-Seq和单核苷酸饱和突变的通量平行测序，精度诠释+51Enh调控TAL1转录的分子机制以及对红系发育分化的影响。研究有助于深刻理解增强子对红系分化的调控意义以及在特异性转录调控中的作用模式，为以特异性增强子为靶向的基因治疗提供理论基础。

TAL1 is known as an important transcription factor in hematopoiesis, particularly in erythropoiesis. However, how TAL1 was translated as a cell type-specific transcription factor remains unclear. Our recent publication on Nucleic Acid Research has revealed a cell type-specific way of regulating the Chromatin-interaction between Enhancer and Promoter at TAL1 locus by chromosome 3D structure. Up to now, the systemic regulatory mechanism of Enhancer in erythropoiesis is still untouched. To address this question, we performed a series of luciferase reporter assays and found that +51Enh was only activated in erythroid K562 cells but not in other hematopoietic cell types, consolidating its promoter in erythroid-specific enhancer feature. Furthermore, using the CRISPR/Cas9 gene-editing system, we knocked out the +51Enh in K562 cells and Jurkat cells. In consistence with the luciferase findings, TAL1 transcription was significantly suppressed in K562 cells while the impact on Jurkat cells was very little. Enlightened by Prof. Daniel E. Bauer that inherited BCL11A enhancer variants confer a higher hemoglobin F level, in turn, affects hemoglobinopathy. We thus raise the scientific question that +51Enh function as an erythroid-specific enhancer through binding erythropoiesis-driven transcription factor, to help TAL1-driven erythropoiesis. Disruption of the +51Enh fine-tune potentially disturbs the normal erythropoiesis. Herein, this project aims to use erythroid cell lines (K562, and Kasumi-1) and human CD36+ cells to define the impact of +51Enh on erythroid development and differentiation, dissect the key transcription factors bound on +51Enh, and systemically interpret the +51Enh regulatory mechanisms and the contribution of single nucleotide variants to TAL1 transcription and erythropoiesis. This study would help understand cell-type-specific TAL1 transcriptional regulation patterns in-depth and provide novel knowledge for gene therapy targeting cell-type-specific enhancers.