Par-4调控T淋巴细胞白血病细胞中染色质重塑子CHD4基因Pre-mRNA选择性剪接的机制及其抗白血病效应

T lymphocyte leukemia (T-ALL) is a hematological malignancy and remains an important challenge in pediatric oncology because of poor outcome. In preliminary study, we found tumor suppressor Par-4 recruited transcription factors THAP1 and WT1 to regulate the alternative pre-mRNA splicing of chromatin remodeler CHD4 in T lymphocyte leukemia cell, which induced cellular apoptosis and mitotic catastrophe. However, the exact molecular mechanisms remain to be elucidated.. In present study, multiple transcription start sites of CHD4 gene will be determined by primer extension analysis and DNase I footprint experiment. We will verify that protein complex of Par-4/THAP1 selectively activates the promoter P2 of CHD4 gene by minigene vector and fluorescence in situ hybridization for RNA. It will be demonstrated that Par-4/WT1 selectively activates the promoter P3 and an enhancer E1 of CHD4 gene, resulting in a splicing isoform with the skipping of exon 17 to 21 and retention of intron 3 under the regulation of splicing factor SRSF1 and SRp55. With mononucleosome disruption assays and chromatin remodeling assays, it will be observed that the effort of Par-4-induced alternative pre-mRNA splicing of CHD4 gene on chromatin remodeling and the adhesion of kinetochore to spindle microtubules in T-ALL cells. This study will provide new ideas for the treatment of T-ALL.

儿童T淋巴细胞白血病(T-ALL)是恶性血液病。我们前期发现抑癌基因Par-4可募集转录因子THAP1和WT1，调控T-ALL细胞中染色质重塑子CHD4基因Pre-mRNA选择性剪接，诱导凋亡和有丝分裂灾变死亡，但机制不明。.本研究拟①利用引物延伸和DNase I足迹实验，确定CHD4基因多个转录起始位点。②利用minigene载体和RNA-FISH，确定Par-4/THAP1选择性激活启动子P2，产生剪接异构体truncated CHD4；确定Par-4/WT1选择性激活启动子P3和增强子E1，协同剪接因子SRSF1和SRp55调控第17至第21外显子跳跃及第3内含子保留，产生非编码RNA异构体ncRNA-CHD4。③采用单个核小体解构实验等，探索Par-4诱导的CHD4基因Pre-mRNA剪接对T-ALL细胞染色质重塑、动粒与纺锤体微管粘附的影响。本研究将为T-ALL治疗提供新思路。

儿童急性T淋巴细胞白血病，是恶性程度较高的血液病。染色质解旋酶DNA结合蛋白4(CHD4)，属于核小体重构和组蛋白脱乙酰酶复合体的重要成员之一，涉及许多致癌过程，与表观遗传密切相关，包括控制细胞周期进展，加速实体肿瘤的转移，发动和维持多种肿瘤抑制基因沉默等。本研究项目的主要鉴定和分析CHD4的启动子结构，分析mRNA剪接异构体，研究其相关转录调控机制，及其对T淋巴细胞白血病细胞增殖与凋亡等生物学功能的影响。研究结果显示，CHD4基因最小活性区域位于转录起始位点-233bp至-13bp之间,其中包含NF-қB、MZF1等转录因子结合位点。NF-қB对CHD4启动子活性具有正向调控作用。CHD4抑制T淋巴细胞白血病细胞Jurkat细胞凋亡，转染siRNA-CHD4的Jurkat细胞在G0/G1期显著升高，而在S期下降；CCK-8检测示CHD4基因对Jurkat细胞的增殖有促进作用。Par-4诱导的CHD4基因Pre-mRNA选择性剪接对T-ALL细胞中微管蛋白纺锤体的装配产生影响。确定截断蛋白CHD4和非编码的CHD4对纺锤体检查点依赖的有丝分裂阻滞作用，导致纺锤体微管几乎消失，染色体排列散乱。本项目采用了一系列分子生物学技术较深入地探索了Par-4调控T淋巴细胞白血病细胞中染色质重塑子CHD4基因转录调控的机制，及其对T淋巴细胞白血病细胞染色质重塑、细胞凋亡和有丝分裂灾变死亡的影响，明确了其抗白血病效应。本研究将为T-ALL的治疗提供新的思路。