【作者】 李莹；

【导师】 赵志壮； 付学奇；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2011， 博士

【摘要】 TAL1/SCL是碱性螺旋-环-螺旋(bHLH)转录因子家族中的一员,具有造血功能特异性。在造血过程中,TAL1/SCL决定着造血干细胞(HSCs)的生成以及维持其自我更新修复的能力,并在红细胞的末端分化过程中起着重要的调控作用。另外,TAL1/SCL在T细胞发育过程中的异常表达会导致T细胞急性淋巴细胞白血病的发生。TAL1的活性主要受与其互作的共激活因子或共抑制因子的调控,这种调控作用对于造血细胞的分化进程具有决定性作用,然而,对于如何调控TAL1与其共抑制因子或共激活因子间的相互作用还不清楚。TAL1/SCL转录因子包含一些苏氨酸(Thr)残基和丝氨酸(Ser)残基,它们可以被蛋白质激酶B(Akt)、丝裂原活化蛋白激酶(MAPK)和蛋白质激酶A(PKA)进行磷酸化修饰。其中,丝氨酸172位点(S172)可以特异性地被PKA磷酸化,这个磷酸化作用可以影响TAL1对其某个靶向依赖性DNA的识别,进而在细胞分化的过程中选择性地发挥转录调控作用。TAL1的这种转录活性的调控机理表明,类似的修饰作用也可能决定TAL1与其转录共抑制因子或共激活因子相互作用的能力。为了揭示TAL1与其共调节因子复合物的作用机制以及这个机制的功能紊乱在T细胞白血病产生过程中的作用,我们对TAL1与其共抑制因子LSD1相互作用的分子机制进行了详细的研究。本研究表明,PKA介导的磷酸化作用可以动态地调控TAL1与H3K4去甲基化酶LSD1复合物间的相互作用。TAL1Ser172位点的磷酸化作用能够特异性地破坏TAL1与LSD1的结合,导致造血细胞中的靶基因启动子甲基化水平上升,从而使其抑制靶基因转录激活。在T-ALL细胞中,下调TAL1或LSD1的表达水平会导致TAL1靶基因的抑制作用失活,并伴随着LSD1在靶基因增强子或启动子区域的结合能力下降,而同一位点上的H3K4甲基化作用上升。同样,在PKA激活剂forskolin处理的T-ALL细胞中,由于LSD1与TAL1的相互作用下降,导致靶基因抑制作用失活,而PKA抑制剂H89却使其仍处于抑制状态。另外,与TAL1在转录作用中的双重调节功能一致,在红细胞分化的过程中,TAL1与组蛋白去甲基化酶LSD1的相互作用下降的同时,与组蛋白甲基化转移酶hSET1的结合上升,同时伴随着靶基因上H3K4甲基化修饰作用的增加。我们的研究发现,TAL1与LSD1互作结构域中的Serine172位点的磷酸化作用能够调节TAL1与LSD1间的相互作用,并且这一过程可以通过PKA的介导来完成。这个磷酸化作用能够动态地调节TAL1对LSD1复合物的招募,并直接影响靶基因的转录,而这些基因对于造血祖细胞的分化和T细胞的发育都是十分必要的。PKA的激活剂forskolin能够通过降低TAL1与LSD1间的结合刺激其靶基因的表达,而PKA的抑制剂H89则可以通过抑制启动子的H3K4甲基化水平进而抑制靶基因转录。因此,我们的结果表明PKA介导的磷酸化作用动态调控着TAL1与组蛋白去甲基化酶LSD1的相互作用,并通过调节启动子H3K4甲基化作用控制TAL1靶基因的转录。本论文对PKA的磷酸化作用在TAL1介导的表观遗传调控过程中所发挥的功能,及其在造血细胞的分化进程和T细胞发育的过程中起到的关键决定性作用进行了研究,揭示了TAL1与组蛋白去甲基化酶LSD1间的互作机制,为进一步了解TAL1这个转录因子在调控红细胞分化以及T细胞白血病的发生中的作用提供了理论依据。更多还原

【Abstract】 TAL1/SCL is a hematopoietic specific member of the basic Helix-Loop-Helix (bHLH) family of transcription factors important for the development of all hematopoietic lineages and pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). TAL1 is required for specification and self-renewal of hematopoietic stem cells (HSCs), as well as for terminal differentiation of erythrocytes. TAL1 activity is regulated by association with transcriptional coactivators and corepressors, and the dynamic regulation of TAL1 and its associated coregulators may determine the onset of the hematopoietic differentiation program. However, it remains unclear how association of TAL1 with corepressors versus coactivators is properly regulated.Several threonine (Thr) and serine (Ser) residues of TAL1 can be phosphorylated by protein kinase B (Akt), mitogen activated protein kinase (MAPK), and protein kinase A (PKA). In particular, serine 172 is phosphorylated in vitro and in vivo by PKA. Phosphorylation of this residue affects TAL1 target-dependent DNA recognition, therefore, potentially discriminating the DNA binding site selection during differentiation. The phosphorylation of TAL1 regulates its DNA binding and transcriptional activities suggesting that phosphorylation may dictate TAL1’s ability to associate with transcriptional corepressors and coactivators.To understand the detailed mechanism that regulates TAL1-associated coregulator complexes and how the dysregulation of this process contributes to T-cell leukemia, we investigated the underlying molecular mechanism that regulates TAL1 and LSD1 interaction. Here we reported that PKA mediated phosphorylation regulates TAL1 interaction with the H3K4 demethylase LSD1. Phosphorylation of serine 172 in TAL1 specifically destabilizes the TAL1-LSD1 interaction that leads to promoter H3K4 hypermethylation and activation of target genes that have been suppressed in normal and malignant hematopoiesis. Knockdown of TAL1 or LSD1 led to a derepression of the TAL1 targets accompanied by reduced recruitment of LSD1 and increased H3K4 methylation at their enhancer/promoter regions. Similarly, treatment of PKA activator forskolin resulted in derepression of target genes by reducing its interaction with LSD1 while PKA inhibitor H89 represses them by suppressing H3K4 methylation levels. Consistent with the dual roles of TAL1 in transcription, TAL1 associated LSD1 is decreased while recruitment of hSET1 is increased at the TAL1 targets during erythroid differentiation. This process is accompanied by a dramatic increase in H3K4 methylation.We summarize that the interaction between TAL1 and LSD1 is modulated by a PKA-mediated serine 172 phosphorylation located at the LSD1 interacting domain of TAL1. Phosphorylation mediated dynamic recruitment of LSD1 complex modulates TAL1 directed transcription of target genes that are essential for hematopoietic progenitor differentiation and T-cell development. Treatment of the PKA activator forskolin stimulates the expression of the TAL1 target genes by reducing its interaction with LSD1 while PKA inhibitor H89 further inhibits the TAL1 targets by suppressing promoter H3K4 methylation levels. Thus, our results suggest that the dynamic regulation of TAL1 and its associated histone demethylase LSD1 by PKA mediated phosphorylation controls the transcription of the TAL1 target genes via modulating the promoter H3K4 methylation.Thus, our data revealed a novel interplay between PKA phosphorylation and TAL1 mediated epigenetic regulation that determines hematopoietic transcription and differentiation programs. Taken together, our study have provided important insights into an underlying mechanism by which phosphorylation regulated demethylase LSD1 recruitment controls the timing of erythroid differentiation and oncogenesis of T-cell leukemia.更多还原