Transcriptional regulatory mechanisms in B cell development and leukemogenesis

描述(申请人提供)：产生抗体的B细胞由造血干细胞通过一系列涉及分级转录因子的发育步骤产生 网络。转录因子E2a通过与其他谱系限制性转录因子的协同作用，在早期B细胞发育中发挥核心作用。尽管人们对E2a的直接遗传靶点和协同作用以及协同调节因子知之甚少，但对它们的研究却很少 已知它们的实际作用机制是通过与作用于一般转录机制或染色质结构的各种共激活因子的相互作用来实现的。E2A对癌症也具有重要意义，因为染色体易位将E2A的激活区域融合到前B细胞白血病因子1(Pbx1)或肝白血病因子(HLF)的DNA结合区域，产生融合蛋白(E2A-Pbx1和E2A-HLF)，通过对致癌靶基因的失调而导致儿童急性淋巴细胞白血病(ALL)，而这些基因在很大程度上是未知的。由于E2a和E2a融合蛋白具有共同的激活结构域(ad1、ad2和一个新发现的ad3)，它们很可能在各自的靶基因上利用一些共同的作用机制。为了了解这些机制，初步研究表明，AD1/2与组蛋白乙酰转移酶/辅活化子p300的KIX结构域以及AD3与起始因子TFIID的TAF4亚单位的TAFH结构域之间存在新的物理和功能相互作用。总的目标是了解E2A和E2A融合蛋白分别在B细胞发育和ALL中的作用机制，我们的具体目标是：(I)使用由纯化因子和DNA模板或相应的重组染色质模板重组的生化定义系统来确定E2A激活域(AD)-辅助激活子相互作用在E2A和E2A融合蛋白激活基因中的作用机制~(Ii)确定AD3-TAFH和Ad1/2-KIX相互作用的高分辨率结构，以确定阻止这些相互作用的突变(用于遗传分析)和设计具有治疗潜力的肽类抑制剂~(III)通过与E2A骨髓细胞的分化试验来研究E2A-辅助激活子相互作用在B细胞发育中的生物学功能-/-小鼠，通过对敲门蛋白E2a(ad1/2/3突变体)的产生和分析，TAF4(TAFH突变)和p300(KIX突变)小鼠；(Iv)采用不同的细胞转化和小鼠模型，通过分析抑制性肽类药物对患者来源白血病细胞的影响，探讨E2APbx1/HLAAD-辅活化子相互作用在B细胞白血病发生(ALL)中的生物学作用。

DESCRIPTION (provided by applicant): Antibody-producing B cells arise from hematopoietic stem cells through a series of developmental steps that involve a hierarchical transcription factor network. The transcription factor E2A plays a central role in early B cell development through synergistic functions with other lineage-restricted transcription factors. Whereas much is known about the direct genetic targets and cooperative functions of E2A and co-regulatory factors, little is known about their actual mechanism of action through interactions with various co-activators that act on the general transcription machinery or on chromatin structure. E2A is also of significance for cancer as a result of chromosomal translocations that fuse the activation domain of E2A to the DNA- binding domain of pre-B-cell leukemia factor 1 (PBX1) or hepatic leukemia factor (HLF), generating fusion proteins (E2A-PBX1 and E2A-HLF) that result in pediatric acute lymphoblastic leukemias (ALL) through dysregulation of oncogenic target genes that are largely unknown. Since E2A and E2A fusion proteins share common activation domains (AD1, AD2 and a newly described AD3), they are likely to utilize some common mechanisms of action on respective target genes. Toward an understanding of these mechanisms, preliminary studies have demonstrated novel physical and functional interactions of AD1/2 with the KIX domain of the histone acetyltransferase/coactivator p300 and of AD3 with the TAFH domain of the TAF4 subunit of initiation factor TFIID. With the general objective of understanding the mechanism of action of E2A and E2A fusion proteins in B cell development and in ALL, respectively, our specific aims are: (i) to employ biochemically defined systems reconstituted with purified factors and either DNA templates or corresponding recombinant chromatin templates to define the mechanism of action of E2A activation domain (AD)-coactivator interactions in gene activation by E2A and E2A fusion proteins~ (ii) to determine high-resolution structures of AD3-TAFH and AD1/2-KIX interactions in order to identify mutations that block these interactions (for genetic analyses) and to design peptidomimetic inhibitors with therapeutic potential~ (iii) To investigate the biological functions of E2A-coactivator interactions in B cell development through differentiation assays with bone marrow cells from E2A-/- mice and through the generation and analysis of knockin E2A (AD1/2/3mutant), TAF4(TAFHmutant) and p300(KIXmutant) mice~ and (iv) to investigate the biological functions of E2A PBX1/HLA AD-coactivator interactions in B cell leukemogenesis (ALL), using various cell transformation and mouse models and by analysis of the effects of inhibitory peptidomimetics on patient-derived leukemic cells.