Transcriptional regulatory mechanisms in B cell development and leukemogenesis

描述（由申请人提供）：产生抗体的B细胞通过涉及分级转录因子的一系列发育步骤由造血干细胞产生 网络转录因子E2 A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。尽管对E2 A和共调节因子的直接遗传靶点和协同功能了解很多， 通过与作用于一般转录机器或染色质结构的各种共激活剂的相互作用，已知它们的实际作用机制。E2 A对于癌症也是重要的，因为染色体易位将E2 A的活化结构域融合到前B细胞白血病因子1（PBX 1）或肝白血病因子（HLF）的DNA结合结构域，产生融合蛋白（E2 A-PBX 1和E2 A-HLF），其通过致癌靶基因的失调导致儿科急性淋巴细胞白血病（ALL），所述致癌靶基因在很大程度上是未知的。 由于E2 A和E2 A融合蛋白共享共同的激活结构域（AD 1、AD 2和新描述的AD 3），因此它们可能利用一些共同的作用机制作用于各自的靶基因。为了理解这些机制，初步研究已经证明了新的物理和功能的相互作用的AD 1/2与组蛋白乙酰转移酶/辅激活因子p300的KIX域和AD 3与TAF 4亚基的TAFH域的起始因子TFIID。为了理解E2 A和E2 A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）采用用纯化的因子和DNA模板或相应的重组染色质模板重构的生物化学确定的系统来确定E2 A激活结构域（AD）的作用机制，E2 A和E2 A融合蛋白基因激活中的辅激活因子相互作用~（ii）确定AD 3-TAFH和AD 1/2-KIX相互作用的高分辨率结构，以鉴定阻断这些相互作用的突变（iii）研究E2 A-1的生物学功能，通过E2 A-/-小鼠骨髓细胞的分化试验以及通过敲入E2 A的产生和分析，在B细胞发育中的辅激活因子相互作用（AD 1/2/3突变体），TAF 4（TAFH突变体）和p300（iv）研究E2 A PBX 1/HLA AD-共激活因子相互作用在B细胞白血病发生（ALL）中的生物学功能，使用各种细胞转化和小鼠模型，并通过分析抑制性肽模拟物对患者来源的白血病细胞的作用。

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.