The role of MTG8 in transcriptional elongation and leukemia

描述(申请人提供)：t(8；21)染色体易位是急性髓系白血病(AML)最常见的易位，其结果是AML1-ETO(RUNX1-MTG8)融合蛋白的表达。AML1-ETO(AE)是由转录调控因子AML1(RUNX1)的N端DNA结合域与ETO(MTG8)的大部分融合而成。Mtg8是包括Mtg16和Mtgr1在内的三个蛋白质家族的成员，它们通过与序列特异的转录因子相互作用，在启动子上组装辅阻遏子复合体，从而介导转录抑制。传统上，AE的表达被认为是通过MTG8转录抑制物在RUNX1靶基因上的异常定位而导致白血病的发生，RUNX1是正常血液发育和干细胞自我更新功能所必需的。有趣的是，对人类AML样本的检查发现，除了全长AE外，还在低水平检测到一种称为AML1-ETO9a(AE9a)的AE剪接变体。AE9a剪接变异体编码C末端截断，与全长AE相反，在小鼠模型中具有高度的白血病致癌性。这些观察表明，AE的C-末端区域可能阻碍白血病的发展。因此，我们进行了酵母双杂交(Y2H)与Mtg蛋白C-末端片段的筛选，以寻找可能解释Mtg C-末端抗白血病功能的新的相互作用伙伴。这一筛选确定了MTG蛋白和一些调节转录延长的因子之间的新的相互作用。重要的是，针对这些伸长因子的siRNAs补充了Mtg8-/-表型，在功能上将这些基因联系在一起，表明调节伸长是mtgs的主要功能。此外，血细胞中Mtg16的失活增加了在延长水平上调节的基因，以调节干细胞的自我更新。基于这些初步数据，我们假设AE的C-末端区域通过负面影响转录延长而起到“抗白血病”区域的作用。去除这个结构域会释放伸长因子来激活目标基因的转录，如HoxA5和HoxA7。我们的建议旨在充分阐明MTG与延长因子相关的分子机制，并定义这些相互作用在“正常”细胞以及白血病发生背景下的功能后果。

DESCRIPTION (provided by applicant): The t(8;21) chromosomal translocation is the most frequently observed translocation in acute myeloid leukemia (AML), the result of which is the expression of the AML1-ETO (RUNX1-MTG8) fusion protein. AML1- ETO (AE) is composed of the N-terminal, DNA binding domain of the transcriptional regulator, AML1 (RUNX1) fused to the majority of the ETO (MTG8). Mtg8 is a member of a three protein family, including Mtg16 and Mtgr1, which function to mediate transcriptional repression through assembly of corepressor complexes at promoters through the interaction with sequence-specific transcription factors. Traditionally, AE expression is thought to contribute to leukemogenesis through the aberrant localization of the MTG8 transcriptional repressor at RUNX1 target genes required for normal blood development and stem cell self-renewal function. Interestingly, examination of human AML samples revealed that in addition to full length AE, an AE splice variant, termed AML1-ETO9a (AE9a) is detected at low levels. The AE9a splice variant encodes a C-terminal truncation, which in contrast to full length AE, is highly leukemogenic in mouse models. Such observations suggest that the C-terminal region of AE may impede leukemia development. Therefore, we carried out a yeast two-hybrid (Y2H) screen with a C-terminal fragment of Mtg protein to identify novel interacting partners that may account for the anti-leukemogenic function of the Mtg C-terminus. This screen identified novel interactions between Mtg proteins and a number of factors that regulate transcriptional elongation. Importantly, siRNAs directed towards these elongation factors complemented Mtg8-/- phenotypes, functionally linking these genes and suggesting that the regulation of elongation is a major function of MTGs. Moreover, inactivation of Mtg16 in blood cells increased genes that are regulated at the level of elongation to regulate stem cell self- renewal. Based on these preliminary data, we hypothesize that the C-terminal region of AE acts as an "anti- leukemogenic" domain by negatively affecting transcriptional elongation. Removal of this domain releases elongation factors to activate transcription at target genes such as HoxA5 and HoxA7. Our proposal seeks to fully elucidate both the molecular mechanism of MTG association with elongation factors as well as define the functional consequences of those interactions both in the context of "normal" cells as well as in the context of leukemogenesis.