The role of MTG8 in transcriptional elongation and leukemia

MTG8 在转录延伸和白血病中的作用

• 批准号: 8456501
• 负责人: Kristy Stengel
• 金额: $ 2.12万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456501
• 关键词: AML1-ETO fusion protein; Accounting; Acute Myelocytic Leukemia; Address; Affect; Binding; Biology; Blood; Blood Cells; C-terminal; CBFA2T1 gene; Chimeric Proteins; Chromatin; Chromosomal Rearrangement; Chromosomal translocation; Complement; Complex; DNA Binding Domain; DNA Polymerase II; Data; Defect; Development; Elongation Factor; Embryo; Enzymes; Excision; Exhibits; Exons; Family; Family member; Fibroblasts; Gene Targeting; Genes; Genetic Transcription; Growth; Hematopoietic; Histone H3; Histones; Human; Length; Link; Maps; Mediating; Molecular; Mus; N-terminal; Normal Cell; Phenotype; Phosphorylation; Polymerase; Positive Transcriptional Elongation Factor B; Protein Family; Proteins; RNA; RNA Splicing; RUNX1 gene; Regulation; Repression; Role; Sampling; Site; Stem Cell Development; Stem cells; Terminator Codon; Testing; Transcription Repressor/Corepressor; Transcriptional Regulation; Variant; Yeasts; base; gene repression; genome-wide; histone methyltransferase; leukemia; leukemogenesis; member; mouse model; negative elongation factor; novel; promoter; protein complex; public health relevance; self-renewal; t(8; 21)(q22; q22); transcription factor; yeast two hybrid system

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.

描述（由申请方提供）：t（8;21）染色体易位是急性髓性白血病（AML）中最常见的易位，其结果是表达AML 1-ETO（RUNX 1-MTG 8）融合蛋白。AML 1- ETO（AE）由N-末端、转录调节因子AML 1（RUNX 1）的DNA结合结构域与大部分ETO（MTG 8）融合组成。Mtg 8是包括Mtg 16和Mtgr 1的三个蛋白质家族的成员，其功能是通过与序列特异性转录因子相互作用在启动子处组装辅阻遏物复合物来介导转录抑制。传统上，AE表达被认为通过MTG 8转录抑制子在正常血液发育和干细胞自我更新功能所需的RUNX 1靶基因处的异常定位而促成白血病发生。有趣的是，对人AML样本的检查显示，除了全长AE外，还检测到低水平的AE剪接变体，称为AML 1-ETO 9a（AE 9a）。AE 9a剪接变体编码C末端截短，与全长AE相反，其在小鼠模型中具有高度致白血病性。这些观察结果表明，AE的C-末端区域可能会阻碍白血病的发展。因此，我们进行了酵母双杂交（Y2 H）筛选与Mtg蛋白的C-末端片段，以确定新的相互作用的合作伙伴，可能占抗白血病功能的Mtg C-末端。该筛选鉴定了Mtg蛋白与调节转录延伸的许多因子之间的新型相互作用。重要的是，针对这些延伸因子的siRNA补充了Mtg 8-/-表型，在功能上连接这些基因，并表明延伸的调节是MTG的主要功能。此外，血细胞中Mtg 16的失活增加了在延长水平上调节以调节干细胞自我更新的基因。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伸而充当“抗白血病”结构域。去除该结构域释放延伸因子以激活靶基因如HoxA 5和HoxA 7的转录。我们的建议旨在充分阐明MTG与延伸因子相关的分子机制，以及在“正常”细胞和白血病发生的背景下定义这些相互作用的功能后果。