Critical roles of GA binding protein in HSC maintenance and ageing

描述（由申请人提供）：造血干细胞（HSC）具有两个定义特征。一个是自我更新的能力，在稳定状态下保持恒定的数量，另一个是在血液中分化成多个谱系的潜力，满足再生需求。HSC稳态和多能性的维持由转录因子如Bmi 1、Zfx和Foxo蛋白规定。然而，目前尚不清楚这些因素是否相互作用，以及它们是否在HSC中形成分级基因调控网络。本研究旨在探讨GA结合蛋白（GABP）在调节HSC稳态和再增殖能力中的作用。我们将进一步描述其与其他转录因子在控制HSC生物学不同方面的相互作用。功能性GABP复合物含有一个DNA结合亚基GABP1和一个反式激活亚基GABP2。两种GABP2亚型有助于形成GABP 1222四聚体。GABP1的失活消除了整个GABP复合物的活性，导致早期胚胎死亡。相比之下，缺失两种四聚体形成GABP2同种型的小鼠是存活的。两种品系的动物均显示HSC频率和数量减少，但在表型的发作和动力学方面不同。我们假设GABP复合物控制调节HSC稳态和功能的不同途径，并且1222个四聚体调节GABP靶基因的子集，并且在调节HSC自我更新和衰老中具有特定作用。我们的长期目标是阐明GABP和其他关键转录因子之间的相互作用，以及这种相互作用如何平衡HSC的自我更新和分化。该项目将产生关键信息，以更好地了解造血干细胞的可塑性，造血恶性肿瘤和干细胞老化。我们将通过以下具体目标来实现这些目标：具体目标1。探讨GABP复合物在肝星状细胞自我更新和再增殖能力中的作用。我们将使用组织特异性GABP1靶向小鼠品系，诱导骨髓中GABP1的失活，并确定对HSC自我更新和再增殖能力的影响。我们还将研究GABP1缺乏是否会影响HSC细胞周期和存活途径。具体目标2.目的：鉴定GABP调控HSC功能的直接靶基因。我们已经获得了一个全基因组的GABP结合图谱在HSC中，并发现直接GABP结合到一些转录因子的调控区域。我们将描述GABP和这些转录因子之间的相互作用，以构建一个维持HSC稳态和多能性的基因调控网络。具体目标3.确定GABP 1222四聚体在DNA损伤修复和HSC老化中的特定作用。我们将使用缺乏形成GABP2四聚体的同种型的小鼠品系，并确定由GABP 1222四聚体控制的HSC生物学的特定方面。我们还将研究四聚体与DNA损伤修复和HSC老化的直接联系。公共卫生相关性：造血干细胞（HSC）负责在人的一生中持续产生所有血细胞，并且HSC具有两个定义特征，自我更新以保持恒定数量和可塑性以产生多种血液谱系。造血干细胞的异常调节可导致血细胞再生缺陷或导致造血系统恶性肿瘤。本项目将研究转录因子GA结合蛋白的关键作用，并表征GA结合蛋白与其他因子在调控HSC自我更新和再增殖能力中的相互作用，这些研究将有助于更好地理解HSC的稳态和多能性是如何控制的，并为如何维持HSC以满足再生需要和防止恶性转化提供分子基础。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) have two defining features. One is the ability to self-renew, maintaining a constant number under steady state, and the other is the potential to differentiate into multiple lineages in blood, meeting the regenerative needs. The maintenance of HSC homeostasis and multipotency is stipulated by transcription factors such as Bmi1, Zfx, and Foxo proteins. However, it is unknown if these factors interact with each other and if they form a hierarchical gene regulatory network in HSCs. In this project, we will investigate the roles of GA binding protein (GABP) in regulating HSC homeostasis and repopulation capacity. We will further characterize its interplay with other transcription factors in controlling different aspects of HSC biology. A functional GABP complex contains one DNA binding subunit, GABP1, and one transactivation subunit, GABP2. Two of GABP2 isoforms contribute to the formation of GABP 1222 tetramers. Inactivation of GABP1 abrogates the activity of entire GABP complex, causing early embryonic lethality. In contrast, mice with deletion of both tetramer-forming GABP2 isoforms were viable. Both strains of animals showed diminished HSC frequency and numbers, but differed in onset and kinetics of the phenotypes. We hypothesize that the GABP complex controls different pathways regulating HSC homeostasis and function and the 1222 tetramers regulate a subset of GABP target gene and have a specific role in regulating HSC self- renewal and ageing. Our long-term goal is to elucidate the interplay between GABP and other key transcription factors and how such interplay balances self-renewal and differentiation of HSCs. This project will generate critical information for a better understanding of HSC plasticity, hematopoietic malignancy, and stem cell ageing. We will approach these objectives through the following specific aims: SPECIFIC AIM 1. To investigate the roles of GABP complex in HSC self-renewal and repopulation capacity. We will use a tissue-specific GABP1-targeted mouse strain, induce inactivation of GABP1 in the bone marrow, and determine the impact on HSC self-renewal and repopulation capacity. We will also investigate if HSC cell cycle and survival pathways are affected by GABP1 deficiency. SPECIFIC AIM 2. To identify GABP direct target genes which mediate its regulation of HSC function. We have obtained a genome-wide GABP binding map in HSCs and found direct GABP binding to regulatory regions of a number of transcription factors. We will characterize the interplay between GABP and these transcription factors to construct a gene regulatory network maintaining HSC homeostasis and multipotency. SPECIFIC AIM 3. To determine a specific role of GABP 1222 tetramers in DNA damage repair and HSC ageing. We will use the mouse strain that is deficient for both tetramer-forming GABP2 isoforms and determine the specific aspect of HSC biology that is controlled by GABP 1222 tetramers. We will also investigate the direct link of tetramers to DNA damage repair and HSC ageing. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cells (HSCs) are responsible for sustained production of all blood cells throughout one's life, and HSCs have two defining features, self-renewal to maintain a constant number and plasticity to generate multiple blood lineages. Aberrant regulation of HSCs can either lead to defective regeneration of blood cells or result in hematopoietic malignancy. This project will investigate critical roles of a transcription factor name GA binding protein and characterize the interplay between GA binding protein and other factors in regulating HSC self-renewal and repopulation capacity, and these studies will lead to a better understanding on how HSC homeostasis and multi-potency are controlled, and provide a molecular basis on how to maintain HSCs to meet regenerative needs and prevent malignant transformation.