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）有两个明确的特征。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是由转录因子如Bmi1、Zfx和Foxo蛋白规定的。然而，这些因素是否相互作用，以及它们是否在造血干细胞中形成一个层次基因调控网络尚不清楚。在本项目中，我们将研究GA结合蛋白（GABP）在调节HSC稳态和再种群能力中的作用。我们将进一步表征其与其他转录因子在控制HSC生物学不同方面的相互作用。一个功能性的GABP复合体包含一个DNA结合亚基GABP1和一个转激活亚基GABP2。GABP2的两个同工异构体有助于GABP 1222四聚体的形成。GABP1的失活使整个GABP复合物的活性丧失，导致胚胎早期死亡。相比之下，缺失两种四聚体形成的GABP2亚型的小鼠可以存活。两株动物的HSC频率和数量都有所减少，但在表型的发病和动力学上有所不同。我们假设GABP复合物控制着调节HSC稳态和功能的不同途径，1222四聚体调节GABP靶基因的一个子集，并在调节HSC自我更新和衰老中发挥特定作用。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造血干细胞的自我更新和分化。该项目将为更好地理解造血干细胞可塑性、造血恶性肿瘤和干细胞老化提供关键信息。我们将通过以下具体目标来实现这些目标：探讨GABP复合物在HSC自我更新和再繁殖能力中的作用。我们将使用组织特异性GABP1靶向小鼠品系，诱导骨髓中GABP1失活，并确定对HSC自我更新和再繁殖能力的影响。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。我们在造血干细胞中获得了全基因组的GABP结合图谱，并发现GABP与许多转录因子的调控区域直接结合。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老化中的特殊作用。我们将使用缺乏GABP2四聚体形成异构体的小鼠品系，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具有两个决定性特征：自我更新以保持恒定数量和可塑性以产生多个血液谱系。造血干细胞的异常调节可能导致血细胞再生缺陷或导致造血恶性肿瘤。本项目将研究一种名为GA结合蛋白的转录因子在调节HSC自我更新和再生能力中的关键作用，并表征GA结合蛋白与其他因子之间的相互作用，这些研究将有助于更好地了解HSC的稳态和多能性是如何被控制的，并为如何维持HSC满足再生需求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.