Genomic Characterization - Differentiation & Homeostasis

描述（由申请人提供）细胞的行为是由一个复杂的遗传相互作用的动力系统所控制的。在理解生命系统的本质、它们在不断变化的环境中的稳定性以及这些系统如何在疾病（如癌症）中失败方面，分化过程起着核心作用。这个项目的目标是从系统的角度来理解这个过程以及细胞的稳态稳定性。这种复杂的非线性动力系统的“状态空间”代表着基因调控网络，由基因活动的所有可能组合组成。调节的相互作用导致这个状态空间中的动态“流”。这种流动或轨迹通常达到活动的循环模式，这构成了系统的吸引子或稳态行为。许多不同的轨迹通常流向同一个吸引子，并构成它的吸引盆地。本研究的目的之一是验证这样一种假设，即这种网络中的吸引子构成了生物体的细胞类型，而分化恰恰是一条途径（基因表达程序），从一个吸引子进入另一个吸引子的吸引池，然后流向新的吸引子。另一个目标是测试在状态空间中有几种不同路径的假设，细胞沿着这些路径走向分化。一个相关的目标是在基因表达水平上描述一个特定的分化过程。第一个具体目标-通过基因表达谱绘制分化途径的分子路径-旨在实现这些目标。最后，另一个目的是在基因表达水平上研究细胞稳态的过程。与此目标相关的特定问题是：细胞是否通过在状态空间中返回其原始状态而在表达水平上表现出稳态，如果是这样，它们是否在返回的过程中沿着相同的轨迹重走？因此，第二个具体目标-研究基因表达水平上的稳态稳定性-被提出来实现这一目标。为实现这些目标而设计的方法包括用不同剂量和持续时间的全反式维甲酸（ATRA）和二甲基亚砜（DMSO）治疗HL60早幼粒细胞白血病细胞，使其分别分化为单核细胞和粒细胞。利用早期分化细胞表面标记（CD11b）和流式细胞术，研究人员将在剂量-持续时间平面上构建位点，这样一个给定的位点对应于一个固定百分比的分化细胞。给定几种不同的处理方法，将细胞放置在同一位点上，细胞将在不同的时间点用微阵列进行分析，以确定它们是否遵循不同的分化路径。通过对未经处理的细胞进行额外的微阵列分析，将揭示不同位点上单核细胞和粒细胞分化的重要基因集。为了研究稳态稳定性，将对细胞进行处理，使其位于50%位点，并在处理期间的不同时间点进行微阵列分析。使用CD11b对细胞进行活分选后，将CD11b阳性和阴性细胞在无诱导分化剂的条件下进行培养。微阵列将使用时间点测量来分析这些细胞群，从而使基因表达水平上的稳态行为表征成为可能。

DESCRIPTION (provided by applicant) A cell's behavior is governed by a complex dynamical system of genetic interactions. A central role in the understanding of the nature of living systems, their stability in a changing environment, and how such systems fail in disease, such as cancer, is played by the process of differentiation. The goal of this project is to understand this process along with cellular homeostatic stability from a systems perspective. The 'state-space' of such complex nonlinear dynamical systems, representing genetic regulatory networks, consists of all possible combinations of gene activities. The regulatory interactions result in a dynamical 'flow' in this state-space. That flow or trajectory typically reaches a recurrent pattern of activities, which constitutes an attractor or the steady-state behavior of the system. Many different trajectories typically flow to the same attractor and constitute its basin of attraction. One objective of this study is to test the hypothesis that the attractors of such networks constitute the cell types of an organism, while differentiation is precisely a route (gene expression program) from one attractor into the basin of attraction of another attractor and subsequent flow to that new attractor. Another objective is to test the hypothesis that there are several distinct paths in the state-space along which cells proceed towards differentiation. A related goal is to characterize a particular differentiation process at the gene expression level. The first specific aim - mapping the molecular paths by gene expression profiling for differentiation pathways - is intended to achieve these objectives. Finally, another objective is to study the process of cellular homeostasis on the gene expression level. The particular questions related to this objective are: do the cells exhibit homeostasis on the expression level by returning to their original states in the state-space and if so, do they retrace the same trajectory on their way back? Thus, the second specific aim - the study of homeostatic stability on the gene expression level - is proposed to realize this objective. The methods designed to achieve these goals include treating HL60 promyelocytic leukemia cells, a well-established differentiation model, with different doses and durations of all-trans retinoic acid (ATRA) and dimethyl sulfoxide (DMSO), to differentiate the cells into monocytes and granulocytes, respectively. Using early differentiation cell surface markers (CD11b) and flow cytometry, the investigators will construct loci on the dose-duration plane such that a given locus corresponds to a fixed percentage of differentiated cells. Given several different treatments that place the cells on the same locus, the cells will be profiled at different time points with microarrays in order to determine whether they follow distinct paths of differentiation. With additional microarray profiling of untreated cells, gene sets important for monocytic and granulocytic differentiation on different loci will be revealed. In order to study homeostatic stability, cells will be treated such that they are on the 50% locus and microarray profiling will be performed at different time points during treatment. After live sorting of the cells using CD11b, the CD11b positive and negative cells will be cultured in the absence of differentiation inducing agents. Microarrays will be used to profile each of these cell populations using time-point measurements, thus making possible the characterization of homeostatic behavior on the gene expression level.