Genomic Characterization - Differentiation & Homeostasis

描述(由申请人提供)细胞的行为由一个复杂的遗传相互作用的动态系统控制。在理解生命系统的性质、它们在不断变化的环境中的稳定性以及这些系统如何在癌症等疾病中失败的过程中，分化起到了核心作用。这个项目的目标是从系统的角度理解这一过程以及细胞内稳态的稳定性。代表遗传调控网络的这种复杂的非线性动力系统的状态空间由所有可能的基因活动组合组成。这种调节的相互作用导致了这个状态空间中的动态“流动”。这种流动或轨迹通常会达到一种循环的活动模式，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，并构成其吸引盆地。这项研究的一个目的是检验这样一个假设，即这种网络的吸引子构成了一个有机体的细胞类型，而分化恰恰是一条从一个吸引子进入另一个吸引子的吸引池并随后流向那个新吸引子的路径(基因表达程序)。另一个目标是检验这一假设，即在状态空间中有几条不同的路径，细胞沿着这些路径进行分化。一个相关的目标是在基因表达水平上描述特定的分化过程。第一个具体目标--通过基因表达谱分析分化途径来绘制分子途径图--旨在实现这些目标。最后，另一个目的是从基因表达水平上研究细胞内稳态的过程。与这一目标相关的具体问题是：细胞是否通过返回到状态空间中的原始状态来在表达水平上表现出动态平衡，如果是的话，它们是否会在返回的过程中回溯相同的轨迹？因此，为了实现这一目标，提出了第二个具体目标--在基因表达水平上研究体内平衡稳定性。为实现这些目标而设计的方法包括处理HL60早幼粒细胞白血病细胞，这是一种成熟的分化模型，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)分别将细胞分化为单核细胞和粒细胞。使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基因座，使给定的基因座对应于固定百分比的分化细胞。给出了几种不同的处理方法，将细胞置于相同的位置，这些细胞将在不同的时间点通过微阵列进行分析，以确定它们是否遵循不同的分化路径。随着对未处理细胞的额外微阵列分析，不同基因座上对单核细胞和粒细胞分化至关重要的基因集将被揭示。为了研究体内平衡的稳定性，将对细胞进行处理，使其处于50%的位置，并在治疗期间的不同时间点进行微阵列分析。用CD11b对细胞进行活体分选后，在不含分化诱导剂的情况下对CD11b阳性和阴性细胞进行培养。微阵列将被用来使用时间点测量来描述这些细胞群体中的每一个，从而使在基因表达水平上表征动态平衡行为成为可能。

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.