Genomic Characterization - Differentiation & Homeostasis

描述（由申请人提供）细胞的行为由遗传相互作用的复杂动力系统控制。在理解生命系统的本质、它们在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，分化过程发挥着核心作用。这个项目的目标是从系统的角度来理解这个过程沿着细胞的稳态稳定性。这种复杂的非线性动力系统的“状态空间”，代表基因调控网络，由所有可能的基因活动的组合。监管的相互作用导致在这个状态空间中的动态“流”。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，并构成其吸引域。本研究的一个目的是检验这样的假设，即这种网络的吸引子构成了生物体的细胞类型，而分化恰恰是从一个吸引子到另一个吸引子的吸引池和随后流向该新吸引子的路线（基因表达程序）。另一个目的是检验在状态空间中有几条不同的路径，细胞沿着这些路径沿着进行分化。一个相关的目标是在基因表达水平上表征特定的分化过程。第一个具体目标--通过分化途径的基因表达谱绘制分子路径--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内稳态的过程。与这个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，它们是否在返回的路上沿着相同的轨迹返回？因此，第二个具体目标-基因表达水平上的稳态稳定性的研究-建议实现这一目标。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）处理HL 60早幼粒细胞白血病细胞（一种成熟的分化模型），以使细胞分别分化为单核细胞和粒细胞。使用早期分化细胞表面标志物（CD 11b）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，使得给定的基因座对应于分化细胞的固定百分比。给定将细胞置于相同位点的几种不同处理，将在不同时间点用微阵列对细胞进行分析，以确定它们是否遵循不同的分化路径。通过额外的未处理细胞的微阵列分析，将揭示不同位点上对单核细胞和粒细胞分化重要的基因组。为了研究稳态稳定性，将处理细胞，使得它们位于50%基因座上，并且将在处理期间的不同时间点进行微阵列分析。在使用CD 11b对细胞进行活体分选后，将在不存在分化诱导剂的情况下培养CD 11b阳性和阴性细胞。微阵列将用于使用时间点测量来分析这些细胞群中的每一个，从而使得在基因表达水平上表征稳态行为成为可能。

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.