Abstract
Dynamic chromatin structure is a fundamental property of gene transcriptional regulation, and has emerged as a critical modulator of physiological processes during cellular differentiation and development. Analysis of chromatin structure using molecular biology and biochemical assays in rare somatic stem and progenitor cells is key for understanding these processes but poses a great challenge because of their reliance on millions of cells. Through the development of a miniaturized genome-scale chromatin immunoprecipitation method (miniChIP–chip), we have documented the genome-wide chromatin states of low abundant populations that comprise hematopoietic stem cells and immediate progeny residing in murine bone marrow. In this report, we describe the miniChIP methodology that can be used for increasing an understanding of the epigenetic mechanisms underlying hematopoietic stem and progenitor cell function. Application of this method will reveal the contribution of dynamic chromatin structure in regulating the function of other somatic stem cell populations, and how this process becomes perturbed in pathological conditions.
Highlights
A unique and defining property for any stem cell population is intrinsic self-renewal throughout the process of cell division, whilst maintaining the capacity to form multiple differentiated and mature cell types over the lifetime of an organism
Genomic regions are packaged into euchromatin, which forms a relaxed structure of large genomic distances between nucleosomes, or a higher degree of DNA compaction known as heterochromatin [7]
This was a refinement of our previously described miniChIP–qPCR method, which allowed histone modifications to be identified at selected genes using 50,000 cells [30]
Summary
A unique and defining property for any stem cell population is intrinsic self-renewal throughout the process of cell division, whilst maintaining the capacity to form multiple differentiated and mature cell types over the lifetime of an organism. Stem cells undergo dramatic changes in morphology, cell cycle status and gene expression during differentiation into specialized progenitor subsets. Such alterations are proposed to result from chromatin reorganization of the genome, allowing for the establishment and maintenance of lineage-specific transcriptional networks [1, 2]. Chromatin structure defines the higher order structure by which DNA is organized within the cell nucleus [4]. It consists of a chain of nucleosomes, representing about 146–147 bp of DNA wrapped around a core of histone octamers [5]. Euchromatin is referred to as active chromatin and is linked to actively transcribed regions of the genome, while genes located in heterochromatin (inactive chromatin) are usually transcriptionally silent
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