RESUMO
Spatio-temporal organization of the cell nucleus adapts to and regulates genomic processes. Microscopy approaches that enable direct monitoring of specific chromatin sites in single cells and in real time are needed to better understand the dynamics involved. In this chapter, we describe the principle and development of ANCHOR, a novel tool for DNA labelling in eukaryotic cells. Protocols for use of ANCHOR to visualize a single genomic locus in eukaryotic cells are presented. We describe an approach for live cell imaging of a DNA locus during the entire cell cycle in human breast cancer cells.
Assuntos
DNA/química , Loci Gênicos/genética , Microscopia Intravital/métodos , Imagem Molecular/métodos , Coloração e Rotulagem/métodos , Ciclo Celular/genética , Humanos , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Microscopia Intravital/instrumentação , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Células MCF-7 , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Molecular/instrumentação , Transfecção/instrumentação , Transfecção/métodos , Transgenes/genéticaRESUMO
Genome dynamics are intimately linked to the regulation of gene expression, the most fundamental mechanism in biology, yet we still do not know whether the very process of transcription drives spatial organization at specific gene loci. Here, we have optimized the ANCHOR/ParB DNA-labeling system for real-time imaging of a single-copy, estrogen-inducible transgene in human cells. Motion of an ANCHOR3-tagged DNA locus was recorded in the same cell before and during the appearance of nascent MS2-labeled mRNA. We found that transcription initiation by RNA polymerase 2 resulted in confinement of the mRNA-producing gene domain within minutes. Transcription-induced confinement occurred in each single cell independently of initial, highly heterogeneous mobility. Constrained mobility was maintained even when inhibiting polymerase elongation. Chromatin motion at constant step size within a largely confined area hence leads to increased collisions that are compatible with the formation of gene-specific chromatin domains, and reflect the assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.
Assuntos
Ciclina D1/biossíntese , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/metabolismo , Ciclina D1/genética , Recuperação de Fluorescência Após Fotodegradação , Loci Gênicos , Humanos , Microscopia de Fluorescência , Imagem Molecular , Movimento (Física) , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Espectrometria de Fluorescência , Transfecção , TransgenesRESUMO
Differentiation of adipose progenitor cells into mature adipocytes entails a dramatic reorganization of the cellular architecture to accommodate lipid storage into cytoplasmic lipid droplets. Lipid droplets occupy most of the adipocyte volume, compressing the nucleus beneath the plasma membrane. How this cellular remodeling affects sub-nuclear structure, including size and number of nucleoli, remains unclear. We describe the morphological remodeling of the nucleus and the nucleolus during in vitro adipogenic differentiation of primary human adipose stem cells. We find that cell cycle arrest elicits a remodeling of nucleolar structure which correlates with a decrease in protein synthesis. Strikingly, triggering cytoskeletal rearrangements mimics the nucleolar remodeling observed during adipogenesis. Our results point to nucleolar remodeling as an active, mechano-regulated mechanism during adipogenic differentiation and demonstrate a key role of the actin cytoskeleton in defining nuclear and nucleolar architecture in differentiating human adipose stem cells.
Assuntos
Adipogenia , Citoesqueleto , Humanos , Células Cultivadas , Citoesqueleto/metabolismo , Adipócitos/metabolismo , Gotículas Lipídicas/metabolismoRESUMO
The long non-coding RNA HOTAIR is the most differentially expressed gene between upper- and lower-body adipose tissue, yet its functional significance in adipogenesis is unclear. We report that HOTAIR expression is transiently induced during early adipogenic differentiation of gluteofemoral adipose progenitors and repressed in mature adipocytes. Upon adipogenic commitment, HOTAIR regulates protein synthesis pathways and cytoskeleton remodeling with a later impact on mature adipocyte lipid storage capacity. Our results support novel and important functions of HOTAIR in the physiological context of adipogenesis.
Assuntos
Adipogenia , RNA Longo não Codificante , Adipócitos/metabolismo , Adipogenia/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Citoesqueleto/genética , Lipídeos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
The mammalian genome is intricately folded in a three-dimensional topology believed to be important for the orchestration of gene expression regulating development, differentiation and tissue homeostasis. Important features of spatial genome conformation in the nucleus are promoter-enhancer contacts regulating gene expression within topologically-associated domains (TADs), short- and long-range interactions between TADs and associations of chromatin with nucleoli and nuclear speckles. In addition, anchoring of chromosomes to the nuclear lamina via lamina-associated domains (LADs) at the nuclear periphery is a key regulator of the radial distribution of chromatin. To what extent TADs and LADs act in concert as genomic organizers to shape the three-dimensional topology of chromatin has long remained unknown. A new study addressing this key question provides evidence of (i) preferred long-range associations between TADs forming TAD "cliques" which organize large heterochromatin domains, and (ii) stabilization of TAD cliques by LADs at the nuclear periphery after induction of terminal differentiation. Here, we review these findings, address the issue of whether TAD cliques exist in single cells and discuss the extent of cell-to-cell heterogeneity in higher-order chromatin conformation. The recent observations provide a first appreciation of changes in 4-dimensional higher-order genome topologies during differentiation.