RESUMEN
The well-established human epidermal keratinocyte (HEK) differentiation model was investigated to determine possible alterations in chromosome territory (CT) association during differentiation. The seven human chromosomes (1, 4, 11, 12, 16, 17, and 18) selected for this analysis are representative of the chromosome size and gene density range of the overall human genome as well as including a majority of genes involved in epidermal development and differentiation (CT1, 12, and 17). Induction with calcium chloride (Ca(2+)) resulted in morphological changes characteristic of keratinocyte differentiation. Combined multi-fluorescence in situ hybridization (FISH) and computational image analysis on the undifferentiated (0 h) and differentiated (24 h after Ca(2+) treatment) HEK revealed that (a) increases in CT volumes correspond to overall nuclear volume increases, (b) radial positioning is gene density-dependent at 0 h but neither gene density- nor size-dependent at 24 h, (c) the average number of interchromosomal associations for each CT is gene density-dependent and similar at both time points, and (d) there are striking differences in the single and multiple pairwise interchromosomal association profiles. Probabilistic network models of the overall interchromosomal associations demonstrate major reorganization of the network during differentiation. Only ~40 % of the CT pairwise connections in the networks are common to both 0 and 24 h HEK. We propose that there is a probabilistic chromosome positional code which can be significantly altered during cell differentiation in coordination with reprogramming of gene expression.
Asunto(s)
Diferenciación Celular/fisiología , Cromosomas Humanos/metabolismo , Epidermis/metabolismo , Queratinocitos/metabolismo , Modelos Biológicos , Células Epidérmicas , Humanos , Queratinocitos/citologíaRESUMEN
Undifferentiated human epidermal keratinocytes are self-renewing stem cells that can be induced to undergo a program of differentiation by varying the calcium chloride concentration in the culture media. We utilize this model of cell differentiation and a 3D chromosome painting technique to document significant changes in the radial arrangement, morphology, and interchromosomal associations between the gene poor chromosome 18 and the gene rich chromosome 19 territories at discrete stages during keratinocyte differentiation. We suggest that changes observed in chromosomal territorial organization provides an architectural basis for genomic function during cell differentiation and provide further support for a chromosome territory code that contributes to gene expression at the global level.
Asunto(s)
Diferenciación Celular , Posicionamiento de Cromosoma , Cromosomas Humanos/metabolismo , Células Epidérmicas , Epidermis/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Biomarcadores/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Cromosomas Humanos Par 18/metabolismo , Cromosomas Humanos Par 19/metabolismo , Humanos , Hibridación Fluorescente in Situ , Tamaño de los OrgánulosRESUMEN
Matrin 3 (matr3), an abundant protein of the internal nuclear matrix, has been linked to a variety of functional events. As a step toward defining its multifunctional nature, we have studied the association of matr3 with chromosome territories and identified potential interacting proteins. A similar staining pattern of matr3 was observed in fixed WI38 fibroblast cells and in live HeLa cells using a matr3-GFP construct. Matr3 was detected throughout autosomal and the active X chromosome territories. Conversely, matr3 was strikingly excluded from the inactive X chromosome as well as within both the perinuclear and perinucleolar heterochromatin. Yeast two hybrid analysis identified matr3 interactions with 33 unique nuclear localized proteins and also revealed its propensity for self association. A majority of these proteins are involved in RNA metabolism and chromatin remodeling while others function in protein translation, DNA replication/repair and apoptosis. Further analysis of a selection of these proteins and scaffold attachment factor A (SAFA) by co-localization and co-immunoprecipitation experiments using HeLa cells confirmed their interactions with matr3.