Riassunto analitico
In the hematopoietic system, the lineage-specific gene expression is controlled by a combined action of transcription factors both widely expressed and specific for cell type.The specific activation of transcription factors leads to differential gene expression that has been investigated during myelopoiesis (Ferrari et al., 2007), starting from the common progenitor (Hematopoietic Stem Cells) up to terminally differentiated cells through gene expression profiling studies. The identification of chromosomal regions stably silenced during myelopoiesis, which partially overlap with regions containing genes which are not related to myeloid differentiation, supports the existence of a relationship between the function and the structure of the genome and it is indicative of the fact that the gene position have a role in the regulation of gene expression.Infact in recent years several studies have shown that gene expression also depends on the subnuclear localization of the DNA sequence and on the interaction with specific subnuclear regions.The aim of this work is the analysis of the intranuclear localization of co-regulate chromatin domains already identified during myelopoiesis through the use of 3DFISH and confocal microscopy, in order to investigate the positional effect on gene expression of the gene clusters and the structural organization of chromatin (heterochromatin and euchromatin) during the myeloid differentiation, in order to improve knowledge about nuclear structure and function and the epigenetic control of myeloid differentation plasticity.In addition, we have analyzed the localization of the clusters already described within chromosome territory (CT).For each chromosome region which we have analised, we have selected a pool of human BAC clones on UCSC Genome Browser. These probes and WCP (Whole Chromosome Painting) specific for the CT selected, were tested on peripheral blood metaphases to verify their localization.Subsequently the probes were amplified, labeled, precipitated and resuspended in the hybridization mix. The cell populations studied were: hematopoietic stem cells, myeloblasts and monoblasts.Stem cells were purified from umbilical cord blood through Ficoll gradient and successive immunomagnetic separation. Myeloblasts and monoblasts were obtained by in vitro differentiation of stem cells and subsequent immunomagnetic separation of the two fractions (positive and negative for the antigen CD14).The cells were made adherent on the cover slips, fixed and permeabilized for the penetration the probe. Hybridization has been carried out for 72 hours and was followed by detection and DAPI staining.The samples were analyzed by confocal microscopy and the images obtained were processed using the software ImageJ. Quantitative evaluation of the position of the clusters in respect to the nuclear volume and the chromosomal territory was carried out by EADS (enhance dAbsolute 3D Distances to Surfaces), an algorithm based on voxels.The results show the different position within the nucleus of the silenced and expressed clusters.The clusters stably silenced have a peripheral location within the nucleus while expressed clusters show a more internal location.These results are in line with the literature data which indicate the correlation between the periphery and the nuclear gene silencing, due to presence of heterochromatin.The analysis of the localization of the clusters with respect to the chromosomal territory shows a functional localization: the silenced clusters are localized within CT, while the expressed clusters are concentrated along the surface, in the vicinity of the inter-chromosomal space containing the transcriptional machine.This is the first study of the nuclear organization in HSC and precursors and highlights the relationship between nuclear architecture, epigenetic control and gene expression during myeloid differentiation.
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