Riassunto analitico
Myelopoiesis is the part of hematopoiesis leading the differentiation of myeloid progenitors, derived from multipotent stem cells, into erythrocytes, megakaryocytes, monocytes and granulocytes. Although this process has been extensively studied, there are still unclear aspects, especially concerning its genetic control. It has become evident that commitment and differentiation of myeloid precursor are regulated by ordered patterns of gene expression, where specific combinations of transcription factors and chromatin remodeling complexes are responsible for the genetic program of each hematopoietic precursor.
Recently, in our laboratory, changes of gene expression in human myelopoiesis have been investigated. Genome distribution of differentially expressed genes in each specific lineage of differentiation, compared with CD34+ cells, was analyzed using an integrative computational approach. Significant clusters up- and down-regulated were identified.
To support the existence of a relationship between the function and the structure of the genome and to confirm that gene position has a role in the regulation of gene expression, we have decided to analyze the intranuclear localization of the differentially expressed gene clusters already identified in myelopoiesis through the use of 3D-FISH and confocal microscopy.
Nuclear organization and higher-order chromatin structure are considered important regulatory factors in gene expression. The nucleus is nonrandom organized and compartmentalized. In interphase, each chromosome takes up a distinct subvolume of the nucleus and exists in the form of a so-called chromosome territory (CT). Although the results of CT positioning in the nucleus indicate comprehensible functional chromatin architecture, the different CTs’ position in different cell types and differentiation stages still cannot be satisfyingly explained.
For this work, differentially expressed gene clusters between CD34+, monoblast and myeloblast were selected for their common location on chromosome 19. Subsequently these clusters were analyzed considering their position in the nucleus and in respect to the CT in each cell type.
CD34+ 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).
Cells were made adherent on coverslip to perform 3D-FISH experiments. The samples were analyzed at a confocal microscope. 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 performed with EADS (Enhanced Absolute 3D Distances to Surfaces), an algorithm based on voxels.
We found that the single up-regulated cluster analyzed in the comparison between CD34+ cells and myeloblasts, has a statistically evident internal location within the nucleus and a peripheral position within the CT19 in myeloblasts, in accord to the expected functional localization.
Instead, considering the four clusters differentially expressed between monoblast and CD34+ cells, we haven't found a significant relocalization within the CT, probably due to the small size of chromosome 19. Indeed in most of the cases the localization per se, in respect to the nucleus and the CT volume, can be correlated with gene expression. Only for the cluster down-regulated the results are different than what expected. Probably in these cases the high gene density of chromosome 19 can play a decisive role in the localization of some region.
This results reveal that regional gene density is a decisive parameter determining the radial position of chromatin in the nucleus, even more than transcriptional activity.
|
