Hematopoiese

Interesse da Investigação

Hematopoietic differentiation
1) The role of 3D genomic organization as an epigenetic mechanism for the regulation of gene expression during hematopoietic differentiation
2) Cell-fate decision genes in hematopoietic differentiation.

Membros do Grupo

Projecto de Investigação

The functional organization of chromatin in the nucleus

This research line investigates the functional implications of the 3-D organization of chromatin in the nucleus of hematopoietic cells. The spatial positions of specific genes as well as heterochromatic centromeric regions are analysed in3D preserved cells using in situ hybridisation and confocal microscopy. We have shown that genes commonly involved in chromosomal translocations in leukaemia have an intrinsic spatial dynamics that is established early in hematopoiesis, and perpetuated differentially in distinct cell lineages, what may facilitate their collision and reciprocal recombination at subsequent stages of hematopoietic differentiation (Neves et al, Blood, 1999, 93:1197), a phenomenon which may be mechanistically relevant for the occurrence of oncogenic gene rearrangements in human leukemia. As to the spatial organization of heterochromatic regions, we observed that chromocenters (associations of centromeres) present in quiescent lymphoid and non-lymphoid peripheral blood cells represent cell-type-specific arrangements of centromeric heterochromatin (Alcobia et al, Blood, 2000, 95:1608). These observations were followed by the analysis of the dynamics of these spatial arrangements during hematopoietic differentiation. The spatial patterns of association of different centromeres were analysed in CD34+ cells and compared with those in early-B, early T cells, mature B and T lymphocytes. Those patterns were shown to change during lymphoid differentiation, with major spatial arrangements taking place at different stages during T and B-cell differentiation. Heritable patterns of centromere association are observed, which can occur either at the level of the common lymphoid progenitor, or in early-T or early-B committed cells. A correlation of the observed patterns of centromere association with the gene content of the respective chromosomes, further suggests that the variation in the composition of these heterochromatic structures may contribute to the dynamic relocation of genes in different nuclear compartments during cell differentiation, what might have functional implications for cell-stage-specific gene expression (Alcobia et al, Experimental Cell Research, 2003).

Funding

POCTI/32576/BCI/2000
The functional organization of the chromatin in the nucleus.

Projecto de Investigação

The role of cell-fate decision genes in human hematopoietic differentiation

The choice between alternative cell-differentiation pathways is regulated by direct intercellular contacts mediated by trans-membrane proteins expressed by adjacent and apparently equivalent stem cells. Two protein families involved in this process are the Notch receptors and their ligands, the Delta and Jagged proteins. Both protein families are phylogenetically conserved and involved in several developmental scenarios including the decision processes underlying the functional divergence of CD4/CD8 T lymphocytes and the choice between αβ and γδ T-cell receptors in mouse thymocytes. Using a cell coculture assay we have recently observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B-cell lineage while promoting the emergence of cells with a phenotype of T-cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B- or T-cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population co-expressing CD4 and CD8 (Jaleco et al, J Exp Med, 2001, 194:991-1001). Recently, the effects of these Notch ligands on myeloid development were investigated. A long-term culture assay was used, where bone marrow stromal cells transduced with human Delta-1 or Jagged-1 cDNAs, were co-cultured with normal human progenitors, followed by the analysis of their differentiation potential in methylcellulose clonogenic assays. We observed that Delta1 and Jagged1, when acting in similar microenvironmental conditions, have different effects on the myeloid clonogenicity of CD34+ cells, both at the level of mature bi- and uni-potent progenitors, and differentially regulate the balance between the granulocytic and monocytic cell compartments of myelopoiesis. Microarray analysis of gene expression profiles of CD34+ cells, emerging upon contact with Delta1 or Jagged1, show that the observed phenotypic and functional differences are associated with distinct gene transcription programs in those cells. The results indicate that both Delta1- and Jagged1-mediated Notch signalling may play an important role in the homeostatic equilibrium of distinct myeloid-cell lineages in the bone marrow.

Funding

POCTI/MGI/44111/2002
The role of Notch ligands Delta1 and Jagged1 in normal hematopoietic differentiation.

Colaboradores

IGC, Oeiras, Portugal
In collaboration with Jocelyne Demegeot

Publicações

Alcobia, I., Quina, A.S., Neves, H., Clode, N. and Parreira, L. (2003). The spatial organization of centromeric heterochromatin during normal human lymphopoiesis: evidence for ontogenically determined spatial patterns. Experimental Cell Research 290 :358-369

Parreira, L., Neves, H., Simões, S. (2003). Notch and Lymphopoiesis. A view from the microenvironment. Seminars in Immunology 15 :81-89

Jaleco, A.C., Neves, H., Hooijberg, E., Gameiro, P., Clode, N., Haury, M., Henrique, D., Parreira, L. (2001). Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation. Journal of Experimental Medicine 194 :991-1001

Alcobia, I., Dilão, R. and Parreira, L. (2000). Spatial associations of centromeres in the nuclei of hematopoietic cells: evidence for cell-type specific organizational patterns. Blood 95 :1608-1615

Neves, H., Ramos, C., Gomes da Silva, M., Parreira, A. and Parreira, L. (1999). The nuclear topography of ABL, BCR, PML and RARa genes: evidence for gene proximity in specific phases of the cell cycle and stages of hematopoietic differentiation. Blood 93 :1197-1207

Parreira, L., Telhada, M., Ramos, C., Hernandez, R., Neves, H., Carmo-Fonseca, M. (1997). The spatial distribution of human immunoglobulin genes within the nucleus: evidence for gene topography independent of cell type and transcriptional activity. Human Genetics 100 :588-594

Carmo-Fonseca, M., Cunha, C., Custódio, N., Carvalho, C., Jordan, P., Ferreira, J., Parreira, L. () . :. (1996). The topography of chromosome and genes in the nucleus. Exp Cell Res 229 :247

Carvalho, C., Telhada, M., Carmo-Fonseca, M. and Parreira, L. (1995). In situ visualization of immunoglobulin genes in normal and malignant lymphoid cells. J Mol Pathol. 48 :M158-M164

Telhada, M., Carvalho, C., Carmo-Fonseca, M. and Parreira, L. (1995). Influence of transcription and replication on the in situ resolution of immunoglobulin heavy-chain constant region genes. An interphase cytogenetics analysis. Human Genetics 96 :552-561