Abstract
The SCL/tal-1 gene (hereafter designated SCL) encodes a basic helix-loop-helix transcription factor which is pivotal for the normal development of all hematopoietic lineages and which is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. The molecular basis for expression of SCL in stem cells and its subsequent modulation during lineage commitment is of fundamental importance for understanding how early "decisions" are made during hematopoiesis. We now compare the activity of SCL promoters 1a and 1b in erythroid cells and in CD34 positive primitive myeloid cells. SCL mRNA expression in CD34 positive myeloid cells did not require GATA-1. Promoter 1a activity was weak or absent in CD34 positive myeloid cells and appeared to correlate with the presence or absence of low levels of GATA-1. However, promoter 1b, which was silent in committed erythroid cells, was strongly active in transient assays using CD34 positive myeloid cells, and functioned in a GATA-independent manner. Interestingly, RNase protection assays demonstrated that endogenous promoter 1b was active in both erythroid and CD34 positive myeloid cells. These results demonstrate that fundamentally different mechanisms regulate the SCL promoter region in committed erythroid cells and in CD34 positive myeloid cells. Moreover these observations suggest that in erythroid, but not in CD34 positive myeloid cells, promoter 1b required integration in chromatin and/or additional sequences for its activity. Stable transfection experiments showed that both core promoters were silent following integration in erythroid or CD34 positive myeloid cells. Our data therefore indicate that additional regulatory elements were necessary for both SCL promoters to overcome chromatin-mediated repression.
Highlights
The SCL/tal-1 gene encodes a basic helix-loop-helix transcription factor which is pivotal for the normal development of all hematopoietic lineages and which is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells
GATA-1 mRNA was undetectable in M1 cells and only very low levels were observed in 416B cells
GATA-1 has been shown to be important for transcriptional regulation of SCL in erythroid cells, the molecular basis for SCL expression in other hematopoietic cell types is unknown
Summary
The SCL/tal-1 gene (hereafter designated SCL) encodes a basic helix-loop-helix transcription factor which is pivotal for the normal development of all hematopoietic lineages and which is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. RNase protection assays demonstrated that endogenous promoter 1b was active in both erythroid and CD34 positive myeloid cells These results demonstrate that fundamentally different mechanisms regulate the SCL promoter region in committed erythroid cells and in CD34 positive myeloid cells. These observations suggest that in erythroid, but not in CD34 positive myeloid cells, promoter 1b required integration in chromatin and/or additional sequences for its activity. Since lineage commitment and differentiation involve alterations in patterns of gene expression, the function and regulation of lineage-restricted transcription factors are of central importance for the behavior and subsequent fate of hematopoietic stem cells [1, 2]. Despite its implicit involvement in several physiological processes, target genes for SCL have not yet been identified
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